"SCREENING ASSAYS, MODULATORS AND MODULATION OF ACTIVATION OF RECEPTOR FOR ADVANCED GLYCATION END-PRODUCTS (RAGE)"

FIELD OF THE INVENTION

[0001 ] This invention relates generally to screening assays for identifying modulators of activation of receptors associated with certain diseases and/or conditions, to such modulators, and to methods of treatment comprising administration of such modulators. More specifically, the invention relates to modulators of activation of the Receptor for Advanced Glycation End-products (RAGE) via RAGE ligand-independent mechanisms by certain co-located, activated G Protein-Coupled Receptors (GPCRs) (also known as RAGE ligand-independent transactivation of RAGE), including activated type 1 angiotensin receptor (AT ₁ R) and activated CC chemokine receptor 2 (CCR2), with or without also modulating activation of RAGE by RAGE ligands, including S100A8/A9, advanced glycation end products (AGEs) and HMGB1 . This invention also relates to screening assays for identifying such modulators, and to methods of treatment of RAGE-related disorders using said modulators.

BACKGROUND OF THE INVENTION

[0002] The Receptor for Advanced Glycation End-products (RAGE) is a multivalent type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily (Neeper et al., 1992). The 50-55 kDa glycosylated RAGE protein is constitutively expressed in a limited range of cells (e.g. vascular endothelium, type II pneumocytes, leukocytes), although RAGE expression may be induced in most cell types and tissues following injury and inflammation (Ballinger et al., 2005). RAGE expression is markedly upregulated in important inflammatory and metabolic disorders including but not limited to cardiovascular disease (CVD), cancer, diabetes, chronic kidney disease (CKD), ischaemic injury and Alzheimer's disease (Yan et al., 2010).

[0003] It has previously been demonstrated that genetic deletion of the AGER gene coding for RAGE results in protection from a number of diseases and disease processes in mice, including some cancers (Malik et al., 2015) and inflammatory disorders (Chuah et al., 2013) including atherosclerosis and diabetic complications. For example, in apolipoprotein E (apoE) knock-out (KO) mice deletion of RAGE results in less plaque accumulation with age and attenuates diabetes-accelerated atherosclerosis (Soro-Paavonen et al., 2008). Similarly, deletion of AGER is able to attenuate renal injury in diabetic mice without affecting glucose control (Thomas et al., 2005).

[0004] Polymorphisms in the AGER gene have been associated with a number of diseases and disease processes in humans, including but not limited to arthritis, atherosclerosis and diabetic complications, cancer risk, obesity, epilepsy, and cognitive impairment including Alzheimer's disease.

[0005] Binding to the ectodomain of RAGE by Advanced Glycation End-products (AGEs) and non-AGE ligands (including members of the S100 calgranulin family of proteins, HMGB1 , amyloid and Mac-1 ) activates a range of signal transduction cascades implicated in inflammation, injury and dysfunction, including nuclear factor kappa B (N FKB) and the renin- angiotensin aldosterone system (RAAS).

[0006] In experimental models, inhibition of ligand-mediated activation of RAGE using a soluble decoy receptor attenuates atherogenesis and vascular injury (Schmidt et al. 1999), implying that the pathological actions of RAGE are partly mediated by ligand-mediated activation of RAGE in these settings.

[0007] The precise molecular mechanisms by which RAGE is activated and asserts all its biological effects are poorly understood, and thus the ability to target these clinically important signalling pathways has not yet occurred in a clinical setting.

[0008] The renin-angiotensin aldosterone system (RAAS) is a key homeostatic pathway that is also implicated in the development and progression of many common diseases and disease processes. Inhibition of the renin-angiotensin aldosterone system (RAAS) with angiotensin-converting enzyme (ACE) inhibitors, or angiotensin II receptor type 1 ( AT ₁ R) blockers (inhibitors) is widely used for the management of many diseases and/or conditions including hypertension, cardiovascular disease (CVD), heart failure, chronic kidney disease (CKD), and diabetic complications. RAAS inhibition has also been shown to have benefits in preventing diabetes (Tikellis et al., 2004), in neuroprotection (Thoene-Reineke et al., 201 1 ), modifying the growth of certain cancers (Shen et al., 2016) and even in ageing, with genetic deletion of AT ₁ R conferring longevity in mice (Benigni et al., 2009).

[0009] These actions of RAAS blockers are additional to and independent of blood pressure lowering conferred by RAAS blockers, as comparable lowering of the blood pressure with other agents does not confer the same benefits (Lee et al., 1993). Specifically, activation of the AT ₁ R by angiotensin II (Ang II) triggers induction of oxidative stress, activation of Nuclear Factor κΒ (N FKB) and inflammation through pathways that are distinct from those that cause vasoconstriction.

[00010] Activation of the renin-angiotensin aldosterone system (RAAS) is known to be an important mediator of atherosclerosis (Lee et al., 1993; and Jacoby et al., 2003). Atherogenesis is increased following an infusion of angiotensin (Ang) II and in experimental models is associated with physiological RAAS activation, including a low salt diet (Tikellis et al., 2012), diabetes (Goldin et al., 2006; and Soro-Paavonen et al., 2008) and genetic deletion of angiotensin converting enzyme 2 (Ace2) (Thomas et al., 2010), independent of its effects on blood pressure homeostasis. Similarly, inhibition of the RAAS has anti- atherosclerotic actions that are additional to and independent of lowering systemic blood pressure (Candido et al., 2002; Candido et al., 2004; and Knowles et al., 2000). Ang II has a number of direct pro-atherosclerotic effects (Daugherty et al., 2000; Ferrario et al., 2006; and Ekholm et al., 2009), including the induction of oxidative stress (Rajagopalan et al., 1996), vascular adhesion (Grafe et al., 1997) and inflammation (Marvar et al., 2010).

[0001 1 ] These pro-atherosclerotic actions are thought to be primarily mediated by activation of the type 1 angiotensin receptor (AT ₁ R) and subsequent induction of reactive oxygen species (ROS) and activation of N FKB signalling (Li et al., 2008). However, the signalling mechanisms that underlie these actions are poorly understood, including their relative independence from conventional vasoconstrictor signalling via the AT ₁ R.

[00012] The pathogenesis of atherosclerosis has also been shown to involve certain chemokine signalling pathways, with the infiltration of macrophages into arterial lesions directly contributing to this aberrant inflammatory disorder (Boisvert et al., 2004). Indeed, all known CC and CXC chemokine receptors, as well as CX3CR1 and XCR1 , have been implicated in inflammation (Murphy et al. 2000; Zlotnik and Yoshie 2000). The primary physiological function of chemokine ligands (CCLs) is the regulation of "cell migration during routine immune surveillance, inflammation and development". (Allen et al., 2007). CCLs are released in response to pro-inflammatory cytokines and selectively bind to a large family of G protein-coupled receptors, which mediate the physiological responses to chemokines. Chemokines were originally referred to as chemotactic cytokines.

[00013] Animal model studies of chronic inflammatory diseases have demonstrated that inhibition of binding between MCP-1 (monocyte chemotactic protein-1 , also known as monocyte chemoattractant protein-1 , monocyte chemotactic and activating factor (MCAF) and chemokine (C-C motif) ligand 2 (CCL2)) and CCR2 (chemokine (C-C motif) receptor 2) by an antagonist suppresses the inflammatory response. The interaction between MCP-1 and its cognate receptor CCR2 has been implicated (Rollins, 1996; Dawson et al., 2003) in inflammatory disease pathologies such as uveitis, atherosclerosis, rheumatoid arthritis, multiple sclerosis, Crohn's Disease, nephritis, organ allograft rejection, fibroid lung, renal insufficiency, diabetes and diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic retinitis, diabetic microangiopathy, tuberculosis, sarcoidosis, invasive staphylococcal infection, inflammation after cataract surgery, allergic rhinitis, allergic conjunctivitis, chronic urticaria, allergic asthma, periodontal diseases, periodonitis, gingivitis, gum disease, diastolic cardiomyopathies, cardiac infarction, myocarditis, chronic heart failure, angiostenosis, restenosis, reperfusion disorders, glomerulonephritis, solid tumours and cancers, chronic lymphocytic leukaemia, chronic myeloid leukaemia, multiple myeloma, malignant myeloma, Hodgkin's disease, and carcinomas of the bladder, breast, cervix, colon, lung, prostate, or stomach.

[00014] Both MCP-1 and CCR2 KO mice have demonstrated that monocyte infiltration into inflammatory lesions is significantly decreased in the absence of these signalling pathways. In addition, such KO mice are resistant to the development of experimental allergic encephalomyelitis (EAE, a model of human Multiple Sclerosis), cockroach allergen- induced asthma, atherosclerosis, and uveitis. Rheumatoid arthritis and Crohn's Disease patients have improved during treatment with TNFa antagonists (e.g., monoclonal antibodies and soluble receptors) at dose levels correlated with decreases in MCP-1 expression and the number of infiltrating macrophages.

[00015] MCP-1 has been implicated in the pathogenesis of seasonal and chronic allergic rhinitis, having been found in the nasal mucosa of most patients with dust mite allergies. MCP-1 has also been found to induce histamine release from basophils in vitro. During allergic conditions, both allergens and histamines have been shown to trigger (i.e. to up- regulate) the expression of MCP-1 and other chemokines in the nasal mucosa of people with allergic rhinitis, suggesting the presence of a positive feedback loop in such patients.

[00016] Kidney disease is associated with chronic inflammation characterised by the accumulation of kidney macrophages. The production of monocyte chemoattractant protein- 1 (MCP-1 /CCL2) by diabetic kidneys has been identified as a major factor influencing macrophage accumulation in the kidney disease arising from diabetic nephropathy (Tesch ei al., 2008). In various animal models inhibition of CCR2 and/or inhibition of specific CCR2 pathways and/or inhibition of the CCR2 ligand MCP-1 has been shown to reduce kidney damage (Tesch et al., 2008; Rao V et a., 2006; Kang et al., 2010; Kitagawa et al., 2004; Park J et al., 2008). [00017] Tesch (2008) notes that selective targeting of MCP-1 has been proven to be an effective treatment in suppressing animal models of kidney disease that include diabetic nephropathy. Treatments including small molecular antagonists of CCR2 (INCB3344, propagermanium, RS-504393) have been shown to suppress inflammation in mouse models of multiple sclerosis, renal ischemia-reperfusion injury, ureteric obstruction, and diabetic nephropathy and in a rat model of arthritis. Engineered biological antagonists of CCR2 have also proven effective. Subcutaneous infusion of cells transfected with a vector expressing a truncated inactive form of MCP-1 has been found to suppress the development of renal inflammation in a mouse model of lupus nephritis. Similarly, muscle transfection with 7ND (a mutant of MCP-1 ) reduces renal inflammation in mouse models of renal ischemia- reperfusion injury, lupus nephritis, and diabetic nephropathy. Human trials of chemokine monotherapies for inflammatory diseases, to date, have not led to drug approvals. Anders HJ et al. considers reasons why single chemokine antagonist treatments have not been effective in disease treatments and discuss possible explanations including redundancy of single chemokine mediators and variable expression patterns of chemokine receptors (Anders HJ et al. 2010). Therefore, there exists a need in the art for an effective treatment of diseases that are caused through activation of CCR2 pathways.

[00018] It is important to note that the concept that RAGE can be activated in a RAGE ligand-independent manner via an activated, co-located GPCR that is the subject of this invention has implications for a number of GPCRs, particularly those associated with inflammation and cellular proliferation.

[00019] It is against this background that the novel functional interactions between certain activated co-located GPCRs, including the AT ₁ R and CCR2, and RAGE in a RAGE ligand-independent manner, are described.

SUMMARY OF THE INVENTION

[00020] It is known that RAGE signalling, the renin angiotensin aldosterone system (RAAS) and certain chemokine signalling pathways interact functionally in pathways implicated in the development and progression of vascular complications. For example, binding of RAGE ligands to RAGE is able to induce pro-inflammatory signalling, which can be reduced by antagonists (inhibitors) of the AT ₁ R (e.g. Fukami et al. 2004). Equally, activation of the AT ₁ R receptor by Ang II increases the formation and release of RAGE ligands, and inhibition of RAGE ligand-binding to RAGE or interventions to reduce RAGE ligands are able to attenuate Ang II-AT1 R induced injury (e.g. Thomas et al. 2005). Some of the downstream signalling pathways and mediators induced following activation of RAGE by RAGE ligands, especially those that lead to inflammation, are also similar to those signalling pathways and mediators induced following activation of AT ₁ R by Ang II (e.g. N FKB activation).

[00021 ] This prior art does not suggest or disclose any evidence for complexing between RAGE and a GPCR, such as an angiotensin receptor, such as AT ₁ R, or certain chemokine receptors, such as CCR2. Nor does it anticipate that activation of a co-located GPCR by that GPCR's cognate ligand, such as an angiotensin receptor by Ang II or CCR2 by MCP-1 , would directly result in activation of RAGE, in particular the cytosolic tail of RAGE, nor the subsequent induction of signalling via RAGE, in the absence of any RAGE ligand, or indeed without requiring the presence of the RAGE ligand-binding ectodomain of RAGE. Consequently, it could not be anticipated that modulation of RAGE ligand-independent activation of the cytosolic tail of RAGE would involve modulation of signalling induced following activation of a certain co-located GPCR, such as by binding of Ang II to the AT ₁ R or MCP-1 to CCR2.

[00022] One of the salient features of RAGE is its activation by multiple ligands at multiple sites on its ectodomain, rather than a single ligand and a single binding site amenable to inhibition. RAGE may be activated by Advanced Glycation End-products (AGEs) and other non-AGE ligands including high mobility group box-1 (HMGB-1 ), S- 100/calgranulins, SAA, Αβ, C3a, heat shock protein 70 (HSP70), the matricellular injury- related glycoprotein secreted protein acidic and rich in cysteine (SPARC), the β2-integrin Mac-1 (CD1 1 b), phosphatidylserine (PS), double-stranded DNA (dsDNA), double stranded RNA (dsRNA), lipopolysaccharides (LPS) and advanced oxidation protein products.

[00023] Activation of the ectodomain of RAGE by RAGE ligands triggers the activation of N FKB and subsequent NFKB-driven gene expression leading to inflammation, oxidative stress, fibrogenesis and cellular proliferation (Bierhaus et al., 2001 ).

[00024] RAGE ligand-induced signalling also triggers a positive feedback loop in which RAGE ligand-receptor interaction increases expression of RAGE via N FKB activation, thereby augmenting subsequent RAGE-induced cellular activation. In fact, the only means the inventors know to strongly downregulate RAGE expression is to reduce activation of RAGE. This situation contrasts with other receptors, such as the low-density lipoprotein (LDL) receptor, in which increased levels of ligand decrease expression of the receptor.

[00025] Importantly, the inventors have shown that following activation of certain co- located GPCRs, such as AT ₁ R by Ang II or CCR2 by MCP-1 , the cytosolic tail of RAGE is activated, independently of any RAGE ligand or the ectodomain of RAGE, initiating downstream signalling leading to activation of N FKB, a key transcription factor implicated in inflammation, oxidative stress, fibrogenesis, cellular proliferation and cellular survival. The absence of RAGE expression, and specifically the absence of expression of a key domain in the RAGE cytosolic tail, independent of expression of the RAGE ectodomain, prevents the induction of N FKB activation following activation of the co-located GPCR, such as AT ₁ R by Ang II or CCR2 by MCP-1 . Without wishing to be bound by theory, the inventors believe that RAGE ligand-independent activation of RAGE cytosolic tail by certain co-located activated GPCRs, including AT ₁ R and CCR2, is a dominant pathway by which RAGE is activated. Moreover, and again without wishing to be bound by theory, the inventors believe the de novo expression of RAGE in cells subjected to injury, stress or hypoxia for example, provides a conduit for pro-inflammatory signalling to occur via activation of established GPCR signalling.

[00026] The inventors have shown that RAGE ligand-independent activation of the RAGE cytosolic tail following activation of certain co-located GPCRs, such as AT ₁ R by Ang II or CCR2 by MCP-1 , also triggers signalling to increase RAGE expression.

[00027] RAGE has been implicated in many aspects of tumour biology including growth, migration and invasion of tumour cells (Malik et al., 2015; Abe et al., 2008). Many cancers have higher levels of RAGE (illustrative examples are breast, colon, kidney and stomach cancer; Taguchi et al., 2000). The exception is lung cancer in which RAGE expression is reduced as RAGE is a normal part of lung function and is lost as lung cells differentiate and become more malignant (Marinakis et al., 2014). In C6 glioma cells, tumour volume is markedly diminished in tumours comprised of cells in which RAGE was blocked. In contrast, tumours overexpressing RAGE grew rapidly and invaded the surrounding tissue very efficiently (Taguchi et al., 2000). Calls for therapeutics to block RAGE signalling as a cancer treatment have been made for many common cancers including but not limited to: glioma/medulloblastoma multiforme (Taguchi et al., 2000); pancreatic cancer (Malik et al., 2015; Leclerc et al., 2015); melanoma (Malik et al., 2015); prostate cancer (Malik et al., 2015); breast cancer (Malik et al., 2015); liver cancer/hepatoma (Logsdon et al., 2007; Volz et al., 2010); and colon cancer (Sparvero et al., 2009).

[00028] RAGE has been implicated in a range of brain disorders including but not limited to Alzheimer's Disease in which preclinical and clinical studies have supported that RAGE inhibitors could be useful in its treatment (Cai et al., 2016). Other brain conditions in which RAGE signalling is implicated include but are not limited to: amylotrophic lateral sclerosis (Ray et a/ 2016); Huntington's Disease (Ray et al 2016); Creutzfeld-Jakob's disease (Ray et al 2016); neurodegenerative conditions such as diabetic neuropathy, familial amyloid polyneuropathy, Charcot neuroarthropathy and vasculitic neuropathy (Ray et al 2016); neuropathic pain (Wan et al., 2016); glioma development and progression (Angelopoulou et al., 2016); and ischaemic brain injury/stroke (Xia et a/ 2010).

[00029] Under healthy conditions, the lungs' expression of RAGE is the highest of all tissues. However, RAGE expression in the lung is normally only seen in type 1 pneumocytes. Upregulation of RAGE signalling in the lung in other cells and at other sites has been implicated in a range of lung disorders including but not limited to: chronic obstructive pulmonary disease (COPD)/emphysema (Sukkar et al., 2012); asthma (Sukkar et al., 2012); injury due to cigarette smoking/pollution; acute lung injury/ Acute Respiratory Distress Syndrome (ARDS) (Guo et al., 2012); and pulmonary fibrosis.

[00030] RAGE is critically involved in a number of inflammatory conditions and is consequently a potential therapeutic target for their treatment. Such conditions include but are not limited to: inflammatory arthritis (Sparvero et al., 2009; Chuah et al., 2013); osteoarthritis (Xie et al., 2013); retinal disease (Barile et al., 2007); atherosclerosis (Soro- Paavonen et al., 2008; Schmidt et al., 1999; Park et al., 1998; Zhou et al., 2003; Yan et al., 2010); vascular calcification (Ott et al., 2014); cardiomyopathy (Volz et al., 2010; Russo et al., 2016); ischaemic cardiac disease/cardiac remodelling/fibrosis (Yan et al., 2010; Ramasamy et al., 2012); heart failure (Ramasamy et al., 2012); diabetic and non-diabetic kidney disease (Fukami et al., 2015; Gugliucci et al., 2014); inflammatory bowel disease (Ott et al., 2014); pre-eclampsia (Daffu et al., 2013); polycystic ovarian syndrome (Garg et al., 2015); hepatic steatosis, fibrosis, ischemic and non-ischemic liver injury (Yamagishi et al., 2015); spinal cord injury (Yamagishi et al., 2015); skin inflammation and ageing (Tong et al., 2014); and keratitis (Tong et al., 2014).

[00031 ] The present invention arises in part from the determination by the inventors that RAGE forms a receptor heteromer complex in the cell membrane with certain co-located GPCRs, including AT ₁ R and CCR2.

[00032] Furthermore, the present invention arises in part from a recognition by the inventors that activation of certain co-located GPCRs, such as the angiotensin receptor, in the form of AT ₁ R, in this case with Ang II, or certain chemokine receptors such as CCR2 in this case with MCP-1 , triggers RAGE ligand-independent activation of the cytosolic tail of RAGE. [00033] The inventors have shown that activation of certain co-located GPCRs, such as the AT ₁ R by Ang II or CCR2 by MCP-1 , results in the activation of a domain of the cytosolic tail of RAGE through a common mechanism. This pathway of transactivation does not require the liberation of RAGE ligands or require their binding to the ectodomain of RAGE (i.e. it is RAGE ligand-independent activation of RAGE).

[00034] Even though there are published data suggesting that the cytosolic tail of RAGE is phosphorylated (Sakaguchi et al., 201 1 ), the inventors have shown that RAGE ligand- independent signalling induced following activation of certain co-located GPCRs, such as the AT ₁ R receptor by Ang II, does not require the cytosolic tail of RAGE to be phosphorylated at Serine391 or any other site in the cytosolic tail of RAGE. Furthermore, the inventors have shown that RAGE ligand-dependent signalling induced following RAGE ligand (e.g. S100A8/A9) binding to the ectodomain of RAGE also does not necessarily require the cytosolic tail of RAGE to be phosphorylated at Serine391 or any other site in the cytosolic tail, as RAGE homologs from other mammals and RAGE mutants devoid of any residues capable of sustaining phosphorylation are still able to be activated and induce signalling in response to RAGE ligand-dependent and RAGE ligand-independent activation of RAGE. Furthermore, the inhibitory functions of N-truncated constructs of RAGE (e.g. S391 A- RAGE362-404) are maintained in the absence of targets for RAGE phosphorylation, confirming that the modulatory effects of the RAGE constructs described by the inventors are independent of phosphorylation of RAGE.

[00035] Prior art demonstrates that inhibitors of ΡΚCζ inhibit RAGE ligand-dependent (e.g. s100-induced) signalling via RAGE, as well as many other PKCζ-dependent pathways. In humans and animals severe illness results from genetic deletion ofΡΚCζ . The investigators have shown that inhibitors of ΡΚCζ also inhibit RAGE ligand-independent (i.e. transactivation-induced) signalling via full-length RAGE. However, the modulatory functions of N-truncated constructs of RAGE (e.g. RAGE362-404) are not affected by inhibition ofΡΚCζ , confirming that the modulatory effects of the RAGE constructs described by the inventors are independent ofΡΚCζ .

[00036] Inhibitors of the shared pathways induced subsequent to RAGE activation (e.g. myD88, TIRAP, interleukin-1 receptor-associated kinase 4 (IRAK4) or N FKB) non-specifically block both RAGE ligand-dependent (e.g. s100-induced) and RAGE ligand-independent (i.e. transactivation-induced) signalling via RAGE. As other receptors (e.g. TLRs) also use these signalling molecules/pathways, inhibition of any of these mediators would not be specific to RAGE signalling and impact on the many other functions of these signalling mediators, which may be deleterious to human health (e.g. genetic deletion of myD88, TIRAP, IRAK4 or N FKB are harmful to humans and animals, unlike RAGE deletion).

[00037] The inventors have further shown that selective modulation, such as inhibition, of RAGE ligand-independent signalling can be achieved by selectively targeting signalling mediated through the cytosolic tail of RAGE, and the inventors' assays and modulators identified therefrom, act upon this transactivation (RAGE ligand-independent activation of RAGE) process.

[00038] The inventors have further shown that dual inhibition of RAGE ligand-dependent activation of RAGE and RAGE ligand-independent transactivation of RAGE signalling can also be achieved through selectively targeting signalling mediated through the cytosolic tail of RAGE, and the inventors' assays and modulators identified therefrom are able to act simultaneously upon both modalities of RAGE activation due to shared mediators. This is in direct distinction to soluble RAGE22-331 , RAGE neutralising antibodies and small molecules that selectively bind to the ectodomain of RAGE and can only potentially inhibit RAGE ligand-dependent activation of RAGE.

[00039] The inventors have further shown that modulation of RAGE ligand-dependent signalling and/or RAGE ligand-independent transactivation of RAGE signalling by selectively targeting signalling mediated through the cytosolic tail of RAGE can be achieved without modulation of the interaction of RAGE and Diaphanous-1 (Diaphl ), that prior art suggests is potentially a modulator of ligand-dependent RAGE activation (Manigrasso, M.B., et al 2016). Furthermore, the modulatory functions of N-truncated constructs of RAGE (e.g. RAGE362-404) are maintained in the absence of Diaphl , confirming that the modulatory effects of the RAGE constructs described by the inventors are independent of Diaphl

[00040] Sakaguchi and co-workers found that common pro-inflammatory adaptor proteins TIRAP, MyD88 and IRAK were co-precipitated with overexpressed RAGE in HEK293 cells predominantly when the cells were treated with RAGE ligands, S100A1 1 , S100A12, HMGB1 or AGEs, resulting in the RAGE ligand-dependent activation of RAGE. These interactions are not specific to RAGE, as TIRAP, MyD88 and IRAK also function as adaptor proteins for all toll-like receptors (TLRs) except TLR-3, to activate the transcription of N FKB.

[00041 ] Following this work, the same group published research proposing S391 E- RAGE ₃ 87-395 (RAGE(E)-I) as an inhibitor of particular aspects of RAGE ligand-dependent signalling (namely inhibition of apoptosis, cell migration and invasion) through mimicking the phosphorylated state of RAGE and sequestering the adaptor protein TIRAP, and thereby preventing endogenous RAGE signalling (Putranto et al., 2013). However, the inventors have shown that phosphorylation is not required for RAGE activation. Moreover, sequestering these common adaptor proteins will also impact on signalling through TLRs (e.g. TLR-2 and TLR-4), some of which may also be activated by RAGE ligands (e.g. s100 proteins) which likely explains the findings of Puranto et al. In the same experiments, they also argued that S391 A-RAGE _387-395 was not a suitable inhibitor as it did not show any appreciable binding to TIRAP and did not attenuate apoptosis induced by RAGE ligand S100B (Putranto et al., 2013). They also noted that S391 E-RAGE _387-395 did not inhibit all RAGE-ligand induced signalling pathways, as the growth of U-87MG cells was not significantly affected as assessed by determining intracellular adenosine triphosphate content (Putranto et al., 2013). Therefore, the RAGE ligand-dependent pathway putatively inhibited by Putranto and co-workers and the fragment of RAGE cytosolic tail that they utilised are demonstrably distinct from the RAGE ligand-independent activation of RAGE by co-located activated GPCR and the modulators that are the subject of this invention. Indeed, their negative findings with S391 A-RAGE387-395 teach away from the current invention. At no point in this publication did Putranto et al contemplate RAGE ligand-independent activation of the cytosolic tail of RAGE by co-located GPCRs.

[00042] EP 1 415 997-A1 details the identification and use of polypeptides to bind directly or indirectly to the cytosolic tail of RAGE and thereby inhibit or augment the signal transduction arising from binding of a ligand to RAGE and subsequent activation of N FKB and downstream pathways arising from its activation. The present invention is distinct from this teaching in a number of ways. Firstly, this teaching does not contemplate RAGE ligand- independent signalling via RAGE or dual inhibition of RAGE ligand-dependent and RAGE ligand-independent signalling via RAGE. Secondly, claims in EP 1 415 997 A1 relate to use of polypeptides to bind to unidentified elements in the cytosolic tail of RAGE. By contrast, the present inventors have demonstrated that a polypeptide coding for the cytosolic tail of RAGE, and mutated forms of such, can be used to selectively bind signalling molecules associated with RAGE ligand-independent signalling via RAGE or RAGE ligand-dependent and RAGE ligand-independent signalling via RAGE leading to the modulation of the subsequent activation of N FKB and downstream pathways arising from its activation. Thirdly, the inventors have demonstrated the ability to modulate RAGE ligand-independent signalling via RAGE using selectively modified polypeptides containing key elements of the RAGE cytosolic tail. Fourthly, no modulation of RAGE ligand-dependent signalling via RAGE is shown in EP 1 415 997-A1 . Moreover, the only polypeptide capable of modulating RAGE ligand-dependent signalling specifically identified in EP 1 415 997-A1 is Ρ,Κ a well -known binding partner and signalling mediator of full length RAGE. The present inventors show that ΡΚCζ is not required for the actions of their modulators.

[00043] RAGE is mono-ubiquitinated by the F-box protein, FBXO10, at cytosolic residue K374 following binding of CpG-DNA, triggering its endocytosis and lysosomal-mediated degradation (Evankovich et al. 2017). Endocytosis and/or RAGE ubiquitination has not been observed with other pro-inflammatory RAGE ligands.

[00044] Ubiquitination of RAGE is partly dependent on S391 , such that S391 A-RAGE mutants are partly resistant to ubiquitination and subsequent degradation following FBXO10 overexpression.

[00045] These data suggest that K374R and S391 A-RAGE mutants may have resistance to ubiquitination in certain circumstances, potentially allowing them to accumulate in higher levels than wild-type RAGE. However, this potentially increased stability/resistance to degradation cannot explain the rapid modulation of RAGE ligand-independent activation of RAGE following activation of a co-located GPCR, as well as inhibition of RAGE ligand- independent activation of RAGE signalling achieved by S391 A-RAGE mutants even in the presence of wild-type RAGE delivered in 1000-fold excess, detailed below, nor that this modulation occurs similarly in the presence and absence of K374.

[00046] Without limiting the generality of the following description of the invention, the inventors have demonstrated that activation of certain co-located GPCRs such as the AT ₁ R, such as by Ang II, or CCR2, such as by MCP-1 , triggers activation of co-located cytosolic tail of RAGE. This activation is able to take place in the absence of the ectodomain of RAGE and therefore is entirely independent of RAGE ligands or their interaction with the ectodomain of RAGE. Without wishing to be bound by theory, the inventors believe that this transactivation of RAGE by certain co-located GPCRs represents the major mechanism of RAGE activation. Consistent with this premise, the inventors demonstrate that selective restoration of RAGE ligand-independent RAGE signalling in AGER apoE double KO (DKO) mice restores atherogenesis to levels not signficiantly different from that observed in apoE KO mice replete in RAGE, even though RAGE-ligand-dependent signalling remains completely absent.

[00047] Many of the adverse signalling events induced by AT ₁ R activation are attenuated when RAGE expression is absent (e.g. genetic deletion or silencing, or in healthy cells not expressing RAGE, or when RAGE ligand-independent activation of RAGE by activated AT ₁ R is prevented or inhibited). [00048] At the same time, RAGE-independent AT ₁ R signalling pathways, such as the Gq signalling pathway induced by AT ₁ R activation leading to the induction of inositol phosphate and calcium influx, which are inhibited by AT ₁ R antagonists, are unaffected by RAGE deletion, silencing of RAGE expression or inhibition of RAGE function.

[00049] As such, modulation, in particular inhibition, of RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs, such as AT ₁ R or CCR2, provides particular advantages for therapeutic interventions targeting pathogenic signalling induced via RAGE following activation of the co-located GPCR. For example, such modulators, in specific embodiments, allow aggressive targeting of the adverse effects of AT ₁ R without compromising blood pressure regulation or inducing feedback "escape" from AT ₁ R inhibition, such as occurs following AT ₁ R inhibition, that limits the use of RAAS inhibitors. Such modulators would also only affect cells and tissues in which this transactivation pathway was constitutively active (e.g. leukocytes, endothelial cells) or induced (e.g. sites of inflammation and injury), leaving the RAAS and other GPCR-mediated signalling unaffected in cells not also expressing RAGE (e.g. healthy smooth muscle cells).

[00050] Activation of AT ₁ R has both hemodynamic and non-hemodynamic effects. Hemodynamic effects are those that lead to changes in blood flow, and include changes in blood volume, blood pressure, flow rate or velocity, resistance, cardiac output, turbulence and wall tension. AT ₁ R blockers (inhibitors) can exhibit both hemodynamic (e.g. lower the blood pressure, alter resistance and cardiac output) as well as non-hemodynamic effects (e.g. trigger oxidative stress and inflammation). In states in which the RAAS is activated (e.g. heart disease, kidney disease, hypertension) both hemodynamic and non-hemodynamic pathways are activated.

[00051 ] RAGE ligand-independent activation of RAGE by activated AT ₁ R is a mediator only of the non-hemodynamic (non-blood flow effects) of AT ₁ R activation. The inventors have observed that total genetic deletion of RAGE has no direct hemodynamic effect (e.g. no effect on blood pressure, vessel resistance or flow, blood volume) and does not modify the hemodynamic effects of AT ₁ R activation or inhibition. The key advantage of targeting RAGE ligand-independent activation of RAGE by activated AT ₁ R is that it is therefore not limited by the constraints of blood pressure regulation, which limit how much blood pressure lowering is possible before adverse hemodynamic effects render said treatment unsafe.

[00052] Moreover, changes in blood flow automatically trigger feedback (homeostatic) responses to maintain blood flow at a constant level. These feedback responses act to offset or escape the hemodynamic effects of inhibition of the RAAS by AT ₁ R inhibition or inhibition of Angiotensin Converting Enzyme (ACE). By contrast, selective inhibition of non- hemodynamic pathways induced following activation of the RAAS achieved through inhibiting RAGE ligand-independent activation of RAGE by activated angiotensin receptor, such as AT ₁ R, is not associated with feedback/escape responses. The absence of such feedback responses supports the durability and efficacy of such inhibition.

Modulators of RAGE ligand-independent activation of RAGE by activated co-located GPCRs

[00053] In one form, the present invention comprises modulators of RAGE activity where such RAGE activity is induced by certain active co-located GPCRs.

[00054] In one form, the present invention comprises modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs.

[00055] In one form, the present invention comprises modulators wherein the modulators are modulators of RAGE-dependent signalling induced by certain activated co-located GPCRs.

[00056] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs act in the absence of any RAGE ligand.

[00057] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs act in the presence of a truncated ectodomain of RAGE.

[00058] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs act in the presence of a truncated ectodomain of RAGE which is not greater than 40, not greater than 20, not greater than 10 or not greater than 5 amino acids in length.

[00059] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs, contain the entire ectodomain of RAGE conjugated to an analogue, fragment or derivative of the transmembrane domain of RAGE which is greater than 5, greater than 10, or greater than 20 amino acids in length.

[00060] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs act in the absence of the RAGE ligand-binding ectodomain of RAGE. [00061 ] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs do not contain the ectodomain of RAGE.

[00062] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs do not contain an analogue, fragment or derivative of the ectodomain of RAGE.

[00063] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs contain a fragment of the ectodomain of RAGE.

[00064] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs contain a fragment of the ectodomain of RAGE, which is not greater than 40, not greater than 20, not greater than 10 or not greater than 5 amino acids in length.

[00065] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs do not bind to the ectodomain of RAGE.

[00066] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit or facilitate signalling that occurs through the C-terminal cytosolic tail of RAGE induced by an activated co-located GPCR.

[00067] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit binding that occurs to the C-terminal cytosolic tail of RAGE.

[00068] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit or facilitate the interaction between the transmembrane domain of RAGE and certain GPCRs.

[00069] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit the interaction between the transmembrane domain of RAGE and certain GPCRs.

[00070] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit or facilitate the capacity of an activated GPCR to modulate RAGE-dependent signalling that is dependent upon proximity of the transmembrane domains of RAGE and the certain GPCR.

[00071 ] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit the capacity of an activated GPCR to modulate RAGE-dependent signalling that is dependent upon proximity of the transmembrane domains of RAGE and the certain GPCR.

[00072] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit or facilitate the capacity of an activated GPCR to modulate RAGE-dependent signalling that is dependent upon proximity of the transmembrane domains of RAGE and the certain GPCR and inhibit or facilitate signalling that occurs through the C-terminal cytosolic tail of RAGE induced by an activated co-located GPCR.

[00073] In one form of the present invention, the modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs inhibit the capacity of an activated GPCR to modulate RAGE-dependent signalling that is dependent upon proximity of the transmembrane domains of RAGE and the certain GPCR and inhibit signalling that occurs through the C-terminal cytosolic tail of RAGE induced by an activated co-located GPCR.

[00074] Throughout this specification, unless the context requires otherwise, a co-located GPCR means a member of the G protein-coupled receptor superfamily (GPCR; also known as seven-transmembrane domain receptors, 7TM receptors, hepta-helical receptors, serpentine receptors, and G protein-linked receptors; some other 7TM proteins have been classified as being members of the G protein-coupled receptor superfamily, including GPR107, GPR137, OR51 E1 , TPRA1 , GPR143 and GPR157) that is co-expressed in the same cell as RAGE, either endogenously or as a result of transfection. Note that not all members of this superfamily couple to G proteins and the term GPCR in this context includes members of the superfamily that do not couple to G protein. Co-expression in the same cell may be demonstrated by a number of techniques known to those skilled in the art, including co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET) and microscopy. A co-located GPCR is preferably a GPCR that is sufficiently proximal to RAGE that functional interaction occurs between the GPCR and RAGE. Even more preferably, a co-located GPCR is a GPCR that is sufficiently proximal to RAGE that a suitable proximity assay is able to detect this proximity. Examples of suitable proximity assays are BRET, FRET, enzyme fragment complementation, split luciferase complementation, split fluorophore complementation, TANGO assay, NanoLuc Binary Technology (NanoBIT) assay, proximity ligation assay (PLA) or any other proximity assay that is able to detect the proximity of two proteins regardless of whether one or more of these proteins is labelled or tagged to facilitate the use of the assay. Such proximity assays can be configured in different ways, and the Receptor-Heteromer Investigation Technology (Receptor-HIT) configuration, and derivations thereof, are preferred configurations of such proximity assays (W02008/055313; Jaeger et al., 2014).

[00075] Throughout this specification, unless the context requires otherwise, an activated GPCR means a GPCR that is in an active state that may result from the binding of an agonist, partial agonist and/or allosteric modulator, and/or as a consequence of constitutive activity that does not necessitate ligand binding.

[00076] Throughout this specification, unless the context requires otherwise, the certain activated co-located GPCRs of the invention are GPCRs that are expressed in the same cell as RAGE and for which an effect on RAGE, indicative of modulation of RAGE activation and/or modulation of induction of RAGE-dependent signalling, is detected upon activation by cognate ligands of the certain co-located GPCRs or when the GPCRs are constitutively active.

[00077] In one embodiment, an effect on RAGE indicative of modulation of RAGE activation is a change in intracellular trafficking such as that detected by a change in proximity of luciferase-conjugated RAGE (such as RAGE/Rluc8) to intracellular compartment markers such as fluorophore-labelled Rabs, such as Rab1 , Rab4, Rab5, Rab6, Rab7, Rab8, Rab9 and/or Rab1 1 (such as Venus-Rab1 , Venus-Rab4, Venus-Rab5, Venus-Rab6, Venus- Rab7, Venus-Rab8, Venus-Rab9 and/or Venus-Rab1 1 ), and/or a plasma membrane marker, such as a fluorophore-conjugated fragment of K-ras (such as Venus-K-ras) using bioluminescence resonance energy transfer (BRET) upon addition of a cognate ligand for the co-located GPCR (Tiulpakov et al., 2016).

[00078] In another embodiment, an effect on RAGE is a change in RAGE-dependent signalling, such as detected by a change in proximity of luciferase-conjugated RAGE (such as RAGE-Rluc8) to a RAGE-interacting group, such as fluorophore-labelled proteins interacting with the cytosolic tail of RAGE, such as IQGAP-1 , protein kinase C zeta (PKCζ, Dock7, MyD88, TIRAP, ERK1/2, (Jules et al., 2013; Ramasamy et al., 2016), olfactory receptor 2T2, ADP/ATP translocase 2, Protein phosphatase 1 G, Intercellular adhesion molecule 1 , Protein DJ-1 (PARK7), Calponin-3, Drebrin, Filamin B, Ras-related protein Rab- 13, Radixin/Ezrin/Moesin, Proteolipid protein 2, Coronin, S100 A1 1 , Succinyl-CoA ligase [GDP-forming] subunit alpha, Hsc70-interacting protein, Apoptosis Inhibitor 5, neuropilin, cleavage stimulation factor, growth factor receptor-bound protein 2, sec61 beta subunit, or Nck1 .

[00079] In another embodiment, an effect on RAGE is a change in RAGE-dependent signalling, such as detected by a change in canonical activation of N FKB upon activation of the certain co-located GPCRs by their cognate ligands as measured by one or more of the following:

• Activity of IkB kinase (IKK) by monitoring in vitro phosphorylation of a substrate, such as GST-ΙκΒα;

• Detection of IkB Degradation Dynamics, including phosphorylation/ubiquitination and/or degradation of IKB and/or ΙκΒ-α;

• Detection of p65(Rel-A) phosphorylation/ubiquitination, such as by using antibodies, gel-shift, EMSA, and/or mass spectroscopy;

• Detection of cytosolic to nuclear shuttling/translocation of N FKB components/subunits, such as p65/phospho-p65;

• Detection of N FKB subunit dimerization/complexation;

• Detection of active N FKB components/subunits by binding to immobilized DNA sequence/oligonucleotide containing the N FKB response element/consensus N FKB binding motif, such as by using electrophoretic mobility shift assay or gel shift assay, SELEX, protein-binding microarray, or sequencing-based approaches;

• Chromatin-immunoprecipitation (ChIP) assays to detect N FKB in situ binding to DNA to the promoters and enhancers of specific genes;

• In vitro kinase assay for N FKB kinase activity;

• Measurement of N FKB transcriptional activity using N FKB reporter assays via transgene expression of reporter constructs, such as LacZ Flue, eGFP SEAP, and NF-gluc, using such approaches as plasmid transfection, reporter cell lines, mini- circles, retrovirus, or lentivirus;

• Measuring changes in expression of downstream targets of N FKB, such as cytokines, growth factors, adhesion molecules and mitochondrial anti-apoptotic genes, by real- time PCR, protein, or functional assays (Note the pleiotropic nature of N FKB is reflected in its transcriptional targets that presently number approximately 500 (see http://www.bu.edu/nf-kb/qene-resources/tarqet-qenes/ as at 2nd August 2017); and

• Measuring changes in function or structure induced by NFKB-dependent signalling, such as POLKADOTS in T-cells, adhesion in endothelial cells, activation in leucocytes, or oncogenicity.

[00080] In another embodiment, an effect on RAGE is a change in RAGE signalling, such as detected by a change in non-canonical activation of N FKB by measuring one or more of the following:

• Detection of NIK (NFKB-lnducing Kinase);

• Detecting IKKa Activation/phosphorylation;

• Detection of NIK kinase activity by ability to autophosphorylate or to phosphorylate a substrate by performing a kinase assay;

• Generation of p52-containing N FKB dimers, such as p52/RelB;

• Detection of Phospho-NFKB2 p100(Ser866/870);

• Detection of partial degradation (called processing) of the precursor p100 into p52;

• Detecting p52/RelB translocation into the nucleus;

• Detecting p52/RelB binding to NFKB sites;

• Measurement of N FKB transcriptional activity using N FKB reporter assays via transgene expression of reporter constructs, such as LacZ Flue, eGFP SEAP, NF- gluc, using such approaches as plasmid transfection, reporter cell lines, mini-circles, retrovirus, or lentivirus; and

• Measuring changes in expression of downstream targets of non-canonical signalling of N FKB, such as CXCL12, by real-time PCR, protein expression or by functional assays. Co-located GPCRs

[00081 ] In one embodiment, the certain activated co-located GPCRs of the invention are those GPCRs that are expressed in the same cell as RAGE and are associated with RAGE- related disorders.

[00082] In one embodiment, the certain activated co-located GPCRs of the invention are those GPCRs that are expressed in the same cell as RAGE, are associated with RAGE- related disorder(s), and upon their removal and/or inhibition result in reduction or alleviation of the RAGE-related disorder(s).

[00083] In one embodiment, the certain activated co-located GPCRs of the invention are those GPCRs that are implicated in inflammation.

[00084] In one embodiment, the certain activated co-located GPCRs of the invention are those GPCRs that are implicated in inflammation, and upon their removal and/or inhibition result in reduction or alleviation of the inflammation.

[00085] In one embodiment, the certain activated co-located GPCRs of the invention are those GPCRs that are implicated in cell proliferation.

[00086] In one embodiment, the certain activated co-located GPCRs of the invention are those GPCRs that are implicated in cell proliferation, and upon their removal and/or inhibition result in reduction or alleviation of the cell proliferation.

[00087] Indeed there is evidence for many GPCRs being involved in inflammation to some degree, and these levels can be differentiated according to the level of evidence:

1 - No evidence found to date;

2 - Receptor structure, or motif within receptor is similar to known

inflammatory/immunological receptor or motif involved in an

inflammatory/immunological process;

3 - Receptor binds a ligand that mediates an inflammatory/immunological process;

4 - Receptor is associated with/involved in an inflammatory/immunological disease;

5 - At least one paper describing direct involvement of receptor in

inflammatory/immunological process; 6 - Receptor is expressed in inflammatory/immune cells; and

7 - Receptor's involvement in inflammatory/immunological processes is well

characterised (as described in http://www.quidetopharmacoloqv.org database).

[00088] Family A GPCRs (except olfactory, vomeronasal, opsins) and the current level of evidence for their involvement in inflammation (see key above):

[00090] Family A vomeronasal and opsin GPCRs and the current level of evidence for their involvement in inflammation (see key above):

[00091 ] Family B GPCRs and the current level of evidence for their involvement in inflammation (see key above):

[00092] Family C GPCRs and the current level of evidence for their involvement in inflammation (see key above):

[00093] Frizzled Family GPCRs and the current level of evidence for their involvement in inflammation (see key above):

[00094] Other 7TM proteins that have been classified as members of the GPCR superfamily and the current level of evidence for their involvement in inflammation (see key above):

[00095] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: ADGRA2, ADGRB2, ADGRB3, ADGRF3, ADGRG4, ADGRV1 , CELSR1 , CELSR2, CELSR3, 0X1 receptor, 0X2 receptor, PTH1 receptor, PTH2 receptor, AMY1 receptor, AMY2 receptor, AMY3 receptor, AM1 receptor, AM2 receptor, GPR63, GPR75, NMU2 receptor, OPN5, V1 B receptor, y6 receptor, 5-HT4 receptor, GPR101 , GPR1 19, GPR135, GPR137, GPR141 , GPR149, GPR150, GPR151 , GPR152, GPR157, GPR19, GPR25, GPR37, GPR37L1 , GPR50, GPR62, LGR5, MRGPRE, MRGPRF, NTS2 receptor, OPN4, OPN4, OR10A7, OR10AG1 , OR10Q1 , OR10W1 , OR12D3, OR13C2, OR13C3, OR13C4, OR13C5, OR13C8, OR13F1 , OR13G1 , OR1 A2, OR1 L1 , OR1 S1 , OR1 S2, OR2AK2, OR2D2, OR2D3, OR4A15, OR4C1 1 , OR4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K15, OR4K17, OR4N5, OR5AC2, OR5AK2, OR5AP2, OR5AR1 , OR5AS1 , OR5B12, OR5B17, OR5B2, OR5B21 , OR5B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1 , OR5I1 , OR5J2, OR5K3, OR5L1 , OR5L2, OR5M1 , OR5M10, OR5M1 1 , OR5M3, OR5M8, OR5M9, OR5R1 , OR5T1 , OR5T2, OR5T3, OR5W2, OR6C74, OR6K6, OR6M1 , OR6Q1 , OR6X1 , OR8H1 , OR8H2, OR8H3, OR8J1 , OR8J3, OR8K1 , OR8K3, OR8K5, OR8U1 , OR8U8, OR9A4, OR9G1 , OR9G4, OR9G9, OR9Q2, TAAR3, TPRA1 , Y4 receptor, 5-HT1 D receptor, 5-HT1 E receptor, ADGRB1 , AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor, GABAB1 , GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER, GPR107, GPR139, GPR156, GPR158, GPR161 , GPR171 , GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor, HCA1 receptor, LPA1 receptor, LPA3 receptor, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1 , MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1 D2, OR2AG1 , OT receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1 A receptor, V2 receptor, a2A-adrenoceptor, a2B-adrenoceptor, a2C-adrenoceptor, β1 -adrenoceptor, β3- adrenoceptor, 5-HT1 B receptor, 5-HT1 F receptor, 5-HT2B receptor, 5-HT2C receptor, 5- HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1 , ADGRG1 , ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1 , FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR11H1, OR2A1, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1 , OR2C3, OR2J3, OR2L13, OR2T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51 B4, OR51B5, OR51 B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1 , OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1 , OR56A3, OR56A4, OR56A5, OR56B1 , OR56B4, OR6V1, OR7D2, OR9A2, oxoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sstl receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenoceptor, a1B-adrenoceptor, a1D-adrenoceptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin receptor, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLTI receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2/ALX, FPR2/ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlul receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y1 1 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PARI , PAR2, PAR3, PAR4, PKR1 , PKR2, S1 P1 receptor, S1 P2 receptor, S1 P3 receptor, S1 P4 receptor, S1 P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1 , β2- adrenoceptor, κ receptor, μ receptor.

[00096] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of CCR4.

[00097] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of CCR5.

[00098] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of CCR4 and CCR5.

[00099] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of CCR4, CCR5, CCR10 and CXCR3.

[000100] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000101 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR1 0, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor. [000102] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adrenergic α1A receptor, CCR3, M2 receptor and 0X1 receptor.

[000103] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adrenergic α1A receptor, CCR3, CCR4, M2 receptor and 0X1 receptor.

[000104] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adrenergic α1A receptor, CCR3, CCR5, M2 receptor and 0X1 receptor.

[000105] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, M2 receptor and 0X1 receptor.

[000106] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, 0X1 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000107] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, M2 receptor and 0X1 receptor.

[000108] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [00095] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, 0X1 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor. [000109] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: 0X1 receptor, 0X2 receptor, PTH1 receptor, PTH2 receptor, AMY1 receptor, AMY2 receptor, AMY3 receptor, AM1 receptor, AM2 receptor, GPR63, GPR75, NMU2 receptor, OPN5, V1 B receptor, y6 receptor, 5-HT4 receptor, GPR101 , GPR1 19, GPR135, GPR137, GPR141 , GPR149, GPR150, GPR151 , GPR152, GPR157, GPR19, GPR25, GPR37, GPR37L1 , GPR50, GPR62, LGR5, MRGPRE, MRGPRF, NTS2 receptor, OPN4, OPN4, OR10A7, OR10AG1 , OR10Q1 , OR10W1 , OR12D3, OR13C2, OR13C3, OR13C4, OR13C5, OR13C8, OR13F1 , OR13G1 , OR1 A2, OR1 L1 , OR1 S1 , OR1 S2, OR2AK2, OR2D2, OR2D3, OR4A15, OR4C1 1 , OR4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K15, OR4K17, OR4N5, OR5AC2, OR5AK2, OR5AP2, OR5AR1 , OR5AS1 , OR5B12, OR5B17, OR5B2, OR5B21 , OR5B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1 , OR5I1 , OR5J2, OR5K3, OR5L1 , OR5L2, OR5M1 , OR5M10, OR5M1 1 , OR5M3, OR5M8, OR5M9, OR5R1 , OR5T1 , OR5T2, OR5T3, OR5W2, OR6C74, OR6K6, OR6M1 , OR6Q1 , OR6X1 , OR8H1 , OR8H2, OR8H3, OR8J1 , OR8J3, OR8K1 , OR8K3, OR8K5, OR8U1 , OR8U8, OR9A4, OR9G1 , OR9G4, OR9G9, OR9Q2, TAAR3, TPRA1 , Y4 receptor, 5-HT1 D receptor, 5-HT1 E receptor, ADGRB1 , AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor, GABAB1 , GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER, GPR107, GPR139, GPR156, GPR158, GPR161 , GPR171 , GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor, HCA1 receptor, LPA1 receptor, LPA3 receptor, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1 , MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1 D2, OR2AG1 , OT receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1 A receptor, V2 receptor, a2A-adrenoceptor, a2B-adrenoceptor, a2C-adrenoceptor, β1 -adrenoceptor, β3- adrenoceptor, 5-HT1 B receptor, 5-HT1 F receptor, 5-HT2B receptor, 5-HT2C receptor, 5- HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1 , ADGRG1 , ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1 , FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1 , GPR1 19, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61 , GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR11H1, OR2A1, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1 , OR2C3, OR2J3, OR2L13, OR2T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51 B4, OR51B5, OR51 B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1 , OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1 , OR56A3, OR56A4, OR56A5, OR56B1 , OR56B4, OR6V1, OR7D2, OR9A2, oxoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sstl receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenoceptor, a1B-adrenoceptor, a1D-adrenoceptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin receptor, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLTI receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2/ALX, FPR2/ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlul receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PARI, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2- adrenoceptor, κ receptor, μ receptor. [0001 10] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of CCR4.

[0001 1 1 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of CCR5.

[0001 12] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of CCR4 and CCR5.

[0001 13] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of CCR4, CCR5, CCR10 and CXCR3.

[0001 14] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0001 15] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0001 16] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: OX1 receptor, OX2 receptor, PTH1 receptor, PTH2 receptor, AMY1 receptor, AMY2 receptor, AMY3 receptor, AM1 receptor, AM2 receptor.

[0001 17] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adrenergic α1A receptor, CCR3, M2 receptor and 0X1 receptor. [0001 18] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adrenergic α1A receptor, CCR3, CCR4, M2 receptor and 0X1 receptor.

[0001 19] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adrenergic α1A receptor, CCR3, CCR5, M2 receptor and 0X1 receptor.

[000120] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, M2 receptor and 0X1 receptor.

[000121 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, 0X1 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000122] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, M2 receptor and 0X1 receptor.

[000123] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000109] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, 0X1 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000124] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: 5-HT4 receptor, GPR101 , GPR1 19, GPR135, GPR137, GPR141 , GPR149, GPR150, GPR151 , GPR152, GPR157, GPR19, GPR25, GPR37, GPR37L1, GPR50, GPR62, LGR5, MRGPRE, MRGPRF, NTS2 receptor, OPN4, OPN4, OR10A7, OR10AG1, OR10Q1, OR10W1, OR12D3, OR13C2, OR13C3, OR13C4, OR13C5, OR13C8, OR13F1, OR13G1, OR1A2, OR1L1, OR1S1, OR1S2, OR2AK2, OR2D2, OR2D3, OR4A15, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K15, OR4K17, OR4N5, OR5AC2, OR5AK2, OR5AP2, OR5AR1, OR5AS1, OR5B12, OR5B17, OR5B2, OR5B21, OR5B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5J2, OR5K3, OR5L1, OR5L2, OR5M1, OR5M10, OR5M11, OR5M3, OR5M8, OR5M9, OR5R1, OR5T1, OR5T2, OR5T3, OR5W2, OR6C74, OR6K6, OR6M1, OR6Q1, OR6X1 , OR8H1, OR8H2, OR8H3, OR8J1, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR8U8, OR9A4, OR9G1, OR9G4, OR9G9, OR9Q2, TAAR3, TPRA1, Y4 receptor, 5-HT1D receptor, 5-HT1E receptor, ADGRB1, AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor, GABAB1, GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER, GPR107, GPR139, GPR156, GPR158, GPR161, GPR171, GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor, HCA1 receptor, LPA1 receptor, LPA3 receptor, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1, MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1D2, OR2AG1, OT receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1A receptor, V2 receptor, a2A-adrenoceptor, a2B-adrenoceptor, a2C-adrenoceptor, β1 -adrenoceptor, β3- adrenoceptor, 5-HT1B receptor, 5-HT1F receptor, 5-HT2B receptor, 5-HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR11H1, OR2A1, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1, OR2C3, OR2J3, OR2L13, OR2T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51 B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51 F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51 L1 , OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J3, OR52K1 , OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2, OR9A2, oxoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sstl receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenoceptor, α1B-adrenoceptor, a1 D-adrenoceptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin receptor, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLTI receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2/ALX, FPR2/ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlul receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PARI, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2-adrenoceptor, κ receptor, μ receptor.

[000125] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of CCR4.

[000126] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of CCR5. [000127] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of CCR4 and CCR5.

[000128] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of CCR4, CCR5, CCR10 and CXCR3.

[000129] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000130] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000131 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adrenergic α1A receptor, CCR3 and M2 receptor.

[000132] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adrenergic α1A receptor, CCR3, CCR4 and M2 receptor.

[000133] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adrenergic α1A receptor, CCR3, CCR5 and M2 receptor. [000134] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5 and M2 receptor.

[000135] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000136] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3 and M2 receptor.

[000137] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000124] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000138] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: 5-HT1 D receptor, 5-HT1 E receptor, ADGRB1 , AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor, GABAB1 , GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER, GPR107, GPR139, GPR156, GPR158, GPR161 , GPR171 , GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor, HCA1 receptor, LPA1 receptor, LPA3 receptor, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1 , MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1 D2, OR2AG1, OT receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1A receptor, V2 receptor, a2A-adrenoceptor, a2B-adrenoceptor, a2C- adrenoceptor, β1 -adrenoceptor, β3 -adrenoceptor, 5-HT1B receptor, 5-HT1F receptor, 5- HT2B receptor, 5-HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, ΟΡΝ3, OR11H1, 0R2A1, 0R2A2, 0R2A4, 0R2A42, 0R2A7, OR2B11, 0R2B6, 0R2C1, 0R2C3, 0R2J3, 0R2L13, 0R2T11, 0R2T34, 0R2W3, 0R3A3, OR4D10, 0R4M1, 0R4Q3, 0R51A2, 0R51A4, 0R51A7, 0R51B2, 0R51B4, 0R51 B5, 0R51B6, OR51D1, OR51E1, OR51E1, 0R51E2, OR51F1, 0R51 F2, OR51G1, 0R51G2, OR51I1, 0R51I2, OR51J1, OR51L1, OR51M1, OR51Q1, OR51S1, 0R51T1, OR51V1, 0R52A1, 0R52A4, 0R52A5, 0R52B2, 0R52B4, 0R52B6, 0R52D1, 0R52E2, 0R52E4, 0R52E5, 0R52E6, 0R52E8, 0R52H1, 0R52I1, 0R52I2, 0R52J3, 0R52K1 , 0R52K2, 0R52L1, 0R52M1, 0R52N1, 0R52N2, 0R52N4, 0R52N5, 0R52R1, 0R52W1, 0R56A1 , 0R56A3, 0R56A4, 0R56A5, 0R56B1, 0R56B4, 0R6V1, 0R7D2, 0R9A2, oxoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sstl receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenoceptor, α1B-adrenoceptor, a1 D-adrenoceptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin receptor, CX3CR1 , CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLTI receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1 , FPR2/ALX, FPR2/ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21 , GPR31 , GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1 , MC1 receptor, MCH1 receptor, mGlul receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y1 1 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PARI , PAR2, PAR3, PAR4, PKR1 , PKR2, S1 P1 receptor, S1 P2 receptor, S1 P3 receptor, S1 P4 receptor, S1 P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1 , β2 -adrenoceptor, κ receptor, μ receptor.

[000139] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of CCR4.

[000140] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of CCR5.

[000141 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of CCR4 and CCR5.

[000142] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of CCR4, CCR5, CCR10 and CXCR3.

[000143] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000144] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000145] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adrenergic α1A receptor, CCR3 and M2 receptor.

[000146] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adrenergic α1A receptor, CCR3, CCR4 and M2 receptor.

[000147] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adrenergic α1A receptor, CCR3, CCR5 and M2 receptor.

[000148] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5 and M2 receptor.

[000149] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000150] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3 and M2 receptor.

[000151 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000138] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2Α receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000152] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: 5-HT1 B receptor, 5-HT1 F receptor, 5-HT2B receptor, 5- HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR11H1, OR2A1, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1, OR2C3, OR2J3, OR2L13, OR2T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51 B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51 F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51 L1 , OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J3, OR52K1 , OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1 , OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2, OR9A2, oxoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sstl receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenoceptor, α1 B-adrenoceptor, α1 D-adrenoceptor, δ receptor, 5-HT1 A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1 , ACKR2, ACKR3, ACKR4, ADGRE1 , ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1 , CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin receptor, CX3CR1 , CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLTI receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1 , FPR2/ALX, FPR2/ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21 , GPR31 , GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1 , MC1 receptor, MCH1 receptor, mGlul receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y1 1 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PARI , PAR2, PAR3, PAR4, PKR1 , PKR2, S1 P1 receptor, S1 P2 receptor, S1 P3 receptor, S1 P4 receptor, S1 P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1 , β2 -adrenoceptor, κ receptor, μ receptor.

[000153] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of CCR4.

[000154] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of CCR5.

[000155] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of CCR4 and CCR5.

[000156] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of CCR4, CCR5, CCR10 and CXCR3.

[000157] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor. [000158] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000159] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adrenergic α1A receptor, CCR3 and M2 receptor.

[000160] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adrenergic α1A receptor, CCR3, CCR4 and M2 receptor.

[000161 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adrenergic α1A receptor, CCR3, CCR5 and M2 receptor.

[000162] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5 and M2 receptor.

[000163] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000164] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3 and M2 receptor.

[000165] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000152] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000166] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: 5-HT1 A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1 , ACKR2, ACKR3, ACKR4, ADGRE1 , ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1 , CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin receptor, CX3CR1 , CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLTI receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1 , FPR2/ALX, FPR2/ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21 , GPR31 , GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1 , MC1 receptor, MCH1 receptor, mGlul receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y1 1 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PARI , PAR2, PAR3, PAR4, PKR1 , PKR2, S1 P1 receptor, S1 P2 receptor, S1 P3 receptor, S1 P4 receptor, S1 P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1 , β2 -adrenoceptor, κ receptor, μ receptor.

[000167] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR4.

[000168] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR5.

[000169] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR4 and CCR5. [000170] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR4, CCR5, CCR10 and CXCR3.

[000171 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, and prostaglandin E4 receptor.

[000172] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, and prostaglandin E4 receptor.

[000173] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR3.

[000174] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR3 and CCR4.

[000175] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR3 and CCR5.

[000176] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR3, CCR4, and CCR5.

[000177] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000178] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of CCR3, CCR4, CCR5, CCRI O and CXCR3.

[000179] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000166] with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[000180] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: angiotensin receptors, including AT ₁ R, and certain chemokine receptors, including CCR1 , CCR2, CCR6, CCR7, CXCR2, CXCR4, CXCR6 and CXCR7.

[000181 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: angiotensin receptors, including AT ₁ R, and certain chemokine receptors, including CCR1 , CCR2, CCR6, CCR7, CXCR1 , CXCR2, CXCR4, CXCR6 and CXCR7.

[000182] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: angiotensin receptors, including AT ₁ R, and certain chemokine receptors, including CCR1 , CCR2, CCR6, CCR7, CXCR1 , CXCR2, CXCR4 and CXCR6.

[000183] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: angiotensin receptors, including AT ₁ R, and certain chemokine receptors, including CCR1 , CCR2, CCR6, CCR7, CXCR2, CXCR4 and CXCR6.

[000184] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: angiotensin receptors, including AT ₁ R, and certain chemokine receptors, including CCR1 , CCR2, CCR6, CCR7, CXCR1 , CXCR2 and CXCR6. [000185] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: angiotensin receptors, including AT ₁ R, and certain chemokine receptors, including CCR1 , CCR2, CCR6, CCR7, CXCR2 and CXCR6.

[000186] In one embodiment, the activated co-located GPCR of the invention is vasopressin receptor 2.

[000187] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE.

[000188] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000189] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000190] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000191 ] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000192] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR1 , CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000193] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 . [000194] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000195] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000196] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000197] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR6, CCR7, CCR8, CCR9, CXCR1 , CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000198] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR1 , CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000199] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000200] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000201 ] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR6, CCR7, CCR8, CCR9, CXCR1 , CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 . [000202] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000203] In one embodiment, certain chemokine receptors are chemokine receptors that are co-expressed in the same cell as RAGE, are implicated in inflammation, and are selected from the group: CCR1 , CCR2, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1 , XCR1 .

[000204] In one form of the invention, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: adenosine 1A receptor, adrenergic α1A receptor, adrenergic α1 B receptor, adrenergic a2B receptor, angiotensin receptor AT ₁ R, bradykinin receptor B2, CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR2, CXCR4, CXCR5, dopamine D1 receptor, endothelin receptor type A, endothelin receptor type B, histamine H3 receptor, muscarinic M1 receptor, muscarinic M2 receptor, muscarinic M3 receptor, neuropeptide Y1 receptor, neurotensin 1 receptor, orexin receptor 1 , orexin receptor 2, prostaglandin E1 receptor, serotonin 5-HT1 a receptor, serotonin 5-HT2a receptor, serotonin 5-HT2b receptor, serotonin 5-HT2c receptor, serotonin 5-HT4b receptor, somatostatin 2 receptor, sphingosine 1 -phosphate receptor S1 P1 , sphingosine 1 -phosphate receptor S1 P3, thyrotrophin-releasing hormone receptor 1 , vasopressin receptor 1 A, vasopressin receptor 1 B or vasopressin receptor 2.

[000205] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of CCR4.

[000206] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of CCR5.

[000207] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of CCR4 and CCR5.

[000208] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of CCR4, CCR5 and CXCR4. [000209] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of adrenergic α1A receptor, CCR3, muscarinic M2 receptor and orexin receptor 1 .

[000210] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of adrenergic α1A receptor, CCR3, CCR4, muscarinic M2 receptor and orexin receptor 1 .

[00021 1 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of adrenergic α1A receptor, CCR3, CCR5, muscarinic M2 receptor and orexin receptor 1 .

[000212] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of adrenergic α1A receptor, CCR3, CCR4, CCR5, muscarinic M2 receptor and orexin receptor 1 .

[000213] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000204] with the exception of adrenergic α1A receptor, CCR3, CXCR4, muscarinic M2 receptor and orexin receptor 1 .

[000214] In a preferred form of the invention, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: adrenergic α1A receptor, adrenergic α1 B receptor, angiotensin receptor AT ₁ R, bradykinin receptor B2, CCR2, CCR3, CCR4, CCR6, CCR9, CXCR4, CXCR5, dopamine D1 receptor, endothelin receptor type B, histamine H3 receptor, muscarinic M2 receptor, neuropeptide Y1 receptor, neurotensin 1 receptor, orexin receptor 1 , orexin receptor 2, prostaglandin E1 receptor, serotonin 5-HT2b receptor, serotonin 5-HT2c receptor, serotonin 5-HT4b receptor, somatostatin 2 receptor, sphingosine 1 -phosphate receptor S1 P3, vasopressin receptor 1 A or vasopressin receptor 1 B.

[000215] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000214] with the exception of CCR4.

[000216] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000214] with the exception of CCR4 and CXCR4.

[000217] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000214] with the exception of adrenergic α1A receptor, CCR3, muscarinic M2 receptor and orexin receptor 1 . [000218] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000214] with the exception of adrenergic α1A receptor, CCR3, CCR4, muscarinic M2 receptor and orexin receptor 1 .

[000219] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000214] with the exception of adrenergic α1A receptor, CCR3, CCR4, CXCR4, muscarinic M2 receptor and orexin receptor 1 .

[000220] In a particularly preferred form of the invention, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: adrenergic α1A receptor, adrenergic α1 B receptor, angiotensin receptor AT ₁ R, bradykinin receptor B2, CCR2, CCR6, CCR9, CXCR4, CXCR5, dopamine D1 receptor, endothelin receptor type B, histamine H3 receptor, muscarinic M2 receptor, neuropeptide Y1 receptor, orexin receptor 1 , orexin receptor 2, prostaglandin E1 receptor, serotonin 5-HT2c receptor, serotonin 5-HT4b receptor, somatostatin 2 receptor, sphingosine 1 -phosphate receptor S1 P3, vasopressin receptor 1 A or vasopressin receptor 1 B.

[000221 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000220] with the exception of CXCR4.

[000222] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000220] with the exception of adrenergic α1A receptor, CCR3, muscarinic M2 receptor and orexin receptor 1 .

[000223] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000220] with the exception of adrenergic α1A receptor, CCR3, CXCR4, muscarinic M2 receptor and orexin receptor 1 .

[000224] In one form of the invention, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: adenosine A1 receptor (ADORA1 ), adrenergic a2B receptor, angiotensin receptor ATi (AT1 R), bradykinin receptor 2 (B2R), CCR1 , CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR2, CXCR4, CXCR5, neuropeptide Y1 receptor (NPY1 R), orexin receptor 2, sphingosine 1 -phosphate receptor 1 (S1 PR1 ), thyrotropin-releasing hormone receptor 1 (TRHR1 ), vasopressin receptor 1 A (V1 aR), vasopressin receptor 1 B (V1 bR) and vasopressin receptor 2 (V2R).

[000225] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000224] with the exception of CCR4. [000226] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000224] with the exception of CCR5.

[000227] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000224] with the exception of CCR4 and CCR5.

[000228] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000224] with the exception of CXCR4.

[000229] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000224] with the exception of CCR4, CCR5 and CXCR4.

[000230] In a preferred form of the invention, the certain activated co-located GPCRs of the invention are GPCRs selected from the group: adrenergic a2B receptor, angiotensin receptor ATi (AT1 R), bradykinin receptor 2 (B2R), CCR1 , CCR2, CCR4, CCR5, CCR6, CCR9, CXCR2, CXCR4, neuropeptide Y1 receptor (NPY1 R), orexin receptor 2, sphingosine 1 -phosphate receptor 1 (S1 PR1 ), thyrotropin-releasing hormone receptor 1 (TRHR1 ), vasopressin receptor 1 A (V1 aR), vasopressin receptor 1 B (V1 bR) and vasopressin receptor 2 (V2R).

[000231 ] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000230] with the exception of CCR4.

[000232] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000230] with the exception of CCR5.

[000233] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000230] with the exception of CCR4 and CCR5.

[000234] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000230] with the exception of CXCR4.

[000235] In one embodiment, the certain activated co-located GPCRs of the invention are GPCRs selected from the group in paragraph [000230] with the exception of CCR4, CCR5 and CXCR4. [000236] In a specific form of the invention, the activated co-located GPCR of the invention is an angiotensin receptor.

[000237] In a specific form of the invention, the activated co-located GPCR of the invention is AT ₁ R.

[000238] In a specific form of the invention, the activated co-located GPCR of the invention is a certain chemokine receptor, selected from the group: CCR1 , CCR2, CCR6, CCR7, CXCR2, CXCR4, CXCR6 and CXCR7.

[000239] In a specific form of the invention, the activated co-located GPCR of the invention is a certain chemokine receptor, selected from the group: CCR1 , CCR2, CCR6, CCR7, CXCR1 , CXCR2 and CXCR6.

[000240] In a specific form of the invention, the activated co-located GPCR of the invention is a certain chemokine receptor, selected from the group: CCR1 , CCR2, CCR6, CCR7, CXCR2 and CXCR6.

[000241 ] In a specific form of the invention, the activated co-located GPCR of the invention is a certain chemokine receptor selected from CCR2 and CCR6.

[000242] In a specific form of the invention, the activated co-located GPCR of the invention is CCR2.

[000243] In a specific form of the invention, the activated co-located GPCR of the invention is CXCR4.

[000244] In one form of the invention, a RAGE ligand is a ligand that interacts with the ectodomain of RAGE to modulate activation of RAGE. Thus, in this form of the invention, RAGE ligand-independent activation of RAGE means activation of RAGE that does not occur by way of a ligand interacting with the ectodomain of RAGE.

[000245] Preferably, a RAGE ligand is a ligand that interacts with the ectodomain of RAGE to modulate activation of RAGE and does not interact with the transmembrane domain or cytosolic tail of RAGE or motifs contained therein. Thus, in this preferred form of the invention, RAGE ligand-independent activation of RAGE means activation of RAGE that does not occur by way of a ligand interacting with the ectodomain of RAGE unless the ligand also interacts with the transmembrane domain or cytosolic tail of RAGE or motifs contained therein. [000246] The ectodomain (also known as the extracellular domain) of RAGE includes three immunoglobulin-like regions: an N-terminal V-type domain followed by two C-type domains (termed C and C or alternatively C1 and C2). The principal ligand-binding portion is the V-domain, however RAGE activation may also be mediated by ligand binding to the C- domains. Most ligands tend to bind to the V domain and/or the C1 domain since ligands tend to be negatively charged, however, there is at least one example of a ligand binding to the C2 domain (S100A6; Leclerc et al., 2007). Though the C1 - and C2-domains may not generally directly bind ligands, they could have important roles in stabilizing the V-domain for mediating its interaction(s) with ligands. RAGE has a single transmembrane-spanning domain and a cytosolic tail. In humans, the cytosolic tail of RAGE is 43 amino acids long (residue 362 to residue 404). This cytosolic tail contains motifs which are critical for RAGE- dependent cellular activation.

[000247] In one form of the invention, a RAGE ligand is a ligand that interacts with the extracellular V, C1 and/or C2 domains of the RAGE ectodomain to activate RAGE. In this form of the invention, RAGE ligand-independent activation of RAGE means activation of RAGE that does not occur by way of a ligand interacting with the extracellular V, C1 or C2 domains of the RAGE ectodomain.

[000248] Preferably, the RAGE ligand does not interact with the transmembrane domain or cytosolic tail of RAGE or motifs contained therein. In this form of the invention, RAGE ligand-independent activation of RAGE means activation of RAGE that does not occur by way of a ligand interacting with the extracellular V, C1 or C2 domains of the RAGE ectodomain unless the ligand also interacts with the transmembrane domain or cytosolic tail of RAGE or motifs contained therein.

[000249] In one form of the invention, a modulator that modulates RAGE ligand- independent activation of RAGE by an activated co-located GPCR, such as activated angiotensin receptor, such as AT ₁ R, or CCR2, also modulates RAGE ligand-dependent activation of RAGE.

[000250] In preferred embodiments of the invention, modulators of the invention do not modulate, or modulate differently, or modulate to a different extent, RAGE-independent signalling pathways associated with the certain activated co-located GPCR.

[000251 ] In a preferred embodiment, modulators of the invention do not inhibit, or inhibit to a lesser extent, one or more RAGE independent certain co-located GPCR signalling pathways. [000252] In one form of the invention, the RAGE-independent certain co-located GPCR signalling pathway is the Gq signalling pathway. In one form of the invention, the RAGE- independent certain co-located GPCR signalling pathway is the Gi/o signalling pathway. In one form of the invention, the RAGE-independent certain co-located GPCR signalling pathway is the Gs signalling pathway. In one form of the invention, the RAGE-independent certain co-located GPCR signalling pathway is the calcium signalling pathway. In one form of the invention, the RAGE-independent certain co-located GPCR signalling pathway is the phospholipase C signalling pathway. In another form of the invention, the RAGE- independent certain co-located GPCR signalling pathway is β-arrestin-mediated extracellular regulated kinase (ERK) signalling.

[000253] In a particularly preferred embodiment, where the activated co-located GPCR is activated AT ₁ R, modulators of the invention do not modulate, or modulate to a lesser extent, one or more RAGE independent AT ₁ R signalling pathways.

[000254] In a particularly preferred embodiment, where the activated co-located GPCR is activated AT ₁ R, modulators of the invention do not inhibit, or inhibit to a lesser extent, one or more RAGE independent AT ₁ R signalling pathways.

[000255] In one form of the invention, the RAGE-independent AT ₁ R signalling pathway is the Gq signalling pathway. In another form of the invention, the RAGE-independent AT ₁ R signalling pathway is β-arrestin-mediated extracellular regulated kinase (ERK) signalling.

[000256] In another particularly preferred embodiment, where the activated co-located GPCR is activated CCR2, modulators of the invention do not modulate, or modulate to a lesser extent, one or more RAGE independent CCR2 signalling pathways.

[000257] In another particularly preferred embodiment, where the activated co-located GPCR is activated CCR2, modulators of the invention do not inhibit, or inhibit to a lesser extent, one or more RAGE independent CCR2 signalling pathways.

[000258] In one form of the invention, the RAGE-independent AT ₁ R signalling pathway is the Gi/o signalling pathway. In another form of the invention, the RAGE-independent CCR2 signalling pathway is β-arrestin-mediated extracellular regulated kinase (ERK) signalling. In another form of the invention, the RAGE-independent CCR2 signalling pathway is the phospholipase C signalling pathway. Modulators

[000259] In one form of the invention, a modulator of the invention is an activator, an inhibitor, an allosteric modulator, or a functional or non-functional substitute for the cytosolic tail of RAGE. A functional substitute is a modulator that takes the place of the cytosolic tail of RAGE in the presence of certain co-located GPCRs and is able to be activated by them to induce downstream RAGE-dependent signalling in the presence or absence of expression of wild-type RAGE. A non-functional substitute is a modulator that takes the place of the cytosolic tail of RAGE in the presence of certain co-located GPCRs, is not able to be activated by them or induce downstream RAGE-dependent signalling, and inhibits signalling that normally occurs through activation of the cytosolic tail of RAGE and RAGE-dependent signalling resulting therefrom.

[000260] In one form of the invention, a modulator of the invention is an activator, an inhibitor, an allosteric modulator, or a non-functional substitute for the transmembrane domain of RAGE or part thereof.

[000261 ] A non-functional substitute is a modulator that takes the place of the transmembrane domain of RAGE in the presence of certain co-located GPCRs, is not able to be activated by them or induce downstream RAGE-dependent signalling, and inhibits signalling that normally occurs through activation of the cytosolic tail of RAGE and RAGE- dependent signalling resulting therefrom.

[000262] In one form of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the RAGE ectodomain.

[000263] In one form of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the cytosolic tail of RAGE.

[000264] In one form of the invention, the modulator comprises a transmembrane domain of RAGE or part thereof and a fragment of the RAGE ectodomain and a fragment of the cytosolic tail of RAGE.

[000265] In one form of the invention, modulators of the invention contain a fragment of the ectodomain of RAGE, which is not greater than 40, not greater than 20, not greater than 10 or not greater than 5 amino acids in length.

[000266] By way of example of a modulator, the inventors have demonstrated that RAGE ₃ 62-404 is a functional substitute for RAGE and is able to be activated by certain co- located GPCRs, such as AT ₁ R and CCR2, and induce downstream RAGE-dependent signalling resulting from RAGE ligand-independent activation of RAGE, in the presence or absence of expression of wild-type RAGE. Furthermore, when RAGE _362-404 is fused to a cell penetrating peptide (TAT) and a marker protein (mCherry), treatment with TAT-mCherry- RAGE ₃ 62-404 oligopeptide following activation of AT1 R by Ang II is able to restore Ang II- dependent inflammation and atherogenesis in Ager Apoe DKO mice in the absence of expression of wild-type RAGE.

[000267] The sequence of RAGE ₃₆₂ - ₄ o4 is SEQ ID NO: 1 :

[L362WQRRQRRGEERKAPENQEEEEERAELNQSEEPEAGESSTGGP404]

[000268] By way of an additional example of a modulator, the inventors have demonstrated that S391 A-RAGE ₃₆ 2- ₄ 04 is a non-functional substitute for RAGE that in the presence of certain co-located GPCRs is not activated by them and inhibits RAGE- dependent signalling. Expression of S391 A-RAGE _362-404 inhibits RAGE ligand-independent activation of wild-type RAGE by activated AT1 R and RAGE ligand-dependent activation of wild-type RAGE by the RAGE ligand S100A8/A9. Furthermore, when S391 A-RAGE _362-404 is fused to a cell penetrating peptide (TAT) and a marker protein (mCherry), treatment with TAT-mCherry-S391 A-RAGE ₃ 62-404 oligopeptide inhibits RAGE ligand-independent activation of RAGE by activated AT1 R to attenuate Ang ll-dependent inflammation and atherogenesis in apolipoprotein E knockout mice. Further examples are provided below.

[000269] The sequence of S391 A-RAGE ₃₆₂ - ₄ o4 is SEQ ID NO: 2:

[L ₃₆ 2WQRRQRRGEERKAPENQEEEEERAELNQAEEPEAGESSTGGP ₄ o4]

[000270] By way of another example of a modulator, the inventors have demonstrated that RAGE ₃₃₈ - ₃ 6i inhibits RAGE ligand-independent activation of wild-type RAGE by activated AT ₁ R. This inhibition is overcome by co-expression of mCherry-RAGE _362-404 .

[000271 ] The sequence of RAGE _338-361 is SEQ ID 3

[000272] [L ₃₃₈ GTLALALGILGGLGTAALLIGVI ₃₆₁ ]

[000273] In one form, the present invention comprises modulators of RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs that modulate transactivation of the cytosolic tail of RAGE triggered by activation of such certain activated co-located GPCRs, such as an angiotensin receptor, such as AT ₁ R, or a chemokine receptor, such as CCR2. [000274] In one form, the present invention comprises modulators of RAGE ligand- independent activation of the cytosolic tail of RAGE by certain activated co-located GPCRs that bind to Ras GTPase-activating-like protein (IQGAP1 ) or other RAGE-associated proteins, including protein kinase C zeta (ΡΚCζ), Dock7, MyD88, TIRAP, IRAK4, ERK1/2, olfactory receptor 2T2, ADP/ATP translocase 2, Protein phosphatase 1 G, Intercellular adhesion molecule 1 , Protein DJ-1 (PARK7), Calponin-3, Drebrin, Filamin B, Ras-related protein Rab-13, Radixin/Ezrin/Moesin, Proteolipid protein 2, Coronin, S100 A1 1 , Succinyl- CoA ligase [GDP-forming] subunit alpha, Hsc70-interacting protein, Apoptosis Inhibitor 5, neuropilin, cleavage stimulation factor, growth factor receptor-bound protein 2, sec61 beta subunit, or Nck1 , or disrupt the binding of these elements to RAGE, in order to modulate RAGE transactivation by certain activated co-located GPCRs, such as an angiotensin receptor, such as AT ₁ R, or a chemokine receptor, such as CCR2.

[000275] In one form of the invention, the modulators of the invention bind to the cytosolic elements of the certain activated co-located GPCR, RAGE and/or elements complexed with either, including IQGAP-1 , ΡΚCζ, Dock7, MyD88, TIRAP, IRAK4, ERK1/2, olfactory receptor 2T2, ADP/ATP translocase 2, Protein phosphatase 1 G, Intercellular adhesion molecule 1 , Protein DJ-1 (PARK7), Calponin-3, Drebrin, Filamin B, Ras-related protein Rab-13, Radixin/Ezrin/Moesin, Proteolipid protein 2, Coronin, S100 A1 1 , Succinyl-CoA ligase [GDP- forming] subunit alpha, Hsc70-interacting protein, Apoptosis Inhibitor 5, neuropilin, cleavage stimulation factor, growth factor receptor-bound protein 2, sec61 beta subunit, or Nck1 to modulate RAGE ligand-independent signalling through the cytosolic tail of RAGE, by modulating these signalling elements required for RAGE transactivation by certain activated co-located GPCRs, such as an angiotensin receptor, such as AT ₁ R, or such as a chemokine receptor, such as CCR2.

[000276] In one form of the invention, modulators of RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs also modulate RAGE ligand-dependent activation of the cytosolic tail of RAGE, by binding to cytosolic elements of RAGE and/or elements that complex with RAGE in the cytosol (such as IQGAP-1 , ΡΚΰζ, Dock7, MyD88, IRAK4, TIRAP, ERK1 /2, olfactory receptor 2T2, ADP/ATP translocase 2, Protein phosphatase 1 G, Intercellular adhesion molecule 1 , Protein DJ-1 (PARK7), Calponin-3, Drebrin, Filamin B, Ras-related protein Rab-13, Radixin/Ezrin/Moesin, Proteolipid protein 2, Coronin, S100 A1 1 , Succinyl-CoA ligase [GDP-forming] subunit alpha, Hsc70-interacting protein, Apoptosis Inhibitor 5, neuropilin, cleavage stimulation factor, growth factor receptor- bound protein 2, sec61 beta subunit, or Nck1 ) to inhibit RAGE ligand-mediated signalling through these elements. [000277] In specific embodiments, the modulator comprises, consists, or consists essentially of an amino acid sequence as set forth in SEQ ID NO: 1 , or an analogue, fragment or derivative thereof.

[000278] In some embodiments, the modulator is introduced by gene delivery (such as by using a virus or artificial non-viral gene delivery such as electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, lipofection, liposomes, nanobubbles and polymeric gene carriers) and the peptide fragment, biologically- active analogue or derivative being generated by the cell as a consequence of transcriptional and translational processes.

[000279] In some embodiments of this aspect, the modulator has a modified capacity to form a complex with certain co-located GPCRs, such as AT ₁ R or CCR2, or elements that complex with them. For example, the RAGE analogue or derivative may be distinguished from a wild-type RAGE polypeptide or fragment sequence by the substitution, addition, or deletion of at least one amino acid residue or addition or substitution of unusual or non- conventional amino-acids or non-amino acid residues.

[000280] In some embodiments, the modulator lacks or has a modification of serine-391 that is normally present in a wild-type human RAGE polypeptide. In illustrative examples of this type, the fragment, analogue or derivative of the cytosolic tail of RAGE lacks a serine at position 391 of the wild-type RAGE sequence (for example, the RAGE370-390 construct is truncated at Glu390). Suitably, the serine at position 391 is deleted or substituted with another amino acid residue, an analogue or derivative, in order to impair or abolish signalling conferred by a serine at this site following activation of a co-located GPCR. In one embodiment, the serine at position 391 is deleted or substituted with another amino acid residue selected from the group: alanine, aspartate, phenylalanine, histidine, lysine, arginine, tyrosine, asparagine, valine, glycine, cysteine or glutamate.

[000281 ] In some embodiments, the modulator retains the serine-391 that is normally present in a wild-type human RAGE polypeptide or be substituted by another amino acid that retains the same function of a serine at the 391 position, or an analogue or derivative thereof. In illustrative examples of this type, the fragment of the cytosolic tail of RAGE retains a serine at position 391 of the wild-type RAGE sequence (for example, the RAGE370-404 construct). Suitably, the serine at position 391 is substituted with another amino acid residue or an analogue or derivative thereof, in order to replicate signalling conferred following activation of a co-located GPCR by RAGE constructs containing a serine at this site. In one embodiment the serine at position 391 is substituted with another amino acid residue selected from the group: proline, glutamine, threonine, leucine, isoleucine, methionine, or tryptophan.

[000282] In some embodiments, the modulator lacks or has an impaired ability to bind Diaphanous 1 (Diaphl ) relative to human wild-type RAGE. In illustrative examples of this type, the peptide, or analogue, fragment or derivative thereof, either lacks the RAGE-Diaph1 binding site (such as RAGE370-390, RAGE374-390, or RAGE379-390) or has an altered Diaphl binding site (such as 366A/367A) in order to abolish or impair this site. Suitably, the residues at 366/367 are deleted or substituted with other residues (such as with alanine) in order to impair or abolish this site, and in doing so, improve affinity for binding to other targets, by reducing constraints induced by wild-type binding to Diaphl .

[000283] In one aspect of the invention, the modulator of the present invention includes isolated or purified peptides which comprise, consist, or consists essentially of an amino acid sequence represented by Formula I:

Z1 M Z2 (I) wherein:

Z1 is absent or is selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues; and

M is the amino acid sequence as set forth in SEQ ID NO: 1 , or an analogue, fragment or derivative thereof; and

Z2 is absent or is a proteinaceous moiety comprising from about 1 to about 50 amino acid residues.

[000284] In some embodiments of the invention described above, the modulator (such as a fragment of the RAGE cytosolic tail, an analogue or derivative thereof as broadly described above and elsewhere herein) is able to penetrate a cell membrane. In non-limiting examples of this type, the RAGE modulator is conjugated, fused or otherwise linked to a cell membrane penetration molecule (e.g., the HIV TAT motif, as set forth in SEQ ID NO: 4 below).

[000285] SEQ ID NO: 4:

[YGRKKRRQRRR]. [000286] In some forms of the invention, the modulator is a non-peptide molecule that shares with the peptide modulator described above the capacity to bind to and/or interfere with elements associated with RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs. These non-peptide modulators may or may not contain structural similarities to functionally important domains contained in peptide modulators.

[000287] In a preferred form, the non-peptide modulator contains structural similarities to functionally important domains contained in the peptide modulators, as represented by the pharmacophore described in paragraph [000318].

[000288] In preferred forms of the invention, the modulator is an inhibitor.

[000289] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE by a certain activated co-located GPCR, the modulator is an inhibitor of the certain co-located GPCR and/or an inhibitor of the certain co-located GPCR signalling pathway.

[000290] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE by a certain activated co-located GPCR, the modulator is an inhibitor of RAGE ligand-dependent activation of RAGE and/or an inhibitor of constitutively-active RAGE and/or an inhibitor of a RAGE signalling pathway.

[000291 ] In certain forms of the invention, where the certain co-located GPCR is AT ₁ R, in addition to being an inhibitor of RAGE ligand-independent activation of RAGE, the modulator is an AT ₁ R inhibitor and/or an inhibitor of an AT ₁ R signalling pathway.

[000292] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE by activated angiotensin receptor, preferably activated AT ₁ R, the modulator is an inhibitor of RAGE ligand-dependent activation of RAGE and/or an inhibitor of constitutively-active RAGE and/or an inhibitor of a RAGE signalling pathway.

[000293] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE by a certain activated co-located GPCR, the modulator is an inhibitor of the certain co-located GPCR and/or an inhibitor of the certain co-located GPCR signalling pathway and an inhibitor of RAGE ligand-dependent activation of RAGE and/or an inhibitor of constitutively-active RAGE and/or an inhibitor of a RAGE signalling pathway. [000294] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE by activated angiotensin receptor, preferably activated AT ₁ R, the modulator is an AT ₁ R inhibitor and/or an inhibitor of an AT ₁ R signalling pathway and an inhibitor of RAGE ligand-dependent activation of RAGE and/or an inhibitor of constitutively-active RAGE and/or an inhibitor of a RAGE signalling pathway.

[000295] In certain forms of the invention, where the certain co-located GPCR is a certain chemokine receptor, preferably CCR2, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE, the modulator is a certain chemokine receptor inhibitor, preferably a CCR2 inhibitor, and/or an inhibitor of a certain chemokine signalling pathway, preferably a CCR2 signalling pathway.

[000296] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE by activated certain chemokine receptor, preferably activated CCR2, the modulator is an inhibitor of RAGE ligand-dependent activation of RAGE and/or an inhibitor of constitutively-active RAGE and/or an inhibitor of a RAGE signalling pathway.

[000297] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand- independent activation of RAGE by activated chemokine receptor, preferably activated CCR2, the modulator is a certain chemokine receptor inhibitor, preferably a CCR2 inhibitor, and/or an inhibitor of a certain chemokine signalling pathway, preferably a CCR2 signalling pathway and an inhibitor of RAGE ligand-dependent activation of RAGE and/or an inhibitor of constitutively-active RAGE and/or an inhibitor of a RAGE signalling pathway.

[000298] In certain forms of the invention, the modulator is a functional substitute for the cytosolic tail of RAGE or a part thereof and is able to be activated by certain co-located GPCRs, such as activated AT ₁ R and activated CCR2, and induce downstream RAGE- dependent signalling in the presence or absence of expression of wild-type RAGE.

[000299] In certain forms of the invention, the modulator is a non-functional substitute for the cytosolic tail of RAGE or a part thereof, which is not able to be activated by a co-located GPCR or facilitate downstream RAGE-dependent signalling and inhibits signalling that occurs through the cytosolic tail of RAGE and RAGE-dependent signalling.

[000300] In certain forms of the invention, the modulator is a non-functional substitute for the transmembrane domain of RAGE or a part thereof, which is not able to be activated by a co-located GPCR or facilitate downstream RAGE-dependent signalling and inhibits signalling that occurs through the cytosolic tail of RAGE and RAGE-dependent signalling. [000301 ] In certain forms of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the RAGE ectodomain. In certain forms of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the cytosolic tail of RAGE.

[000302] In certain forms of the invention, the modulator comprises a transmembrane domain of RAGE or part thereof and a fragment of the RAGE ectodomain and a fragment of the cytosolic tail of RAGE.

[000303] In certain forms of the invention, the modulators of RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs contain a fragment of the ligand- binding ectodomain of human wild-type RAGE, which is not greater than 40, not greater than 20, not greater than 10 or not greater than 5 amino acids in length.

[000304] The inventors have further discovered that a peptide comprising residues 370- 390 of the cytosolic tail of RAGE (see SEQ ID NO: 5) is an inhibitory peptide, inhibiting both RAGE ligand-independent and RAGE ligand-dependent activation of wild-type RAGE.

[000305] SEQ ID NO: 5:

[G370EERKAPENQEEEEERAELNQ390].

[000306] A solution NMR structure exists for RAGE ₃₆ 3-404 (Rai V et al., 2012) showing that the N-terminus (residues 363-376) of this peptide is ordered. A Rosetta-derived model exists for RAGE "362-404 (model4) which is consistent with the NMR structure (http://www.rcsb.orq/pdb/explore/explore.do?structureld=2LMB , accessed 25 August 2016)) and also suggests that the remainder of the peptide forms an alpha helix.

[000307] An initial model of RAGE370-390 was constructed by truncating model4 (model4_37o-39o). Model4 is a theoretical model of the RAGE cytosolic tail, generated by inputting the sequence into the l-Tasser web server (http://zhanglab.ccmb.med.umich.edu/l- TASSER/). See also Yang et al (2015), Roy et al (2010) and Y Zhang (2008). All five models presented by the l-Tasser server predicted the region 370-390 to form a helix. The models and the NMR structure were aligned by the C-alpha carbons of the backbones of the peptide sequences. Model 4 was selected as the preferred model, as the predicted structure of the region corresponding to the Diaphanous 1 binding site in model4 was closest to the documented NMR structure for this region. [000308] A 20 ns molecular dynamics simulation of model4 in water was run using GROMACS (Hess et al., 2008). The molecular dynamics simulation suggests that the alpha helix region of model4_ ₃ 7o-39o is stable. Strong interactions are observed between a number of charged side chains, suggesting that these interactions stabilise the folded structure and that any conservation of these residues might result from their role in stabilising the peptide structure.

[000309] A Blast search was used to identify homologous sequences for RAGE370-390. The sequences were aligned as follows:

[000310] This analysis identified a number of strongly conserved residues in RAGE370-390 marked with as follows: * (asterisk) indicates positions which have a single, fully conserved residue. : (colon) indicates conservation between groups of strongly similar properties - scoring > 0.5 in the Gonnet PAM 250 matrix. . (period) indicates conservation between groups of weakly similar properties - scoring =< 0.5 in the Gonnet PAM 250 matrix:

[00031 1 ] Highly conserved residues are likely to play a structural role. Residues underlined are located on one face of the helix and likely represent the binding pharmacophore.

[000312] Examination of the model4_RAGE ₃ 7o-39o structure and the molecular dynamics simulation results shows that a number of salt bridges are present in the structure. The molecular dynamics simulations show that these interactions are important structural features. Structural function is a likely reason for the conserved nature of these amino acids.

[000313] A number of strongly conserved amino acids are not involved in salt-bridge formation. These are present on one face of the RAGE370-390 helix and likely represent the binding interface. These are Glu380, Glu384, Glu387 and Leu388. Another highly conserved residue, Glu377 is also present on this face of the peptide and may also be involved in binding, in addition to forming an alpha-helix-stabilising salt bridge to Lys374.

[000314] Replacement of the key hydrophobic residue L388 with alanine (e.g. L388A- RAGE370-390) results in the loss of the inhibition achieved by RAG E370-390 when acting on wild type RAGE. N-Truncation of RAGE eliminating both 380 and 384 (e.g. RAGE ₃₈ 5-39o and RAGE385-404) results in the loss of the modulatory actions of these RAGE contructs. By contrast, N-truncation of RAGE eliminating both 374 & 377 does not result in a loss of function of RAGE peptides either as an inhibitor (RAGE379-390) or as a functional substitute of wild type RAGE (e.g. RAGE379-404) , implying that these conserved residues (374 and 377) are not essential for modulatory activity, even though they may play a role stabilising the alpha helical tertiary structure of the cytosolic tail of RAGE.

[000315] Consistent with the conserved nature of these four amino acids that represent the binding interface, the inventors have further discovered that a peptide comprising only residues 379-390 of the cytosolic tail of RAGE (i.e. RAGE379-390) is an inhibitory peptide, inhibiting both ligand-independent and ligand-dependent activation of wild-type RAGE, and that RAGE379-404 is able to be activated by certain co-located GPCRs in CHO cells.

[000316] In a preferred form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a peptide Q379EEEEERAELNQ390 as set forth in SEQ ID NO: 6, or a derivative thereof.

[000317] SEQ ID NO: 6:

[Q379EEEEERAELNQ390]

[000318] A pharmacophore for RAGE379-390 peptide derived from the structure model4_RAGE ₃ 7o-39o is represented below:

[000319] H4 is a hydrophobic residue, and P1 -P3 are polar residues, and distances are shown in Angstroms. A matrix of distances between site points is as follows, where P represents a polar site point (hydrogen bonding or charged), and H represents a hydrophobic site point. Distances are in Angstroms. A tolerance should be applied to the position of each point.

[000320] The molecular dynamics simulations show that the interacting groups of RAGE379-390 are mobile and a tolerance should be applied to the position of each group of up to ± 10A provided the distances between the site points is positive in magnitude.

[000321 ] As would be understood by a person skilled in the art, additional, smaller pharmacophores can be generated by taking subsets of the above, and the present invention encompasses such pharmacophores, methods for using such to identify compounds, and compounds so identified.

[000322] In one form, the present invention further comprises a modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR comprising two or more features selected from the group: a first charged or hydrogen bonding group (A), a second charged or hydrogen bonding group (B), a third charged or hydrogen bonding group (C), and a hydrophobic group (D) wherein the distances between the site points of the features are as follows, within a tolerance of up to ±10 A, provided the distances between the site points is positive in magnitude:

[000323] In a preferred form of the invention, the tolerance is up to ±5 A, provided the distances between the site points is positive in magnitude. In a preferred form of the invention, the tolerance is up to ±2 A, provided the distances between the site points is positive in magnitude. In a preferred form of the invention, the tolerance is up to ±1 A, provided the distances between the site points is positive in magnitude.

[000324] In a preferred form of the invention, the modulator comprises three or more features selected from the above-specified group.

[000325] In a preferred form of the invention, the modulator comprises four features from the above-specified group.

[000326] In one form of the invention, there is provided a modulator characterised in that the modulator comprises at least two features chosen from one of the following combinations: AB, AC, AD, BC, BD, and CD.

[000327] In one form of the invention, there is provided a modulator, characterised in that the modulator comprises at least three features chosen from one of the following combinations: ABC, ABD, ACD, and BCD.

[000328] In one form of the invention, there is provided a modulator characterised in that the modulator comprises at least four features chosen from one of the following combinations: ABCD.

[000329] In one form of the invention, there is provided a modulator characterised in that the modulator comprises an additional charged or hydrogen bonding group (P1 ), consistent with the conserved stabilizing actions of E377 in RAGE370-390, and therefore comprises two or more features selected from the group: a first charged or hydrogen bonding group (A), a second charged or hydrogen bonding group (B), a third charged or hydrogen bonding group (C), a fourth charged or hydrogen group (D), and a hydrophobic group (E) wherein the distances between the site points of the features are as follows, within a tolerance of ±10 A:

[000330] The modulator of RAGE ligand-independent activation of RAGE may be a peptide, or a non-peptidyl compound.

[000331 ] In one form of the invention, the hydrophobic group is an amino acid residue selected from the group: Ala, Val, Leu, lie, Phe, Trp, Tyr.

[000332] In one form of the invention, the hydrophobic group is a chemical moiety selected from the group: C _1-8 alkyl, C _1-8 alkenyl, C ₃ - ₆ cycloalkyl, aryl, substituted aryl, alkyl aryl, heteroaryl, alkyl heteroaryl.

[000333] "Alkyl" means an aliphatic hydrocarbon group, which may be straight or branched and comprising about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups contain about 1 to about 12 carbon atoms in the chain. More preferred alkyl groups contain about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkyl chain.

[000334] "Lower alkyl" means a group having about 1 to about 6 carbon atoms in the chain which may be straight or branched. The alkyl group may be optionally substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkyl, aryl, cycloalkyi, cyano, hydroxy, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), -N(alkyl) ₂ , carboxy and - C(0)0-alkyl. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl and t-butyl.

[000335] "Alkenyl" means an aliphatic hydrocarbon group containing at least one carbon- carbon double bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkenyl chain.

[000336] "Lower alkenyl" means about 2 to about 6 carbon atoms in the chain which may be straight or branched. Non-limiting examples of suitable alkenyl groups include ethenyl, propenyl, 2-butenyl and 3- methylbutenyl. The term "substituted alkenyl" means that the alkenyl group may be substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of alkyl, aryl and cycloalkyi.

[000337] "Alkynyl" means an aliphatic hydrocarbon group containing at least one carbon- carbon triple bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkynyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkynyl chain.

[000338] "Lower alkynyl" means about 2 to about 6 carbon atoms in the chain which may be straight or branched. Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl. The term "substituted alkynyl" means that the alkynyl group may be substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of alkyl, aryl and cycloalkyi.

[000339] "Aliphatic" means and includes straight or branched chains of paraffinic, olefinic or acetylenic carbon atoms. The aliphatic group can be optionally substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of H, halo, halogen, alkyl, aryl, cycloalkyi, cycloalkylamino, alkenyl, heterocyclic, alkynyl, cycloalkylaminocarbonyl, hydroxyl, thio, cyano, hydroxy, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), - N(alkyl)2) carboxyl, -C(0)0-alkyl, heteroaryl, aralkyl, alkylaryl, aralkenyl, heteroaralkyl, alkylheteroaryl, heteroaralkenyl, heteroalkyl, carbonyl, hydroxyalkyl, aryloxy, aralkoxy, acyl, aroyl, nitro, amino, amido, ester, carboxylic acid aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkenyl, heterocyclyl, heterocyclenyl, carbamate, urea, ketone, aldehyde, cyano, sulfonamide, sulfoxide, sulfone, sulfonyl urea, sulfonyl, hydrazide, hydroxamate, S(alkyl)Y1 Y2N-alkyl-, Y1 Y2N-alkyl-, Y1 Y2NC(0)- and Y1 Y2NS0 ₂ -, wherein Y1 and Y2 can be the same or different and are independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl.

[000340] "Heteroaliphatic" means an otherwise aliphatic group that contains at least one heteroatom (such as oxygen, nitrogen or sulfur). The term heteroaliphatic includes substituted heteroaliphatic.

[000341 ] "Aryl" means an aromatic monocyclic or multicyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein. Non-limiting examples of suitable aryl groups include phenyl and naphthyl.

[000342] "Heteroalkyl" means an alkyl as defined above, wherein one or more hydrogen atoms are substituted by a heteroatom selected from N, S, or O.

[000343] "Heteroaryl" means an aromatic monocyclic or multicyclic ring system comprising about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. Preferred heteroaryls contain about 5 to about 6 ring atoms. The "heteroaryl" can be optionally substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein. The prefix aza, oxa or thia before the heteroaryl root name means that at least a nitrogen, oxygen or sulfur atom respectively, is present as a ring atom. A nitrogen atom of a heteroaryl can be optionally oxidized to the corresponding N-oxide. Non-limiting examples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1 ,2,4- thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[1 ,2- a]pyridinyl, imidazo[2,1 -b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1 ,2,4-triazinyl, benzothiazolyl and the like. The term "heteroaryl" also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like.

[000344] "Aralkyl" or "arylalkyl" means an aryl-alkyl- group in which the aryl and alkyl are as previously described. Preferred aralkyls comprise a lower alkyl group. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyl and naphthalenylmethyl. The bond to the parent moiety is through the alkyl.

[000345] "Alkylaryl" means an alkyl-aryl- group in which the alkyl and aryl are as previously described. Preferred alkylaryls comprise a lower alkyl group. Non-limiting example of a suitable alkylaryl group is tolyl. The bond to the parent moiety is through the aryl.

[000346] "Cycloalkyi" means a non-aromatic mono- or multi-cyclic ring system comprising about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred cycloalkyi rings contain about 5 to about 7 ring atoms. The cycloalkyi can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined above. Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Non-limiting examples of suitable multicyclic cycloalkyls include 1 -decalinyl, norbornyl, adamantyl and the like, as well as partially saturated species such as, for example, indanyl, tetrahydronaphthyl and the like. "Halogen" means fluorine, chlorine, bromine, or iodine. Preferred are fluorine, chlorine and bromine.

[000347] "Ring system substituent" means a substituent attached to an aromatic or non- aromatic ring system which, for example, replaces an available hydrogen on the ring system. Ring system substituents may be the same or different, each being independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyi, heterocyclyl, -C(=N-CN)-NH ₂ , -C(=NH)-NH ₂ , -C(=NH)-NH(alkyl), Y1 Y2N-, Y1 Y2N-alkyl-, Y1 Y2NC(0)-, Y1 Y2NS0 ₂ - and - SO2NYI Y2, wherein Y1 and Y2 can be the same or different and are independently selected from the group consisting of hydrogen, alkyl, aryl, cycloalkyi, and aralkyl. "Ring system substituent" may also mean a single moiety which simultaneously replaces two available hydrogens on two adjacent carbon atoms (one H on each carbon) on a ring system. Examples of such moieties are methylene dioxy, ethylenedioxy, -C(CH ₃ )2- and the like which form moieties such as, for example:

[000348] It should be noted that in hetero-atom containing ring systems of this invention, there are no hydroxyl groups on carbon atoms adjacent to a N, O or S1 as well as there are no N or S groups on carbon adjacent to another heteroatom. Thus, for example, in the ring:

there is no -OH attached directly to carbons marked 2 and 5.

[000349] It should also be noted that tautomeric forms such as, for example, the moieties: are considered equivalent in certain embodiments of this invention.

[000350] "Alkynylalkyl" means an alkynyl-alkyl- group in which the alkynyl and alkyl are as previously described. Preferred alkynylalkyls contain a lower alkynyl and a lower alkyl group. The bond to the parent moiety is through the alkyl. Non-limiting examples of suitable alkynylalkyl groups include propargylmethyl.

[000351 ] "Heteroaralkyl" means a heteroaryl-alkyl- group in which the heteroaryl and alkyl are as previously described. Preferred heteroaralkyls contain a lower alkyl group. Non- limiting examples of suitable aralkyl groups include pyridylmethyl, and quinolin-3-ylmethyl. The bond to the parent moiety is through the alkyl.

[000352] "Hydroxyalkyi" means a HO-alkyl- group in which alkyl is as previously defined. Preferred hydroxyalkyls contain lower alkyl. Non-limiting examples of suitable hydroxyalkyi groups include hydroxymethyl and 2-hydroxyethyl. [000353] "Acyl" means an H-C(O)-, alkyl-C(O)- or cycloalkyl-C(O)-, group in which the various groups are as previously described. The bond to the parent moiety is through the carbonyl. Preferred acyls contain a lower alkyl. Non-limiting examples of suitable acyl groups include formyl, acetyl and propanoyl.

[000354] "Aroyl" means an aryl-C(O)- group in which the aryl group is as previously described. The bond to the parent moiety is through the carbonyl. Non-limiting examples of suitable groups include benzoyl and 1 -naphthoyl.

[000355] "Alkoxy" means an alkyl-O- group in which the alkyl group is as previously described. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, n- propoxy, isopropoxy and n-butoxy. The bond to the parent moiety is through the ether oxygen.

[000356] "Aryloxy" means an aryl-O- group in which the aryl group is as previously described. Non-limiting examples of suitable aryloxy groups include phenoxy and naphthoxy. The bond to the parent moiety is through the ether oxygen.

[000357] "Alkylthio" means an alkyl-S- group in which the alkyl group is as previously described. Non-limiting examples of suitable alkylthio groups include methylthio and ethylthio. The bond to the parent moiety is through the sulfur.

[000358] "Arylthio" means an aryl-S- group in which the aryl group is as previously described. Non-limiting examples of suitable arylthio groups include phenylthio and naphthylthio. The bond to the parent moiety is through the sulfur.

[000359] "Aralkylthio" means an aralkyl-S- group in which the aralkyl group is as previously described. Non-limiting example of a suitable aralkylthio group is benzylthio. The bond to the parent moiety is through the sulfur.

[000360] "Alkoxycarbonyl" means an alkyl-O-CO- group. Non-limiting examples of suitable alkoxycarbonyl groups include methoxycarbonyl and ethoxycarbonyl. The bond to the parent moiety is through the carbonyl.

[000361 ] "Aralkoxycarbonyl" means an aralkyl-O-C(O)- group. Non-limiting example of a suitable aralkoxycarbonyl group is benzyloxycarbonyl. The bond to the parent moiety is through the carbonyl.

[000362] "Alkylsulfonyl" means an alkyl-S(0 ₂ )- group. Preferred groups are those in which the alkyl group is lower alkyl. The bond to the parent moiety is through the sulfonyl. [000363] "Arylsulfonyl" means an aryl-S(0 ₂ )- group. The bond to the parent moiety is through the sulfonyl.

[000364] The term "substituted" means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

[000365] By "stable compound" or "stable structure" is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[000366] The term "optionally substituted" means optional substitution with the specified groups, radicals or moieties.

[000367] When a functional group in a compound is termed "protected", this means that the group is in modified form to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those with ordinary skill in the art as well as by reference to standard textbooks such as, for example, Greene et a/ (1991 ).

[000368] When any variable (e.g., aryl, heterocycle, R2) occurs more than one time in any constituent or in the present invention, its definition on each occurrence is independent of its definition at every other occurrence.

[000369] In one form of the invention, each of the charged or hydrogen bonding groups is an amino acid residue selected, independently, from the group: Asp, Glu.

[000370] In one form of the invention, each of the charged or hydrogen bonding groups is an amino acid residue having a carboxylic acid moiety.

[000371 ] In one form of the invention, each of the charged or hydrogen bonding groups is a chemical moiety selected, independently, from the group: carboxylic acid, Hydroxaymic acids, phosphonic and phosphinic acids, sulfonic and sulfinic acids, sulphonamides, acylsulfonamides and sulfonylureas, 2,2,2-Trifluoroethan-1 -ol and Trifluoromethylketones, tetrazoles, 5-Oxo-1 ,2,4-oxadiazole and 5-Oxo-1 ,2,4-thiadiazoles, Thiazolidinedione, Oxazolidinedione, and Oxadiazolidine-diones, 3-Hydroxyisoxazole and 3- Hydroxyisothiazoles, substituted phenols, squaric acids, 3- and 4-Hydroxyquinolin-2-ones, Tetronic and Tetramic Acids, Cyclopentane-1 ,3-diones and other cyclic and acyclic structures, including boronic acids, mercaptoazoles, and sulfonimidamides (Ballatore et al., 2013).

[000372] In one form, the invention provides a method for identifying a modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, said method comprising the steps of: (1 ) comparing the three dimensional structure of the compound with a pharmacophore comprising two or more features selected from the group: a first charged or hydrogen bonding group (A), a second charged or hydrogen bonding group (B), a third charged or hydrogen bonding group (C), and a hydrophobic group (D) wherein the distances in between the features are as follows, within a tolerance of ±10 A:

and (2) selecting a compound with hydrophobic and/or charged or hydrogen bonding chemical moieties so located.

[000373] The modulator of RAGE ligand-independent activation of RAGE identified by the abovementioned method comprising comparison to a pharmacophore may be a peptide, or a non-peptidyl compound.

[000374] In a preferred form of the invention, the tolerance is up to ±5 A, provided the distances between the site points is positive in magnitude. In a preferred form of the invention, the tolerance is up to ±2 A, provided the distances between the site points is positive in magnitude. In a preferred form of the invention, the tolerance is up to ±1 A, provided the distances between the site points is positive in magnitude.

[000375] In a preferred form of the invention, the modulator comprises three or more features selected from the above-specified group.

In a preferred form of the invention, the modulator comprises four features from the above- specified group.

[000376] In one form of the invention, comparison of the three dimensional structure of the compound with the pharmacophore involves comparison of a minimum energy structure of the compound with the pharmacophore. [000377] An efficient means to select a compound from a potentially large number of compounds involves comparing compounds against the pharmacophore of the invention using a computer program, for example Catalyst (MSI), to screen one or more computerised databases of three dimensional chemical structures of compounds.

[000378] In one form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a peptide that has an amino acid sequence as set forth in SEQ ID NO: 1 , or an analogue, fragment or derivative thereof that contains at least residues 379-390.

[000379] In one form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a peptide of the formula SEQ ID NO: 1 , or an analogue or derivative thereof.

[000380] In one form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a peptide of the formula SEQ ID NO: 2, or an analogue or derivative thereof.

[000381 ] In one form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a peptide of the formula SEQ ID NO: 5, or an analogue or derivative thereof.

[000382] In one form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a peptide of the formula SEQ ID NO: 6, or an analogue or derivative thereof.

[000383] In one form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a S391 A-E392X-RAGE peptide as set forth in SEQ ID NO: 7, or an analogue or derivative thereof.

[000384] SEQ ID NO: 7:

[L362WQRRQRRGEERKAPENQEEEEERAELNQA391] [000385] In one form of the invention, the modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, is a S391 X-RAGE peptide as set forth in of SEQ ID NO: 8, or an analogue or derivative thereof.

[000386] SEQ ID NO: 8:

[L362WQRRQRRGEERKAPENQEEEEERAELNQ390]

[000387] Preferred specific derivatives include Q379EEEEERAELNR390, as set forth in SEQ ID NO: 9, Q379EEEEERAELNK390 as set forth in SEQ ID NO: 10, K379EEEEERAELNQ390 as set forth in SEQ ID NO: 1 1 , K379EEEERAELNK390 as set forth in SEQ ID NO: 12, and K379EEEEERAELNR390 as set forth in SEQ ID NO: 13 below.

[000388] SEQ ID NO: 9: [Q379EEEEERAELNR390]

[000389] SEQ ID NO: 10: [Q379EEEEERAELN K390]

[000390] SEQ ID NO: 1 1 : [K379EEEEERAELNQ390]

[000391 ] SEQ ID NO: 12: [K379EEEEERAELNK390]

[000392] SEQ ID NO: 13: [K379EEEEERAELNR390]

[000393] The term "derivative" as used herein in connection with modulators of the invention, such as SEQ ID NO: 1 , 2, 5 to 13, refers to a modulator characterised in that its primary structure is taken from or owes its derivation to the C-terminal cytosolic tail of RAGE or fragment thereof, but which includes amino acid additions, substitutions, truncations, chemical and/or biochemical modifications (acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, side chain methylation), labelling with radionucleotides or halogens, unusual or artificial amino acids (such as D-amino acids, N-methylated amino acids, fefra-substitution, β-peptides, pyroglutamic acid; 2-Aminoadipic acid; 3-Aminoadipic acid; beta-Alanine; beta-Aminopropionic acid; 2-Aminobutyric acid; 4-Aminobutyric acid; Piperidinic acid; 6-Aminocaproic acid; 2-Aminoheptanoic acid; 2-Aminoisobutyric acid; 3- Aminoisobutyric acid; 2-Aminopimelic acid; 2,4-Diaminobutyric acid; Desmosine; 2,2"- Diaminopimelic acid; 2,3-Diaminopropionic acid; N-Ethylglycine; N-Ethylasparagine; Hydroxylysine; allo-Hydroxylysine; 3-Hydroxyproline; 4-Hydroxyproline; Isodesmosine; allo- Isoleucine; N-Methylglycine; Sarcosine; N-Methylisoleucine; N-Methylvaline; Norvaline; Norleucine; Ornithine; Statine), retroinverted sequences, cyclic peptides, peptoids, or linkage to a non-peptide drug, non-peptide label, non-peptide carrier, or non-peptide resin. [000394] The inventors have further discovered that a peptide comprising residues 343- 361 of wild-type RAGE (SEQ ID NO: 14) is an inhibitory peptide, that inhibits both RAGE ligand-independent and RAGE ligand-dependent activation of RAGE.

[000395] Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a polypeptide is replaced with another naturally-occurring amino acid of similar character either in relation to polarity, side chain functionality, or size, for example

It is to be understood that some non-conventional amino acids may also be suitable replacements for the naturally occurring amino acids. For example ornithine, homoarginine and dimethyllysine are related to His, Arg and Lys.

[000396] Substitutions encompassed by the present invention may also be "non- conservative", in which an amino acid residue which is present in a polypeptide is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (e.g. substituting a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

[000397] Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed. Preferably, amino acid substitutions are conservative.

[000398] Additions encompass the addition of one or more naturally occurring or non- conventional amino acid residues. Deletion encompasses the deletion of one or more amino acid residues.

[000399] As stated above the present invention includes peptides in which one or more of the amino acids has undergone sidechain modifications. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ₄ ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ₄ . [000400] The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[000401 ] The carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. In a preferred form of the invention, any modification of cysteine residues must not affect the ability of the peptide to form the necessary disulfide bonds. It is also possible to replace the sulphydryl groups of cysteine with selenium equivalents such that the peptide forms a di-selenium bond in place of one or more of the disulfide bonds.

[000402] Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

[000403] Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Proline residues may be modified by, for example, hydroxylation in the 4-position.

[000404] A list of some amino acids having modified side chains and other unnatural amino acids is shown in the following table:

[000405] These types of modifications may be important to stabilise the peptide if administered to an individual or for use as a diagnostic reagent. [000406] Conservative amino acid substitutions, as used herein, may include amino acid residues within a group which have sufficiently similar physicochemical properties, so that a substitution between members of the group will preserve the biological activity of the molecule (see for example Grantham, R., 1974). Particularly, conservative amino acid substitutions are preferably substitutions in which the amino acids originate from the same class of amino acids (e.g. basic amino acids, acidic amino acids, polar amino acids, amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chains, amino acids the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function). Conservative substitutions are in the present case for example substituting a basic amino acid residue (Lys, Arg, His) for another basic amino acid residue (Lys, Arg, His), substituting an aliphatic amino acid residue (Gly, Ala, Val, Leu, lie) for another aliphatic amino acid residue, substituting an aromatic amino acid residue (Phe, Tyr, Trp) for another aromatic amino acid residue, substituting threonine by serine or leucine by isoleucine. Further conservative amino acid exchanges will be known to the person skilled in the art. The isomer form should preferably be maintained, e.g. K is preferably substituted for R or H, while k is preferably substituted for r and h.

[000407] When considering replacement amino acids, preferred replacements of the present invention are those described as having a D of less than 100 in Grantham, R. (1974), the contents of which are incorporated by reference. Most preferred replacements are those described as having a D of less than 50.

[000408] Peptide modulators of the present invention include retro inverso isomers of, or modified or substituted variants of, SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13, or peptides formed by additions or deletions (Li et al., 2010).

Methods for treating, preventing or managing RAGE-related disorders

[000409] In another related aspect, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, the method comprising administration of an effective amount of a modulator of RAGE ligand- independent activation of RAGE by a certain activated co-located GPCR of the invention.

[000410] In another aspect, the present invention comprises use of a modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR for the manufacture of a medicament for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment. [00041 1 ] In another aspect, the present invention comprises use of a modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment.

[000412] In a preferred form of the invention, the certain co-located GPCR is an angiotensin receptor. In a preferred form of the invention, the certain co-located GPCR is

[000413] In a preferred form of the invention, the certain co-located GPCR is a certain chemokine receptor. In a preferred form of the invention, the certain co-located GPCR is CCR2.

[000414] Furthermore, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, the method comprising administration of an effective amount of a combination of a modulator of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR of the invention with a modulator of the certain co-located GPCR and/or a modulator of the certain co-located GPCR signalling pathway.

[000415] In a preferred form of the invention, the certain co-located GPCR is an angiotensin receptor. In a preferred form of the invention, the certain co-located GPCR is

[000416] In a preferred form of the invention, the certain co-located GPCR is a certain chemokine receptor. In a preferred form of the invention, the certain co-located GPCR is CCR2.

[000417] The method may comprise administration of an effective amount of a combination of a modulator of RAGE ligand-independent activation of RAGE by activated certain co-located GPCR of the invention with a modulator of the certain co-located GPCR and/or a modulator of the certain co-located GPCR signalling pathway, wherein the modulator of the certain co-located GPCR and/or the modulator of the certain co-located GPCR signalling pathway is administered at a lower dose than normally administered for the treatment of a disorder related to the certain co-located GPCR.

[000418] The method may comprise administration of an effective amount of a combination of a modulator of RAGE ligand-independent activation of RAGE by activated certain co-located GPCR of the invention with a modulator of the certain co-located GPCR and/or a modulator of the certain co-located GPCR signalling pathway, wherein the modulator of the certain co-located GPCR and/or the modulator of the certain co-located GPCR signalling pathway is administered at a lower dose than normally administered for the treatment of a disorder related to RAGE.

[000419] In a particularly preferred form of the invention, the method comprises administration of an effective amount of a combination of a modulator of RAGE ligand- independent activation of RAGE by activated angiotensin receptor of the invention with a modulator of AT ₁ R and/or a modulator of an AT ₁ R signalling pathway, wherein the modulator of the AT ₁ R and/or the modulator of an AT ₁ R signalling pathway is administered at a lower dose than normally administered for the treatment of an AT ₁ R -related disorder.

[000420] In another particularly preferred form of the invention, the method comprises administration of an effective amount of a combination of a modulator of RAGE ligand- independent activation of RAGE by activated certain chemokine receptor of the invention with a modulator of CCR2 and/or a modulator of a CCR2 signalling pathway, wherein the modulator of the CCR2 and/or the modulator of a CCR2 signalling pathway is administered at a lower dose than normally administered for the treatment of a CCR2-related disorder.

[000421 ] Furthermore, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, the method comprising administration of an effective amount of a combination of a modulator of RAGE ligand-independent activation of RAGE by activated certain co-located GPCR of the invention with a modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or a modulator of a RAGE signalling pathway.

[000422] In a particularly preferred form of the invention, the method comprises administration of an effective amount of a combination of a modulator of RAGE ligand- independent activation of RAGE by activated certain co-located GPCR of the invention with a modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or a modulator of a RAGE signalling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or the modulator of a RAGE signalling pathway is administered at a lower dose than normally administered for the treatment of a RAGE- related disorder.

[000423] Furthermore, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, the method comprising administration of an effective amount of a combination of a modulator of RAGE ligand-independent activation of RAGE by activated certain co-located GPCR of the invention with a modulator of the certain co-located GPCR and/or a modulator of the certain co-located GPCR signalling pathway.

[000424] For example, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, the method comprising administration of an effective amount of a combination of a modulator of RAGE ligand-independent activation of RAGE by activated angiotensin receptor of the invention with a modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or a modulator of a RAGE signalling pathway, and a modulator of AT ₁ R and/or a modulator of an AT ₁ R signalling pathway.

[000425] For example, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, the method comprising administration of an effective amount of a combination of a modulator of RAGE ligand-independent activation of RAGE by activated certain chemokine receptor of the invention with a modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or a modulator of a RAGE signalling pathway, and a modulator of CCR2 and/or a modulator of a CCR2 signalling pathway.

[000426] In a particularly preferred form of the invention, the method comprises administration of an effective amount of a combination of a modulator of RAGE ligand- independent activation of RAGE by activated certain co-located GPCR of the invention with a modulator of the certain co-located GPCR and/or a modulator of a signalling pathway of the certain co-located GPCR and a modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or a modulator of a RAGE signalling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or the modulator of a RAGE signalling pathway is administered at a lower dose than normally administered for the treatment of a RAGE-related disorder, and/or the modulator of the certain co-located GPCR and/or the modulator of a signalling pathway of the certain co-located GPCR is administered at a lower dose than normally administered for the treatment of a disorder related to the GPCR.

[000427] In a particularly preferred form of the invention, the method comprises administration of an effective amount of a combination of a modulator of RAGE ligand- independent activation of RAGE by activated angiotensin receptor of the invention with a modulator of AT ₁ R and/or a modulator of an AT ₁ R signalling pathway and a modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or a modulator of a RAGE signalling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or the modulator of a RAGE signalling pathway is administered at a lower dose than normally administered for the treatment of a RAGE-related disorder, and/or the modulator of AT ₁ R and/or the modulator of an AT ₁ R signalling pathway is administered at a lower dose than normally administered for the treatment of an AT ₁ R -related disorder.

[000428] In a particularly preferred form of the invention, the method comprises administration of an effective amount of a combination of a modulator of RAGE ligand- independent activation of RAGE by activated certain chemokine receptor of the invention with a modulator of CCR2 and/or a modulator of a CCR2 signalling pathway and a modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or a modulator of a RAGE signalling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE and/or a modulator of constitutively-active RAGE and/or the modulator of a RAGE signalling pathway is administered at a lower dose than normally administered for the treatment of a RAGE-related disorder, and/or the modulator of CCR2 and/or the modulator of a CCR2 signalling pathway is administered at a lower dose than normally administered for the treatment of a CCR2-related disorder.

[000429] A RAGE-related disorder is defined as a disorder that is dependent upon the expression of RAGE. It does not exclude disorders related to the certain co-located GPCR, such as AT ₁ R-related disorders or CCR2-related disorders, that are also dependent upon the expression of RAGE. Indeed a disorder can be both RAGE-related and related to the certain co-located GPCR, including AT ₁ R -related or CCR2-related.

[000430] A disorder related to the certain co-located GPCR is defined as a disorder that is dependent upon the expression of the certain co-located GPCR. It does not exclude RAGE- related disorders that are also dependent upon the expression of the certain co-located GPCR. Indeed a disorder can be both RAGE-related and related to the certain co-located GPCR, including AT ₁ R -related or CCR2-related.

[000431 ] In one form of the invention, a RAGE-related disorder is a disorder selected from the group: cardiovascular disorders; digestive disorders; cancers; neurological disorders, respiratory disorders, connective tissue disorders, kidney disorders, genital disorders, skin disorders, eye disorders and endocrine disorders.

[000432] In one form of the invention, the RAGE-related disorder is a cardiovascular disorder selected from the group: atherosclerosis, ischaemic heart disease, myocarditis, endocarditis, cardiomyopathy, acute rheumatic fever, chronic rhematic heart disease, cerebrovascular disease/stroke, heart failure, vascular calcification, peripheral vascular disease, and lymphangitis.

[000433] In one form of the invention, the RAGE-related disorder is a digestive system disorder selected from the group: periodontitis, oesophagitis, gastritis, gastro-duodenal ulceration, Crohn disease, ulcerative colitis, ischaemic colitis, enteritis and enterocolitis, peritonitis, alcoholic liver disease, hepatitis, toxic liver disease, biliary cirrhosis, hepatic fibrosis/cirrhosis, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), liver trauma and recovery from liver injury, trauma or surgery.

[000434] In one form of the invention, the RAGE-related disorder is a cancer selected from the group: malignant neoplasms of lip, oral cavity and pharynx, malignant neoplasms of digestive organs, malignant neoplasms of respiratory and intrathoracic organs, malignant neoplasms of bone and articular cartilage, melanoma and other malignant neoplasms of skin, malignant neoplasms of mesothelial and soft tissue, malignant neoplasm of breast, malignant neoplasms of female genital organs, malignant neoplasms of male genital organs, malignant neoplasms of urinary tract, malignant neoplasms of eye, brain and other parts of central nervous system, malignant neoplasms of thyroid and other endocrine glands, malignant neoplasms of lymphoid, haematopoietic and related tissue, malignant neoplasms of ill-defined, secondary and/or unspecified sites.

[000435] In one form of the invention, the RAGE-related disorder is a neurological disorder and is selected from the group: inflammatory diseases of the central nervous system, systemic atrophies primarily affecting the central nervous system, extrapyramidal and movement disorders, Parkinson's disease, demyelinating diseases of the central nervous system, Alzheimer's disease, circumscribed brain atrophy, Lewy body disease, epilepsy, migraine, neuropathic pain, diabetic neuropathy, polyneuropathies, glioma development and progression, spinal cord trauma, and ischaemic brain injury/stroke, brain trauma and recovery from brain injury, trauma or surgery.

[000436] In one form of the invention, the RAGE-related disorder is a mental disorder and is selected from the group: dementia, Alzheimer's disease, vascular dementia, addiction, schizophrenia, major affective disorder, depression, mania, bipolar disorder, and anxiety disorder.

[000437] In one form of the invention, the RAGE-related disorder is a respiratory (pulmonary) disorder and is selected from the group: Acute upper respiratory infections, rhinitis, nasopharyngitis, sinusitis, laryngitis, influenza and pneumonia, acute bronchitis, acute bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, emphysema, chronic lung diseases due to external agents, Acute Respiratory Distress Syndrome (ARDS), pulmonary eosinophilia, and pleuritic, lung trauma and recovery from lung injury, trauma or surgery.

[000438] In one form of the invention, the RAGE-related disorder is a connective tissue disorder and is selected from the group: osteoarthritis, infectious arthritis, rheumatoid arthritis, psoriatic and enteropathic arthropathies, juvenile arthritis, gout and other crystal arthropathies, diabetic arthropathy, polyarteritis nodosa, Churg-Strauss, mucocutaneous lymph node syndrome [Kawasaki], hypersensitivity angiitis, Goodpasture syndrome, thrombotic microangiopathy, Wegener granulomatosis, Aortic arch syndrome [Takayasu], giant cell arteritis, polymyalgia rheumatica, microscopic polyangiitis, hypocomplementaemic vasculitis, systemic lupus erythematosus, dermatopolymyositis, polymyositis, systemic sclerosis, CR(E)ST syndrome, Sicca syndrome [Sjogren], mixed connective tissue disease, Behget disease, traumatic muscle damage, sprain, strain, and fracture.

[000439] In one form of the invention, the RAGE-related disorder is a kidney disorder and is selected from the group: glomerulonephritis, nephritis, diabetic kidney disease, interstitial nephritis, obstructive and reflux nephropathy, acute renal failure, and chronic kidney disease.

[000440] In one form of the invention, the RAGE-related disorder is a genital disorder and is selected from the group: prostatitis, prostatic hypertrophy, prostatic dysplasia, salpingitis, oophoritis, pelvic inflammatory disease (PID), polycystic ovarian syndrome, cervicitis, cervical dysplasia, vaginitis, vulvitis.

[000441 ] In one form of the invention, the RAGE-related disorder is a skin disorder selected from the group: dermatitis, eczema, pemphigus/pemphygoid, psoriasis, pityriasis rosea, lichen planus, urticarial, erythrema multiforme, erythema nordosum, sunburn, keratosis, photoageing skin ulceration, superficial skin injury, and open wound.

[000442] In one form of the invention, the RAGE-related disorder is an eye disorder selected from the group: keratitis, conjunctivitis, retinitis, glaucoma, scleritis, episcleritis, chorioretinal inflammation, diabetic retinopathy, macular oedema, retinopathy of prematurity, and optic neuritis, eye trauma and recovery from eye injury, trauma or surgery.

[000443] In one form of the invention, the RAGE-related disorder is an endocrine disorder selected from the group: diabetes mellitus, insulin resistance, impaired glucose tolerance and thyroiditis. [000444] In one form of the invention, the inhibitor that inhibits AT ₁ R or inhibits an AT ₁ R signalling pathway is selected from the group: Eprosartan (market name Teveten®, Abbott Laboratories USA), Losartan (market name Cozaar®, Merck & Co), Valsartan (market name Diovan®, Novartis), Telmisartan (market name Micardis®, Boehringer Ingelheim), Irbesartan (market name Avapro®, SanofiAventis), Olmesartan (market name Benicar®, Daiichi Sankyo Inc), Azilsartan (market name Edarbi, Takeda), Candesartan (market name Atacand®, AstraZeneca), ZD-71 15, Saralasin ((Sar1 -Ala8)Angll), Sarthran ((Sar1 - Thr8)Angll) and DuP753. This list also includes pro-drugs of these inhibitors, including Candesartan (Candesartan cilexetil), Azilsartan (Azilsartan medoxomil) and Olmesartan (Olmesartan medoxomil), that may be the form in which they are administered, as well as active metabolites (such as EXP-3174, the active metabolite of Losartan). Note a partial agonist can act to inhibit endogenous Ang II as a partial agonist does not result in maximal efficacy even though it exhibits agonism, and so therapeutically may act as an inhibitor.

[000445] In one form of the invention, the inhibitor that inhibits the certain chemokine receptor or inhibits the certain chemokine signalling pathway is selected from the group: Propagermanium (also known as 3-[(2-Carboxyethyl-oxogermyl)oxy-oxogermyl]propanoic acid, proxigermanium, Ge-132, bis (2-carboxyethylgermanium) sesquioxide (CEGS), 2- carboxyethylgermasesquioxane, SK-818, organic germanium, germanium sesquioxide, 3,3 ^' - (1 ,3-dioxo-1 ,3-digermanoxanediyl) bispropionic acid, 3-oxygermylpropionic acid polymer, poly-trans-(2-carboxyethyl) germasesquioxane, proxigermanium, repagermanium and Serocion; CCR2), BMS CCR2 22 (CCR2), resveratrol (CCR2), RS504393 (CCR2), RS102895 (CCR2), MLN-1202 (Millennium Pharmaceuticals; CCR2), INCB8696 (Incyte Pharmaceuticals; CCR2), MK-0812 (Merck; CCR2), CCX140 (ChemoCentryx; CCR2), PF- 4136309 (Pfizer; CCR2), BMS-741672 (Bristol-Myers Squibb; CCR2); Repertaxin (CXCR2), TAK-779 (CCR5), TAK-220 (CCR5), TAK-652 (CCR5), AK692 (CCR5), CMPD167 (CCR5), BX-471 (CCR1 ), AMD3100 (CXCR4), AMD1 1070 (CXCR4), FC131 (CXCR4), MLN3897 (CCR1 ), CP-481715 (CCR1 ), GW-873140 (CCR5), SB 225002 (CXCR2) and SB 265610 (CXCR2).

[000446] In one form of the invention, the inhibitor that inhibits CCR2 or inhibits a CCR2 signalling pathway is selected from the group: Propagermanium (also known as 3-[(2- Carboxyethyl-oxogermyl)oxy-oxogermyl]propanoic acid, proxigermanium, Ge-132, bis (2- carboxyethylgermanium) sesquioxide (CEGS), 2-carboxyethylgermasesquioxane, SK-818, organic germanium, germanium sesquioxide, 3,3 ^' -(1 ,3-dioxo-1 ,3-digermanoxanediyl) bispropionic acid, 3-oxygermylpropionic acid polymer, poly-trans-(2-carboxyethyl) germasesquioxane, proxigermanium, repagermanium and Serocion), BMS CCR2 22 (CCR2), resveratrol (CCR2), RS504393, RS102895, MLN-1202 (Millennium Pharmaceuticals), INCB8696 (Incyte Pharmaceuticals), MK-0812 (Merck), CCX140 (ChemoCentryx), PF- 4136309 (Pfizer), BMS-741672 (Bristol-Myers Squibb).

[000447] In one form of the invention, the inhibitor of RAGE ligand-dependent activation of RAGE and/or an inhibitor of constitutively-active RAGE and/or the inhibitor of a RAGE signalling pathway is selected from the group: Azeliragon (TTP488/ PF-04494700) (an oral, small-molecule inhibitor of RAGE-ligand interactions targeting the V-domain); TTP4000 (a soluble fusion protein inhibitor of RAGE, using the ligand-binding ectodomains of RAGE linked to a human Ig Fc domain as described in US 7981423); antibodies that bind specifically to RAGE and RAGE-binding fragments thereof as described in WO2007109747; FPS-ZM127 (a tertiary amide that blocks Αβ/RAGE interaction with high affinity); peptides that antagonise RAGE ligand-induced signalling as described in US 20100249038; lysophosphatidic acid (LPA) antagonists as described in WO2012109569; 2- aminopyrimidines as described in Han et a/ (2012); pyrazole-5-carboxamides as described in Han et al (2014); 4,6-bisphenyl-2-(3-alkoxyanilino)pyrimidine as described in Han et al (2015); small molecule inhibitors of Ligand-Stimulated RAGE-DIAPH1 Signal Transduction as described in Manigrasso et al (2016); polypeptides consisting essentially of all or a portion of the cytosolic tail of RAGE, or consisting essentially of a portion of Diaphanous-1 that binds to the cytosolic tail of RAGE as described in US20090220484.

[000448] In specific embodiments, the modulator is administered to the subject on the basis that it is identified as a modulator of RAGE ligand-independent activation of RAGE by activated certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or a certain chemokine receptor, such as CCR2, using the screening methods or methods for identifying modulators broadly described herein.

[000449] An AT ₁ R-related disorder is defined as a disorder that is dependent upon the expression of AT ₁ R. It does not exclude RAGE-related disorders that are also dependent upon expression of AT ₁ R. Indeed a disorder can be both RAGE-related and AT ₁ R-related.

[000450] A certain chemokine receptor-related disorder is defined as a disorder that is dependent upon the expression of a certain chemokine receptor. It does not exclude RAGE- related disorders that are also dependent upon expression of a certain chemokine receptor. Indeed a disorder can be both RAGE-related and certain chemokine receptor-related.

[000451 ] A CCR2-related disorder is defined as a disorder that is dependent upon the expression of CCR2. It does not exclude RAGE-related disorders that are also dependent upon the expression of CCR2. Indeed a disorder can be both RAGE-related and CCR2- related.

[000452] The following doses are "normally" administered for the combination agents.

Methods of screening candidate agents

[000453] In one form, the present invention comprises methods of screening candidate agents for their ability to modulate RAGE activity where such RAGE activity is induced by an active co-located GPCR, the method comprising the steps of: contacting a RAGE polypeptide with a GPCR polypeptide in the presence of a candidate agent where the GPCR polypeptide is constitutively active and/or is activated by addition of an agonist, partial agonist or allosteric modulator of that GPCR; and detecting whether the candidate agent is a modulator of RAGE ligand-independent activation of RAGE by activated co-located GPCR by detecting an effect indicative of modulation of RAGE activation by the presence of the candidate agent and/or by detecting RAGE-dependent signalling that is modulated by the presence of the candidate agent.

[000454] In one form, the present invention comprises methods of screening candidate agents for their ability to modulate (i.e. activate, inhibit or allosterically modulate), RAGE ligand-independent activation of RAGE by, activated certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 (also known as RAGE ligand-independent transactivation of RAGE). These methods generally comprise, consist or consist essentially of: a. contacting a RAGE polypeptide with a GPCR polypeptide in the presence of a candidate agent where the GPCR polypeptide is constitutively active and/or is activated by addition of an agonist, partial agonist or allosteric modulator of that GPCR; and b. detecting whether the candidate agent is a modulator of RAGE ligand- independent activation of RAGE by activated co-located GPCR by detecting an effect indicative of modulation of RAGE activation by the presence of the candidate agent and/or by detecting RAGE-dependent signalling that is modulated by the presence of the candidate agent.

[000455] In some embodiments, the screening methods further comprise detecting whether the candidate agent is a modulator (such as activator, inhibitor or allosteric modulator) of the certain co-located GPCR, such as angiotensin receptor, such as an AT ₁ R or certain chemokine receptor, such as CCR2, or a signalling pathway of the certain co- located GPCR, such as an angiotensin receptor signalling pathway, such as an AT ₁ R signalling pathway or such as a certain chemokine receptor signalling pathway, such as a CCR2 signalling pathway, in the presence or absence of RAGE. In some embodiments, the candidate agent that results in greater modulation of the signal when the RAGE polypeptide is present compared to when it is absent is selective for modulating RAGE-ligand independent activation of RAGE by activated co-located GPCR over RAGE-independent signalling resulting from activation of the co-located GPCR.

[000456] In one form, the invention comprises peptides idenitifed as modulators by said methods. In one form, the invention comprises compounds identified as modulators by said methods.

[000457] In some embodiments, the screening methods further comprise detecting whether the candidate agent is a modulator (such as activator, inhibitor, allosteric modulator or functional substitute) of RAGE or a RAGE signalling pathway in the presence or absence of the certain co-located GPCR, such as an angiotensin receptor, such as AT ₁ R or such as a certain chemokine receptor, such as CCR2. In some embodiments, the candidate agent that results in greater modulation of the RAGE-dependent signal when the GPCR polypeptide is present compared to when it is absent is selective for modulating RAGE-ligand independent activation of RAGE by activated co-located GPCR.

[000458] In some embodiments, the screening methods further comprise detecting whether the candidate agent is a modulator (such as activator, inhibitor, allosteric modulator or functional substitute) of a RAGE polypeptide or a RAGE signalling pathway as well as the certain co-located GPCR, such as angiotensin receptor, such as an AT ₁ R or certain chemokine receptor, such as CCR2, or a signalling pathway of the certain co-located GPCR, such as an angiotensin receptor signalling pathway, such as an AT ₁ R signalling pathway or such as a certain chemokine receptor signalling pathway, such as a CCR2 signalling pathway.

[000459] In some embodiments, the screening method further comprises the step of using an inhibitor of RAGE ligand binding to the RAGE ectodomain that as such inhibits activation of RAGE in a RAGE ligand-dependent manner.

[000460] In some embodiments, the screening method further comprises use of a RAGE polypeptide that is mutated and/or truncated such that it is not able to bind RAGE ligands to its ectodomain and as such is not able to be activated in a RAGE ligand-dependent manner.

[000461 ] In some embodiments, binding of RAGE ligands to the ectodomain of RAGE is impaired by exposing the cell to a modulator that modulates the binding of RAGE ligands to RAGE. [000462] In some embodiments the use of a RAGE polypeptide that is mutated and/or truncated such that it is not able to bind RAGE ligands and as such is not able to be activated in a RAGE ligand-dependent manner occurs before, after or in parallel with a screen involving a RAGE polypeptide that is able to bind RAGE ligands.

[000463] Suitably, a candidate agent or a derivative of a candidate agent, which modulates RAGE ligand-independent activation of RAGE by activated certain co-located GPCR, such as angiotensin receptor, such as ATiR, or certain chemokine receptor, such as CCR2, and that suitably modulates a certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 and/or a signalling pathway of the certain co-located GPCR, such as an angiotensin receptor signalling pathway, such as an AT ₁ R signalling pathway or such as a certain chemokine receptor signalling pathway, such as a CCR2 signalling pathway and/or that inhibits RAGE ligand-dependent activation of RAGE and/or inhibits constitutively-active RAGE and/or a RAGE signalling pathway, is particularly useful for treating, preventing or managing a RAGE-related disorder.

[000464] In certain embodiments of the screening method of the invention wherein if the candidate agent modulates the RAGE-dependent signal detected when a RAGE polypeptide is contacted with a GPCR polypeptide, the method further comprises determining whether, and/or the extent to which the candidate agent modulates the RAGE-dependent signal in the absence of the GPCR polypeptide such that the candidate agent that results in greater modulation of the RAGE-dependent signal when the GPCR polypeptide is present is selective for modulating RAGE-ligand independent activation of RAGE by activated co- located GPCR.

[000465] In certain embodiments of the screening method of the invention wherein if the candidate agent modulates the signal detected when a RAGE polypeptide is contacted with a GPCR polypeptide, the method further comprises determining whether, and/or the extent to which the signal is generated in the absence of the RAGE polypeptide and if the signal is generated in the absence of the RAGE polypeptide, determining whether, and/or the extent to which the candidate agent modulates the signal in the absence of the RAGE polypeptide such that the candidate agent that results in greater modulation of the signal when the RAGE polypeptide is present is selective for modulating RAGE-ligand independent activation of RAGE by activated co-located GPCR over RAGE-independent signalling resulting from activation of the co-located GPCR.

[000466] In certain embodiments, the screening method assesses proximity of the RAGE polypeptide to the certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 using a proximity screening assay. In illustrative examples of this type, the RAGE polypeptide is coupled (e.g., conjugated or otherwise linked) to a first reporter component and the certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 is coupled (e.g., conjugated or otherwise linked) to a second reporter component. Proximity of the first and second reporter components generates a signal capable of detection by the detector. The first and second reporter components constitute a complementary pair, in the sense that the first reporter component may be interchanged with the second reporter component without appreciably affecting the functioning of the invention. The first and second reporter components can be the same or different.

[000467] In one embodiment, the proximity screening assay is that described in patent WO2008055313 (Dimerix Bioscience Pty Ltd; also US8283127, US8568997, EP2080012, CA2669088, CN101657715), also known as Receptor Heteromer Investigation Technology or Receptor-HIT (Jaeger et al., 2014). With this method, RAGE is coupled to a first reporter component, the certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2, is unlabeled with respect to the proximity screening assay, and a GPCR-interacting group is linked to the complementary second reporter component, whose interaction with the complex is modulated upon binding a ligand selective for the unlabeled GPCR or the heteromer complex specifically. Preferred examples of GPCR-interacting groups are arrestins, G proteins and ligands. Alternatively, the certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2, is coupled to a first reporter component, RAGE is unlabeled with respect to the proximity screening assay, and a RAGE-interacting group is linked to the complementary second reporter component, whose interaction with the complex is modulated upon binding a ligand selective for the unlabeled RAGE or the heteromer complex specifically. Preferred examples of RAGE-interacting groups are proteins interacting with the cytosolic tail of RAGE, such as IQGAP-1 , Diaphanous 1 , Dock7, MyD88, TIRAP, IRAK4, ERK1/2, and ΡΚCζ (Jules et al., 2013; Ramasamy et al., 2016).

[000468] Reporter components can include enzymes, luminescent or bioluminescent molecules, fluorescent molecules, and transcription factors or other molecules coupled to RAGE, the certain co-located GPCR or the interacting group by linkers incorporating enzyme cleavage sites. In short any known molecule, organic or inorganic, proteinaceous or non- proteinaceous or complexes thereof, capable of emitting a detectable signal as a result of their spatial proximity. [000469] Preferably, signal generated by the proximity of the first and second reporter components in the presence of the reporter component initiator is selected from the group consisting of: luminescence, fluorescence and colorimetric change.

[000470] In some embodiments, the luminescence is produced by a bioluminescent protein selected from the group consisting of luciferase, galactosidase, lactamase, peroxidase, or any protein capable of luminescence in the presence of a suitable substrate.

[000471 ] Preferable combinations of first and second reporter components include those detailed in US Patent 8,283,127, however, useful combinations of first and second reporter components are by no means limited to such.

[000472] In some embodiments, the screening methods further comprise detecting proximity of the first and second reporter components to one another to thereby determine whether the candidate agent modulates the interaction between the RAGE polypeptide and the certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2. Generally, this is achieved when proximity of the first and second reporter components generates a proximity signal that is altered by the modulation by the candidate agent of the proximity between the RAGE polypeptide and the certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2.

[000473] One or both of the RAGE and certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 may be in soluble form or expressed on the cell surface.

[000474] In some embodiments, the RAGE and certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 are located in, partially in, or on a single membrane; for example, both are expressed at the surface of a host cell.

[000475] In another embodiment of the invention, the certain co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 is pre- assembled with RAGE in a pre-formed complex at the cell membrane.

[000476] In another embodiment of the invention, following activation of the certain co- located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 by engagement of cognate ligand, such as Ang II for AT ₁ R or MCP-1 for CCR2, signalling is triggered that involves the cytosolic tail of RAGE. [000477] In one embodiment of the invention, activation of the cytosolic tail of RAGE is associated with changes in its structural conformation and/or affinity for binding partners.

[000478] In one embodiment of the invention, monitoring of the structural conformation of RAGE and/or affinity for binding partners occurs when the cytosolic tail of RAGE has been mutated and/or truncated such that it can no longer be activated by RAGE ligands or by RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs.

[000479] In one embodiment of the invention, monitoring structural conformation and/or affinity for binding partners occurs in the presence of agents that inhibit binding and/or activation of RAGE by RAGE ligands or RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs.

[000480] In one embodiment of the invention, monitoring recruitment of binding partners occurs prior to activation of RAGE by RAGE ligands or RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs.

[000481 ] In one embodiment of the invention, monitoring recruitment and activation of signalling mediators and/or binding partners to the RAGE cytosolic tail occurs subsequent to activation of RAGE by RAGE ligands or RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs.

[000482] In one embodiment of the invention, monitoring recruitment of binding partners following activation of RAGE by RAGE ligands or RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs occurs in the presence of agents that inhibit binding and/or activation of RAGE by RAGE ligands.

[000483] Further embodiments of the invention comprise methods of screening candidate agents for their ability to modulate (such as activate, inhibit or otherwise modulate) RAGE ligand-independent activation of RAGE by a certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2, by detecting modulation of the RAGE-mediated signalling. Such methods may include the step of measuring canonical activation of N FKB, by measuring one or more of the following:

• Activity of IkB kinase (IKK) by monitoring in vitro phosphorylation of a substrate, such as GST-ΙκΒα;

• Detection of IkB Degradation Dynamics including phosphorylation/ubiquitination and/or degradation of I KB and/or ΙκΒ-α; • Detection of p65(Rel-A) phosphorylation/ubiquitination, such as by using antibodies, gel-shift, EMSA, or mass spectroscopy;

• Detection of cytoplasmatic to nuclear shuttling/translocation of N FKB components/subunits, such as p65/phospho-p65;

• Detection of N FKB subunit dimerization/complexation;

• Detection of active N FKB components/subunits by binding to immobilized DNA sequence/oligonucleotide containing the N FKB response element/consensus N FKB binding, such as by using Electrophoretic mobility shift assay or gel shift assay, SELEX, protein-binding microarray, or sequencing-based approaches;

• Chromatin-immunoprecipitation (ChIP) assays to detect N FKB in situ binding to DNA to the promoters and enhancers of specific genes;

• In vitro kinase assay for N FKB kinase activity;

• Measurement of N FKB transcriptional activity using N FKB reporter assays via transgene expression of reporter constructs, such as LacZ Flue, eGFP SEAP, NF- gluc, using approaches such as plasmid transfection, reporter cell lines, mini-circles, retrovirus, or lentivirus;

• Measuring changes in expression of downstream targets of N FKB (such as cytokines, growth factors, adhesion molecules and mitochondrial anti-apoptotic genes by realtime PCR, protein, or functional assays) (Note the pleiotropic nature of N FKB is reflected in its transcriptional targets that presently number over 500 (see http://www.bu.edu/nf-kb/qene-resources/tarqet-qenes/ accessed 2 ^nd August 2017) and;

• Measuring changes in function or structure induced by NFKB-dependent signalling, such as POLKADOTS in T-cells, adhesion in endothelial cells, activation in leucocytes, or oncogencity.

[000484] Additionally or alternately, such methods may include measuring signals arising from the non-canonical actions of NF-κΒ, by measuring one or more of the following:

• Detection of NIK (NFKB-lnducing Kinase);

• Detecting IKKa Activation/phosphorylation;

• Detection of NIK kinase activity by ability to autophosphorylate or to phosphorylate a substrate by performing a kinase assay;

• Generation of p52-containing N FKB dimers, such as p52/RelB;

• Detection of Phospho-NFKB2 p100(Ser866/870);

• Detection of partial degradation (called processing) of the precursor p100 into p52;

• Detecting p52/RelB translocation into the nucleus; • Detecting p52/RelB binding to κΒ sites;

• Measurement of N FKB transcriptional activity using N FKB reporter assays via transgene expression of reporter constructs, such as LacZ Flue, eGFP SEAP, NF- gluc, using approaches such as plasmid transfection, reporter cell lines, mini-circles, retrovirus, or lentivirus;

• Measuring changes in expression of downstream targets of non-canonical signalling of N FKB (such as CXCL12) by real-time PCR, protein expression or by functional assays.

[000485] In another aspect, the present invention provides methods of identifying a modulator (such as activator, inhibitor, allosteric modulator or functional substitute) that modulates (i.e., activates, inhibits or otherwise modulates) RAGE ligand-independent activation of RAGE following activation of a certain co-located GPCR by a cognate ligand, such as AT ₁ R by Angll, or CCR2 by MCP-1 , or if the certain co-located GPCR is constitutively active, and that suitably modulates a certain co-located GPCR, such as an angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 and/or that modulates a RAGE polypeptide or a RAGE signalling pathway. In a preferred form of the invention, such a modulator is an inhibitor of one or both of the RAGE or certain co- located GPCR, such as an angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2 or of the RAGE signalling pathway. In a particularly preferred form of the invention, the modulation of the RAGE signalling pathway is distinct from and/or occurs to a significantly different extent to the modulation of classical certain co-located GPCR signalling pathways, such as AT ₁ R signalling pathways, such as the Gq signalling pathway, or CCR2 signalling pathways, such as the Gi signalling pathway. In a particularly preferred form of the invention, the inhibition of the RAGE signalling pathway is distinct from and/or greater than the inhibition of classical certain co-located GPCR signalling pathways, such as AT ₁ R signalling pathways, such as the Gq signalling pathway, or CCR2 signalling pathways, such as the Gi signalling pathway.

Constructs

[000486] In a related aspect, the present invention provides construct systems for identifying modulators of proximity between RAGE and certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2.

[000487] In some embodiments, these construct systems comprise a first construct that comprises a regulatory sequence that is operably connected to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence that encodes a polypeptide corresponding to a RAGE polypeptide and a nucleic acid sequence that encodes a proximity signal or energy donor molecule; and a second construct that comprises a regulatory sequence that is operably connected to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence that encodes a polypeptide corresponding to a certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2, and a nucleic acid sequence that encodes a proximity signal or energy acceptor molecule. In specific embodiments, the energy donor molecule is a bioluminescent or fluorescent molecule and the energy acceptor molecule is a fluorescent acceptor molecule.

[000488] In other embodiments, the construct systems of the present invention comprise a first construct that comprises a regulatory sequence that is operably connected to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence that encodes polypeptide corresponding to a certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2, and a nucleic acid sequence that encodes a proximity signal or energy donor molecule; and a second construct that comprises a regulatory sequence that is operably connected to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence that encodes a polypeptide corresponding to a RAGE polypeptide and a nucleic acid sequence that encodes a proximity signal or energy acceptor molecule. In specific embodiments, the energy donor molecule is a bioluminescent or fluorescent molecule and the energy acceptor molecule is a fluorescent acceptor molecule.

[000489] In other embodiments, the construct systems of the present invention comprise a first construct that comprises a regulatory sequence that is operably connected to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence that encodes polypeptide corresponding to a certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2; and a second construct that comprises a regulatory sequence that is operably connected to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence that encodes a polypeptide corresponding to a RAGE polypeptide whereby the RAGE polypeptide has deleted one or more of the ectodomains of the native sequence.

Methods for modulating RAGE ligand-independent activation of RAGE

[000490] In a related aspect, the present invention provides methods for modulating RAGE ligand-independent activation of RAGE by an activated certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2, in a cell or tissue of an animal or of animal origin (which may or may not be of a human or of human origin).

Methods for specifically modulating RAGE ligand-independent activation of RAGE

[000491 ] In another related aspect, the present invention provides methods for specifically modulating RAGE ligand-independent activation of RAGE by activated certain co-located GPCR, such as angiotensin receptor, such as ATiR, or certain chemokine receptor, such as CCR2, and subsequent downstream signalling pathways in a cell. These methods comprise truncating or mutating RAGE such that it is unable to bind RAGE ligands to its ectodomain, or that binding RAGE ligands to its ectodomain is impaired by exposing the cell to a modulator that modulates the binding of RAGE ligands to RAGE.

[000492] In a preferred form of the invention, the modulation of the RAGE ligand- independent signalling pathway, is distinct from and/or significantly more than the modulation of the RAGE ligand-dependent signalling pathway.

[000493] In a particularly preferred form of the invention, the inhibition of the RAGE ligand- independent signalling pathway, is distinct from and/or significantly more than the inhibition of the RAGE ligand-dependent signalling pathway.

Methods for modulating both RAGE ligand-dependent and RAGE ligand-independent activation of RAGE

[000494] In another related aspect, the present invention provides methods for inhibiting RAGE ligand-dependent activation of RAGE by RAGE ligands, (including AGE-modified proteins, lipids or DNA, members of the S100 calgranulin family of proteins, HMGB1 , amyloid and Mac-1 ) and subsequent downstream signalling pathways in a cell, tissue or animal in addition to modulating RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs.

[000495] In one aspect of the invention, these methods comprise using a modulator as described herein, including fragments, analogues or derivatives of the cytosolic tail of RAGE, to take the place of the cytosolic tail of RAGE in binding interactions and therein prevent activation of both RAGE-ligand dependent activation of RAGE and RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs. In one aspect of the invention, RAGE-dependent signalling is impaired by exposing the cell to an inhibitor that inhibits the binding of signalling elements to the cytosolic tail of RAGE resulting in inhibition of both RAGE ligand-mediated activation of RAGE and RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs.

[000496] In one aspect of the invention, these methods comprise using a modulator as described herein, including fragments, analogues or derivatives of the transmembrane domain of RAGE, to take the place of the transmembrane domain of RAGE and therein prevent activation of both RAGE-ligand dependent activation of RAGE and RAGE ligand- independent activation of RAGE by certain activated co-located GPCRs. In one aspect of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the RAGE ectodomain. In one aspect of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the cytosolic tail of RAGE. In one aspect of the invention, the modulator comprises a transmembrane domain of RAGE or part thereof and a fragment of the RAGE ectodomain and a fragment of the cytosolic tail of RAGE.

[000497] In one aspect of the invention, the modulators of RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs contain a fragment of the ligand- binding ectodomain of RAGE, which is not greater than 40, not greater than 20, not greater than 10 or not greater than 5 amino acids in length.

[000498] In one aspect, inhibition of the RAGE ligand-dependent activation of RAGE occurs at the same time as inhibition of the RAGE ligand-independent activation of RAGE by certain activated co-located GPCR.

[000499] In one aspect, these methods comprise silencing, truncating, modifying or mutating RAGE such that RAGE, or analogues, fragments or derivatives thereof, are a nonfunctional substitute for the cytosolic tail of wild type RAGE or a part thereof, which are unable to be activated by either RAGE ligand-dependent or RAGE ligand-independent pathways (such as S391 A-RAGE mutation) or facilitate downstream RAGE-dependent signalling and so inhibit signalling that occurs through the cytosolic tail of RAGE and RAGE- dependent signalling.

[000500] In one aspect, these methods comprise silencing, truncating, modifying or mutating RAGE such that RAGE, or analogues, fragments or derivatives thereof, are a nonfunctional substitute for the transmembrane domain of wild type RAGE or a part thereof, which are unable to be activated by either RAGE ligand-dependent or RAGE ligand- independent pathways or facilitate downstream RAGE-dependent signalling and so inhibit signalling that occurs through the cytosolic tail of RAGE and RAGE-dependent signalling. In one aspect of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the RAGE ectodomain. In one aspect of the invention, the modulator comprises a transmembrane domain of RAGE or a part thereof and a fragment of the cytosolic tail of RAGE. In one aspect of the invention, the modulator comprises a transmembrane domain of RAGE or part thereof and a fragment of the RAGE ectodomain and a fragment of the cytosolic tail of RAGE.

[000501 ] In one aspect, the modulators of RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs contain a fragment of the ligand-binding ectodomain of RAGE, which is not greater than 40, not greater than 20, not greater than 10 or not greater than 5 amino acids in length.

[000502] In one aspect, these methods comprise silencing, truncating, modifying or mutating RAGE such that RAGE, or analogues, fragments or derivatives thereof, modulate common elements involved in signalling mediated by the cytosolic tail of RAGE (such as ΡΚCζ, Diaphl , MyD88, TIRAP, N FKB). Association with activation of RAGE by either RAGE ligand-dependent or RAGE ligand-independent activation pathways.

[000503] In one aspect, these methods comprise the use of a modulator that modulates RAGE ligand-independent activation of RAGE by activated certain co-located GPCR, such as angiotensin receptor, such as AT ₁ R, or certain chemokine receptor, such as CCR2, in addition to a modulator that modulates RAGE ligand-dependent activation of RAGE (such as by a modulator that modulates the binding of RAGE ligands to the RAGE ectodomain).

Methods for modulating RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs while also modulating RAGE-independent signalling via certain co- located GPCRs.

[000504] In one aspect, the invention provides a method for modulating a RAGE- independent, certain co-located GPCR signalling pathway induced following activation by a cognate ligand as well as modulating RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR.

[000505] In one form, the invention provides a method for modulating a RAGE- independent, certain co-located GPCR signalling pathway induced following activation by a cognate ligand at the same time as modulating RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR. [000506] In one form, the RAGE-independent, certain co-located GPCR signalling pathway induced following activation by a cognate ligand is the Gq signalling pathway, such as for ATiR activated by Ang II. In another form, the RAGE-independent, certain co-located GPCR signalling pathway is the Gi signalling pathway, such as for CCR2 activated by MCP- 1 . In another form, the RAGE-independent, certain co-located GPCR signalling pathway is beta-arrestin-mediated extracellular regulated kinase (ERK) signalling. In another form, the RAGE-independent, certain co-located GPCR signalling pathway is changes in intracellular signalling intermediates (such as inositol phosphate or calcium).

BRIEF DESCRIPTION OF THE DRAWINGS

EXAMPLE 1 .

[000507] Figure 1 A. Quantitated plaque area expressed as the percentage of the aortic arch surface area staining positive to Sudan IV following a four-week infusion of Ang II (1μg/kg/min) or vehicle control in male apoE KO mice and AGER/apoE double knockout (DKO) mice.

[000508] Figure 1 B. The expression of pro-atherosclerotic mediators including AGER itself, adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines (TNFa, MCP- 1 and IL-6) and the macrophage marker (Mac- 1/Cd11b), as measured by real time RT- PCR in aortic homogenates from apoE KO mice and AGER/apoE DKO mice following a four-week infusion of Ang II (1μg/kg/min) or vehicle control.

[000509] Figure 1 C. Markers of oxidative stress following a 4-week infusion of Ang II (1μg/kg/min) or vehicle control in apoE KO mice and AGER/apoE DKO mice, as estimated by (i) plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage and (ii) induction of the gene expression of the NADPH oxidase subunits, NOX- 1 and NOX-4 in the aortae of apoE KO mice and AGER/apoE DKO mice, as estimated by real time RT-PCR in aortic homogenates.

[000510] Figure 1 D. The expression of RAGE ligands, including (i) circulating plasma levels of S100A8/A9, as measured by commercial ELISA, (ii) plasma AGE levels, as measured by in-house ELISA, (iii) the circulating AGE-precursor, methylglyoxal levels as measured by HPLC in apoE KO mice and AGER/apoE DKO mice following a 4-week infusion of Ang II (1 μg/kg/min) or vehicle. [00051 1 ] Figure 1 E. Systolic blood pressure as measured by tail-cuff plethysmography following a 4-week infusion of Ang II (1μg/kg/min) or vehicle control in apoE KO mice and AGER/apoE DKO mice.

[000512] Data are mean ±SEM; n=8 per group, *vs control apoE KO mice, # vs apoE KO +Ang II, p<0.05.

EXAMPLE 2.

[000513] Figure 2A. Quantitated plaque area expressed as the percentage of the aortic arch surface area staining positive to Sudan IV in apoE KO mice and AGER/apoE DKO mice following six-weeks of a 0.05% (low) sodium diet or normal chow.

[000514] Figure 2B. The expression of pro-atherosclerotic mediators including AGER itself, adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines ( TNFa, MCP- 1 and IL-6) and the macrophage marker (Mac- 1/Cd11b), as measured by real time RT- PCR in aortic homogenates of apoE KO mice and AGER/apoE DKO mice following six- weeks of a 0.05% (low) sodium diet or normal chow.

[000515] Figure 2C. Markers of oxidative stress in apoE KO mice and AGER/apoE DKO mice following six-weeks of a 0.05% (low) sodium diet or normal chow, as estimated by (i) plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage and (ii) induction of the gene expression of the NADPH oxidase subunits, NOX-1 and NOX-4 in the aortae of apoE KO mice and AGER/apoE DKO mice, as estimated by real time RT-PCR in aortic homogenates.

[000516] Figure 2D. Circulating levels of soluble MCP-1 and ICAM-1 , as measured by ELISA in apoE KO mice and AGER/apoE DKO mice exposed to a 0.05% (low) sodium diet or normal chow for six weeks.

[000517] Figure 2E. The number of labelled-leucocytes adhering ex vivo to the aortic surface of apoE KO mice and AGER/apoE DKO mice subsequent to 1 -week prior exposure to a low sodium diet or normal chow, as measured by dynamic flow assay.

[000518] Figure 2F. The expression of RAGE ligands, including (i) circulating plasma levels of S100A8/A9, as measured by commercial ELISA, (ii) plasma AGE levels, as measured by in-house ELISA, and (iii) the circulating AGE-precursor, methylglyoxal levels as measured by HPLC in apoE KO mice and AGER/apoE DKO mice following six-weeks of a 0.05% (low) sodium diet or normal chow. [000519] Figure 2G. Systolic blood pressure as measured by tail-cuff plethysmography in apoE KO mice and AGER/apoE DKO mice following six-weeks of a 0.05% (low) sodium diet or normal chow.

[000520] Figure 2H. Markers of activation of the RAAS including (i) reduced sodium excretion, (ii) increased plasma renin activity and (iii) increased plasma aldosterone levels as measured by radioimmunoassay in apoE KO mice and AGER/apoE DKO mice following six- weeks of a 0.05% (low) sodium diet or normal chow.

[000521 ] Data are mean ±SEM; n=8 per group, *vs apoE KO mice on normal chow, p<0.05. # vs apoE KO mice + low sodium.

EXAMPLE 3.

[000522] Figure 3A. Circulating Ang II concentrations in apoE KO mice and AGER/apoE KO mice with or without genetic Ace2 deficiency, as measured by radioimmunoassay.

[000523] Figure 3B. Systolic blood pressure as measured by tail-cuff plethysmography in 18-week old apoE KO mice and AGER/apoE KO mice with or without genetic Ace2 deficiency.

[000524] Figure 3C. Quantitated plaque area expressed as the percentage of the aortic arch surface area staining positive to Sudan IV in 18-week old apoE KO mice and AGER/apoE KO mice with or without genetic Ace2 deficiency.

[000525] Figure 3D. The aortic expression of pro-atherosclerotic mediators including AGER itself, adhesion molecules (ICAM- 1, VCAM- 1), inflammatory cytokines and chemokines (TNFa, MCP- 1 and IL-6) and the macrophage marker (Mac- 1/Cd11b), as measured by real time RT-PCR in aortic homogenates from apoE KO mice in the presence or absence of Ace2 and or RAGE.

[000526] Figure 3E. Oxidative stress in 18-week old apoE KO mice, Ace2/apoE DKO mice, AGER/apoE DKO and Ace2/ AGER/apoE triple KO (TKO) mice, as estimated by plasma 8- hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage.

[000527] Figure 3F. The expression of RAGE ligands, including (i) circulating plasma levels of S100A8/A9, as measured by commercial ELISA, (ii) plasma AGE levels, as measured by in-house ELISA, in 18-week old apoE KO mice, Ace2/apoE DKO mice, AGER/apoE DKO and Ace2/A GER/apoE T KO mice. [000528] Data are mean ± SEM; n=8 per group, *vs apoE KO control; # vs Ace2/apoE DKO mice.

EXAMPLE 4.

[000529] Figure 4A. The aortic expression of pro-atherosclerotic mediators including AGER itself, adhesion molecules ( ICAM- 1, VCAM- 1), inflammatory cytokines and chemokines (TNFa, MCP-1 and IL-6) and the macrophage marker (Mac-1/Cd11b) in apoE KO mice and AGER/apoE DKO, as measured by real time RT-PCR in aortic homogenates following their exposure ex vivo to Ang II or vehicle. Data are mean ±SEM; n=6 per group, *vs untreated apoE KO control; # vs apoE KO + Ang II; p<0.05

[000530] Figure 4B. The number of labelled-leucocytes adhering to the aortic surface of apoE KO mice and AGER/apoE DKO as a marker of endothelial activation after four-hours ex vivo exposure to Ang II (1 μΜ) or vehicle control, as measured by dynamic flow assay. Data are mean ±SEM; n=6 per group, *vs untreated apoE KO control; # AGER/apoE DKO + Ang II vs apoE KO + Ang II; p<0.05

[000531 ] Figure 4C. The number of labelled THP-1 monocytes adherent to a monolayer of primary murine aortic endothelial cells (PMAEC) from C57bl6 or AGER KO mice in the presence or absence of pre-treatment with Ang II (1 μΜ for 2 hours).

[000532] Figure 4D. The expression of pro-atherosclerotic mediators including AGER itself, key adhesion molecules (ICAM-1, VCAM- 1), inflammatory cytokines and chemokines ( TNFa and MCP-1), as measured by real time RT-PCR in primary murine aortic endothelial cells (PMAEC) from C57bl6 and PMAEC from AGER KO mice following exposure to Ang II (1 μΜ) or vehicle control.

[000533] Figure 4E. Markers of oxidative stress following exposure to Ang II or vehicle control in PMAEC from c57bl6 mice and AGER KO mice, as estimated by (i) the induction of DCFH fluorescence in a flow chamber assay, and (ii) levels of the GTP-activated NADPH oxidase subunit, Rac-1 and (iii) levels of oxidized glutathione.

[000534] Figure 4F. The gene expression of VCAM- 1 and markers of non-canonical and canonical signalling via N FKB (CXCL 12 and CXCL2, respectively) in a monolayer of primary murine aortic endothelial cells (PMAEC) from C57bl6 and AGER KO mice following exposure to Ang II. PMAEC, as measured by real time RT-PCR. TNFa is shown as a canonical specific control. VCAM-1 is shown as a target-specific control, replicating data in Figure 4D. [000535] Figure 4G. The expression of pro-atherosclerotic mediators including adhesion molecules (ICAM-1, VCAM-1), and inflammatory cytokines and chemokines ( TNFa and MCP- 1), in primary murine aortic endothelial cells (PMAEC) from C57bl6 and AGER KO mice treated with the RAGE ligand, S100A8/A9 (5ng/ml_), as measured by real time RT-PCR. Data are mean ±SEM; n=6 per group, symbols denote *vs untreated wild type PMAEC; # vs S100A8/A9-treated wild type PMAEC, p<0.05.

[000536] Figure 4H. The gene expression of key adhesion protein, VCAM- 1 in a monolayer of PMAEC, in which the expression of RAGE or the N FKB subunit p65 has been selectively silenced using siRNA or unaltered (scrambled RNA control) and subsequently exposed to Ang II (1 μΜ) or the RAGE ligand, S100A8/A9 (5ng/ml_), as estimated by real time RT-PCR.

[000537] Figure 4I. Markers of Gq-mediated signalling induced following activation of the AT ₁ R by Ang II (1 μΜ) in a monolayer of PMAEC from c57bl6 mice and AGER KO mice, including (i) induction of inositol phosphate synthesis as estimated by IP-1 and (ii) downstream induction of the early growth response gene (EGR1 ).

[000538] Data are mean ±SEM; n=6 per group, * vs untreated wild type control PMAEC and # vs Ang II treated wild type control unless otherwise stated, p<0.05.

EXAMPLE 5.

[000539] Figure 5A. The induction of inositol phosphate synthesis in response to Ang II (1 μΜ), a marker of classical responsiveness to exogenous Ang II, as estimated by IP-1 levels in CHO cells in the presence or absence of expression of human AT ₁ R, with or without the additional expression of full length human RAGE.

[000540] Figure 5B. The induction of EGR1 expression in response to Ang II (1 μΜ), a marker of responsiveness to exogenous Ang II, as estimated by downstream induction of the EGR1 gene in CHO cells in the presence or absence of expression of human AT ₁ R, with or without the additional expression of full length human RAGE.

[000541 ] Figure 5C. NFKB activation following exposure to Ang II (1 μΜ), as measured by (i) chemiluminescent SEAP reporter gene assay and (ii) the induction in the gene expression of the N FKB subunit, p65, in the presence or absence of expression in CHO cells of human AT ₁ R, with or without the additional expression of full length human RAGE and (iii) following exposure to the RAGE ligand, S100A8/A9 (5ng/ml_), as a control for the integrity of RAGE signalling in CHO cells. [000542] Figure 5D. N FKB activation following exposure to Ang I I (1 μΜ) in AT1 R-CHO cells, as measured by the induction in the gene expression of the N FKB subunit, p65, and chemiluminescent SEAP reporter gene assay for N FKB activity in the presence or absence of expression of the full-length human RAGE and N-truncated mCherry-RAGE constructs. Data are mean ± SEM, n=6 per group, *vs vector (neo)-transfected AT1 R-CHO, p<0.05.

[000543] Figure 5E. N FKB activation following exposure to Ang I I (1 μΜ) in AT1 R-CHO as estimated by the induction in the gene expression of the N FKB subunit, p65, and chemiluminescent SEAP reporter gene assay for N FKB activity in the presence or absence of expression of the full-length human RAGE and C-truncated mCherry-RAGE constructs. Data are mean ± SEM, n=6 per group, *vs vector (neo)-transfected AT1 R-CHO, p<0.05.

[000544] Figure 5F. N FKB activation following exposure to S100A8/A9 (5ng/ml_) or Ang I I (1 μΜ) in AT1 R-CHO as estimated by the induction in the gene expression of the N FKB subunit, p65, in the presence or absence of expression of the full-length human RAGE and N-truncated mCherry-RAGE constructs. Data are mean ± SEM, n=6 per group, *vs vector (neo)-transfected AT1 R-CHO, p<0.05.

[000545] Figure 5G. N FKB activation following exposure to S100A8/A9 (5ng/ml_) or Ang I I (1 μΜ) in AT1 R-CHO as estimated by the induction in the gene expression of the N FKB subunit, p65, in the presence or absence of expression of the full-length human RAGE and C-truncated mCherry-RAGE constructs. Data are mean ± SEM, n=6 per group, *vs vector (neo)-transfected AT1 R-CHO, p<0.05.

[000546] Figure 5H. N FKB activation following exposure to Ang I I (1 μΜ) in AT1 R-CHO as estimated by the induction in the gene expression of the N FKB subunit, p65, in the presence of expression of the N-truncated RAGE constructs without fusion to mCherry.

[000547] Data are mean ± SEM, n=6 per group, *vs untreated AT1 R-CHO unless otherwise stated, p<0.05.

EXAMPLE 6.

[000548] Figure 6A. A RAGE neutralizing antibody targeting the ectodomain of RAGE (RAGEab) or a decoy receptor with ligand-binding affinity (soluble RAGE22-331 ) inhibit the induction of pro-inflammatory signalling by the RAGE ligand S100A8/A9 but not by Ang I I (1 μΜ) in RAGE-AT1 R-CHO cells, as estimated by the expression of the N FKB subunit, p65 as measured by RT-PCR. Data show mean ±SEM; n=6 per group * vs vehicle alone, p<0.05. [000549] Figure 6B. A RAGE neutralizing antibody targeting the ectodomain of RAGE (RAGEab) or a decoy receptor with ligand-binding affinity (soluble RAGE22-331 ; sRAGE) do not inhibit the induction of pro-inflammatory signalling by Ang II (1 μΜ) in PMAEC from wild type mice, as estimated by the induction of key adhesion genes, ICAM- 1 and VCAM- 1, and an inflammatory chemokine gene (MCP- 1). Data from AGER KO mice are shown as a negative control. Data show mean ±SEM; n=6 per group * vs control cells treated with vehicle alone (white bar), # vs control cells treated with Ang II alone, p<0.05.

[000550] Figure 6C. A RAGE neutralizing antibody targeting the ectodomain of RAGE (RAGEab) or a decoy receptor with ligand-binding affinity (soluble RAGE22-331 ) inhibit the induction of pro-inflammatory signalling by the RAGE ligand S100A8/A9 in PMAEC from wild type mice, as estimated by the induction of key adhesion gene, ICAM-1 . Data show mean ±SEM; n=6 per group * vs control cells treated with vehicle alone (white bar), # vs control cells treated with S100A8/A9 alone, p<0.05.

EXAMPLE 7.

[000551 ] Figure 7A. The induction of pro-inflammatory signalling by the RAGE ligand S100A8/A9 (5ng/ml; grey bars) or Ang II (1 μΜ; black bars) in AT1 R-CHO cells also expressing full-length wild type RAGE22-404 or selected S391 -RAGE ₂ 2-404 mutants, as measured by the induction in the gene expression of the N FKB subunit, p65. Data show mean ±SEM; n=6-8 per group * vs vehicle treated AT1 R-CHO cell expressing full length RAGE, p<0.05.

[000552] Figure 7B. The induction of pro-inflammatory signalling by the RAGE ligand S100A8/A9 (5ng/ml) or Ang II (1 μΜ) in AT1 R-CHO cells also expressing chimeric RAGE lacking phosphorylatable motifs in the cytosolic tail other than S391 (Chimeric RAGE; cRAGE), and S391 cRAGE mutants completely lacking any phosphorylatable motifs in the cytosolic tail, as measured by the induction in the gene expression of the N FKB subunit, p65, in AT1 R-CHO cells. Data show mean ±SEM; n=6-8 per group * vs vehicle treated AT1 R-CHO cells expressing full length chimeric RAGE; p<0.05.

[000553] Figure 7C. The induction of pro-inflammatory signalling by Ang II (1 μΜ) in AT1 R- CHO cells also expressing full length or N-truncated S391 A-RAGE mutants, as measured by the induction in the gene expression of the N FKB subunit, p65. Data show mean ±SEM; n=6- 8 per group * vs vector transfected AT1 R-CHO cells, p<0.05.

[000554] Figure 7D. The induction of pro-inflammatory signalling by Ang II (1 μΜ) in the presence of wild type mCherry-RAGE ₃ 62-404 in AT1 R-CHO cells also expressing S391 - RAGE ₃ 62-404 mutants, as measured by the induction in the gene expression of the N FKB subunit, p65. Data show mean ±SEM; n=6-8 per group * vs vehicle control; p<0.05.

EXAMPLE 8.

[000555] Figure 8A. The effect of selective suppression of MyD88 expression using siRNA or scrambled control on the induction of RAGE ligand-dependent induction of proinflammatory signalling by the RAGE ligand S100A8/A9 (5ng/ml) in a monolayer of PMAEC from C57bl6 mice, as estimated by the expression of ICAM- 1 measured by real time RT- PCR. The selective suppression of p65 expression, another downstream mediator of RAGE signalling, using siRNA is shown as a positive control.

[000556] Figure 8B. The effect of selective suppression of MyD88 expression using siRNA or scrambled control in a monolayer of PMAEC from C57bl6 mice on the induction of RAGE ligand-independent induction of pro-inflammatory signalling by Ang II (1 μΜ), as estimated by the expression of ICAM-1 measured by real time RT-PCR.

[000557] Figure 8C. The effect of selective suppression of MyD88 expression using siRNA or scrambled control in a monolayer of HMEC on the induction of RAGE ligand-independent induction of pro-inflammatory signalling by Ang II (1 μΜ) in the presence and absence of RAGE ₃ 62-404, as estimated by the expression of MCP- 1 measured by real time RT-PCR.

[000558] Data are mean ±SEM; n=6-8 per group, * vs scrambled control, p<0.05.

EXAMPLE 9.

[000559] Figure 9A. The effect of selective suppression of ΡΚCζ using a pseudo-substrate for ΡΚCζ (iPKCz), or siRNA targeted against the expression of ΡΚCζ (siPKCz) or RAGE (siRAGE) or scrambled control, in a monolayer of PMAEC from C57bl6 mice on the induction of RAGE ligand-dependent signalling by the RAGE ligand S100A8/A9, as estimated by the expression of ICAM-1 measured by real time RT-PCR.

[000560] Figure 9B. The effect of selective suppression ofΡΚCζ expression using a pseudo-substrate for ΡΚCζ (i ΡΚCζ , or siRNA targeted against ΡΚCζ (sίΡΚCζ), in a monolayer of PMAEC from C57bl6 mice on RAGE ligand-independent induction of proinflammatory signalling by Ang II (1 μΜ), as estimated by the gene expression of ICAM- 1 measured by real time RT-PCR. Columns 1 and 2 contain scrambled siRNA control.

[000561 ] Figure 9C. The effect of selective suppression of ΡΚCζ expression using a pseudo-substrate for ΡΚCζ ( ΡΚCζi) on RAGE ligand-independent induction of p65 and PCNA by Ang II (1 μΜ), in CHO cells expressing chimeric RAGE lacking phosphorylatable motifs in the cytosolic tail other than S391 (cRAGE) as well as cRAGE also containing the S391 Q-RAGE mutation (S319Q-cRAGE) thereby removing all phosphorylation sites in the cytosolic tail, as estimated by the gene expression of RelA/p65 and PCNA measured by real time RT-PCR.

[000562] Figure 9D. The effect of selective suppression of ΡΚC eζxpression using siRNA or scrambled control in a monolayer of HMEC on the induction of RAGE ligand-independent induction of pro-inflammatory signalling by Ang II (1 μΜ) in the presence and absence of RAGE ₃ 62-404, as estimated by the expression of MCP- 1 measured by real time RT-PCR.

[000563] Data are mean ±SEM; n=6-8 per group, * vs un-treated control, p<0.05.

EXAMPLE 10.

[000564] Figure 10A. The effect of selective suppression of Diaphi expression using siRNA in a monolayer of PMAEC from C57bl6 mice on the induction of RAGE ligand- dependent signalling by the RAGE ligand S100A8/A9, as estimated by the expression of ICAM- 1 measured by real time RT-PCR. Data with siRAGE are included as a control.

[000565] Figure 10B. The differential effect of a R366A-Q367A-RAGE mutation that selectively disrupts a charged patch through which Diaphi and RAGE putatively interact, on signalling induced by the RAGE ligand, S100A8/A9, and RAGE ligand-independent signalling induced by Ang II in AT1 R-CHO cells.

[000566] Figure 10C. The effect of selective suppression of Diaphi expression using siRNA in a monolayer of PMAEC from C57bl6 mice on the induction of RAGE ligand- independent signalling induced by Ang II (1 μΜ) as estimated by the expression of ICAM- 1 and VCAM-1 as measured by real time RT-PCR. Data are mean ±SEM; n=6 per group, * vs scrambled control, p<0.05.

[000567] Figure 10D. Selective suppression of Diaphi or AGER expression using siRNA, in contrast to scrambled control, in a monolayer of SVEC on the induction of leukocyte adhesion to an endothelial monolayer following exposure to Ang II. Data are mean ±SEM; n=6-8 per group, * vs scrambled control, p<0.05.

[000568] Figure 10E. The effect of transfection with R366A-Q367A-RAGE mutants in which the charged patch through which Diaphi and RAGE putatively interact is disrupted, or deleted, on signalling induced by Ang II as measured by the induction in the gene expression of the N FKB subunit, p65, in AT ₁ R-CHO cells. Data are mean ±SEM; n=6 per group, * vs mCherry control, p<0.05.

[000569] Figure 10F. The effect of selective suppression of Diaphl expression using siRNA or scrambled control in a monolayer of HMEC on the induction of RAGE ligand- independent induction of pro-inflammatory signalling by Ang II (1 μΜ) in the presence and absence of RAGE362-404, as estimated by the expression of MCP-1 measured by real time RT-PCR. Data are mean ±SEM; n=6-8 per group, * vs vehicle control treated with scrambled siRNA, p<0.05.

[000570] Figure 10G. The effect of transfection with full length RAGE, truncated RAGE or RAGE mutants in AT1 R-CHO cells pre-treated with the inhibitory peptide, S391 A-RAGE ₃₆ 2-404, on signalling induced by Ang II as measured by the induction in the gene expression of the N FKB subunit, p65. Data are mean ±SEM; n=6-8 per group, * vs mCherry control, p<0.05

[000571 ] Data are mean ±SEM; n=6-8 per group, * vs vehicle control, p<0.05, unless stated otherwise.

EXAMPLE 1 1 .

[000572] Figure 1 1 A. The effect of selective suppression of IQGAP- 1 expression using siRNA targeting IQGAP-1 , in contrast to scrambled control, in a monolayer of PMAEC from C57bl6 mice on the induction of RAGE ligand-independent induction of pro-inflammatory signalling by Ang II (1 μΜ), as estimated by the expression of ICAM-1 measured by real time RT-PCR.

[000573] Figure 1 1 B. The effect of selective suppression of IQGAP- 1 expression using siRNA, in contrast to scrambled control, in a monolayer of PMAEC from C57bl6 mice on the induction of RAGE ligand-dependent induction of pro-inflammatory signalling by the RAGE ligand S100A8/A9, as estimated by the expression of ICAM-1 and VCAM-1 measured by real time RT-PCR.

[000574] Figure 1 1 C. Pull down of a protein identified as IQGAP-1 from other cytosolic components using a column coated with the mutant RAGE cytosolic tail (S391 A-RAGE362-404) , as well as IQGAP-1 associated proteins, ezrin/radixin/moesin and the GPCR olfactory receptor 2T2.

[000575] Figure 1 1 D. The effect of selective suppression of IQGAP-1 expression using siRNA or scrambled control in a monolayer of HMEC on the induction of RAGE ligand- independent induction of pro-inflammatory signalling by Ang II (1 μΜ) in the presence and absence of RAGE ₃ 62-404, as estimated by the expression of MCP-1, as measured by real time RT-PCR.

[000576] Data are mean ±SEM; n=6-8 per group, *vs scrambled control, p<0.05, # vs scrambled + ligand (Ang II or S100A8/A9 as applicable), p<0.05.

EXAMPLE 12.

[000577] Figure 12A. The effect of transfection of murine SVEC with RAGE or RAGE mutants on the induction of ICAM-1 by Ang II compared to vector alone (pc-Neo) as a control, as measured by RT-PCR. Data are mean ±SEM; n=6-8 per group. * vs untreated control, # vs neo + Ang II, p<0.05.

[000578] Figure 12B. The effect of transfection with truncated RAGE mutants on the induction of ICAM-1 by Ang II in murine SVEC to identify the smallest fragments with inhibitory activity. Data are mean ±SEM; n=6-8 per group, * vs no treatment control, p<0.05.

[000579] Figure 12C. Inhibition of N FKB activation following exposure to Ang II (1 μΜ) in RAGE-AT ₁ R-CHO as estimated by the induction in the gene expression of the N FKB subunit, p65, in the presence of expression of mutant and N-truncated RAGE constructs without fusion to mCherry. Data are mean ±SEM; n=6-8 per group, * vs no treatment control, p<0.05.

[000580] Figure 12D. The effect of transfection with single site-specific alanine or lysine mutants of RAGE370-390 on the induction of ICAM- 1 by Ang II in murine SVEC. Data are mean ±SEM; n=6-8 per group, * vs no treatment control, p<0.05.

[000581 ] Figure 12E. Sequence homology between RAGE379-390 and anti-inflammatory proteins in Streptomyces and proteins from other microorganisms.

EXAMPLE 13.

[000582] Figure 13A. The effects of TAT-mCherry-RAGE ₃₆ 2-404 ( 0.4ng/ml) without or with a S391 A-RAGE mutation, on the induction of gene expression of the N FKB subunit, p65 by Ang II (1 μΜ; black bars) in AT1 R-CHO cells when compared to TAT-Cherry alone ^g). Data are mean ±SEM; n=6-8 per group, * vs no treatment control, p<0.05.

[000583] Figure 13B. The inhibition of signalling achieved by the S391 A-RAGE ₃₆ 2-404 peptide on Ang ll-dependent induction of gene expression of the N FKB subunit, p65 in AT ₁ R- CHO cells is not reversed by pre-treatment with the wild-type RAGE _362-404 peptide. Data are mean ±SEM; n=6-8 per group, * vs no treatment control; p<0.05.

[000584] Figure 13C. The inhibition of signalling achieved by the S391 A-RAGE _362-404 peptide on Ang ll-dependent induction of gene expression of the N FKB subunit, p65 in AT1 R- CHO cells is observed regardless of subsequent treatment with the wild-type RAGE _362-404 peptide in a thousand-fold excess. Data are mean ±SEM; n=6-8 per group, * vs no treatment control, p<0.05.

[000585] Figure 13D. The inhibitory effects of S391 A-RAGE _362-404 peptide on induction of p65 and PCNA gene expression in response to Ang II in AT1 R-CHO cells transfected with full length S391 Q-cRAGE with no available targets for phosphorylation. Data are mean ±SEM; n=6-8 per group, * vs Ang II # versus full length wild type RAGE; p<0.05.

[000586] Figure 13E. The effects of the wild-type RAGE _362-404 peptide (0.4ng/ml) on induction of pro-inflammatory gene expression in RAGE-deficient PMAECs in response to Ang II. Data are mean ±SEM; n=6-8 per group, * vs no treatment control, p<0.05.

[000587] Figure 13F. The effect of S391 A-RAGE _362-404 peptide on induction of proinflammatory VCAM- 1, CXCL2 and CXCL 12 gene expression in PMAECs in response to Ang II, with response to TNFa as a control. Data are mean ±SEM; n=6-8 per group, * vs vehicle and control (TAT) treated, # vs Ang II and control (TAT) treated; p<0.05.

[000588] Figure 13G. The effect of S391 A-RAGE _362-404 peptide and the AT1 R blocker, irbesartan, on induction of pro-inflammatory gene expression in response to Ang II in HAECs.

[000589] Figure 13H. (i) The inhibitory effect of the S391 A-RAGE _362-404 peptide on induction of pro-inflammatory gene expression (p65) in response to the RAGE ligand S100A8/A9 in AT1 R-CHO cells also expressing full length RAGE, with AT1 R-CHO cells expressing the inactive full length S391 A-RAGE mutant shown as a control, (ii) The inhibitory effect of S391 A-RAGE _362-404 peptide on induction of pro-inflammatory gene expression ( VCAM-1) in response to Ang II or the RAGE ligand S100A8/A9 in PMAEC endogenously replete in RAGE.

[000590] Data are mean ±SEM; n=6-8 per group, * vs vehicle control, p<0.05, # vs control + Ang II, p<0.05, unless stated otherwise.

EXAMPLE 14. [000591 ] Figure 14A. The effect of S391 A-RAGE _362-404 on Ang ll-dependent induction of pro-inflammatory markers following ex vivo exposure of whole aortae from apoE KO mice to Ang II (1 μΜ). Data are mean ±SEM; n=6 per group, * vs apoE KO + vehicle + TAT-mCherry control, p<0.05, # vs apoE KO + TAT-mCherry control + Ang II, p<0.05.

[000592] Figure 14B. The effect of wild-type RAGE _362-404 on Ang ll-dependent induction of pro-inflammatory markers following ex vivo exposure of whole aortae from AGERIapoE KO mice to Ang II (1 μΜ). Data are mean ±SEM; n=8 per group, * vs apoE KO + vehicle + TAT- mCherry control, p<0.05.

EXAMPLE 15.

[000593] Figure 15A. The pro-atherosclerotic effect of TAT-mCherry-RAGE ₃ 62-404 comprising the C-terminal 42 amino acids of RAGE tagged with mCherry fluorescent protein and an HIV-TAT motif to facilitate cellular penetration, on Ang ll-dependent induction of aortic atherosclerosis in apoE KO and Ace2/AGER/apoE triple KO mice. This is compared to the anti-atherosclerotic effect of TAT-mCherry-S391 A-RAGE _362-404 on Ang ll-dependent induction of aortic atherosclerosis in Ace lapoE DKO mice. Data are mean ±SEM; n=8 per group; * vs apoE KO control; # vs Ace2/apoE DKO control; p<0.05.

[000594] Figure 15B. The pro-atherosclerotic effect of TAT-mCherry-RAGE ₃ 62-404 peptide, comprising the C-terminal 42 amino acids of RAGE tagged with mCherry fluorescent protein and an HIV-TAT motif to facilitate cellular penetration, on Ang ll-dependent induction of aortic atherosclerosis in diabetic apoE KO and diabetic AGER/apoE DKO mice. This is compared to the anti-atherosclerotic effect of TAT-mCherry-S391 A-RAGE ₃ 62-404 on Ang ll- dependent induction of aortic atherosclerosis in diabetic apoE KO mice. Data are mean ±SEM; n=8 per group; * vs apoE KO control # vs diabetic apoE DKO control; p<0.05.

[000595] Figure 15C. The lack of effect of TAT-mCherry-RAGE _362-404 and TAT-mCherry- S391 A-RAGE ₃₆ 2-404 on systolic blood pressure in mice and diabetic apoE KO mice with or without AGER expression, as shown.

EXAMPLE 16.

[000596] Figure 16A. BRET saturation curves with ATi/Rluc8 and RAGE/Venus generated 60 minutes following addition of Ang II or vehicle with or without soluble RAGE ₂ 2- ₃₃ i (sRAGE). Data are combined from 3 independent experiments. [000597] Figure 16B. Ang ll-induced recruitment of β arrestin2/Venus to ATi/Rluc8 and CCL22-induced recruitment of β-arrestin2/Venus to CCR4/Rluc8 as controls.

[000598] Figure 16C. Ang ll-induced recruitment of -arrestin2/Venus proximal to RAGE/Rluc8 in the presence of ATi receptor following exposure to Ang II, and not in the presence of CCR4 following exposure to CCL22.

[000599] Figure 16D. CCL22-induced BRET signal is observed when Gai/Nluc and GY2/Venus are co-expressed in the presence of untagged CCR4.

EXAMPLE 17.

[000600] Figure 17A. Ang ll-induced recruitment of β-arrestin2/Venus (β -arr2/Venus) proximal to RAGE/Rluc8 in the presence, but not in the absence, of AT ₁ receptor. Ang ll- induced recruitment of β-arrestin2/Venus to AT ₁ /Rluc8 included as a control.

[000601 ] Figure 17B. Thyrotrophin-releasing hormone (TRH)-induced weak recruitment of β-arrestin2/Venus (β -arr2/Venus) proximal to RAGE/Rluc8 in the presence, but not in the absence, of TRH receptor 1 (TRHR1 ). TRH-induced recruitment of β-arrestin2/Venus to TRHR1 /Rluc8 included as a control. Inset shows same data with expanded y-axis scale.

[000602] Figure 17C. Orexin A (OxA)-induced recruitment of β-arrestin2/Venus (β- arr2/Venus) proximal to RAGE/Rluc8 in the presence, but not in the absence, of orexin receptor 1 (OxR1 ). OxA-induced recruitment of β arrestin2/Venus to OxR1/Rluc8 included as a control.

[000603] Figure 17D. Bradykinin (BDK)-induced weak recruitment of β-arrestin2/Venus (β- arr2/Venus) proximal to RAGE/Rluc8 in the presence, but not in the absence, of BDK receptor 2 (BDKR). BDK-induced recruitment of β arrestin2/Venus to BDKR/Rluc8 included as a control. Inset shows same data with expanded y-axis scale.

[000604] Figure 17E. Arginine vasopressin (AVP)-induced recruitment of β- arrestin2/Venus (β- arr2/Venus) proximal to RAGE/Rluc8 in the presence, but not in the absence, of vasopressin receptor 2 (V2R). AVP-induced recruitment of β-arrestin2/Venus to V2R/Rluc8 included as a control.

[000605] Figure 17F. CCL2 (MCPI )-induced recruitment of β-arrestin2/Venus (β- arr2/Venus) proximal to RAGE/Rluc8 in the presence, but not in the absence, of CCR2. MCP1 -induced recruitment of β-arrestin2/Venus to CCR2/Rluc8 included as a control. [000606] Figure 17G. CCL4 (MlP^)-induced particularly weak recruitment of β- arrestin2/Venus (β-arr2/Venus) proximal to RAGE/Rluc8 in the presence of CCR5 following exposure to ΜΙΡ1 β, especially compared to the control in the absence of CCR5. ΜΙΡ1 β- induced recruitment of βarrestin2/Venus to CCR5/Rluc8 included as a further control. Inset shows same data with expanded y-axis scale.

[000607] All data are mean ±SEM of 3 independent experiments.

EXAMPLE 18.

[000608] Figure 18A. CCL3-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CCR1 .

[000609] Figure 18B. CCL2-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CCR2.

[000610] Figure 18C. Lack of CCL22-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CCR4.

[00061 1 ] Figure 18D. Lack of CCL4-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CCR5.

[000612] Figure 18E. CCL20-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CCR6.

[000613] Figure 18F. CCL19-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CCR7.

[000614] Figure 18G. Lack of CCL27-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CCR10.

[000615] Figure 18H. Weak CXCL8-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CXCR1 .

[000616] Figure 181. CXCL8-induced recruitment ofβ-arr2/Venus proximal to RAGE/Rluc8 in the presence of CXCR2.

[000617] Figure 18J. Lack of CXCL1 1 -induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CXCR3. [000618] Figure 18K. CXCL12-induced reduction in -arr2/Venus proximity to RAGE/Rluc8 in the presence of CXCR4.

[000619] Figure 18L. CXCL16-induced recruitment of β-arr2/Venus proximal to RAGE/Rluc8 in the presence of CXCR6.

[000620] All data are mean ±SEM of 3 independent experiments. EXAMPLE 19.

[000621 ] Figure 19A-00. Proximity of Rluc8-tagged RAGE to Venus-tagged indicated subcellular compartment markers, in the presence of the indicated non-BRET tagged GPCR activated by the indicated ligand at the indicated concentration at time zero.

EXAMPLE 20.

[000622] Figure 20A. Activation of N FKB, as measured by the induction in gene expression of the N FKB subunit, p65, by CCL2 (MCP-1 ) in CHO cells expressing CCR2 in the presence or absence of RAGE co-expression.

[000623] Figure 20B. Activation of N FKB, as measured by the induction in gene expression of the N FKB subunit, p65, by CXCL2 (IL-8) in CHO cells expressing CXCR2 in the presence or absence of RAGE co-expression.

[000624] Figure 20C. Activation of N FKB, as measured by the induction in gene expression of the N FKB subunit, p65, by CCL2 (MCP-1 ) in bone marrow-derived primary macrophages in the presence or absence of the peptide inhibitor of RAGE activation, S391 A-RAGE ₃₆₂ -404-

[000625] Figure 20D. Activation of N FKB, as measured by the auto-induction in gene expression of MCP-1 , by CCL2 (MCP-1 ) in HMEC in the presence or absence of the peptide inhibitor of RAGE activation, S391 A-RAGE _362-404 .

[000626] Figure 20E. Activation of N FKB, as measured by the induction in gene expression of MCP-1 , by IL-8 in HMEC expressing CXCR2, in the presence or absence of the peptide inhibitor of RAGE activation, S391 A-RAGE _362-404 .

EXAMPLE 21

[000627] Figure 21 A. BRET between mCherry/RAGE ₃₃₈ - ₃₆ i and Nluc/ATi is increased with Ang II. Data are presented as mean ± SEM; n=3-5. [000628] Figure 21 B. BRET saturation curves with ATi/Rluc8 and RAGE/Venus, where cells were also transfected with either 200 ng or 400 ng of mCherry/RAGE _338-361 cDNA, generated 60 minutes following addition of vehicle or Ang II as indicated. Data are combined from 3 independent experiments.

[000629] Figure 21 C. Ang ll-induced modulation of BRET signal between ATi/Rluc8 and RAGE/Venus where cells were also transfected with 0, 50, 100, 200, 300 or 400 ng of mCherry/RAGE _338-361 cDNA or pcDNA3 control plasmid as indicated. Data are presented as mean ± SEM; n=3-4. Filters: Venus 550 nm / Rluc8 450 nm.

[000630] Figure 21 D. Lack of Ang ll-induced BRET signal between ATi/Rluc8 and mCherry/RAGE _338-361 where cells were transfected with 50 ng ATi/Rluc8 cDNA, 300 ng RAGE/Venus cDNA and 0, 50, 100, 200, 300 or 400 ng of mCherry/RAGE ₃₃ 8-36i cDNA or pcDNA3 control plasmid as indicated. Data are presented as mean ± SEM; n=3-4. Filters: mCherry 650 nm / Rluc8 450 nm.

[000631 ] Figure 21 E. Luminescence from ATi/Rluc8, fluorescence from RAGE/Venus and fluorescence from mCherry/RAGE _338-361 in experiments shown in Figure 21 C and 21 D. Data are presented as mean ± SEM; n=3-4.

[000632] Figure 21 F. Ligand-induced modulation of BRET signal between GPCR/Rluc8 and RAGE/Venus where cells were also transfected with mCherry/RAGE _338-361 cDNA or pcDNA3 control plasmid as indicated. Data are presented as mean ± SEM; n=2-4. Filters: Venus 550 nm / Rluc8 450 nm. Amounts of cDNA transfected: 50 ng GPCR/Rluc8 + 300 ng RAGE/Venus + 400 ng of mCherry/RAGE ₃₃ 8-36i or pcDNA3. Indicated Rluc8-tagged GPCR activated by indicated ligand, at indicated concentration, at time zero.

[000633] Figure 21 G. Angll-mediated pro-inflammatory signaling (ICAM-1 expression) in HMEC1 inhibited by mCherry/RAGE _338-361 is rescued by mCherry/RAGE ₃ 62-404. Data are presented as mean ± SEM; n=6-8.

[000634] Figure 21 H. The induction of proinflammatory signalling by Ang II in HMEC cells is inhibited by overexpression of the RAGE transmembrane domain RAGE343-361 with or without an N-terminal mCherry fusion to monitor expression, as denoted by the expression of ICAM1 as measured using real time RT-PCR. Data are presented as mean ± SEM; n=6-8.

[000635] Figure 21 1. The induction of proinflammatory signalling by the RAGE ligand, S100A8/A9, in RAGE-CHO cells is inhibited by overexpression of the RAGE transmembrane domain RAGE343-361 alone, or RAGE370-390, as denoted by the expression of p65 as measured using real time RT-PCR. Data are presented as mean ± SEM; n=6-8.

[000636] Figure 21 J. The induction of proinflammatory signalling by Ang II in HMEC cells is inhibited by siRNA targeting Diaphi or PKCz, as denoted by the expression of ICAM-1 as measured by real time RT-PCR. This inhibition is rescued by RAGE362-404, but not RAGE343- 404. Data are presented as mean ± SEM; n=6-8.

[000637] BRIEF DESCRIPTION OF THE SEQUENCES

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[000638] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[000639] A "receptor heteromer" is defined as a "macromolecular complex composed of at least two (functional) receptor units with biochemical properties that are demonstrably different from those of its individual components." (Ferre et al., 2009).

[000640] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[000641 ] By "about" is meant a measurement, quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference measurement, quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[000642] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[000643] The term "agent", "candidate agent", "modulatory agent", "modulator" "substitute", "functional substitute", "non-functional substitute" or "inhibitor" includes a chemical compound, a mixture of chemical compounds, a biological macromolecule, an extract made from biological materials, a biological organism or part thereof, or other material, which induces a desired pharmacological and/or physiological effect. These terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogues and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, and analogues. The term "agent", "modulator" "substitute" or "inhibitor" is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogues thereof as well as cellular agents. The term "agent", "modulator" "substitute" or "inhibitor" includes a cell that is capable of producing and secreting a polypeptide referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes that polypeptide. Thus, the term "agent", "modulator" "substitute" or "inhibitor" extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells.

[000644] The term "inhibitor" is used in its broadest sense, and includes any compound including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kDa), which decreases at least one aspect of the activity, activation or function of another molecule. For example, an inhibitor may decrease the activity, activation or function of RAGE and/or a certain co-located GPCR, such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, and/or suitably RAGE ligand-independent activation of RAGE by activated certain co-located GPCR such as angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2. Thus, an "inhibitor of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR" refers to an agent that is capable of substantially reducing, inhibiting, antagonising, blocking, negatively modulating and/or mitigating RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR. Inhibition of RAGE ligand- independent activation of RAGE by a certain activated co-located GPCR by an inhibitor suitably reduces or inhibits the biological effect thereof, including the production of proinflammatory mediators including pro-inflammatory cytokines by the cell or the modulation of other cellular elements that are associated with aberrant RAGE ligand-independent activation of RAGE disease symptoms. Note a partial agonist can act as an inhibitor as it does not result in maximal efficacy even though it exhibits agonism. Therefore, by competing for or modulating the agonistic activity of a more efficacious agonist, such as the endogenous agonist, it may effectively act as an inhibitor of a receptor polypeptide and/or its signalling pathway as it reduces the signalling output compared to if it was absent. Consequently a partial agonist may act in a therapeutic setting as an inhibitor. An inhibitor does not necessarily inhibit all aspects of the activity, activation or function of another molecule and indeed, may inhibit some aspects while activating other aspects and/or not modulating yet further aspects. Therefore an inhibitor may exhibit ligand bias.

[000645] The term "ligand bias" refers to the phenomenon whereby, for the same receptor, distinct ligand-stabilised receptor states can exist that selectively promote or inhibit activation of different signalling pathways (Mustafa et al., 2010). This phenomenon has been given multiple names, including but not limited to: ligand-biased signalling, ligand-induced biased signalling, agonist trafficking of receptor signals, cell-based functional selectivity, receptor active-state based selectivity, stimulus trafficking, biased agonism, collateral efficacy and ligand-induced selective signalling (Mustafa et al., 2010). By way of example, this includes the concept that not all agonists activate all signalling pathways normally activated by a reference agonist, which is often the/an endogenous agonist. An agonist may activate some pathways but not others, relative to the reference, thereby exhibiting bias. Furthermore, an antagonist or inverse agonist or inhibitor may only inhibit some pathways but not others and may act at the orthosteric ligand-binding site and/or an allosteric binding site. Orthosteric and allosteric binding sites are defined as commonly known in the art, and allosterism can occur across complexes from one receptor to another such that binding of a ligand to one receptor can result in allosteric modulation of another receptor in the same macromolecular complex. Allosteric modulators can also exhibit ligand bias and modulate some signalling pathways but not others. It is also known in the art that ligands can potentially, for example, act as an agonist for one signalling pathway while acting as an inhibitor for another signalling pathway and/or not affect a third signalling pathway. Indeed, multiple variations and combinations of signalling modulatory effects can occur. Ligand bias is also not absolute in that a ligand may, for example, reduce signalling via one pathway without completely inhibiting it, and/or incompletely activate another signalling pathway. Pathways can be modulated to different extents and this can be measured by multiple parameters, including but not limited to differences in potency and/or efficacy and/or temporal aspects of signalling and/or spatial aspects of signalling.

[000646] The term "functional substitute" is used in its broadest sense, and includes any compound including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kDa), which in taking the place of another molecule is able to mimic or increases at least one aspect of the activity, activation or function of that molecule. For example, a functional substitute of RAGE may replicate the activity, activation or function of RAGE and/or a certain co-located GPCR, such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, and/or suitably RAGE ligand-independent activation of RAGE by activated certain co-located GPCR such as angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, in a system otherwise devoid of RAGE expression. Thus, "a functional substitute" of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR" refers to an agent that is capable of substantially increasing, augmenting, agonising, and/or positively modulating RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR. Restoring signalling capability of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR by a functional substitute suitably restores or augments the biological effect thereof, including the production of pro-inflammatory mediators including pro-inflammatory cytokines by the cell or the modulation of other cellular elements that are associated with RAGE ligand-independent activation of RAGE disease symptoms. A functional substitute does not necessarily mimic all aspects of the activity, activation or function of another molecule and indeed, may inhibit some aspects while activating other aspects and/or not modulate yet further aspects. Therefore a functional substitute may also exhibit ligand bias.

[000647] The term "non functional substitute" is used in its broadest sense, and includes any compound including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kDa), which in taking the place of another molecule is able to inhibit, antagonise or reduce at least one aspect of the activity, activation or function of that molecule. For example, a non functional substitute of RAGE may inhibit the activity, activation or function of RAGE and/or a certain co-located GPCR, such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, and/or suitably RAGE ligand-independent activation of RAGE by activated certain co-located GPCR such as angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2. Thus, "a non functional substitute" of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR" refers to an agent that is capable of substantially reducing, inhibiting, antagonising, and/or negatively modulating RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR. Reducing signalling capability of RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR by a non functional substitute suitably reduces or inhibits the biological effect thereof, including the production of pro-inflammatory mediators including pro-inflammatory cytokines by the cell or the modulation of other cellular elements that are associated with RAGE ligand- independent activation of RAGE disease symptoms. Note a partial agonist can also act as an inhibitor as it does not result in maximal efficacy even though it exhibits agonism. Therefore, by competing for or modulating the agonistic activity of a more efficacious agonist, such as the endogenous agonist, it may effectively act as an inhibitor of a receptor polypeptide and/or its signalling pathway as it reduces the signalling output compared to if it was absent. Consequently a partial agonist may act in a therapeutic setting as an inhibitor. A non functional substitute does not necessarily reduce all aspects of the activity, activation or function of another molecule and indeed, may inhibit some aspects while facilitating other aspects and/or not modulating yet further aspects. Therefore a non functional substitute may also exhibit ligand bias.

[000648] The term "binding" and its grammatical equivalents refer to a physical association between molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions under physiological conditions, and includes interactions such as salt bridges and water bridges, as well as any other conventional means of binding. Binding may occur directly or via interactions with one or more other intermediary molecules.

[000649] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[000650] The term "construct" refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1 ) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be contained within two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1 , 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[000651 ] By "corresponds to" or "corresponding to" is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence (e.g., at least about 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 97, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to all or a portion of the reference nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence (e.g., at least 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 97, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to all or a portion of the reference amino acid sequence).

[000652] By "effective amount", in the context of treating or preventing a condition is meant the administration of an amount of an agent or modulator, such as an inhibitor, or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[000653] As used herein, the terms "encode," "encoding" and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode," "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[000654] The term "expression" with respect to a gene sequence refers to transcription of the gene to produce a RNA transcript (e.g. mRNA, antisense RNA, siRNA, shRNA, miRNA) and, as appropriate, translation of a resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a coding sequence results from transcription and translation of the coding sequence. Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence. "Expression" may also refer to the location of a gene product in, on or outside a cell, including cell compartments or structures, illustrative examples of which include cytoplasm, nucleus, ribosome, lysosome and cell membrane or surface.

[000655] By "expression vector" is meant any genetic element capable of directing the transcription of a polynucleotide contained within the vector and suitably the synthesis of a peptide or polypeptide encoded by the polynucleotide. Such expression vectors are known to practitioners in the art.

[000656] As used herein, the term "function" refers to a biological, enzymatic, or therapeutic function. [000657] As used herein, the term "gene" refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and 5' and 3' untranslated regions).

[000658] The term "host cell" refers to a cell into which a polynucleotide or nucleic acid construct is introduced. Host cells of the invention include, but need not be limited to, bacterial, yeast and animal (including vertebrate animals falling within the scope of the term "subject" as defined herein). Host cells can be unicellular, or can be grown in tissue culture as liquid cultures, monolayers or the like. Host cells may also be derived directly or indirectly from tissues or may exist within an organism including animals (including vertebrate animals falling within the scope of the term "subject" as defined herein).

[000659] The term "interaction" refers to the effect based on intermolecular force between two molecules, and examples thereof include ion-to-ion interaction, interaction by a hydrogen bond, dipole-dipole interaction, hydrophobic interaction, and combinations thereof, and includes binding between molecules. Interaction can occur directly or via one or more other intermediary molecules.

[000660] The term "functional interaction" refers to one molecule affecting the function of another molecule and does not necessarily mean that the two molecules are physically interacting with each other.

[000661 ] The term "luciferase" means a polypeptide corresponding to a luciferase protein, that emits light upon oxidation of its substrate. An exemplary luciferase protein is Renilla luciferase or a derivative thereof, that oxidises coelenterazine. Another example luciferase protein is NanoLuc.

[000662] The term "analogue" refers to a small molecule that, because of its structural analogy to the endogenous peptide, is capable of mimicking at least one biological function of the peptide to which it is analogous. A "peptide analogue" is an analogue that is a peptide in nature as it contains peptide bonds and has a modular structure composed of amino acids or their derivatives. A "non-peptide analogue" is an analogue that is not a peptide in nature as it does not contain peptide bonds and does not have a modular structure composed of amino acids or their derivatives as building blocks. [000663] By way of illustration, a RAGE analogue may mimic the ability of RAGE to form a heteromer complex with AT ₁ R but suitably inhibits transactivation of RAGE by an AT ₁ R ligand acting via AT ₁ R.

[000664] The term "derivative" refers to a molecule that its primary structure is taken from or owes its derivation to the cytosolic tail of RAGE or fragment thereof, but which includes amino acid additions, substitutions, truncations, chemical and/or biochemical modifications, retro- inverted sequences, cyclic peptides, peptoids, β-peptides, or linkage to a non-peptide drug, non-peptide label, non-peptide carrier, or non-peptide resin.

[000665] As used herein, the terms "modulating", "regulating" and their grammatical equivalents refer to an effect of altering a biological activity or effect (e.g., cytokine production, cellular adhesion, GPCR signalling, RAGE signalling). For example, an agonist, antagonist, inverse agonist or allosteric modulator of a particular biomolecule modulates the activity of that biomolecule, e.g., a receptor, by either increasing / stimulating (e.g., agonist, activator, positive allosteric modulator), or decreasing / inhibiting (e.g., antagonist, inhibitor, inverse agonist, negative allosteric modulator) the activity or effect (e.g., cytokine production, cellular adhesion, receptor signalling) of the biomolecule, such as a receptor.

[000666] The term "operably connected" or "operably linked" as used herein refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence "operably linked" to a coding sequence refers to positioning and/or orientation of the regulatory sequence relative to the coding sequence to permit expression of the coding sequence under conditions compatible with the regulatory sequence.

[000667] By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent or modulator, such as an inhibitor, without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, colouring agents, wetting or emulsifying agents, pH buffering agents, preservatives, transfection agents and the like.

[000668] Similarly, a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that is not biologically or otherwise undesirable.

[000669] The terms "polynucleotide," "nucleic acids" and "nucleotide sequence" include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.

[000670] The terms "polypeptide," "proteinaceous molecule," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.

[000671 ] The term "polypeptide variant" refers to polypeptides in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter. These terms also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acids.

[000672] As used herein, the terms "prevent," "prevented," or "preventing," refer to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. These terms also include within their scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it.

[000673] Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Preferred promoters according to the invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a cell, and/or to alter the timing of expression of a structural gene to which it is operably connected.

[000674] By "proximity" refers to the distance between two integers (generally, polypeptides). For example, the proximity of a RAGE polypeptide and a GPCR polypeptide is meant the relative distance between the two polypeptides. If two polypeptides directly interact, they are considered to be spatially near one another, or described as being in close proximity. If two polypeptides are part of the same heteromer and/or macromolecular complex but are not directly interacting they are also considered to be in close proximity. More specifically, for the purposes of the present invention a distance of around 30 nm or less, 10 nm or less, or 5 nm or less would be considered to be within close proximity.

[000675] By "proximity screening assay" is meant an assay, system or experimental approach that comprises the bringing of a first and second reporter component into proximity to generate a detectable proximity signal. Proximity of the first and second reporter components generates a proximity signal capable of detection by the detector. The first and second reporter components constitute a complementary pair, in the sense that the first reporter component may be interchanged with the second reporter component (i.e. the first reporter component coupled to the RAGE polypeptide and the second reporter component coupled to the GPCR polypeptide or the first reporter component coupled to the GPCR polypeptide and the second reporter component coupled to the RAGE polypeptide) without appreciably affecting the functioning of the invention. Direct physical contact between the RAGE polypeptide and the GPCR polypeptide or between the reporter components is not required and may be mediated by one or more linkage molecule(s). Preferably, the proximity signal generated by the proximity of the first and second reporter components in the presence of the reporter component initiator is selected from the group consisting of: luminescence, fluorescence and colorimetric change. In some embodiments, the luminescence is produced by a bioluminescent protein selected from the group consisting of luciferase, galactosidase, lactamase, peroxidase, or any protein capable of luminescence in the presence of a suitable substrate.

[000676] The term "reporter component" can be any known compound, organic or inorganic, proteinaceous or non-proteinaceous or complex thereof, that when in proximity to another reporter component is capable of resulting in the emission of a detectable proximity signal. In some embodiments, the reporter component is selected from the group comprising, consisting or consisting essentially of an enzyme, a luminescent molecule or part thereof, a fluorescent molecule or part thereof and a transcription factor or other molecule coupled to the RAGE polypeptide and/or the certain co-located GPCR polypeptide, such as angiotensin receptor such as AT ₁ R or certain chemokine receptors such as CCR2. In some embodiments, reporter components can include enzymes, luminescent or bioluminescent molecules, fluorescent molecules, and transcription factors or other molecules coupled to the RAGE polypeptide and/or the certain co-located GPCR polypeptide by linkers incorporating enzyme cleavage sites.

[000677] In some aspects of the present invention, the proximity reporter system involves combinations of pairs of reporter components, capable of being a donor and/or acceptor molecule. Accordingly, the reporter components that can be used according to the present invention can be selected based on the physical properties thereof, as is known in the art of resonance energy transfer (RET), the two being selected so that they together comprise the donor and acceptor molecules of a RET pair. If one of the reporter components within a RET pair is a bioluminescent protein, the RET is known as bioluminescence RET (BRET). If both reporter components forming a RET pair are fluorophores the resulting RET is known as fluorescence RET (FRET). Examples of known suitable donor and acceptor pairs include: Renilla luciferase (or variant thereof) and yellow fluorescent protein; Renilla luciferase (or variant thereof) and green fluorescent protein (or variant thereof); NanoLuc and yellow fluorescent protein; NanoLuc and mCherry fluorescent protein; NanoLuc and HaloTag (with suitable HaloTag ligand); Cyan fluorescent protein and yellow fluorescent protein; fluorescein and tetramethylrhodamine; 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS) and fluorescein (See generally R. Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed. 1995). One or both of the fluorophores can be a fluorescent protein such as green fluorescent protein, and it is particularly advantageous to employ a fluorescent protein as the fluorophore by preparing a fusion protein of the RAGE polypeptide or the certain co-located GPCR polypeptide and a fluorescent protein.

[000678] Preferable combinations of first and second reporter components include a luminescent reporter component with a fluorescent reporter component, a luminescent reporter component with a non-fluorescent quencher, a fluorescent reporter component with a non-fluorescent quencher, first and second fluorescent reporter components capable of resonance energy transfer. However, useful combinations of first and second reporter components are by no means limited to such. Alternate combinations of first and second reporter components that may be utilised by the present invention include those exemplified in US6893827 (Applera Corporation); US6800445 (Applera Corporation); US7049076 (Sentigen Biosciences, Inc., and The Trustees of Columbia University of the City of New York); US61 10693 (Duke University); US5891646 (Duke University); WO/2005/031309 (ODYSSEY THERA INC.); and US8101373 (DiscoveRx Corporation).

[000679] The terms "coupled", "coupled directly" and "coupled indirectly" as used herein means that the reporter component is attached to or associated with the RAGE polypeptide and/or certain co-located GPCR polypeptide to form an entity that is capable of being analysed or detected. The direct or indirect coupling of the reporter components to the RAGE polypeptide and/or certain co-located GPCR polypeptide may be by any known covalent or non-covalent means of coupling two molecules, including chemical cross-linking, chemical modification of proteins, chemical modification of amino acids, chemical modification of nucleic acids, chemical modification of carbohydrates, chemical modification of lipids, chemical modification of any other organic or inorganic molecule, biotin-avidin interactions, antigen-antibody interaction and nucleic acid hybridisation. In one form of the invention, the reporter components are coupled indirectly to the RAGE polypeptide and/or certain co-located GPCR polypeptide by a linker. In some embodiments, the linker comprises an enzyme cleavage site. An example of a direct method of coupling a RAGE polypeptide and/or certain co-located GPCR polypeptide and a proteinaceous reporter component is genetic fusion, wherein the genes encoding the RAGE polypeptide and/or the certain co-located GPCR polypeptide and the bioluminescent or fluorescent protein are fused to produce a single polypeptide chain. Another example of a direct coupling method is conjugation, wherein the coupling of the RAGE polypeptide and/or the certain co-located GPCR polypeptide with the reporter component uses enzymes such as ligases, hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.

[000680] In a preferred embodiment of the invention, the RAGE polypeptide forms a single polypeptide chain with a reporter component and/or the certain co-located GPCR polypeptide forms a single polypeptide chain with a reporter component. In a particularly preferred form of the invention, the reporter components are different. Additional functionality may form part of the same polypeptide chain. For example, the single polypeptide chain additionally comprises: a sequence coding for a peptide sequence used for affinity purification of a fusion construct; and/or a sequence coding for a peptide sequence which directs the fusion construct to a subcellular compartment of a eukaryotic cell; and/or a sequence coding for a peptide sequence that facilitates the penetration of a eukaryotic cell membrane; and/or a sequence enabling expression levels to be assessed by the use of antibodies or otherwise. [000681 ] By "regulatory sequence" is meant a nucleic acid sequence (e.g., DNA) that expresses an operably linked nucleotide sequence (e.g., a coding sequence) in a particular host cell. The regulatory sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a cis-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or trailing sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.

[000682] The terms "salts," "derivatives" and "prodrugs" includes any pharmaceutically acceptable salt, ester, hydrate, or any other compound which, upon administration to the recipient, is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. Also, basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts and prodrugs and derivatives can be carried out by methods known in the art. For example, metal salts can be prepared by reaction of a compound of the invention with a metal hydroxide. An acid salt can be prepared by reacting an appropriate acid with a compound of the invention.

[000683] The term "selective" refers to compounds that modulate an interaction, suitably a physical interaction (e.g., binding) between a RAGE polypeptide and certain co-located GPCR polypeptide without substantially modulating another biological interaction of the RAGE polypeptide or the certain co-located GPCR polypeptide. Accordingly, a compound that is selective for the direct RAGE- certain co-located GPCR interaction exhibits a selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to modulation of another binding activity of RAGE or of the certain co- located GPCR to another substrate or binding partner.

[000684] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[000685] "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined below.

[000686] Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[000687] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1 ) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wl, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons Inc, 1994-1998, Chapter 15.

[000688] As used herein a "small molecule" refers to a composition that has a molecular weight of less than 3 kilodaltons (kDa), and typically less than 1 .5 kilodaltons, and more preferably less than about 1 kilodalton. Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. As those skilled in the art will appreciate, based on the present description, extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity. A "small organic molecule" is an organic compound (or organic compound complexed with an inorganic compound (e.g., metal)) that has a molecular weight of less than 3 kDa, less than 1 .5 kDa, or even less than about 1 kDa.

[000689] "Stringency" as used herein, refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization and washing procedures. The higher the stringency, the higher the degree of complementarity will be between immobilized target nucleotide sequences and probe nucleotide sequences that remain hybridized to the target after washing. Stringency conditions include low, medium, high and very high stringency conditions, which describe certain conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al., (1998, supra), Sections 6.3.1 -6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used. Reference herein to low stringency conditions include and encompass from at least about 1 % v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C. Low stringency conditions also may include 1 % Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 x SSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6 x sodium chloride/sodium citrate (SSC) at about 45DC, followed by two washes in 0.2 x SSC, 0.1 % SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions). Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42°C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55°C. Medium stringency conditions also may include 1 % Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 xx SSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at 60-65°C. One embodiment of medium stringency conditions includes hybridizing in 6 xx SSC at about 45°C, followed by one or more washes in 0.2 xx SSC, 0.1 % SDS at 60°C. High stringency conditions include and encompass from at least about 31 % v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42°C, and about 0.01 M to about 0.02 M salt for washing at 55°C. High stringency conditions also may include 1 % BSA, 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 0.2 xx SSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 1 % SDS for washing at a temperature in excess of 65°C. One embodiment of high stringency conditions includes hybridizing in 6 xx SSC at about 45°C, followed by one or more washes in 0.2 xx SSC, 0.1 % SDS at 65°C. In certain embodiments, a peptide or polypeptide is encoded by a polynucleotide that hybridizes to a reference nucleotide sequence under very high stringency conditions. One embodiment of very high stringency conditions includes hybridizing 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2 x SSC, 1 % SDS at 65°C. Other stringency conditions are well known in the art and a skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1 .101 to 1.104. While stringent washes are typically carried out at temperatures from about 42°C to 68°C, one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rate typically occurs at about 20°C to 25°C below the Tm for formation of a DNA-DNA hybrid. It is well known in the art that the Tm is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating Tm are well known in the art (see Ausubel et al., supra at page 2.10.8). In general, the Tm of a perfectly matched duplex of DNA may be predicted as an approximation by the formula:

Tm = 81 .5 + 16.6 (log 10 M) + 0.41 (%G+C) - 0.63 (% formamide) - (600/length) wherein:

M is the concentration of Na+, preferably in the range of 0.01 molar to 0.4 molar; %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex. The Tm of a duplex DNA decreases by approximately 1 °C with every increase of 1 % in the number of randomly mismatched base pairs. Washing is generally carried out at Tm - 15°C for high stringency, or Tm - 30 ^oo C for moderate stringency. In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 42 ⁰⁰ C in a hybridization buffer (50% deionized formamide, 5 x SSC, 5 x Denhardt's solution (0.1 % ficoll, 0.1 % polyvinylpyrrolidone and 0.1 % bovine serum albumin), 0.1 % SDS and 200 mg/mL denatured salmon sperm DNA) containing labeled probe. The membrane is then subjected to two sequential medium stringency washes (i.e., 2 x SSC, 0.1 % SDS for 15 min at 45°C, followed by 2 x SSC, 0.1 % SDS for 15 min at 50°C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1 % SDS for 12 min at 55°C followed by 0.2 x SSC and 0.1 % SDS solution for 12 min at 65-68°C.

[000690] The terms "subject," "host", "individual" or "patient," used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards), and fish. In specific embodiments, the subject is a primate such as a human. However, it will be understood that the terms "patient," "subject," "host" or "individual" do not imply that symptoms are present.

[000691 ] As used herein, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or condition (e.g., a hematologic malignancy) and/or adverse effect attributable to the disease or condition. These terms also cover any treatment of a condition or disease in a mammal, particularly in a human, and include: (a) inhibiting the disease or condition, i.e., arresting its development; or (b) relieving the disease or condition, i.e., causing regression of the disease or condition.

[000692] The term "variant" is employed broadly to refer to a protein that differs in some way from a reference wild-type protein, where the protein may retain biological properties of the reference wild-type protein, or may have biological properties that differ from the reference wild-type protein. The term "biological property" of the subject proteins includes, but is not limited to, spectral properties, such as emission maximum, quantum yield, brightness, and the like; in vivo and/or in vitro stability (e.g. half-life); and the like. Variants can include single amino acid changes (substitutions), deletions of one or more amino acids (point deletions), N-terminal truncations, C-terminal truncations, insertions, and the like. Polypeptide variants can be generated using standard techniques well known in molecular biology.

[000693] By "vector" is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integratable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, or two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[000694] Unless the context requires otherwise, "RAGE" or "RAGE polypeptide" shall indicate the protein product or products generated from transcription and translation and/or alternative splicing of the AGER gene, also known as the AGER nucleotide or AGER polynucleotide, or the RAGE nucleotide or RAGE polynucleotide. [000695] Unless the context requires otherwise, "AT ₁ R" or "AT ₁ R polypeptide" or "ATi receptor", or "AT ₁ R" shall indicate the protein product or products generated from transcription and translation and/or alternative splicing of the AGTR1 ,Agtr1 a or Agtri b genes, also known as AT ₁ R nucleotide or ATiR polynucleotide. For example, in humans, one gene codes for AT ₁ R located on chromosome 3q21 -q25 (IUPHAR database, www.guidetopharmacology.org). In mouse, two genes code for AT ₁ R, Agtr1 a located on chromosome 13 16.0 cM and Agtri b located on chromosome 3 7.6 cM (IUPHAR database, www.guidetopharmacology.org). In rat, two genes code for AT ₁ R, Agtr1 a located on chromosome 17q12 and Agtri b located on chromosome 2q24 (IUPHAR database, www.quidetopharmacoloqy.org).

[000696] In some instances, the protein product or products of the Agtr1 a gene may be referred to as ATi _a R and the protein product or products of the Agtri b gene may be referred to as AT _{1 b} R, however, the protein product or products of the Agtr1 a and Agtri b genes may both be referred to as AT ₁ R.

[000697] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

2. Abbreviations

[000698] The following abbreviations are used throughout the application: aa = amino acid(s);

d = day;

h = hour;

kb = kilobase(s) or kilobase pair(s);

kDa = kilodalton(s);

nm = nanometre;

nt = nucleotide;

nts = nucleotides;

s = seconds

3. Receptor Polypeptides, as well as constructs and nucleotide sequences encoding polypeptides

[000699] In some embodiments a polynucleotide encoding a receptor polypeptide is operably connected to a nucleic acid sequence encoding a bioluminescent or fluorescent donor molecule or fluorescent acceptor molecule or other suitable reporter component of a proximity screening assay. Once coding sequences for the desired polypeptides have been isolated or synthesised they can be cloned into any suitable construct for expression (e.g., transient or stable expression). Numerous constructs including cloning and expression vectors are known to those of skill in the art, and the selection of appropriate constructs is well within the skill of a practitioner in the field.

[000700] In some embodiments, the receptor polynucleotide coding sequences are inserted into locations within a suitable construct using routine technologies and molecular biological methods including DNA synthesis, PCR mutagenesis, and restriction endonuclease digestion followed by PCR ligation. Any coding sequence for a core protein is suitable for preparing nucleic acid sequences encoding the chimeric polypeptides of the present invention.

[000701 ] General texts which describe molecular biological techniques, which are applicable to the present invention such as cloning, mutation, and the like, include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning— A Laboratory Manual (3rd Ed.), Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., ("Ausubel"). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the cloning and expression of chimeric core proteins.

3. 1 Receptor for Advanced Glycation End-products (RAGE) Polypeptides

[000702] A significant role of RAGE is to bind to advanced glycation end-products (AGE), including glycated proteins on which amino groups have been modified non-enzymatically through the Maillard reaction. RAGE is often referred to as a pattern recognition receptor due to its ability to detect a class of ligands through a common motif.

[000703] In specific embodiments of the present invention, the RAGE polypeptide comprises a RAGE protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a RAGE protein sequence. In some embodiments, the RAGE protein sequence corresponds to a mammalian RAGE protein sequence. In some embodiments the RAGE protein sequence is derived from a mammal selected from the group comprising human (UniProtKB Accession No. Q15109), mouse (UniProtKB Accession No. Q62151 ), cow (UniProtKB Accession No. Q28173), rat (UniProtKB Accession No. Q63495), macaca (UniProtKB Accession No. G7P2Q8), and dog (UniProtKB Accession No. Q20JV7). In preferred embodiments, the RAGE protein sequence corresponds to a human wild-type RAGE protein sequence (for example, UniProtKB Accession No. Q15109) or a functional fragment of this sequence. The human full-length wild-type protein sequence, as defined by UniProtKB Accession No. Q15109, is set forth as follows:

[000704] MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKL

NTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETK SN

YRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEK GVSV

KEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVW EPV

PLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLPPSPVLILPE IGPQD

QGTYSCVATHSSHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILGGLG TAALLI

GVILWQRRQRRGEERKAPENQEEEEERAELNQSEEPEAGESSTGGP [SEQ ID NO: 14].

[000705] The wild-type RAGE comprises five distinct domains. The ectodomain of RAGE bears three immunoglobulin-like (Ig) domains, the V, C1 and C2 domains (residues 23-342 of SEQ ID NO: 14). These Ig domains belong to the V-, C- and S-type, respectively. The fourth domain comprises a single transmembrane (TM) spanning helix (residues 343-361 of SEQ ID NO: 14), and finally a cytosolic tail (residues 362-404 of SEQ ID NO: 14, as set forth in SEQ ID NO: 1 ) that is devoid of kinase activity and presumed to be unstructured (see, Neepers, 1992). In addition, the human wild-type RAGE protein sequence comprises a 22 amino acid N-terminal signal peptide, which has previously been shown to play a role in trafficking the RAGE protein to the plasma membrane.

[000706] Throughout this specification, unless the context requires otherwise, reference to the ectodomain of RAGE means a reference to amino acid residues 23-342 of wild-type RAGE (SEQ ID NO: 14).

[000707] Throughout this specification, unless the context requires otherwise, reference to the transmembrane domain (or TM) of RAGE means a reference to amino acid residues 343-361 of wild-type RAGE (SEQ ID NO: 14).

[000708] Throughout this specification, unless the context requires otherwise, reference to the cytosolic tail of RAGE means a reference to amino acid residues 362-404 of wild-type RAGE (SEQ ID NO: 14).

[000709] In one form of the invention, the RAGE polypeptide comprises a truncated form of a mammalian wild-type RAGE protein sequence. For example, the RAGE polypeptide sequence may comprise the human wild-type RAGE protein sequence in which one or more of the ligand-binding regions (V, C or S-type Ig domains) of the ectodomain of RAGE are mutated or deleted so as to impair RAGE ligand induced signalling. By way of a non-limiting illustrative example, a RAGE polypeptide suitable for using with the present invention comprised amino acid residues 362 to 404 of the human wild-type RAGE protein sequence as set forth in SEQ ID NO: 1 .

[000710] The RAGE cytosolic tail (residues 362-404 of SEQ ID NO: 1 ) is responsible for signal transduction. In some embodiments the RAGE polypeptide of the present invention comprises, consists or consists essentially of the RAGE cytosolic tail, or a fragment thereof or mutated, truncated or fusion fragments. For example, the RAGE polypeptide may comprise, consist or consist essentially of residues 362 to 404 of the human wild-type RAGE as set forth in SEQ ID NO: 1 .

3.2 Vectors and nucleotide sequences encoding RAGE polypeptides.

[000711] The present invention also encompasses isolated polynucleotide sequences and constructs encoding RAGE polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000712] In some embodiments, the polynucleotide sequences comprise a sequence that correspond to a mouse AGER nucleotide sequence, as set forth, for example, in Genbank Accession No. L33412.1 , EU570247.1 , EU570246.1 , EU570245.1 , EU570244.1 , EU570242.1 , EU570241 .1 , EU570240.1 .

[000713] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a bovine AGER nucleotide sequence as set forth for example in GenBank Accession Nos.M91212.1 , or NM_173982.3.

[000714] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a rat AGER nucleotide sequence as set forth for example in GenBank Accession Nos.L33413.1 , NM_053336.2.

[000715] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a macaca AGER nucleotide sequence as set forth for example in GenBank Accession Nos. CM001279.1 , NM_0012051 17.1 , GU139408.1 , GU139409.1 , GU139410.1 , GU13941 1 .1 , GU139412.1 , GU139413.1 , GU139414.1 , GU139415.1 , GU139416.1 , GU139417.1 , GU139418.1 , GU139419.1 , GU139420.1 , GU139421 .1 , GU139422.1 .

[000716] In some embodiments, the polynucleotide sequences comprise a sequence that correspond to a canine AGER nucleotide sequence as set forth, for example, in Genbank Accession Nos. DQ125936.1 , DQ125937.1 , DQ125938.1 , DQ125940.1 , or DQ125939. [000717] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human AGER nucleotide sequence as set forth for example in GenBank Accession Nos. AF001095.1 , AB061668.1 , AJ012753.1 , Y18060.1 , EU826620.1 , EU826618.1 , EU826617.1 , EU826616.1 , AF065213.1 , AF065212.1 , AF065210.1 , CU690977.1 , CU690976.1 , AJ238896.1 , Y18762.1 . In some embodiments of this type, the polynucleotide comprises a AGER nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000718] In some embodiments, an AGER polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian RAGE protein, or a fragment thereof. In some embodiments, the AGER polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian RAGE protein, or a fragment thereof under low, medium or high stringency conditions.

3.3 Type-1 angiotensin II receptor (ATiR) polypeptides

[000719] The G protein-dependent signalling by AT ₁ R is vital for normal cardiovascular homeostasis, yet detrimental in chronic dysfunction, which associates with cell death and tissue fibrosis, and leads to cardiac hypertrophy and heart failure (Ma et al., 2010).

[000720] Despite its high medical relevance and decades of research, the structure of AT ₁ R and the binding mode of well established AT ₁ R blockers (ARBs) were only recently elucidated (Zhang et al., 2015). The structure indicated that the extracellular part of AT ₁ R consists of the N-terminal segment ECL1 (Glu91 -Phe96 of the human AT ₁ R) linking helices II and III, ECL2 (His166 to Ile191 of the human AT ₁ R) linking helices IV and V, and ECL3 (Ile270 to Cys274 of the human AT ₁ R) linking helices VI to VII. Two disulphide bonds help to shape the extracellular side of AT ₁ R with Cys18-Cys 274 connecting the N terminus and ECL3, and Cys101 -Cys180 connecting helix III and ECL2 (similar to the chemokine receptor CXCR4, which shares around 36% sequence identity with AT ₁ R).

[000721 ] In specific embodiments of the present invention, the AT ₁ R polypeptide comprises a AT ₁ R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an AT ₁ R protein sequence. [000722] In some embodiments, the AT ₁ R protein sequence corresponds to a mammalian AT1 R protein sequence. Suitable AT ₁ R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P30556), sheep (UniProtKB Accession No. 077590), cow (UniProtKB Accession No. P25104), rabbit (UniProtKB Accession No. P34976), guinea pig (UniProtKB Accession No. Q9WV26), pig (UniProtKB Accession No. P30555), chimpanzee (UniProtKB Accession No. Q9GLN9), gerbil (UniProtKB Accession No. 035210, rat (UniProtKB Accession No. P29089), mouse (UniProtKB Accession No. P29754), cat (UniProtKB Accession No. M3VVA2), Tasmanian devil (UniProtKB Accession No. G3W0M6), horse (UniProtKB Accession No. F7D1 N0), and panda (UniProtKB Accession No. D2HWD9).

[000723] In some preferred embodiments, the AT ₁ R protein sequence corresponds to a human AT ₁ R protein sequence. In some embodiments, the AT ₁ R polypeptide comprises a human full-length wild-type AT ₁ R protein sequence (UniProtKB Accession No. P30556), as set forth below, or a functional fragment of the wild-type AT ₁ R protein sequence.

[000724] MILNSSTEDGIKRIQDDCPKAGRHNYIFVMIPTLYSIIFVVGIFGNSLVVIVIYFYMK LKTVASVFLLNLALADLCFLLTLPLWAVYTAMEYRWPFGNYLCKIASASVSFNLYASVFL LTC LSIDRYLAIVHPMKSRLRRTMLVAKVTCIIIWLLAGLASLPAIIHRNVFFIENTNITVCA FHYESQ NSTLPIGLGLTKNILGFLFPFLIILTSYTLIWKALKKAYEIQKNKPRNDDIFKIIMAIVL FFFFSWIP HQIFTFLDVLIQLGIIRDCRIADIVDTAMPITICIAYFNNCLNPLFYGFLGKKFKRYFLQ LLKYIPP KAKSHSNLSTKMSTLSYRPSDNVSSSTKKPAPCFEVE [SEQ ID NO: 16].

[000725] In one form of the invention, the AT ₁ R polypeptide comprises a truncated form of a mammalian wild-type AT ₁ R protein sequence. For example, the AT ₁ R polypeptide sequence may comprise the human wild-type AT ₁ R protein sequence with a C-terminal truncation (e.g., amino acid residues 320-359 may be truncated). Alternatively or in addition, the AT ₁ R polypeptide sequence may comprise the wild-type AT ₁ R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type AT ₁ R protein sequence (e.g., amino acid residues 7-16 may be truncated). By way of a non-limiting illustrative example, a AT ₁ R polypeptide suitable for using with the present invention comprised amino acid residues 2-6 and 17-319 of the human wild-type AT ₁ R protein sequence as set forth in SEQ ID NO: 16.

Constructs and nucleotide sequences encoding ATiR polypeptides. [000726] The present invention also encompasses isolated polynucleotide sequences and constructs encoding AT ₁ R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000727] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human AT ₁ R nucleotide (i.e., corresponding to the AGTR1 gene) sequence as set forth for example in GenBank Accession Nos. KR71 1424.1 , KR71 1423.1 , KR71 1422.1 , KR71 1421 .1 , KJ896399.1 , KJ896398.1 , NM_032049.3, NM_031850.3, NM_004835.4, NM_000685.4, NM_009585.3, DQ895601 .2, BC068494.1 , BC022447.1 , DQ892388.2, and AK291541 .1 . In representative examples of this type, the polynucleotide comprises an AT ₁ R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000728] In some embodiments, an AT ₁ R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian AT ₁ R polynucleotide, or a fragment thereof. In some embodiments, the AT ₁ R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian AT ₁ R protein, or a fragment thereof under low, medium or high stringency conditions.

3.5 Adenosine 1 receptor (A 1R) polypeptides

[000729] The A1 R is an important regulator of the cardiovascular system, and also has various other roles such as those in neurological, metabolic and skeletal processes (Fredholm et al., 201 1 ).

[000730] To date, a crystal structure of the A1 R has not yet been published, so the structural and ligand-binding features of the receptor has to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of A1 R consists of the N-terminal segment (Met1 -Ser4 of the human A1 R), ECL1 (Pro73-Thr75 of the human A1 R) linking helices II and III, ECL2 (Trp146-lle175 of the human A1 R) linking helices IV and V, and ECL3 (Pro261 -Lys265 of the human A1 R) linking helices VI to VII. It is believed that four disulphide bonds may form on the extracellular side of A1 R (as seen in the crystal structure of A2A) which help to stabilise the ligand-binding cleft (Piirainen et al., 201 1 ).

[000731 ] In specific embodiments of the present invention, the A1 R polypeptide comprises a A1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an A1 R protein sequence.

[000732] In some embodiments, the A1 R protein sequence corresponds to a mammalian A1 R protein sequence. Suitable A1 R sequences may suitably be from mammal selected from the group comprising chimpanzee (UniProtKB Accession No. H2Q0X8), cow (UniProtKB Accession No. P28190), dog (UniProtKB Accession No. P1 1616), guinea pig (UniProtKB Accession No. P47745), horse (UniProtKB Accession No. F6RAN5), human (UniProtKB Accession No. P30542), mouse (UniProtKB Accession No. Q60612), panda (UniProtKB Accession No. G1 M0W2), pig (UniProtKB Accession No. I3LEN5), rabbit (UniProtKB Accession No. P34970), rat (UniProtKB Accession No. 008766), rat (UniProtKB Accession No. P25099), sheep (UniProtKB Accession No. W5NSY0), Tasmanian devil (UniProtKB Accession No. G3WVA4).

[000733] In some preferred embodiments, the A1 R protein sequence corresponds to a human A1 R protein sequence. In some embodiments, the A1 R polypeptide comprises a human full-length wild-type A1 R protein sequence (UniProtKB Accession No. P30542), as set forth below, or a functional fragment of the wild-type A1 R protein sequence.

[000734] MPPSISAFQAAYIGIEVLIALVSVPGNVLVIWAVKVNQALRDATFCFIVSLAVADV

AVGALVIPLAILINIGPQTYFHTCLMVACPVLILTQSSILALLAIAVDRYLRVKIPL RYKMVVTPR

RAAVAIAGCWILSFVVGLTPMFGWNNLSAVERAWAANGSMGEPVIKCEFEKVISMEY MVYF

NFFVWVLPPLLLMVLIYLEVFYLIRKQLNKKVSASSGDPQKYYGKELKIAKSLALIL FLFALSW

LPLHILNCITLFCPSCHKPSILTYIAIFLTHGNSAMNPIVYAFRIQKFRVTFLKIWN DHFRCQPA

PPIDEDLPEERPDD [SEQ ID NO: 17].

[000735] In one form of the invention, the A1 R polypeptide comprises a truncated form of a mammalian wild-type A1 R protein sequence. For example, the A1 R polypeptide sequence may comprise the human wild-type A1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 315-326 may be truncated). Alternatively or in addition, the A1 R polypeptide sequence may comprise the wild-type A1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type A1 R protein sequence (e.g., amino acid residues 154-161 may be truncated). By way of a non-limiting illustrative example, an A1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-153 and 162-314 of the human wild-type A1 R protein sequence as set forth in SEQ ID NO: 17. 3.6 Constructs and nucleotide sequences encoding A 1 R polypeptides.

[000736] The present invention also encompasses isolated polynucleotide sequences and constructs encoding A1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000737] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human A1 R nucleotide (i.e., corresponding to the ADORA1 gene) sequence as set forth for example in GenBank Accession Nos. CR541749.1 , NM 000674.2, NM_001048230.1 , NG_052917.1 , KC884744.1 , KC881 108.1 , L22214.1 , AY136746.1 and BC026340.1 . In representative examples of this type, the polynucleotide comprises an A1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000738] In some embodiments, an A1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian A1 R polynucleotide, or a fragment thereof. In some embodiments, the A1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian A1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.7 Adrenergic a 1A receptor (α1A -AR) polypeptides

[000739] The α1A-AR is involved in cardiovascular system regulation, and in particular appears to mediate increases in blood pressure and cardiac contractility (Chen & Minneman, 2005).

[000740] To date, a crystal structure of the α1A-AR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of α1A-AR consists of the N-terminal segment (Met1 -Asn22 of the human α1A-AR), ECL1 (Gly90- Phe94 of the human α1A-AR) linking helices II and III, ECL2 (Trp165-Glu180 of the human α1A-AR) linking helices IV and V, and ECL3 (Pro299-Pro303 of the human α1A-AR) linking helices VI to VII. Like most GPCR's, the α1A-AR likely contains a disulphide bond connecting ECL1 and ECL2 (Finch et al, 2006). [000741 ] In specific embodiments of the present invention, the α1A-AR polypeptide comprises a α1A-AR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an α1A-AR protein sequence.

[000742] In some embodiments, the α1A-AR protein sequence corresponds to a mammalian α1A-AR protein sequence. Suitable α1A-AR sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3WRU5), chimpanzee (UniProtKB Accession No. H2R0T2), cow (UniProtKB Accession No. F1 MYI6), dog (UniProtKB Accession No. A0A0A0MPB9), gerbil (UniProtKB Accession No. E0AE55), gorilla (UniProtKB Accession No. G3QE17), guinea pig (UniProtKB Accession No. Q9WU25), horse (UniProtKB Accession No. F6UTQ3), human (UniProtKB Accession No. E7EW16), mouse (UniProtKB Accession No. Q8BUE5), rabbit (UniProtKB Accession No. G1 TM07), rat (UniProtKB Accession No. P70648), rhesus (UniProtKB Accession No. F7DLK7), sheep (UniProtKB Accession No. W5PJ34).

[000743] In some preferred embodiments, the α1A-AR protein sequence corresponds to a human α1A-AR protein sequence. In some embodiments, the α1A-AR polypeptide comprises a human full-length wild-type α1A-AR protein sequence (UniProtKB Accession No. E7EW16), as set forth below, or a functional fragment of the wild-type α1A-AR protein sequence.

[000744] MVFLSGNASDSSNCTQPPAPVNISKAILLGVILGGLILFGVLGNILVILSVACHRH LHSV THYYIVNLAVADLLLTSTVLPFSAIFEVLGYWAFGRVFCNIWAAVDVLCCTASIMGLCII SIDRYIGVSYPLRYPTIVTQRRGLMALLCVWALSLVISIGPLFGWRQPAPEDETICQINE EPGYVLFSALGSFYLPLAIILVMYCRVYVVAKRESRGLKSGLKTDKSDSEQVTLRIHRKN APAGGSGMASAKTKTHFSVRLLKFSREKKAAKTLGIVVGCFVLCWLPFFLVMPIGSFFPD FKPSETVFKIVFWLGYLNSCINPIIYPCSSQEFKKAFQNVLRIQCLCRKQSSKHALGYTL HPPSQAVEGQHKDMVRIPVGSRETFYRISKTDGVCEWKFFSSMPRGSARITVSKDQSSCT TARSSGF [SEQ ID NO: 18].

[000745] In one form of the invention, the α1A-AR polypeptide comprises a truncated form of a mammalian wild-type α1A-AR protein sequence. For example, the α1A-AR polypeptide sequence may comprise the human wild-type α1A-AR protein sequence with a C-terminal truncation (e.g., amino acid residues 342-413 may be truncated). Alternatively or in addition, the α1A-AR polypeptide sequence may comprise the wild-type α1A-AR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type α1A-AR protein sequence (e.g., amino acid residues 174-185 may be truncated). By way of a non-limiting illustrative example, a α1A-AR polypeptide suitable for using with the present invention comprised amino acid residues 2-173 and 186-341 of the human wild-type α1A-AR protein sequence as set forth in SEQ ID NO: 18.

3.8 Constructs and nucleotide sequences encoding ct1A-AR polypeptides.

[000746] The present invention also encompasses isolated polynucleotide sequences and constructs encoding α1A-AR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000747] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human α1A-AR nucleotide (i.e., corresponding to the ADRA1 A gene) sequence as set forth for example in GenBank Accession Nos. AY389505.1 , NG_029395.1 , NM_001322504.1 , NM_001322502.1 , NM_033304.3, NM_033303.4, NM_033302.3, NR_136343.1 , NM_000680.3, NM_001322503.1 , AY491781 .1 , AY491780.1 , AY491779.1 , AY491778.1 , AY491777.1 and AY491776.1 . In representative examples of this type, the polynucleotide comprises an α1A-AR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000748] In some embodiments, an α1A-AR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian α1A-AR polynucleotide, or a fragment thereof. In some embodiments, the α1A-AR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian α1A-AR protein, or a fragment thereof under low, medium or high stringency conditions.

3.9 Adrenergic α 1B receptor ( α 1B-AR) polypeptides

[000749] The α1 B-AR is involved in cardiovascular system regulation, locomotor activity and glucose metabolism (Chen & Minneman, 2005).

[000750] To date, a crystal structure of the α1 B-AR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of α1 B-AR consists of the N-terminal segment (Met1 -Thr36 of the human α1 B-AR), ECL1 (Gly109- Leu1 13 of the human α1 B-AR) linking helices II and III, ECL2 (Lys185-Glu199 of the human α1 B-AR) linking helices IV and V, and ECL3 (Ser321 -Pro325 of the human α1 B-AR) linking helices VI to VII. Like most GPCR's, the α1 B-AR likely contains a disulphide bond connecting ECL1 and ECL2 (Finch et al 2.,006).

[000751 ] In specific embodiments of the present invention, the α1 B-AR polypeptide comprises a α1 B-AR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an α1 B-AR protein sequence.

[000752] In some embodiments, the α1 B-AR protein sequence corresponds to a mammalian α1 B-AR protein sequence. Suitable α1 B-AR sequences may suitably be from mammal selected from the group comprising chimpanzee (UniProtKB Accession No. H2QRX4), cow (UniProtKB Accession No. F1 MGA6), dog (UniProtKB Accession No. F1 PK71 ), gorilla (UniProtKB Accession No. G3S977), guinea pig (UniProtKB Accession No. H0WBQ9), hamster (UniProtKB Accession No. P18841 ), horse (UniProtKB Accession No. F7ATM9), human (UniProtKB Accession No. P35368), marmoset (UniProtKB Accession No. U3DR86), mouse (UniProtKB Accession No. P97717), pig (UniProtKB Accession No. F1 RR36), rabbit (UniProtKB Accession No. U3KM59), rat (UniProtKB Accession No. P15823), Tasmanian devil (UniProtKB Accession No. G3VWX9).

[000753] In some preferred embodiments, the α1 B-AR protein sequence corresponds to a human α1 B-AR protein sequence. In some embodiments, the α1 B-AR polypeptide comprises a human full-length wild-type α1 B-AR protein sequence (UniProtKB Accession No. P35368), as set forth below, or a functional fragment of the wild-type α1 B-AR protein sequence.

[000754] MNPDLDTGHNTSAPAHWGELKNANFTGPNQTSSNSTLPQLDITRAISVGLVLG

AFILFAIVGNILVILSVACNRHLRTPTNYFIVNLAMADLLLSFTVLPFSAALEVLGY WVLGRIFC

DIWAAVDVLCCTASILSLCAISIDRYIGVRYSLQYPTLVTRRKAILALLSVWVLSTV ISIGPLLG

WKEPAPNDDKECGVTEEPFYALFSSLGSFYIPLAVILVMYCRVYIVAKRTTKNLEAG VMKEM

SNSKELTLRIHSKNFHEDTLSSTKAKGHNPRSSIAVKLFKFSREKKAAKTLGIVVGM FILCWL

PFFIALPLGSLFSTLKPPDAVFKVVFWLGYFNSCLNPIIYPCSSKEFKRAFVRILGC QCRGRG

RRRRRRRRRLGGCAYTYRPWTRGGSLERSQSRKDSLDDSGSCLSGSQRTLPSASPSP GY

LGRGAPPPVELCAFPEWKAPGALLSLPAPEPPGRRGRHDSGPLFTFKLLTEPESPGT DGG

ASNGGCEAAADVANGQPGFKSNMPLAPGQF [SEQ ID NO: 19].

[000755] In one form of the invention, the α1 B-AR polypeptide comprises a truncated form of a mammalian wild-type α1 B-AR protein sequence. For example, the α1 B-AR polypeptide sequence may comprise the human wild-type α1 B-AR protein sequence with a C-terminal truncation (e.g., amino acid residues 365-520 may be truncated). Alternatively or in addition, the α1 B-AR polypeptide sequence may comprise the wild-type α1 B-AR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type α1 B-AR protein sequence (e.g., amino acid residues 186-196 may be truncated). By way of a non-limiting illustrative example, a α1 B-AR polypeptide suitable for using with the present invention comprised amino acid residues 2-185 and 197-364 of the human wild-type α1 B-AR protein sequence as set forth in SEQ ID NO: 19.

3. 10 Constructs and nucleotide sequences encoding α 1B-AR polypeptides.

[000756] The present invention also encompasses isolated polynucleotide sequences and constructs encoding α1 B-AR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000757] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human α1 B-AR nucleotide (i.e., corresponding to the ADRA1 B gene) sequence as set forth for example in GenBank Accession Nos. NM 000679.3, AY530191 .1 , EU332831 .1 , BC136569.1 and BC136568.1 . In representative examples of this type, the polynucleotide comprises an α1 B-AR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000758] In some embodiments, an α1 B-AR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian α1 B-AR polynucleotide, or a fragment thereof. In some embodiments, the α1 B-AR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian α1 B-AR protein, or a fragment thereof under low, medium or high stringency conditions.

3. 1 1 Adrenergic a2B receptor (a2B-AR) polypeptides

[000759] The a2B-AR is a poorly characterised GPCR, however it appears to be involved in blood pressure regulation as well as having a role in developmental processes (Kable et al., 2000). [000760] To date, a crystal structure of the a2B-AR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of a2B-AR consists of the N-terminal segment (Met1 -Ser8 of the human a2B-AR), ECL1 (Gly76- Phe80 of the human a2B-AR) linking helices II and III, ECL2 (Lys151 -Gln168 of the human a2B-AR) linking helices IV and V, and ECL3 (Pro395-Val400 of the human a2B-AR) linking helices VI to VII.

[000761 ] In specific embodiments of the present invention, the a2B-AR polypeptide comprises a a2B-AR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an a2B-AR protein sequence.

[000762] In some embodiments, the a2B-AR protein sequence corresponds to a mammalian a2B-AR protein sequence. Suitable a2B-AR sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3X1 F7), chimpanzee (UniProtKB Accession No. H2RHZ6), cow (UniProtKB Accession No. G3X6S2), dog (UniProtKB Accession No. F1 Q1 Q4), gorilla (UniProtKB Accession No. G3RMW6), guinea pig (UniProtKB Accession No. Q60475), horse (UniProtKB Accession No. F7BW54), human (UniProtKB Accession No. P18089), marmoset (UniProtKB Accession No. F7IPR3), mouse (UniProtKB Accession No. P30545), pig (UniProtKB Accession No. Q38PT0), rabbit (UniProtKB Accession No. G1 TQL5), rat (UniProtKB Accession No. P19328), Tasmanian devil (UniProtKB Accession No. G3VWZ4).

[000763] In some preferred embodiments, the a2B-AR protein sequence corresponds to a human a2B-AR protein sequence. In some embodiments, the a2B-AR polypeptide comprises a human full-length wild-type a2B-AR protein sequence (UniProtKB Accession No. P18089), as set forth below, or a functional fragment of the wild-type a2B-AR protein sequence.

[000764] MDHQDPYSVQATAAIAAAITFLILFTIFGNALVILAVLTSRSLRAPQNLFLVSLAAA

DILVATLIIPFSLANELLGYWYFRRTWCEVYLALDVLFCTSSIVHLCAISLDRYWAV SRALEYN

SKRTPRRIKCIILTVWLIAAVISLPPLIYKGDQGPQPRGRPQCKLNQEAWYILASSI GSFFAPC

LIMILVYLRIYLIAKRSNRRGPRAKGGPGQGESKQPRPDHGGALASAKLPALASVAS AREVN

GHSKSTGEKEEGETPEDTGTRALPPSWAALPNSGQGQKEGVCGASPEDEAEEEEEEE EE

EEECEPQAVPVSPASACSPPLQQPQGSRVLATLRGQVLLGRGVGAIGGQWWRRRAQL TR

EKRFTFVLAVVIGVFVLCWFPFFFSYSLGAICPKHCKVPHGLFQFFFWIGYCNSSLN PVIYTIF

NQDFRRAFRRILCRPWTQTAW [SEQ ID NO: 20]. [000765] In one form of the invention, the a2B-AR polypeptide comprises a truncated form of a mammalian wild-type a2B-AR protein sequence. For example, the a2B-AR polypeptide sequence may comprise the human wild-type a2B-AR protein sequence with a C-terminal truncation (e.g., amino acid residues 440-447 may be truncated). Alternatively or in addition, the a2B-AR polypeptide sequence may comprise the wild-type a2B-AR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type a2B-AR protein sequence (e.g., amino acid residues 153-161 may be truncated). By way of a non-limiting illustrative example, a a2B-AR polypeptide suitable for using with the present invention comprised amino acid residues 2-152 and 162-439 of the human wild-type a2B-AR protein sequence as set forth in SEQ ID NO: 20.

3. 12 Constructs and nucleotide sequences encoding a2B-AR polypeptides.

[000766] The present invention also encompasses isolated polynucleotide sequences and constructs encoding a2B-AR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000767] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human a2B-AR nucleotide (i.e., corresponding to the ADRA2B gene) sequence as set forth for example in GenBank Accession Nos. NM 000682.6, HF583494.1 , EU332847.1 , NG_032950.1 , AF316895.1 , DQ057076.1 , BC136537.1 , BC133021 .1 and AY548167.1 . In representative examples of this type, the polynucleotide comprises an a2B- AR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000768] In some embodiments, an a2B-AR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian a2B-AR polynucleotide, or a fragment thereof. In some embodiments, the a2B-AR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian a2B-AR protein, or a fragment thereof under low, medium or high stringency conditions.

3. 13 Brady kinin receptor B2 (B2R) polypeptides [000769] The B2R is most commonly associated with having hypotensive actions through its mediation of vasodilation and water and salt loss, however it also involved in inflammation, proliferation, apoptosis and angiogensis (Blaes & Girolami, 2013).

[000770] To date, a crystal structure of the B2R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of B2R consists of the N-terminal segment (Met1 -Gly54 of the human B2R), ECL1 (Asn120- Phe125 of the human B2R) linking helices II and III, ECL2 (Thr197-lle219 of the human B2R) linking helices IV and V, and ECL3 (Ile300 -Ser302 of the human B2R) linking helices VI to VII. Like most GPCRs, the B2R likely contains a disulphide bond connecting ECL1 and ECL2 (Leeb- Lundberg et al., 2005).

[000771 ] In specific embodiments of the present invention, the B2R polypeptide comprises a B2R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a B2R protein sequence.

[000772] In some embodiments, the B2R protein sequence corresponds to a mammalian B2R protein sequence. Suitable B2R sequences may suitably be from mammal selected from the group comprising bat (UniProtKB Accession No. G1 Q158), cat (UniProtKB Accession No. M3WR05), chimpanzee (UniProtKB Accession No. H2Q8W1 ), cow (UniProtKB Accession No. F1 MWK0), dog (UniProtKB Accession No. Q9BDQ4), guinea pig (UniProtKB Accession No. 070526), human (UniProtKB Accession No. P3041 1 ), marmoset (UniProtKB Accession No. U3CVS9), mouse (UniProtKB Accession No. P32299), panda (UniProtKB Accession No. G1 LAZ3), pig (UniProtKB Accession No. Q9GLX8), rabbit (UniProtKB Accession No. Q28642), rat (UniProtKB Accession No. P25023), sheep (UniProtKB Accession No. W5Q382).

[000773] In some preferred embodiments, the B2R protein sequence corresponds to a human B2R protein sequence. In some embodiments, the B2R polypeptide comprises a human full-length wild-type B2R protein sequence (UniProtKB Accession No. P3041 1 ), as set forth below, or a functional fragment of the wild-type B2R protein sequence.

[000774] MFSPWKISMFLSVREDSVPTTASFSADMLNVTLQGPTLNGTFAQSKCPQVEWL GWLNTIQPPFLWVLFVLATLENIFVLSVFCLHKSSCTVAEIYLGNLAAADLILACGLPFW AITIS NNFDWLFGETLCRVVNAIISMNLYSSICFLMLVSIDRYLALVKTMSMGRMRGVRWAKLYS LV IWGCTLLLSSPMLVFRTMKEYSDEGHNVTACVISYPSLIWEVFTNMLLNVVGFLLPLSVI TFC TMQIMQVLRNNEMQKFKEIQTERRATVLVLVVLLLFIICWLPFQISTFLDTLHRLGILSS CQDE RIIDVITQIASFMAYSNSCLNPLVYVIVGKRFRKKSWEVYQGVCQKGGCRSEPIQMENSM GT LRTSISVERQIHKLQDWAGSRQ [SEQ ID NO: 21 ].

[000775] In one form of the invention, the B2R polypeptide comprises a truncated form of a mammalian wild-type B2R protein sequence. For example, the B2R polypeptide sequence may comprise the human wild-type B2R protein sequence with a C-terminal truncation (e.g., amino acid residues 351 -391 may be truncated). Alternatively or in addition, the B2R polypeptide sequence may comprise the wild-type B2R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type B2R protein sequence (e.g., amino acid residues 199-208 may be truncated). By way of a non-limiting illustrative example, a B2R polypeptide suitable for using with the present invention comprised amino acid residues 2-198 and 209-350 of the human wild-type B2R protein sequence as set forth in SEQ ID NO: 21 .

3. 14 Constructs and nucleotide sequences encoding B2R polypeptides.

[000776] The present invention also encompasses isolated polynucleotide sequences and constructs encoding B2R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000777] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human B2R nucleotide (i.e., corresponding to the BDKRB2 gene) sequence as set forth for example in GenBank Accession Nos. HF583430.1 , AY275465.1 , NM_000623.3, KJ950628.1 , AF378542.2, BC074894.2 and BC074895.2. In representative examples of this type, the polynucleotide comprises a B2R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000778] In some embodiments, a B2R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian B2R polynucleotide, or a fragment thereof. In some embodiments, the B2R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian B2R protein, or a fragment thereof under low, medium or high stringency conditions.

3. 15 CC chemokine receptor CCR1 (CCR1) polypeptides [000779] Like all chemokine receptors, the CCR1 is involved in the mediation of inflammatory processes. It is found in T cells, monocytes, eosinophils and basophils and is associated with Rheumatoid arthritis and multiple sclerosis (Charo & Ransohoff, 2006).

[000780] To date, a crystal structure of the CCR1 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CCR1 consists of the N-terminal segment (Met1 -Asn28 of the human CCR1 ), ECL1 (Asp 97- Phe101 of the human CCR1 ) linking helices II and III, ECL2 (Lys173-Ser191 of the human CCR1 ) linking helices IV and V, and ECL3 (Thr270 -His271 of the human CCR1 ) linking helices VI to VII. Like all chemokine receptors the N-terminus appears to be crucial for ligand binding and receptor activation (Kufareva et al., 2015).

[000781 ] In specific embodiments of the present invention, the CCR1 polypeptide comprises a CCR1 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR1 protein sequence.

[000782] In some embodiments, the CCR1 protein sequence corresponds to a mammalian CCR1 protein sequence. Suitable CCR1 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. B6DXF4), chimpanzee (UniProtKB Accession No. H2QMG4), cow (UniProtKB Accession No. A0JN72), dog (UniProtKB Accession No. Q2V0Q7), gorilla (UniProtKB Accession No. G3RL29), horse (UniProtKB Accession No. F6U0D1 ), human (UniProtKB Accession No. P32246), marmoset (UniProtKB Accession No. Q9MYJ8), mouse (UniProtKB Accession No. P51675), pig (UniProtKB Accession No. Q6YST0), rabbit (UniProtKB Accession No. Q9MYJ9), rat (UniProtKB Accession No. Q9JLY8), rhesus (UniProtKB Accession No. F6QJR2), sheep (UniProtKB Accession No. W5PXX5).

[000783] In some preferred embodiments, the CCR1 protein sequence corresponds to a human CCR1 protein sequence. In some embodiments, the CCR1 polypeptide comprises a human full-length wild-type CCR1 protein sequence (UniProtKB Accession No. P32246), as set forth below, or a functional fragment of the wild-type CCR1 protein sequence.

[000784] METPNTTEDYDTTTEFDYGDATPCQKVNERAFGAQLLPPLYSLVFVIGLVGNIL VVLVLVQYKRLKNMTSIYLLNLAISDLLFLFTLPFWIDYKLKDDWVFGDAMCKILSGFYY TGL YSEIFFIILLTIDRYLAIVHAVFALRARTVTFGVITSIIIWALAILASMPGLYFSKTQWE FTHHTCS LHFPHESLREWKLFQALKLNLFGLVLPLLVMIICYTGIIKILLRRPNEKKSKAVRLIFVI MIIFFLF WTPYNLTILISVFQDFLFTHECEQSRHLDLAVQVTEVIAYTHCCVNPVIYAFVGERFRKY LRQ LFHRRVAVHLVKWLPFLSVDRLERVSSTSPSTGEHELSAGF [SEQ ID NO: 22].

[000785] In one form of the invention, the CCR1 polypeptide comprises a truncated form of a mammalian wild-type CCR1 protein sequence. For example, the CCR1 polypeptide sequence may comprise the human wild-type CCR1 protein sequence with a C-terminal truncation (e.g., amino acid residues 318-355 may be truncated). Alternatively or in addition, the CCR1 polypeptide sequence may comprise the wild-type CCR1 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR1 protein sequence (e.g., amino acid residues 174-182 may be truncated). By way of a non-limiting illustrative example, a CCR1 polypeptide suitable for using with the present invention comprised amino acid residues 2-173 and 183-317 of the human wild-type CCR1 protein sequence as set forth in SEQ ID NO: 22.

3. 16 Constructs and nucleotide sequences encoding CCR1 polypeptides.

[000786] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR1 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000787] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR1 nucleotide (i.e., corresponding to the CCR1 gene) sequence as set forth for example in GenBank Accession Nos. NM_001295.2, AF051305.1 , BC064991 .1 and BC051306.1 . In representative examples of this type, the polynucleotide comprises a CCR1 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000788] In some embodiments, a CCR1 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR1 polynucleotide, or a fragment thereof. In some embodiments, the CCR1 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR1 protein, or a fragment thereof under low, medium or high stringency conditions.

3. 17 CC chemokine receptor CCR2 (CCR2) polypeptides [000789] Like all chemokine receptors, the CCR2 is involved in the mediation of inflammatory processes. It is found in monocytes, dendritic cells and memory T cells and is associated with atherosclerosis, rheumatoid arthritis, multiple sclerosis, resistance to intracellular pathogens and type 2 diabetes mellitus (Charo & Ransohoff, 2006).

[000790] The crystal structure of the CCR2 has recently been published (Zheng et al., 2016), giving insight to its ligand-binding mode and activation process. The extracellular part of CCR2 consists of the N-terminal segment (Met1 -D36 of the human CCR2), ECL1 (Glu105- Phe108 of the human CCR2) linking helices II and III, ECL2 (Lys180-Phe194 of the human CCR2) linking helices IV and V, and ECL3 (Leu274-Ser275 of the human CCR2) linking helices VI to VII. Like all chemokine receptors, the CCR2 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016).

[000791 ] In specific embodiments of the present invention, the CCR2 polypeptide comprises a CCR2 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR2 protein sequence.

[000792] In some embodiments, the CCR2 protein sequence corresponds to a mammalian CCR2 protein sequence. Suitable CCR2 sequences may suitably be from mammal selected from the group comprising chimpanzee (UniProtKB Accession No. H2QMG5), cow (UniProtKB Accession No. D9ZDD9), gorilla (UniProtKB Accession No. G3S6T3), guinea pig (UniProtKB Accession No. H0VII5), horse (UniProtKB Accession No. B3SPX1 ), human (UniProtKB Accession No. P41597), marmoset (UniProtKB Accession No. F7DT70), mouse (UniProtKB Accession No. P51683), panda (UniProtKB Accession No. G1 MMF1 ), pig (UniProtKB Accession No. Q6YT42), rabbit (UniProtKB Accession No. G1 SK57), rat (UniProtKB Accession No. 055193), rhesus (UniProtKB Accession No. 018793), sheep (UniProtKB Accession No. W5PXU4).

[000793] In some preferred embodiments, the CCR2 protein sequence corresponds to a human CCR2 protein sequence. In some embodiments, the CCR2 polypeptide comprises a human full-length wild-type CCR2 protein sequence (UniProtKB Accession No. P41597), as set forth below, or a functional fragment of the wild-type CCR2 protein sequence.

[000794] MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIF GFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKL FTGL YHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFT KCQKED SVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVI FTIM IVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVG EKFR SLFHIALGCRIAPLQKPVCGGPGVRPGKNVKVTTQGLLDGRGKGKSIGRAPEASLQDKEG A

[SEQ ID NO: 23].

[000795] In one form of the invention, the CCR2 polypeptide comprises a truncated form of a mammalian wild-type CCR2 protein sequence. For example, the CCR2 polypeptide sequence may comprise the human wild-type CCR2 protein sequence with a C-terminal truncation (e.g., amino acid residues 322-374 may be truncated). Alternatively or in addition, the CCR2 polypeptide sequence may comprise the wild-type CCR2 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR2 protein sequence (e.g., amino acid residues 182-199 may be truncated). By way of a non-limiting illustrative example, a CCR2 polypeptide suitable for using with the present invention comprised amino acid residues 2-181 and 200-321 of the human wild-type CCR2 protein sequence as set forth in SEQ ID NO: 23.

3. 18 Constructs and nucleotide sequences encoding CCR2 polypeptides.

[000796] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR2 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000797] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR2 nucleotide (i.e., corresponding to the CCR2 gene) sequence as set forth for example in GenBank Accession Nos. NM_001 123396.1 , NM_001 123041 .2, NG_021428.1 , KC248079.1 , KC248078.1 , KC248077.1 , KC248076.1 , KC248075.1 , KC248074.1 , KC248073.1 , KC248072.1 , KC248071 .1 , KC248070.1 , AF545480.1 , BC126452.1 and BC095540.1 . In representative examples of this type, the polynucleotide comprises a CCR2 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000798] In some embodiments, a CCR2 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR2 polynucleotide, or a fragment thereof. In some embodiments, the CCR2 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR2 protein, or a fragment thereof under low, medium or high stringency conditions.

3. 19 CC chemokine receptor CCR3 (CCR3) polypeptides

[000799] Like all chemokine receptors, the CCR3 is involved in the mediation of inflammatory processes. It is found in eosinophils, basophils, mast cells, Th2 cells and platelets and is associated with allergic asthma and rhinitis (Charo & Ransohoff, 2006).

[000800] To date, a crystal structure of the CCR3 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CCR3 consists of the N-terminal segment (Met1 -Asp28 of the human CCR3), ECL1 (His97-Phe101 of the human CCR3) linking helices II and III, ECL2 (Glu173-Thr191 of the human CCR3) linking helices IV and V, and ECL3 (Gly270-Asn271 of the human CCR3) linking helices VI to VII. Like all chemokine receptors, the CCR3 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016).

[000801 ] In specific embodiments of the present invention, the CCR3 polypeptide comprises a CCR3 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR3 protein sequence.

[000802] In some embodiments, the CCR3 protein sequence corresponds to a mammalian CCR3 protein sequence. Suitable CCR3 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3WZ27), chimpanzee (UniProtKB Accession No. H2R0Z4), cow (UniProtKB Accession No. F1 MP16), dog (UniProtKB Accession No. Q64H34), guinea pig (UniProtKB Accession No. Q9Z2I3), horse (UniProtKB Accession No. F6UH12), human (UniProtKB Accession No. P51677), marmoset (UniProtKB Accession No. F7DTA4), mouse (UniProtKB Accession No. Q8BHB8), panda (UniProtKB Accession No. G1 MMC8), pig (UniProtKB Accession No. Q75ZH4), rabbit (UniProtKB Accession No. B5SU39), rat (UniProtKB Accession No. 054814), sheep (UniProtKB Accession No. W5PXW1 ).

[000803] In some preferred embodiments, the CCR3 protein sequence corresponds to a human CCR3 protein sequence. In some embodiments, the CCR3 polypeptide comprises a human full-length wild-type CCR3 protein sequence (UniProtKB Accession No. P51677), as set forth below, or a functional fragment of the wild-type CCR3 protein sequence. [000804] MTTSLDTVETFGTTSYYDDVGLLCEKADTRALMAQFVPPLYSLVFTVGLLGNVV

VVMILIKYRRLRIMTNIYLLNLAISDLLFLVTLPFWIHYVRGHNWVFGHGMCKLLSG FYHTGLY

SEIFFIILLTIDRYLAIVHAVFALRARTVTFGVITSIVTWGLAVLAALPEFIFYETE ELFEETLCSA

LYPEDTVYSWRHFHTLRMTIFCLVLPLLVMAICYTGIIKTLLRCPSKKKYKAIRLIF VIMAVFFIF

WTPYNVAILLSSYQSILFGNDCERSKHLDLVMLVTEVIAYSHCCMNPVIYAFVGERF RKYLR

HFFHRHLLMHLGRYIPFLPSEKLERTSSVSPSTAEPELSIVF [SEQ ID NO: 24].

[000805] In one form of the invention, the CCR3 polypeptide comprises a truncated form of a mammalian wild-type CCR3 protein sequence. For example, the CCR3 polypeptide sequence may comprise the human wild-type CCR3 protein sequence with a C-terminal truncation (e.g., amino acid residues 318-355 may be truncated). Alternatively or in addition, the CCR3 polypeptide sequence may comprise the wild-type CCR3 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR3 protein sequence (e.g., amino acid residues 175-187 may be truncated). By way of a non-limiting illustrative example, a CCR3 polypeptide suitable for using with the present invention comprised amino acid residues 2-174 and 188-317 of the human wild-type CCR3 protein sequence as set forth in SEQ ID NO: 24.

3.20 Constructs and nucleotide sequences encoding CCR3 polypeptides.

[000806] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR3 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000807] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR3 nucleotide (i.e., corresponding to the CCR3 gene) sequence as set forth for example in GenBank Accession Nos. NM_178329.2, NM_001837.3, NM_001 164680.1 , NM_178328.1 , AH009867.2, AF247361 .1 , AF262303.1 , AF262302.1 , AF262301 .1 , AF262300.1 , AF262299.1 , AH010691 .2, AF224495.1 , AF247360.1 , AF247359.1 and AF026535.1 ln representative examples of this type, the polynucleotide comprises a CCR3 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000808] In some embodiments, a CCR3 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR3 polynucleotide, or a fragment thereof. In some embodiments, the CCR3 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR3 protein, or a fragment thereof under low, medium or high stringency conditions.

3.21 CC chemokine receptor CCR4 (CCR4) polypeptides

[000809] Like all chemokine receptors, the CCR4 is involved in the mediation of inflammatory processes. It is found in T cells (Th2), dendritic cells (mature), basophils, macrophages and platelets and is associated with parasitic infection, graft rejection and T- cell homing to skin (Charo & Ransohoff, 2006).

[000810] To date, a crystal structure of the CCR4 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CCR4 consists of the N-terminal segment (Met1 -Gly33 of the human CCR4), ECL1 (Gln102- Phe105 of the human CCR4) linking helices II and III, ECL2 (Thr177-Asn194 of the human CCR4) linking helices IV and V, and ECL3 (Leu273-Gln274 of the human CCR4) linking helices VI to VII. Like all chemokine receptors, the CCR4 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016).

[00081 1 ] In specific embodiments of the present invention, the CCR4 polypeptide comprises a CCR4 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR4 protein sequence.

[000812] In some embodiments, the CCR4 protein sequence corresponds to a mammalian CCR4 protein sequence. Suitable CCR4 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3VZE0), chimpanzee (UniProtKB Accession No. H2QM91 ), cow (UniProtKB Accession No. A6QLA5), dog (UniProtKB Accession No. Q8MJW8), gorilla (UniProtKB Accession No. G3RC17), guinea pig (UniProtKB Accession No. H0V3A3), horse (UniProtKB Accession No. F6RJT2), human (UniProtKB Accession No. P51679), marmoset (UniProtKB Accession No. F7F2B0), mouse (UniProtKB Accession No. P51680), pig (UniProtKB Accession No. I3L552), rabbit (UniProtKB Accession No. G1 TXV0), rat (UniProtKB Accession No. Q91 ZH4), sheep (UniProtKB Accession No. W5Q1 A7).

[000813] In some preferred embodiments, the CCR4 protein sequence corresponds to a human CCR4 protein sequence. In some embodiments, the CCR4 polypeptide comprises a human full-length wild-type CCR4 protein sequence (UniProtKB Accession No. P51679), as set forth below, or a functional fragment of the wild-type CCR4 protein sequence.

[000814] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSLVFVFGLLG

NSVVVLVLFKYKRLRSMTDVYLLNLAISDLLFVFSLPFWGYYAADQWVFGLGLCKMI SWMY

LVGFYSGIFFVMLMSIDRYLAIVHAVFSLRARTLTYGVITSLATWSVAVFASLPGFL FSTCYTE

RNHTYCKTKYSLNSTTWKVLSSLEINILGLVIPLGIMLFCYSMIIRTLQHCKNEKKN KAVKMIF

AVVVLFLGFWTPYNIVLFLETLVELEVLQDCTFERYLDYAIQATETLAFVHCCLNPI IYFFLGE

KFRKYILQLFKTCRGLFVLCQYCGLLQIYSADTPSSSYTQSTMDHDLHDAL [SEQ ID NO:

25].

[000815] In one form of the invention, the CCR4 polypeptide comprises a truncated form of a mammalian wild-type CCR4 protein sequence. For example, the CCR4 polypeptide sequence may comprise the human wild-type CCR4 protein sequence with a C-terminal truncation (e.g., amino acid residues 321 -360 may be truncated). Alternatively or in addition, the CCR4 polypeptide sequence may comprise the wild-type CCR4 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR4 protein sequence (e.g., amino acid residues 179-189 may be truncated). By way of a non-limiting illustrative example, a CCR4 polypeptide suitable for using with the present invention comprised amino acid residues 2-178 and 190-320 of the human wild-type CCR4 protein sequence as set forth in SEQ ID NO: 25.

3.22 Constructs and nucleotide sequences encoding CCR4 polypeptides.

[000816] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR4 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000817] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR4 nucleotide (i.e., corresponding to the CCR4 gene) sequence as set forth for example in GenBank Accession Nos. AY322539.1 , NC_000005.10, NM_005508.4, EF064759.1 , BC074935.2, BC071751 .1 and BC069139.1 .In representative examples of this type, the polynucleotide comprises a CCR4 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences. [000818] In some embodiments, a CCR4 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR4 polynucleotide, or a fragment thereof. In some embodiments, the CCR4 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR4 protein, or a fragment thereof under low, medium or high stringency conditions.

3.23 CC chemokine receptor CCR5 (CCR5) polypeptides

[000819] Like all chemokine receptors, the CCR5 is involved in the mediation of inflammatory processes. It is found in T cells and monocytes and is associated with HIV-1 infection and transplant rejection (Charo & Ransohoff, 2006).

[000820] The crystal structure of the CCR5 has been solved (Tan et a/., 2013), giving insight to its ligand-binding mode and activation process. The extracellular part of CCR5 consists of the N-terminal segment (Met1 -Asn24 of the human CCR5), ECL1 (Gln93- Phe96 of the human CCR5) linking helices II and III, ECL2 (Arg168-Gln186 of the human CCR5) linking helices IV and V, and ECL3 (Leu266-Asn267 of the human CCR5) linking helices VI to VII. As expected for all chemokine receptors, the CCR5 has two conserved disulfide bonds in its ectodomains (Tan et al., 2013).

[000821 ] In specific embodiments of the present invention, the CCR5 polypeptide comprises a CCR5 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR5 protein sequence.

[000822] In some embodiments, the CCR5 protein sequence corresponds to a mammalian CCR5 protein sequence. Suitable CCR5 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. A4ZY83), chimpanzee (UniProtKB Accession No. P56440), cow (UniProtKB Accession No. Q2HJ17), dog (UniProtKB Accession No. Q5ECR9), goat (UniProtKB Accession No. F5B6L8), gorilla (UniProtKB Accession No. P56439), horse (UniProtKB Accession No. A4ZZ54), human (UniProtKB Accession No. P51681 ), marmoset (UniProtKB Accession No. Q6WN98), mouse (UniProtKB Accession No. P51682), pig (UniProtKB Accession No. Q6YT41 ), rabbit (UniProtKB Accession No. Q1 ZY22), rat (UniProtKB Accession No. 008556), sheep (UniProtKB Accession No. B7T903). [000823] In some preferred embodiments, the CCR5 protein sequence corresponds to a human CCR5 protein sequence. In some embodiments, the CCR5 polypeptide comprises a human full-length wild-type CCR5 protein sequence (UniProtKB Accession No. P51681 ), as set forth below, or a functional fragment of the wild-type CCR5 protein sequence.

[000824] MDYQVSSPIYDINYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNMLVILILIN

CKRLKSMTDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFGNTMCQLLTGLYFIGFFS GIFFIILL

TIDRYLAVVHAVFALKARTVTFGVVTSVITWVVAVFASLPGIIFTRSQKEGLHYTCS SHFPYS

QYQFWKNFQTLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMI VYFLFWAP

YNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCINPIIYAFVGEKFRNY LLVFFQ

KHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL [SEQ ID NO: 26].

[000825] In one form of the invention, the CCR5 polypeptide comprises a truncated form of a mammalian wild-type CCR5 protein sequence. For example, the CCR5 polypeptide sequence may comprise the human wild-type CCR5 protein sequence with a C-terminal truncation (e.g., amino acid residues 314-352 may be truncated). Alternatively or in addition, the CCR5 polypeptide sequence may comprise the wild-type CCR5 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR5 protein sequence (e.g., amino acid residues 171 -181 may be truncated). By way of a non-limiting illustrative example, a CCR5 polypeptide suitable for using with the present invention comprised amino acid residues 2-170 and 182-313 of the human wild-type CCR5 protein sequence as set forth in SEQ ID NO: 26.

3.24 Constructs and nucleotide sequences encoding CCR5 polypeptides.

[000826] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR5 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000827] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR5 nucleotide (i.e., corresponding to the CCR5 gene) sequence as set forth for example in GenBank Accession Nos. AY463215.1 , GQ121035.1 , AH005786.2, NM_000579.3, NM_001 100168.1 , AF056019.1 , AB182990.1 , AB182984.1 , AB182986.1 , AF01 1537.1 , AF01 1532.1 , AF01 1525.1 , AF01 1521 .1 , JQ291232.1 , EF202089.1 , AF052539.1 . In representative examples of this type, the polynucleotide comprises a CCR5 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000828] In some embodiments, a CCR5 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR5 polynucleotide, or a fragment thereof. In some embodiments, the CCR5 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR5 protein, or a fragment thereof under low, medium or high stringency conditions.

3.25 CC chemokine receptor CCR6 (CCR6) polypeptides

[000829] Like all chemokine receptors, the CCR6 is involved in the mediation of inflammatory processes. It is found in T cells (T regulatory and memory), B cells and dendritic cells and is associated with mucosal humoral immunity, allergic asthma, and intestinal T-cell homing (Charo & Ransohoff, 2006).

[000830] To date, a crystal structure of the CCR4 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CCR6 consists of the N-terminal segment (Met1 -Glu40 of the human CCR6), ECL1 (Gly109- Phe1 13 of the human CCR6) linking helices II and III, ECL2 (Gln187-Glu206 of the human CCR6) linking helices IV and V, and ECL3 (Asn285-Arg286 of the human CCR6) linking helices VI to VII. Like all chemokine receptors, the CCR6 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016).

[000831 ] In specific embodiments of the present invention, the CCR6 polypeptide comprises a CCR6 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR6 protein sequence.

[000832] In some embodiments, the CCR6 protein sequence corresponds to a mammalian CCR6 protein sequence. Suitable CCR6 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3WPJ2), chimpanzee (UniProtKB Accession No. H2QU13), cow (UniProtKB Accession No. F1 MT56), dog (UniProtKB Accession No. J9P4Z8), gorilla (UniProtKB Accession No. G3R6S8), guinea pig (UniProtKB Accession No. H0WBT8), horse (UniProtKB Accession No. F6PP77), human (UniProtKB Accession No. P51684), marmoset (UniProtKB Accession No. F6PMZ8), mouse (UniProtKB Accession No. 054689), pig (UniProtKB Accession No. I3LBI5), rabbit (UniProtKB Accession No. G1 U041 ), rat (UniProtKB Accession No. Q5BK58), sheep (UniProtKB Accession No. W5P4D1 ).

[000833] In some preferred embodiments, the CCR6 protein sequence corresponds to a human CCR6 protein sequence. In some embodiments, the CCR6 polypeptide comprises a human full-length wild-type CCR6 protein sequence (UniProtKB Accession No. P51684), as set forth below, or a functional fragment of the wild-type CCR6 protein sequence.

[000834] MSGESMNFSDVFDSSEDYFVSVNTSYYSVDSEMLLCSLQEVRQFSRLFVPIAY

SLICVFGLLGNILVVITFAFYKKARSMTDVYLLNMAIADILFVLTLPFWAVSHATGA WVFSNAT

CKLLKGIYAINFNCGMLLLTCISMDRYIAIVQATKSFRLRSRTLPRSKIICLVVWGL SVIISSSTF

VFNQKYNTQGSDVCEPKYQTVSEPIRWKLLMLGLELLFGFFIPLMFMIFCYTFIVKT LVQAQN

SKRHKAIRVIIAVVLVFLACQIPHNMVLLVTAANLGKMNRSCQSEKLIGYTKTVTEV LAFLHCC

LNPVLYAFIGQKFRNYFLKILKDLWCVRRKYKSSGFSCAGRYSENISRQTSETADND NASSF

TM [SEQ ID NO: 27].

[000835] In one form of the invention, the CCR6 polypeptide comprises a truncated form of a mammalian wild-type CCR6 protein sequence. For example, the CCR6 polypeptide sequence may comprise the human wild-type CCR6 protein sequence with a C-terminal truncation (e.g., amino acid residues 333-374 may be truncated). Alternatively or in addition, the CCR6 polypeptide sequence may comprise the wild-type CCR6 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR6 protein sequence (e.g., amino acid residues 191 -202 may be truncated). By way of a non-limiting illustrative example, a CCR6 polypeptide suitable for using with the present invention comprised amino acid residues 2-190 and 203-332 of the human wild-type CCR6 protein sequence as set forth in SEQ ID NO: 27.

3.26 Constructs and nucleotide sequences encoding CCR6 polypeptides.

[000836] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR6 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000837] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR6 nucleotide (i.e., corresponding to the CCR6 gene) sequence as set forth for example in GenBank Accession Nos. NM_004367.5, NM_031409.3, AY242126.1 , BC037960.1 and U45984.1 . In representative examples of this type, the polynucleotide comprises a CCR6 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000838] In some embodiments, a CCR6 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR6 polynucleotide, or a fragment thereof. In some embodiments, the CCR6 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR6 protein, or a fragment thereof under low, medium or high stringency conditions.

3.27 CC chemokine receptor CCR7 (CCR7) polypeptides

[000839] Like all chemokine receptors, the CCR7 is involved in the mediation of inflammatory processes. It is found in T cells and mature dendritic cells and is associated with transport of T cells and dendritic cells to lymph node, antigen presentation, and cellular immunity (Charo & Ransohoff, 2006).

[000840] To date, a crystal structure of the CCR7 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CCR7 consists of the N-terminal segment (Met1 -Asp52 of the human CCR7), ECL1 (Ser121 - Phe124 of the human CCR7) linking helices II and III, ECL2 (Asp198-Val217 of the human CCR7) linking helices IV and V, and ECL3 (Thr294-Ser296 of the human CCR7) linking helices VI to VII. Like all chemokine receptors, the CCR7 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016).

[000841 ] In specific embodiments of the present invention, the CCR7 polypeptide comprises a CCR7 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR7 protein sequence.

[000842] In some embodiments, the CCR7 protein sequence corresponds to a mammalian CCR7 protein sequence. Suitable CCR7 sequences may suitably be from mammal selected from the group comprising mouse (UniProtKB Accession No. P47774), human (UniProtKB Accession No. P32248), rat (UniProtKB Accession No. Q6U2D6), pig (UniProtKB Accession No. Q861 S1 ), cow (UniProtKB Accession No. A0JNA6), dog (UniProtKB Accession No. J9P893), rabbit (UniProtKB Accession No. G1 TKJ1 ), marmoset (UniProtKB Accession No. F6R3A6), chimpanzee (UniProtKB Accession No. H2R9P0), tasmanian devil (UniProtKB Accession No. G3WAF3), sheep (UniProtKB Accession No. W5PZG4), guinea pig (UniProtKB Accession No. H0VL65), cat (UniProtKB Accession No. M3WBU1 ), horse (UniProtKB Accession No. F6SPH5).

[000843] In some preferred embodiments, the CCR7 protein sequence corresponds to a human CCR7 protein sequence. In some embodiments, the CCR7 polypeptide comprises a human full-length wild-type CCR7 protein sequence (UniProtKB Accession No. P32248), as set forth below, or a functional fragment of the wild-type CCR7 protein sequence.

[000844] MDLGKPMKSVLVVALLVIFQVCLCQDEVTDDYIGDNTTVDYTLFESLCSKKDVR

NFKAWFLPIMYSIICFVGLLGNGLVVLTYIYFKRLKTMTDTYLLNLAVADILFLLTL PFWAYSAA

KSWVFGVHFCKLIFAIYKMSFFSGMLLLLCISIDRYVAIVQAVSAHRHRARVLLISK LSCVGIWI

LATVLSIPELLYSDLQRSSSEQAMRCSLITEHVEAFITIQVAQMVIGFLVPLLAMSF CYLVIIRT

LLQARNFERNKAIKVIIAVVVVFIVFQLPYNGVVLAQTVANFNITSSTCELSKQLNI AYDVTYSL

ACVRCCVNPFLYAFIGVKFRNDLFKLFKDLGCLSQEQLRQWSSCRHIRRSSMSVEAE TTTT

FSP [SEQ ID NO: 28].

[000845] In one form of the invention, the CCR7 polypeptide comprises a truncated form of a mammalian wild-type CCR7 protein sequence. For example, the CCR7 polypeptide sequence may comprise the human wild-type CCR7 protein sequence with a C-terminal truncation (e.g., amino acid residues 358-378 may be truncated). Alternatively or in addition, the CCR7 polypeptide sequence may comprise the wild-type CCR7 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR7 protein sequence ( e.g., amino acid residues 201 -209 may be truncated). By way of a non-limiting illustrative example, a CCR7 polypeptide suitable for using with the present invention comprised amino acid residues 2-200 and 210-357 of the human wild-type CCR7 protein sequence as set forth in SEQ ID NO: 28.

3.28 Constructs and nucleotide sequences encoding CCR7 polypeptides

[000846] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR7 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000847] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR7 nucleotide i.e( ., corresponding to the CCR7 gene) sequence as set forth for example in GenBank Accession Nos. NM_001301718.1 , NM_001301717.1 , NM_001301716.1 , NM_001301714.1 , NM_001838.3, EF064758.1 , NC_000017.1 1 , NC_018928.2, CM000268.1 , BC035343.1 . In representative examples of this type, the polynucleotide comprises a CCR7 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000848] In some embodiments, a CCR7 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR7 polynucleotide, or a fragment thereof. In some embodiments, the CCR7 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR7 protein, or a fragment thereof under low, medium or high stringency conditions.

3.29 CC chemokine receptor CCR9 (CCR9) polypeptides

[000849] Like all chemokine receptors, the CCR9 is involved in the mediation of inflammatory processes. It is found in T cells and lgA+ plasma cells and is associated with homing of T cells and lgA+ plasma cells to the intestine and inflammatory bowel disease (Charo & Ransohoff, 2006).

[000850] To date, a crystal structure of the CCR9 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CCR9 consists of the N-terminal segment (Met1 -Asn42 of the human CCR9), ECL1 (Gln1 1 1 - Phe1 14 of the human CCR9) linking helices II and III, ECL2 (Gln188-Glu206 of the human CCR9) linking helices IV and V, and ECL3 (Phe285-Ser287 of the human CCR9) linking helices VI to VII. Like all chemokine receptors, the CCR9 is expected to have two conserved disulphide bonds in its ectodomains (Zheng et al., 2016).

[000851 ] In specific embodiments of the present invention, the CCR9 polypeptide comprises a CCR9 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CCR9 protein sequence.

[000852] In some embodiments, the CCR9 protein sequence corresponds to a mammalian CCR9 protein sequence. Suitable CCR9 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3W6K0), chimpanzee (UniProtKB Accession No. H2QMG2), cow (UniProtKB Accession No. F6RMK7), dog (UniProtKB Accession No. E2RQ21 ), gorilla (UniProtKB Accession No. G3QQ18), guinea pig (UniProtKB Accession No. H0VWP5), horse (UniProtKB Accession No. F6X7N4), human (UniProtKB Accession No. P51686), marmoset (UniProtKB Accession No. F7F023), mouse (UniProtKB Accession No. Q9WUT7), panta (UniProtKB Accession No. G1 M0T5), pig (UniProtKB Accession No. Q6YT47), rabbit (UniProtKB Accession No. G1 SUQ1 ), sheep (UniProtKB Accession No. Q1WLP9).

[000853] In some preferred embodiments, the CCR9 protein sequence corresponds to a human CCR9 protein sequence. In some embodiments, the CCR9 polypeptide comprises a human full-length wild-type CCR9 protein sequence (UniProtKB Accession No. P51686), as set forth below, or a functional fragment of the wild-type CCR9 protein sequence.

[000854] MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFASHFLPP

LYWLVFIVGALGNSLVILVYWYCTRVKTMTDMFLLNLAIADLLFLVTLPFWAIAAAD QWKFQT

FMCKVVNSMYKMNFYSCVLLIMCISVDRYIAIAQAMRAHTWREKRLLYSKMVCFTIW VLAAA

LCIPEILYSQIKEESGIAICTMVYPSDESTKLKSAVLTLKVILGFFLPFVVMACCYT IIIHTLIQAK

KSSKHKALKVTITVLTVFVLSQFPYNCILLVQTIDAYAMFISNCAVSTNIDICFQVT QTIAFFHS

CLNPVLYVFVGERFRRDLVKTLKNLGCISQAQWVSFTRREGSLKLSSMLLETTSGAL SL

[SEQ ID NO: 29].

[000855] In one form of the invention, the CCR9 polypeptide comprises a truncated form of a mammalian wild-type CCR9 protein sequence. For example, the CCR9 polypeptide sequence may comprise the human wild-type CCR9 protein sequence with a C-terminal truncation (e.g., amino acid residues 334-369 may be truncated). Alternatively or in addition, the CCR9 polypeptide sequence may comprise the wild-type CCR9 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CCR9 protein sequence (e.g., amino acid residues 190-204 may be truncated). By way of a non-limiting illustrative example, a CCR9 polypeptide suitable for using with the present invention comprised amino acid residues 2-189 and 205-333 of the human wild-type CCR9 protein sequence as set forth in SEQ ID NO: 29.

3.30 Constructs and nucleotide sequences encoding CCR9 polypeptides.

[000856] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CCR9 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs. [000857] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CCR9 nucleotide (i.e., corresponding to the CCR9 gene) sequence as set forth for example in GenBank Accession Nos. AY242127.1 , NM_001256369.1 , NM_031200.2, NM_006641 .3, AF145207.1 , NG_029472.1 , AF145440.1 , AF145439.1 , BC095516.1 , BC069678.1 and AJ132337.1 . In representative examples of this type, the polynucleotide comprises a CCR9 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000858] In some embodiments, a CCR9 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CCR9 polynucleotide, or a fragment thereof. In some embodiments, the CCR9 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CCR9 protein, or a fragment thereof under low, medium or high stringency conditions.

3.31 CXC chemokine receptor CXCR2 (CXCR2) polypeptides

[000859] Like all chemokine receptors, the CXCR2 is involved in the mediation of inflammatory processes. It is found in neutrophils, monocytes and micro-vascular endothelial cells and is associated with inflammatory lung disease, COPD and is angiogenic for tumor growth (Charo & Ransohoff, 2006).

[000860] To date, a crystal structure of the CXCR2 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CXCR2 consists of the N-terminal segment (Met1 -Glu42 of the human CXCR2), ECL1 (Gly1 1 1 - Phe1 14 of the human CXCR2) linking helices II and III, ECL2 (Arg185-Ala205 of the human CXCR2) linking helices IV and V, and ECL3 (Val281 -Glu284 of the human CXCR2) linking helices VI to VII. Like all chemokine receptors, the CXCR2 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016).

[000861 ] In specific embodiments of the present invention, the CXCR2 polypeptide comprises a CXCR2 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CXCR2 protein sequence. [000862] In some embodiments, the CXCR2 protein sequence corresponds to a mammalian CXCR2 protein sequence. Suitable CXCR2 sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P25025), mouse (UniProtKB Accession No. P35343), rat (UniProtKB Accession No. P35407), rabbit (UniProtKB Accession No. P35344), dog (UniProtKB Accession No. 097571 ), cow (UniProtKB Accession No. Q28003), chimpanzee (UniProtKB Accession No. Q28807), gorilla (UniProtKB Accession No. Q28422), marmoset (UniProtKB Accession No. F6YI18), bat (UniProtKB Accession No. G1 P3F4), cat (UniProtKB Accession No. M3XFH4), guinea pig (UniProtKB Accession No. Q810T4), pig (UniProtKB Accession No. F1 SS05), sheep (UniProtKB Accession No. W5QDX4).

[000863] In some preferred embodiments, the CXCR2 protein sequence corresponds to a human CXCR2 protein sequence. In some embodiments, the CXCR2 polypeptide comprises a human full-length wild-type CXCR2 protein sequence (UniProtKB Accession No. P25025), as set forth below, or a functional fragment of the wild-type CXCR2 protein sequence.

[000864] MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIY

ALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGW IFGTFLC

KVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLL ALPVLLFR

RTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKA HMGQ

KHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEIL GILHSCL

NPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL [SEQ ID NO: 30].

[000865] In one form of the invention, the CXCR2 polypeptide comprises a truncated form of a mammalian wild-type CXCR2 protein sequence. For example, the CXCR2 polypeptide sequence may comprise the human wild-type CXCR2 protein sequence with a C-terminal truncation (e.g., amino acid residues 346-360 may be truncated). Alternatively or in addition, the CXCR2 polypeptide sequence may comprise the wild-type CXCR2 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CXCR2 protein sequence (e.g., amino acid residues 187-202 may be truncated). By way of a non-limiting illustrative example, a CXCR2 polypeptide suitable for using with the present invention comprised amino acid residues 2-186 and 203-345 of the human wild-type CXCR2 protein sequence as set forth in SEQ ID NO: 30.

Constructs and nucleotide sequences encoding CXCR2 polypeptides [000866] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CXCR2 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000867] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CXCR2 nucleotide (i.e., corresponding to the CXCR2 gene) sequence as set forth for example in GenBank Accession Nos. NM 001 168298.1 , NM_001557.3, AB032734.1 , AB032733.1 , NG_052975.1 , XM_017003992.1 , XM_017003991 .1 , XM_017003990.1 , XM_005246530.3. In representative examples of this type, the polynucleotide comprises a CXCR2 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000868] In some embodiments, a CXCR2 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CXCR2 polynucleotide, or a fragment thereof. In some embodiments, the CXCR2 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CXCR2 protein, or a fragment thereof under low, medium or high stringency conditions.

3.33 CXC chemokine receptor CXCR4 (CXCR4) polypeptides

[000869] Like all chemokine receptors, the CXCR4 is involved in the mediation of inflammatory processes. It is widely expressed throughout the immune system and is associated with HIV-infection, tumour metastases and hematopoiesis (Charo & Ransohoff, 2006).

[000870] To date, two studies have reported solving the crystal structure of the CXCR4, providing insight into how it binds its ligands (Wu et al., 2010; Qin et al., 2015). The extracellular part of CXCR4 consists of the N-terminal segment (Met1 -Phe29 of the human CXCR4), ECL1 (Asn101 -Phe104 of the human CXCR4) linking helices II and III, ECL2 (Asn176-Asn192 of the human CXCR4) linking helices IV and V, and ECL3 (Ne269-Gln272 of the human CXCR4) linking helices VI to VII. As expected for most chemokine receptors, the CXCR4 has two conserved disulfide bonds in its ectodomain (Wu et al., 2010).

[000871 ] In specific embodiments of the present invention, the CXCR4 polypeptide comprises a CXCR4 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CXCR4 protein sequence.

[000872] In some embodiments, the CXCR4 protein sequence corresponds to a mammalian CXCR4 protein sequence. Suitable CXCR4 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. P56498), chimpanzee (UniProtKB Accession No. P61072), cow (UniProtKB Accession No. P25930), dog (UniProtKB Accession No. Q3LSL6), hedgehog (UniProtKB Accession No. A0A1 S2ZV52), horse (UniProtKB Accession No. F6Y9M7), human (UniProtKB Accession No. P61073), lion (UniProtKB Accession No. B5LVX6), marmoset (UniProtKB Accession No. Q8HZU1 ), mouse (UniProtKB Accession No. A0A0R4J0N8), pig (UniProtKB Accession No. Q764M9), rabbit (UniProtKB Accession No. G1 SGF1 ), rat (UniProtKB Accession No. A0A0G2K9U1 ), sheep (UniProtKB Accession No. W5PLA4).

[000873] In some preferred embodiments, the CXCR4 protein sequence corresponds to a human CXCR4 protein sequence. In some embodiments, the CXCR4 polypeptide comprises a human full-length wild-type CXCR4 protein sequence (UniProtKB Accession No. P61073), as set forth below, or a functional fragment of the wild-type CXCR4 protein sequence.

[000874] MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGN

GLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVH VIYTV

NLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFA NVSEADDR

YICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKT TVILILAFF

ACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLG AKFKTSAQH

ALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS [SEQ ID NO: 31 ].

[000875] In one form of the invention, the CXCR4 polypeptide comprises a truncated form of a mammalian wild-type CXCR4 protein sequence. For example, the CXCR4 polypeptide sequence may comprise the human wild-type CXCR4 protein sequence with a C-terminal truncation (e.g., amino acid residues 319-352 may be truncated). Alternatively or in addition, the CXCR4 polypeptide sequence may comprise the wild-type CXCR4 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CXCR4 protein sequence (e.g., amino acid residues 179-188 may be truncated). By way of a non-limiting illustrative example, a CXCR4 polypeptide suitable for using with the present invention comprised amino acid residues 2-178 and 189-318 of the human wild-type CXCR4 protein sequence as set forth in SEQ ID NO: 31 . 3.34 Constructs and nucleotide sequences encoding CXCR4 polypeptides.

[000876] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CXCR4 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000877] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CXCR4 nucleotide (i.e., corresponding to the CXCR4 gene) sequence as set forth for example in GenBank Accession Nos. AY242129.1 , AJ224869.1 , NM_003467.2, NM_001008540.2, NM_001348056.1 , NM_001348060.1 , NM_001348059.1 , AF147204.2, AY826773.1 , KU245647.1 , KU245646.1 , Y14739.1 , AF025375.1 , AF052572.1 , AF348491 .1 and U81003.1 . In representative examples of this type, the polynucleotide comprises a CXCR4 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000878] In some embodiments, a CXCR4 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CXCR4 polynucleotide, or a fragment thereof. In some embodiments, the CXCR4 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CXCR4 protein, or a fragment thereof under low, medium or high stringency conditions.

3.35 CXC chemokine receptor CXCR5 (CXCR5) polypeptides

[000879] Like all chemokine receptors, the CXCR5 is involved in the mediation of inflammatory processes. It is expressed in B cells and follicular helper T cells and is involved in the formation of B-cell follicles (Charo & Ransohoff, 2006).

[000880] To date, a crystal structure of the CCR9 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CXCR5 consists of the N-terminal segment (Met1 -Glu42 of the human CXCR5), ECL1 (Gly1 14- Leu1 17 of the human CXCR5) linking helices II and III, ECL2 (Lys189-Glu21 1 of the human CXCR5) linking helices IV and V, and ECL3 (Ala289-Asn292 of the human CXCR5) linking helices VI to VII. Like all chemokine receptors, the CCR9 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016). [000881 ] In specific embodiments of the present invention, the CXCR5 polypeptide comprises a CXCR5 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CXCR5 protein sequence.

[000882] In some embodiments, the CXCR5 protein sequence corresponds to a mammalian CXCR5 protein sequence. Suitable CXCR5 sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3XAS4), chimpanzee (UniProtKB Accession No. H2Q4W8), cow (UniProtKB Accession No. G3N3U0), dog (UniProtKB Accession No. E2RR03), gorilla (UniProtKB Accession No. G3RDW5), guinea pig (UniProtKB Accession No. H0W6I1 ), human (UniProtKB Accession No. P32302), marmoset (UniProtKB Accession No. F7A1 S1 ), mouse (UniProtKB Accession No. Q04683), panda (UniProtKB Accession No. G1 KZK8), pig (UniProtKB Accession No. F1 SAJ4), rabbit (UniProtKB Accession No. G1 TNG1 ), rat (UniProtKB Accession No. P34997), sheep (UniProtKB Accession No. W5PPW1 ).

[000883] In some preferred embodiments, the CXCR5 protein sequence corresponds to a human CXCR5 protein sequence. In some embodiments, the CXCR5 polypeptide comprises a human full-length wild-type CXCR5 protein sequence (UniProtKB Accession No. P32302), as set forth below, or a functional fragment of the wild-type CXCR5 protein sequence.

[000884] MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVF

VPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAE GSVGWVL

GTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIW LVGFLLAL

PEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYV GVV

HRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGS LPVAIT

MCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSLSE SENA

TSLTTF [SEQ ID NO: 32].

[000885] In one form of the invention, the CXCR5 polypeptide comprises a truncated form of a mammalian wild-type CXCR5 protein sequence. For example, the CXCR5 polypeptide sequence may comprise the human wild-type CXCR5 protein sequence with a C-terminal truncation (e.g., amino acid residues 339-372 may be truncated). Alternatively or in addition, the CXCR5 polypeptide sequence may comprise the wild-type CXCR5 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CXCR5 protein sequence (e.g., amino acid residues 193-208 may be truncated). By way of a non-limiting illustrative example, a CXCR5 polypeptide suitable for using with the present invention comprised amino acid residues 2-192 and 209-338 of the human wild-type CXCR5 protein sequence as set forth in SEQ ID NO: 32.

3.36 Constructs and nucleotide sequences encoding CXCR5 polypeptides.

[000886] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CXCR5 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000887] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CXCR5 nucleotide (i.e., corresponding to the CXCR5 gene) sequence as set forth for example in GenBank Accession Nos. NM 001716.4, NM 032966.2 and BC1 10352.1 . In representative examples of this type, the polynucleotide comprises a CXCR5 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000888] In some embodiments, a CXCR5 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CXCR5 polynucleotide, or a fragment thereof. In some embodiments, the CXCR5 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CXCR5 protein, or a fragment thereof under low, medium or high stringency conditions.

3.37 CXC chemokine receptor CXCR6 (CXCR6) polypeptides

[000889] Like all chemokine receptors, the CXCR6 is involved in the mediation of inflammatory processes. It is found in CD8+ T cells, natural killer cells, and memory CD4+ T cells and is associated with inflammatory liver disease and atherosclerosis (Charo & Ransohoff, 2006).

[000890] To date, a crystal structure of the CXCR6 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CXCR6 consists of the N-terminal segment (Met1 -Asp25 of the human CXCR6), ECL1 (Glu94- Phe97 of the human CXCR6) linking helices II and III, ECL2 (Asn171 -Glu185 of the human CXCR6) linking helices IV and V, and ECL3 (Trp258-Ala262 of the human CXCR6) linking helices VI to VI I. Like all chemokine receptors, the CXCR6 is expected to have two conserved disulfide bonds in its ectodomains (Zheng et al., 2016).

[000891 ] In specific embodiments of the present invention, the CXCR6 polypeptide comprises a CXCR6 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CXCR6 protein sequence.

[000892] In some embodiments, the CXCR6 protein sequence corresponds to a mammalian CXCR6 protein sequence. Suitable CXCR6 sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. 000574), chimpanzee (UniProtKB Accession No. Q9TV16), mouse (UniProtKB Accession No. Q9EQ16), rat (UniProtKB Accession No. A7ISD5), pig (UniProtKB Accession No. Q6YT44), cow (UniProtKB Accession No. Q5EA94), dog (UniProtKB Accession No. F1 PMU3), tasmanian devil (UniProtKB Accession No. G3W5Z9), marmoset (UniProtKB Accession No. F7DTR0), cat (UniProtKB Accession No. M3W6K1 ), bat (UniProtKB Accession No. G1 P8C6), rabbit (UniProtKB Accession No. G1 SLJ0), guinea pig (UniProtKB Accession No. H0W823), horse (UniProtKB Accession No. F6XQ88).

[000893] In some preferred embodiments, the CXCR6 protein sequence corresponds to a human CXCR6 protein sequence. In some embodiments, the CXCR6 polypeptide comprises a human full-length wild-type CXCR6 protein sequence (UniProtKB Accession No. 000574), as set forth below, or a functional fragment of the wild-type CXCR6 protein sequence.

[000894] MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLVVFVCGLVGNSLV

LVISIFYHKLQSLTDVFLVNLPLADLVFVCTLPFWAYAGIHEWVFGQVMCKSLLGIY TINFYTS

MLILTCITVDRFIVVVKATKAYNQQAKRMTWGKVTSLLIWVISLLVSLPQI IYGNVFNLDKLICG

YHDEAISTVVLATQMTLGFFLPLLTMIVCYSVI IKTLLHAGGFQKHRSLKI IFLVMAVFLLTQMP

FNLMKFIRSTHWEYYAMTSFHYTIMVTEAIAYLRACLNPVLYAFVSLKFRKNFW

KLVKDIGCLPYLGVSHQWKSSEDNSKTFSASHNVEATSMFQL [SEQ ID NO: 33].

[000895] In one form of the invention, the CXCR6 polypeptide comprises a truncated form of a mammalian wild-type CXCR6 protein sequence. For example, the CXCR6 polypeptide sequence may comprise the human wild-type CXCR6 protein sequence with a C-terminal truncation (e.g., amino acid residues 31 8-342 may be truncated). Alternatively or in addition, the CXCR6 polypeptide sequence may comprise the wild-type CXCR6 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CXCR6 protein sequence e( .g., amino acid residues 172-180 may be truncated). By way of a non-limiting illustrative example, a CXCR6 polypeptide suitable for using with the present invention comprised amino acid residues 2-171 and 181 -317 of the human wild-type CXCR6 protein sequence as set forth in SEQ ID NO: 33.

3.38 Constructs and nucleotide sequences encoding CXCR6 polypeptides

[000896] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CXCR6 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000897] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CXCR6 nucleotide (i.e., corresponding to the CXCR6 gene) sequence as set forth for example in GenBank Accession Nos. NM 006564.1 , XM_005264809.2, XM_01 1533291 .2, XM_01 1533290.2, EU076974.1 , EF064741 .1 , AK313754.1 ,. In representative examples of this type, the polynucleotide comprises a CXCR6 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000898] In some embodiments, a CXCR6 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CXCR6 polynucleotide, or a fragment thereof. In some embodiments, the CXCR6 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CXCR6 protein, or a fragment thereof under low, medium or high stringency conditions.

3.39 CXC chemokine receptor CXCR7 (also known as atypical chemokine receptor 3; ACKR3) polypeptides

[000899] Like all chemokine receptors, the CXCR7 is involved in the mediation of inflammatory processes. It is found in reticulocytes, post capillary venules, epithelial cells of the kidneys and lungs, and cerebellar neurons. The CXCR7 has been associated with various autoimmune diseases, transplant complications, HIV infection, pre-eclampsia and malignancy (Home & Woolley, 2009).

[000900] To date, a crystal structure of the CXCR7 has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of CXCR7 consists of the N-terminal segment (Met1 -Ser41 of the human CXCR7), ECL1 (His107- Met1 12 of the human CXCR7) linking helices II and III, ECL2 (Lys184-Glu207 of the human CXCR7) linking helices IV and V, and ECL3 (Tyr282-Phe285 of the human CXCR7) linking helices VI to VII. The extracellular amino-terminal domain harbors three potential N- glycosylation sites at residues 16, 27, and 33 (Czerwinski et al., 2007).

[000901 ] In specific embodiments of the present invention, the CXCR7 polypeptide comprises a CXCR7 protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a CXCR7 protein sequence.

[000902] In some embodiments, the CXCR7 protein sequence corresponds to a mammalian CXCR7 protein sequence. Suitable CXCR7 sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P25106), rat (UniProtKB Accession No. 089039), mouse (UniProtKB Accession No. P56485), dog (UniProtKB Accession No. P 1 1613), cow (UniProtKB Accession No. A4IF77), macaque (UniProtKB Accession No. F7GTL4), marmoset (UniProtKB Accession No. F7HZX1 ), chimpanzee (UniProtKB Accession No. H2QJP1 ), mink (UniProtKB Accession No. U6DE19).

[000903] In some preferred embodiments, the CXCR7 protein sequence corresponds to a human CXCR7 protein sequence. In some embodiments, the CXCR7 polypeptide comprises a human full-length wild-type CXCR7 protein sequence (UniProtKB Accession No. P25106), as set forth below, or a functional fragment of the wild-type CXCR7 protein sequence.

[000904] MDLHLFDYSEPGNFSDISWPCNSSDCIVVDTVMCPNMPNKSVLLYTLSFIYIFIF

VIGMIANSVVVWVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQWPM GELTC

KVTHLIFSINLFGSIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRVVCILVWLLAFC VSLPDTY

YLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIIAVFYFLLARA ISASSDQ

EKHSSRKIIFSYVVVFLVCWLPYHVAVLLDIFSILHYIPFTCRLEHALFTALHVTQC LSLVHCCV

NPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSETEYSALEQS TK [SEQ ID NO:

34].

[000905] In one form of the invention, the CXCR7 polypeptide comprises a truncated form of a mammalian wild-type CXCR7 protein sequence. For example, the CXCR7 polypeptide sequence may comprise the human wild-type CXCR7 protein sequence with a C-terminal truncation (e.g., amino acid residues 339-362 may be truncated). Alternatively or in addition, the CXCR7 polypeptide sequence may comprise the wild-type CXCR7 protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type CXCR7 protein sequence (e.g., amino acid residues 187-204 may be truncated). By way of a non-limiting illustrative example, a CXCR7 polypeptide suitable for using with the present invention comprised amino acid residues 2-186 and 205-338 of the human wild-type CXCR7 protein sequence as set forth in SEQ ID NO: 34.

3.40 Constructs and nucleotide sequences encoding CXCR7 polypeptides

[000906] The present invention also encompasses isolated polynucleotide sequences and constructs encoding CXCR7 polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000907] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human CXCR7 nucleotide (i.e., corresponding to the ACKR3 gene) sequence as set forth for example in GenBank Accession Nos. NM 02031 1 .2, XM_017004516.1 , XM_005246098.3, XM_005246097.2, BC036661 .2. In representative examples of this type, the polynucleotide comprises a CXCR7 nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000908] In some embodiments, a CXCR7 polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian CXCR7 polynucleotide, or a fragment thereof. In some embodiments, the CXCR7 polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian CXCR7 protein, or a fragment thereof under low, medium or high stringency conditions.

3.41 Dopamine D 1 receptor (D1R) polypeptides

[000909] In the central nervous system (CNS) the D1 R has numerous functions, including stimulation of locomotor activity, involvement in reward and reinforcement mechanisms, and roles in learning, memory and other cognitive functions. Outside of the CNS, the D1 R regulates renin secretion and maintenance of renal function (Beaulieu & Gainetdinov, 201 1 ).

[000910] To date, a crystal structure of the D1 R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of D1 R consists of the N-terminal segment (Met1 -Leu14 of the human D1 R), ECL1 (Ala87-Phe92 of the human D1 R) linking helices II and III, ECL2 (Trp163-Leu190 of the human D1 R) linking helices IV and V, and ECL3 (Gly299-Pro305 of the human D1 R) linking helices VI to VII.

[00091 1 ] In specific embodiments of the present invention, the D1 R polypeptide comprises a D1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a D1 R protein sequence.

[000912] In some embodiments, the D1 R protein sequence corresponds to a mammalian D1 R protein sequence. Suitable D1 R sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3VVD1 ), chimpanzee (UniProtKB Accession No. H2QS18), cow (UniProtKB Accession No. Q95136), dog (UniProtKB Accession No. F1 PI21 ), gorilla (UniProtKB Accession No. G3RXE8), guinea pig (UniProtKB Accession No. H0VES0), human (UniProtKB Accession No. P21728), mouse (UniProtKB Accession No. Q61616), panda (UniProtKB Accession No. G1 LK07), pig (UniProtKB Accession No. P50130), rabbit (UniProtKB Accession No. G1 SML2), rat (UniProtKB Accession No. P18901 ), sheep (UniProtKB Accession No. W5PPK6), Tasmanian devil (UniProtKB Accession No. G3VDD8).

[000913] In some preferred embodiments, the D1 R protein sequence corresponds to a human D1 R protein sequence. In some embodiments, the D1 R polypeptide comprises a human full-length wild-type D1 R protein sequence (UniProtKB Accession No. P21728), as set forth below, or a functional fragment of the wild-type D1 R protein sequence.

[000914] MRTLNTSAMDGTGLVVERDFSVRILTACFLSLLILSTLLGNTLVCAAVIRFRHLRS

KVTNFFVISLAVSDLLVAVLVMPWKAVAEIAGFWPFGSFCNIWVAFDIMCSTASILN LCVISV

DRYWAISSPFRYERKMTPKAAFILISVAWTLSVLISFIPVQLSWHKAKPTSPSDGNA TSLAETI

DNCDSSLSRTYAISSSVISFYIPVAIMIVTYTRIYRIAQKQIRRIAALERAAVHAKN CQTTTGNG

KPVECSQPESSFKMSFKRETKVLKTLSVIMGVFVCCWLPFFILNCILPFCGSGETQP FCIDS

NTFDVFVWFGWANSSLNPIIYAFNADFRKAFSTLLGCYRLCPATNNAIETVSINNNG AAMFS

SHHEPRGSISKECNLVYLIPHAVGSSEDLKKEEAAGIARPLEKLSPALSVILDYDTD VSLEKIQ

PITQNGQHPT [SEQ ID NO: 35].

[000915] In one form of the invention, the D1 R polypeptide comprises a truncated form of a mammalian wild-type D1 R protein sequence. For example, the D1 R polypeptide sequence may comprise the human wild-type D1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 348-446 may be truncated). Alternatively or in addition, the D1 R polypeptide sequence may comprise the wild-type D1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type D1 R protein sequence (e.g., amino acid residues 166-186 may be truncated). By way of a non-limiting illustrative example, a D1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-165 and 187-347 of the human wild-type D1 R protein sequence as set forth in SEQ ID NO: 35.

3.42 Constructs and nucleotide sequences encoding D1R polypeptides.

[000916] The present invention also encompasses isolated polynucleotide sequences and constructs encoding D1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000917] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human D1 R nucleotide (i.e., corresponding to the DRD1 gene) sequence as set forth for example in GenBank Accession Nos. CR541922.1 , EU249297.1 , KR712133.1 , KR712132.1 , KR712131 .1 , KR712130.1 , KJ896726.1 , NM_000794.3, and NG_01 1802.1 . In representative examples of this type, the polynucleotide comprises a D1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000918] In some embodiments, a D1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian D1 R polynucleotide, or a fragment thereof. In some embodiments, the D1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian D1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.43 Endothelin receptor type A (ETAR) polypeptides

[000919] The ETAR mediates many functions including vasoconstriction, cardiovascular remodelling, cell proliferation, cell differentiation, extracellular matrix production, and control of water and sodium secretion. It is also implicated in the development of various diseases such as pulmonary hypertension, atherosclerosis, diabetes, and cardiac remodelling after myocardial ischemia (Horinouchi et al., 2013). [000920] To date, a crystal structure of the ETAR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of ETAR consists of the N-terminal segment (Met1 -Cys69 of the human ETAR), ECL1 (Gly144- Phe148 of the human ETAR) linking helices II and III, ECL2 (Val225-Ser245 of the human ETAR) linking helices IV and V, and ECL3 (Asn334-Asn339 of the human ETAR) linking helices VI to VII.

[000921 ] In specific embodiments of the present invention, the ETAR polypeptide comprises a ETAR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an ETAR protein sequence.

[000922] In some embodiments, the ETAR protein sequence corresponds to a mammalian ETAR protein sequence. Suitable ETAR sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3WMJ5), chimpanzee (UniProtKB Accession No. H2QQ94), cow (UniProtKB Accession No. P21450), dog (UniProtKB Accession No. Q5KSU9), horse (UniProtKB Accession No. F7CHF1 ), human (UniProtKB Accession No. P25101 ), marmoset (UniProtKB Accession No. F6YN68), mouse (UniProtKB Accession No. Q61614), panda (UniProtKB Accession No. G1 L5F1 ), pig (UniProtKB Accession No. Q29010), rabbit (UniProtKB Accession No. A5A8K3), rat (UniProtKB Accession No. P26684), sheep (UniProtKB Accession No. Q95L55), Tasmanian devil (UniProtKB Accession No. G3VXK8).

[000923] In some preferred embodiments, the ETAR protein sequence corresponds to a human ETAR protein sequence. In some embodiments, the ETAR polypeptide comprises a human full-length wild-type ETAR protein sequence (UniProtKB Accession No. P25101 ), as set forth below, or a functional fragment of the wild-type ETAR protein sequence.

[000924] METLCLRASFWLALVGCVISDNPERYSTNLSNHVDDFTTFRGTELSFLVTTHQP TNLVLPSNGSMHNYCPQQTKITSAFKYINTVISCTIFIVGMVGNATLLRIIYQNKCMRNG PNAL IASLALGDLIYVVIDLPINVFKLLAGRWPFDHNDFGVFLCKLFPFLQKSSVGITVLNLCA LSVD RYRAVASWSRVQGIGIPLVTAIEIVSIWILSFILAIPEAIGFVMVPFEYRGEQHKTCMLN ATSKF MEFYQDVKDWWLFGFYFCMPLVCTAIFYTLMTCEMLNRRNGSLRIALSEHLKQRREVAKT V FCLVVIFALCWFPLHLSRILKKTVYNEMDKNRCELLSFLLLMDYIGINLATMNSCINPIA LYFVS KKFKNCFQSCLCCCCYQSKSLMTSVPMNGTSIQWKNHDQNNHNTDRSSHKDSMN [SEQ ID NO: 36]. [000925] In one form of the invention, the ETAR polypeptide comprises a truncated form of a mammalian wild-type ETAR protein sequence. For example, the ETAR polypeptide sequence may comprise the human wild-type ETAR protein sequence with a C-terminal truncation (e.g., amino acid residues 387-427 may be truncated). Alternatively or in addition, the ETAR polypeptide sequence may comprise the wild-type ETAR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type ETAR protein sequence (e.g., amino acid residues 228-242 may be truncated). By way of a non-limiting illustrative example, a ETAR polypeptide suitable for using with the present invention comprised amino acid residues 2-227 and 243-386 of the human wild-type ETAR protein sequence as set forth in SEQ ID NO: 36.

3.44 Constructs and nucleotide sequences encoding ETAR polypeptides.

[000926] The present invention also encompasses isolated polynucleotide sequences and constructs encoding ETAR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000927] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human ETAR nucleotide (i.e., corresponding to the EDNRA gene) sequence as set forth for example in GenBank Accession Nos. NM 001957.3, NG_013343.1 , NR_045958.1 , NM_001256283.1 , NM_001 166055.1 , AY275462.1 , L06622.1 and AY422989.1 . In representative examples of this type, the polynucleotide comprises an ETAR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000928] In some embodiments, an ETAR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian ETAR polynucleotide, or a fragment thereof. In some embodiments, the ETAR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian ETAR protein, or a fragment thereof under low, medium or high stringency conditions.

3.45 Endothelin receptor type B (ETBR) polypeptides [000929] The ETBR is believed to mediate vasodilation through endothelial production of nitric oxide and prostacyclin. In addition, it is thought to attenuate many of the pathological actions of the ETAR by depleting levels of endogenous ligands (Horinouchi et al., 2013).

[000930] The crystal structure of the ETBR has recently been solved, giving insight to its ligand-binding mode and activation process (Shihoya et al., 2016). The extracellular part of ETBR consists of the N-terminal segment (Met1 -Cys90 of the human ETBR), ECL1 (Glu165- Phe169 of the human ETBR) linking helices II and III, ECL2 (Asp241 -Thr263 of the human ETBR) linking helices IV and V, and ECL3 (Asn351 -Asn356 of the human ETBR) linking helices VI to VII. The ETBR contains two disulphide bonds in its ectodomains (Shihoya et al., 2016).

[000931 ] In specific embodiments of the present invention, the ETBR polypeptide comprises a ETBR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an ETBR protein sequence.

[000932] In some embodiments, the ETBR protein sequence corresponds to a mammalian ETBR protein sequence. Suitable ETBR sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3WRP2), chimpanzee (UniProtKB Accession No. K7DEA2), cow (UniProtKB Accession No. P28088), dog (UniProtKB Accession No. P56497), fox (UniProtKB Accession No. I6UG97), gorilla (UniProtKB Accession No. G3RES6), horse (UniProtKB Accession No. 062709), human (UniProtKB Accession No. P24530), mouse (UniProtKB Accession No. P48302), panda (UniProtKB Accession No. G1 LGC8), pig (UniProtKB Accession No. P35463), rabbit (UniProtKB Accession No. Q9N0W7), rat (UniProtKB Accession No. P21451 ), sheep (UniProtKB Accession No. W5NPP6).

[000933] In some preferred embodiments, the ETBR protein sequence corresponds to a human ETBR protein sequence. In some embodiments, the ETBR polypeptide comprises a human full-length wild-type ETBR protein sequence (UniProtKB Accession No. P24530), as set forth below, or a functional fragment of the wild-type ETBR protein sequence.

[000934] MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPTKTL

WPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETFKYINTVVSCL VFVLGII

GNSTLLRIIYKNKCMRNGPNILIASLALGDLLHIVIDIPINVYKLLAEDWPFGAEMC KLVPFIQK

ASVGITVLSLCALSIDRYRAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIG FDIITMDY

KGSYLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEMLRKK SGMQI ALNDHLKQRREVAKTVFCLVLVFALCWLPLHLSRILKLTLYNQNDPNRCELLSFLLVLDY IGIN MASLNSCINPIALYLVSKRFKNCFKSCLCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNF R SSNKYSSS [SEQ ID NO: 37].

[000935] In one form of the invention, the ETBR polypeptide comprises a truncated form of a mammalian wild-type ETBR protein sequence. For example, the ETBR polypeptide sequence may comprise the human wild-type ETBR protein sequence with a C-terminal truncation (e.g., amino acid residues 404-442 may be truncated). Alternatively or in addition, the ETBR polypeptide sequence may comprise the wild-type ETBR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type ETBR protein sequence (e.g., amino acid residues 245-260 may be truncated). By way of a non-limiting illustrative example, a ETBR polypeptide suitable for using with the present invention comprised amino acid residues 2-244 and 261 -403 of the human wild-type ETBR protein sequence as set forth in SEQ ID NO: 37.

3.46 Constructs and nucleotide sequences encoding ETBR polypeptides.

[000936] The present invention also encompasses isolated polynucleotide sequences and constructs encoding ETBR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000937] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human ETBR nucleotide (i.e., corresponding to the EDNRB gene) sequence as set forth for example in GenBank Accession Nos. AY275463.1 , NM 0001 15.4, NM_001 122659.2, NM_003991 .3, NM_001201397.1 , L06623.1 , NG_01 1630.2, AY547312.1 , AY275463.1 , AB209198.1 and BC014472.1 . In representative examples of this type, the polynucleotide comprises an ETBR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000938] In some embodiments, an ETBR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian ETBR polynucleotide, or a fragment thereof. In some embodiments, the ETBR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian ETBR protein, or a fragment thereof under low, medium or high stringency conditions. 3.47 Histamine H3 receptor (H3R) polypeptides

[000939] The H3R regulates serotonergic, noradrenergic, cholinergic, and dopaminergic neurotransmitter release and is involved in locomotion, mammalian hibernation and gastric acid secretion. It may also have roles in immune responses and multiple sclerosis (Panula et al., 2015).

[000940] To date, a crystal structure of the H3R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of H3R consists of the N-terminal segment (Met1 -Ser30 of the human H3R), ECL1 (Gly98-Phe102 of the human H3R) linking helices II and III, ECL2 (Trp174-Tyr194 of the human H3R) linking helices IV and V, and ECL3 (His385-Val389 of the human H3R) linking helices VI to VII.

[000941 ] In specific embodiments of the present invention, the H3R polypeptide comprises a H3R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an H3R protein sequence.

[000942] In some embodiments, the H3R protein sequence corresponds to a mammalian H3R protein sequence. Suitable H3R sequences may suitably be from mammal selected from the group comprising chimpanzee (UniProtKB Accession No. H2R2N9), cow (UniProtKB Accession No. F1 MML8), dog (UniProtKB Accession No. F1 PSJ0), gorilla (UniProtKB Accession No. G3SJN4), guinea pig (UniProtKB Accession No. Q9JI35), horse (UniProtKB Accession No. F6UMY9), human (UniProtKB Accession No. Q9Y5N1 ), marmoset (UniProtKB Accession No. U3EPT7), mouse (UniProtKB Accession No. P58406), orangutan (UniProtKB Accession No. H2P2I0), platypus (UniProtKB Accession No. F6VA72), rat (UniProtKB Accession No. Q9QYN8), sheep (UniProtKB Accession No. W5PY09), Tasmanian devil (UniProtKB Accession No. G3WQV4).

[000943] In some preferred embodiments, the H3R protein sequence corresponds to a human H3R protein sequence. In some embodiments, the H3R polypeptide comprises a human full-length wild-type H3R protein sequence (UniProtKB Accession No. Q9Y5N1 ), as set forth below, or a functional fragment of the wild-type H3R protein sequence.

[000944] MERAPPDGPLNASGALAGEAAAAGGARGFSAAWTAVLAALMALLIVATVLGNA LVMLAFVADSSLRTQNNFFLLNLAISDFLVGAFCIPLYVPYVLTGRWTFGRGLCKLWLVV DY LLCTSSAFNIVLISYDRFLSVTRAVSYRAQQGDTRRAVRKMLLVWVLAFLLYGPAILSWE YLS GGSSIPEGHCYAEFFYNWYFLITASTLEFFTPFLSVTFFNLSIYLNIQRRTRLRLDGARE AAG PEPPPEAQPSPPPPPGCWGCWQKGHGEAMPLHRYGVGEAAVGAEAGEATLGGGGGGG SVASPTSSSGSSSRGTERPRSLKRGSKPSASSASLEKRMKMVSQSFTQRFRLSRDRKVAK SLAVIVSIFGLCWAPYTLLMIIRAACHGHCVPDYWYETSFWLLWANSAVNPVLYPLCHHS FR RAFTKLLCPQKLKIQPHSSLEHCWK [SEQ ID NO: 38].

[000945] In one form of the invention, the H3R polypeptide comprises a truncated form of a mammalian wild-type H3R protein sequence. For example, the H3R polypeptide sequence may comprise the human wild-type H3R protein sequence with a C-terminal truncation (e.g., amino acid residues 429-445 may be truncated). Alternatively or in addition, the H3R polypeptide sequence may comprise the wild-type H3R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type H3R protein sequence (e.g., amino acid residues 177-192 may be truncated). By way of a non-limiting illustrative example, a H3R polypeptide suitable for using with the present invention comprised amino acid residues 2-176 and 193-428 of the human wild-type H3R protein sequence as set forth in SEQ ID NO: 38.

3.48 Constructs and nucleotide sequences encoding H3R polypeptides.

[000946] The present invention also encompasses isolated polynucleotide sequences and constructs encoding H3R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000947] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human H3R nucleotide (i.e., corresponding to the HRH3 gene) sequence as set forth for example in GenBank Accession Nos. NM_007232.2, AF346904.1 , AF346903.1 , AF321913.1 , AF321912.1 , AF32191 1 .1 , AF321910.1 , AF363791 .1 , BC096840.1 , AJ296652.1 and AJ278250.1 .In representative examples of this type, the polynucleotide comprises an H3R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000948] In some embodiments, an H3R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian H3R polynucleotide, or a fragment thereof. In some embodiments, the H3R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian H3R protein, or a fragment thereof under low, medium or high stringency conditions.

3.49 Muscarinic M1 receptor (M1R) polypeptides

[000949] The M1 R plays important roles in learning, memory and cognition. It is viewed as a promising target for a variety of CNS disorders, such as Alzheimer's disease, schizophrenia, and drug addiction (Thai et al., 2016).

[000950] The crystal structure of the M1 R has recently been solved, giving insight to its ligand-binding mode and activation process (Thai et al., 2016). The extracellular part of M1 R consists of the N-terminal segment (Met1 -Gly21 of the human M1 R), ECL1 (Gly89-Leu93 of the human M1 R) linking helices II and III, ECL2 (Glu170-Ser184 of the human M1 R) linking helices IV and V, and ECL3 (Lys392-Val395 of the human M1 R) linking helices VI to VII. It contains a disulphide bridge in its ectodomains (Thai et al., 2016).

[000951 ] In specific embodiments of the present invention, the M1 R polypeptide comprises a M1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a M1 R protein sequence.

[000952] In some embodiments, the M1 R protein sequence corresponds to a mammalian M1 R protein sequence. Suitable M1 R sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3VUK2), chimpanzee (UniProtKB Accession No. H2Q3X5), cow (UniProtKB Accession No. F1 N5C5), dog (UniProtKB Accession No. F1 PT23), gorilla (UniProtKB Accession No. G3RJP5), guinea pig (UniProtKB Accession No. H0W4R2), hedgehog (UniProtKB Accession No. A0A1 S3WFM0), horse (UniProtKB Accession No. F6SLG5), human (UniProtKB Accession No. P1 1229), marmoset (UniProtKB Accession No. F7IPY9), mouse (UniProtKB Accession No. P12657), pig (UniProtKB Accession No. P04761 ), rabbit (UniProtKB Accession No. G1 SR55), rat (UniProtKB Accession No. P08482).

[000953] In some preferred embodiments, the M1 R protein sequence corresponds to a human M1 R protein sequence. In some embodiments, the M1 R polypeptide comprises a human full-length wild-type M1 R protein sequence (UniProtKB Accession No. P1 1229), as set forth below, or a functional fragment of the wild-type M1 R protein sequence.

[000954] MNTSAPPAVSPNITVLAPGKGPWQVAFIGITTGLLSLATVTGNLLVLISFKVNTEL KTVNNYFLLSLACADLIIGTFSMNLYTTYLLMGHWALGTLACDLWLALDYVASNASVMNL LLI SFDRYFSVTRPLSYRAKRTPRRAALMIGLAWLVSFVLWAPAILFWQYLVGERTVLAGQCY I

QFLSQPIITFGTAMAAFYLPVTVMCTLYWRIYRETENRARELAALQGSETPGKGGGS SSSS

ERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGS EV

VIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKGRDRAGKGQKPRGKEQLAKRKTFSL VKE

KKAARTLSAILLAFILTWTPYNIMVLVSTFCKDCVPETLWELGYWLCYVNSTINPMC YALCNK

AFRDTFRLLLLCRWDKRRWRKIPKRPGSVHRTPSRQC [SEQ ID NO: 39].

[000955] In one form of the invention, the M1 R polypeptide comprises a truncated form of a mammalian wild-type M1 R protein sequence. For example, the M1 R polypeptide sequence may comprise the human wild-type M1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 435-460 may be truncated). Alternatively or in addition, the M1 R polypeptide sequence may comprise the wild-type M1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type M1 R protein sequence (e.g., amino acid residues 171 -182 may be truncated). By way of a non-limiting illustrative example, a M1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-170 and 183-434 of the human wild-type M1 R protein sequence as set forth in SEQ ID NO: 39.

3.50 Constructs and nucleotide sequences encoding M 1 R polypeptides.

[000956] The present invention also encompasses isolated polynucleotide sequences and constructs encoding M1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000957] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human M1 R nucleotide (i.e., corresponding to the CHRM1 gene) sequence as set forth for example in GenBank Accession Nos. NM_000738.2, BC007740.2, BC022984.1 , BT007166.1 and AF385587.1 . In representative examples of this type, the polynucleotide comprises a M1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000958] In some embodiments, a M1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian M1 R polynucleotide, or a fragment thereof. In some embodiments, the M1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian M1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.51 Muscarinic M2 receptor (M2R) polypeptides

[000959] The M2R plays an important role in the regulation of cardiac function, and is also involved in many CNS processes, including cognition and pain perception (Kruse et al., 2013).

[000960] To date, two crystal structures of the M2R have been published (Haga et al., 2012; Kruse et al., 2013), providing insight into the ligand-binding and activation mechanisms of the receptor. The extracellular part of M2R consists of the N-terminal segment (Met1 -Tyr18 of the human M2R), ECL1 (Gly87-Leu91 of the human M2R) linking helices II and III, ECL2 (Val168-Ser182 of the human M2R) linking helices IV and V, and ECL3 (Ala414-lle417 of the human M2R) linking helices VI to VII. It contains two disulphide bridges in its ectodomains (Haga et al, 2012).

[000961 ] In specific embodiments of the present invention, the M2R polypeptide comprises a M2R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a M2R protein sequence.

[000962] In some embodiments, the M2R protein sequence corresponds to a mammalian M2R protein sequence. Suitable M2R sequences may suitably be from mammal selected from the group comprising bat (UniProtKB Accession No. G1 PWZ4), cat (UniProtKB Accession No. M3WSG4), chimpanzee (UniProtKB Accession No. H2RAP9), cow (UniProtKB Accession No. P41985), dog (UniProtKB Accession No. F1 PWR7), gorilla (UniProtKB Accession No. G3QKB8), human (UniProtKB Accession No. P08172), marmoset (UniProtKB Accession No. F7APE0), mouse (UniProtKB Accession No. Q9ERZ4), pig (UniProtKB Accession No. P06199), rabbit (UniProtKB Accession No. G1 TBX1 ), rat (UniProtKB Accession No. P10980), sheep (UniProtKB Accession No. W5NSW2), Tasmanian devil (UniProtKB Accession No. G3VX75).

[000963] In some preferred embodiments, the M2R protein sequence corresponds to a human M2R protein sequence. In some embodiments, the M2R polypeptide comprises a human full-length wild-type M2R protein sequence (UniProtKB Accession No. P08172), as set forth below, or a functional fragment of the wild-type M2R protein sequence. [000964] MNNSTNSSNNSLALTSPYKTFEVVFIVLVAGSLSLVTIIGNILVMVSIKVNRHLQT

VNNYFLFSLACADLIIGVFSMNLYTLYTVIGYWPLGPVVCDLWLALDYVVSNASVMN LLIISFD

RYFCVTKPLTYPVKRTTKMAGMMIAAAWVLSFILWAPAILFWQFIVGVRTVEDGECY IQFFS

NAAVTFGTAIAAFYLPVIIMTVLYWHISRASKSRIKKDKKEPVANQDPVSPSLVQGR IVKPNN

NNMPSSDDGLEHNKIQNGKAPRDPVTENCVQGEEKESSNDSTSVSAVASNMRDDEIT QDE

NTVSTSLGHSKDENSKQTCIRIGTKTPKSDSCTPTNTTVEVVGSSGQNGDEKQNIVA RKIVK

MTKQPAKKKPPPSREKKVTRTILAILLAFIITWAPYNVMVLINTFCAPCIPNTVWTI GYWLCYI

NSTINPACYALCNATFKKTFKHLLMCHYKNIGATR [SEQ ID NO: 40].

[000965] In one form of the invention, the M2R polypeptide comprises a truncated form of a mammalian wild-type M2R protein sequence. For example, the M2R polypeptide sequence may comprise the human wild-type M2R protein sequence with a C-terminal truncation (e.g., amino acid residues 457-466 may be truncated). Alternatively or in addition, the M2R polypeptide sequence may comprise the wild-type M2R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type M2R protein sequence (e.g., amino acid residues 172-177 may be truncated). By way of a non-limiting illustrative example, a M2R polypeptide suitable for using with the present invention comprised amino acid residues 2-171 and 178-456 of the human wild-type M2R protein sequence as set forth in SEQ ID NO: 40.

3.52 Constructs and nucleotide sequences encoding M2R polypeptides.

[000966] The present invention also encompasses isolated polynucleotide sequences and constructs encoding M2R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000967] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human M2R nucleotide (i.e., corresponding to the CHRM2 gene) sequence as set forth for example in GenBank Accession Nos. AF498916.1 , NM_001006632.1 , NM_001006631 .1 , NM_001006630.1 , NM_001006629.1 , NM_001006628.1 , NM_001006627.1 , NM_001006626.1 , NM_000739.2, NG_01 1846.2 and M16404.1 . In representative examples of this type, the polynucleotide comprises a M2R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences. [000968] In some embodiments, a M2R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian M2R polynucleotide, or a fragment thereof. In some embodiments, the M2R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian M2R protein, or a fragment thereof under low, medium or high stringency conditions.

3.53 Muscarinic M3 receptor (M3R) polypeptides

[000969] The M3R is widely distributed both in the CNS and in peripheral tissues. It is a potential therapeutic target for several diseases, including Sjogren's syndrome, type 2 diabetes, obesity, peptic ulcer disease, overactive bladder, chronic obstructive pulmonary disease, irritable bowel syndrome and gastrointestinal spasms (Wess et al., 2007).

[000970] To date, a crystal structure of the M3R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of M3R consists of the N-terminal segment (Met1 -Thr64 of the human M3R), ECL1 (Asn132-Leu136 of the human M3R) linking helices II and III, ECL2 (Lys213-Ser227 of the human M3R) linking helices IV and V, and ECL3 (Asp518-lle521 of the human M3R) linking helices VI to VII.

[000971 ] In specific embodiments of the present invention, the M3R polypeptide comprises a M3R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a M3R protein sequence.

[000972] In some embodiments, the M3R protein sequence corresponds to a mammalian M3R protein sequence. Suitable M3R sequences may suitably be from mammal selected from the group comprising bat (UniProtKB Accession No. G1 Q9Z8), cat (UniProtKB Accession No. M3X7A6), chimpanzee (UniProtKB Accession No. Q9N2A4), cow (UniProtKB Accession No. P41984), dog (UniProtKB Accession No. F1 PGZ2), gorilla (UniProtKB Accession No. Q9N2A3), human (UniProtKB Accession No. P20309), marmoset (UniProtKB Accession No. U3D417), mouse (UniProtKB Accession No. Q9ERZ3), panda (UniProtKB Accession No. G1 MP56), pig (UniProtKB Accession No. P1 1483), rabbit (UniProtKB Accession No. G1 U308), rat (UniProtKB Accession No. P08483), sheep (UniProtKB Accession No. W5Q8R1 ). [000973] In some preferred embodiments, the M3R protein sequence corresponds to a human M3R protein sequence. In some embodiments, the M3R polypeptide comprises a human full-length wild-type M3R protein sequence (UniProtKB Accession No. P20309), as set forth below, or a functional fragment of the wild-type M3R protein sequence.

[000974] MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPD

GTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSL ACADLIIGV

ISMNLFTTYIIMNRWALGNLACDLWLAIDYVASNASVMNLLVISFDRYFSITRPLTY RAKRTTK

RAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAF YMPVTI

MTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPTGSSRSCSSYELQQQSMKRS NRRK

YGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGSETRAI YSI

VLKLPGHSTILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSF SKLPI

QLESAVDTAKTSDVNSSVGKSTATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKE KKAAQ

TLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCN KTFRTTF

KMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL [SEQ ID NO: 41 ].

[000975] In one form of the invention, the M3R polypeptide comprises a truncated form of a mammalian wild-type M3R protein sequence. For example, the M3R polypeptide sequence may comprise the human wild-type M3R protein sequence with a C-terminal truncation (e.g., amino acid residues 560-590 may be truncated). Alternatively or in addition, the M3R polypeptide sequence may comprise the wild-type M3R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type M3R protein sequence (e.g., amino acid residues 216-221 may be truncated). By way of a non-limiting illustrative example, a M3R polypeptide suitable for using with the present invention comprised amino acid residues 2-215 and 222-559 of the human wild-type M3R protein sequence as set forth in SEQ ID NO: 41 .

3.54 Constructs and nucleotide sequences encoding M3R polypeptides.

[000976] The present invention also encompasses isolated polynucleotide sequences and constructs encoding M3R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000977] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human M3R nucleotide (i.e., corresponding to the CHRM3 gene) sequence as set forth for example in GenBank Accession Nos. AF498917.1 , NG_032046.2, NM_001347716.1 , NM_000740.3, AH01 1672.2, AF385589.1 , U29589.1 , AB041395.1 , BC096844.1 and BC121026.2. In representative examples of this type, the polynucleotide comprises a M3R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000978] In some embodiments, a M3R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian M3R polynucleotide, or a fragment thereof. In some embodiments, the M3R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian M3R protein, or a fragment thereof under low, medium or high stringency conditions.

3.55 Neuropeptide Y1 receptor (Y1R) polypeptides

[000979] The Y1 R is expressed in a variety of tissues including the brain, heart, kidney, and gastrointestinal tract, and early studies showed that it mediated vasoconstriction and anxiolytic effects (Michel et al., 1998). It has been implicated in a variety of diseases, such as obesity, anxiety and depression, alcohol dependence, bone metabolism, pain, cancer, cardiovascular disease, intestinal disease, circadian disorders and Alzheimer's disease (Brothers & Wahlestedt, 2010).

[000980] To date, a crystal structure of the Y1 R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of Y1 R consists of the N-terminal segment (Met1 -A38 of the human Y1 R), ECL1 (Glu104-Phe108 of the human Y1 R) linking helices II and III, ECL2 (Val178-Ser204 of the human Y1 R) linking helices IV and V, and ECL3 (His290-Ala294 of the human Y1 R) linking helices VI to VII.

[000981 ] In specific embodiments of the present invention, the Y1 R polypeptide comprises a Y1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a Y1 R protein sequence.

[000982] In some embodiments, the Y1 R protein sequence corresponds to a mammalian Y1 R protein sequence. Suitable Y1 R sequences may suitably be from mammal selected from the group comprising bat (UniProtKB Accession No. G1 P047), cat (UniProtKB Accession No. M3W4X9), chimpanzee (UniProtKB Accession No. H2QQD5), cow (UniProtKB Accession No. Q1 RMU8), dog (UniProtKB Accession No. 002813), gorilla (UniProtKB Accession No. G3QP89), guinea pig (UniProtKB Accession No. Q9WVD0), human (UniProtKB Accession No. P25929), marmoset (UniProtKB Accession No. F7I8A1 ), mouse (UniProtKB Accession No. Q04573), pig (UniProtKB Accession No. 002835), rabbit (UniProtKB Accession No. B6VRS4), rat (UniProtKB Accession No. P21555), sheep (UniProtKB Accession No. W5PW25).

[000983] In some preferred embodiments, the Y1 R protein sequence corresponds to a human Y1 R protein sequence. In some embodiments, the Y1 R polypeptide comprises a human full-length wild-type Y1 R protein sequence (UniProtKB Accession No. P25929), as set forth below, or a functional fragment of the wild-type Y1 R protein sequence.

[000984] MNSTLFSQVENHSVHSNFSEKNAQLLAFENDDCHLPLAMIFTLALAYGAVIILGV

SGNLALIIIILKQKEMRNVTNILIVNLSFSDLLVAIMCLPFTFVYTLMDHWVFGEAM CKLNPFV

QCVSITVSIFSLVLIAVERHQLIINPRGWRPNNRHAYVGIAVIWVLAVASSLPFLIY QVMTDEP

FQNVTLDAYKDKYVCFDQFPSDSHRLSYTTLLLVLQYFGPLCFIFICYFKIYIRLKR RNNMMD

KMRDNKYRSSETKRINIMLLSIVVAFAVCWLPLTIFNTVFDWNHQIIATCNHNLLFL LCHLTAM

ISTCVNPIFYGFLNKNFQRDLQFFFNFCDFRSRDDDYETIAMSTMHTDVSKTSLKQA SPVAF

KKINNNDDNEKI [SEQ ID NO: 42].

[000985] In one form of the invention, the Y1 R polypeptide comprises a truncated form of a mammalian wild-type Y1 R protein sequence. For example, the Y1 R polypeptide sequence may comprise the human wild-type Y1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 336-384 may be truncated). Alternatively or in addition, the Y1 R polypeptide sequence may comprise the wild-type Y1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type Y1 R protein sequence (e.g., amino acid residues 183-195 may be truncated). By way of a non-limiting illustrative example, a Y1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-182 and 196-335 of the human wild-type Y1 R protein sequence as set forth in SEQ ID NO: 42.

3.56 Constructs and nucleotide sequences encoding Y1R polypeptides.

[000986] The present invention also encompasses isolated polynucleotide sequences and constructs encoding Y1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[000987] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human Y1 R nucleotide (i.e., corresponding to the NPY1 R gene) sequence as set forth for example in GenBank Accession Nos. AY548168.1 , NM_000909.5, BC071720.1 , BC036657.2 and AK312578.1 . In representative examples of this type, the polynucleotide comprises a Y1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000988] In some embodiments, a Y1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian Y1 R polynucleotide, or a fragment thereof. In some embodiments, the Y1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian Y1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.57 Neurotensin 1 receptor (NTS1R) polypeptides

[000989] The NTS1 R mediates most of the known effects of neurotensin, such as the modulation of dopamine neurotransmission, hypothermia, anti-nociception, and promotion of the growth of cancer cells. It has also been implicated in Parkinson's disease and the pathogenesis of schizophrenia (White et al., 2012).

[000990] A crystal structure of the NTS1 R has been published (White et al., 2012), providing insight into the ligand-binding and activation mechanisms of the receptor. The extracellular part of NTS1 R consists of the N-terminal segment (Met1 -Asp59 of the human NTS1 R), ECL1 (His131 -Phe136 of the human NTS1 R) linking helices II and III, ECL2 (Gly208-His229 of the human NTS1 R) linking helices IV and V, and ECL3 (Ser330-Trp334 of the human NTS1 R) linking helices VI to VII. It has a disufide bond linking helices ECL2 and III (White et al., 2012).

[000991 ] In specific embodiments of the present invention, the NTS1 R polypeptide comprises a NTS1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a NTS1 R protein sequence.

[000992] In some embodiments, the NTS1 R protein sequence corresponds to a mammalian NTS1 R protein sequence. Suitable NTS1 R sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3X9J3), chimpanzee (UniProtKB Accession No. H2QKR3), cow (UniProtKB Accession No. F1 MWG2), dog (UniProtKB Accession No. E2RN63), gorilla (UniProtKB Accession No. G3RLF9), guinea pig (UniProtKB Accession No. H0W4P3), hedgehog (UniProtKB Accession No. A0A1 S2ZJT3), horse (UniProtKB Accession No. F7BSR0), human (UniProtKB Accession No. P30989), marmoset (UniProtKB Accession No. U3D2G1 ), mouse (UniProtKB Accession No. 088319), rat (UniProtKB Accession No. P20789), sheep (UniProtKB Accession No. W5PSM7), Tasmanian devil (UniProtKB Accession No. G3VE69).

[000993] In some preferred embodiments, the NTS1 R protein sequence corresponds to a human NTS1 R protein sequence. In some embodiments, the NTS1 R polypeptide comprises a human full-length wild-type NTS1 R protein sequence (UniProtKB Accession No. P30989), as set forth below, or a functional fragment of the wild-type NTS1 R protein sequence.

[000994] MRLNSSAPGTPGTPAADPFQRAQAGLEEALLAPGFGNASGNASERVLAAPSS ELDVNTDIYSKVLVTAVYLALFVVGTVGNTVTAFTLARKKSLQSLQSTVHYHLGSLALSD LLT LLLAMPVELYNFIWVHHPWAFGDAGCRGYYFLRDACTYATALNVASLSVERYLAICHPFK A KTLMSRSRTKKFISAIWLASALLAVPMLFTMGEQNRSADGQHAGGLVCTPTIHTATVKVV IQ VNTFMSFIFPMVVISVLNTIIANKLTVMVRQAAEQGQVCTVGGEHSTFSMAIEPGRVQAL RH GVRVLRAVVIAFVVCWLPYHVRRLMFCYISDEQWTPFLYDFYHYFYMVTNALFYVSSTIN PI LYNLVSANFRHIFLATLACLCPVWRRRRKRPAFSRKADSVSSNHTLSSNATRETLY [SEQ ID NO: 43].

[000995] In one form of the invention, the NTS1 R polypeptide comprises a truncated form of a mammalian wild-type NTS1 R protein sequence. For example, the NTS1 R polypeptide sequence may comprise the human wild-type NTS1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 380-418 may be truncated). Alternatively or in addition, the NTS1 R polypeptide sequence may comprise the wild-type NTS1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type NTS1 R protein sequence (e.g., amino acid residues 213-223 may be truncated). By way of a non-limiting illustrative example, a NTS1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-212 and 224-379 of the human wild-type NTS1 R protein sequence as set forth in SEQ ID NO: 43.

3.58 Constructs and nucleotide sequences encoding NTS 1 R polypeptides.

[000996] The present invention also encompasses isolated polynucleotide sequences and constructs encoding NTS1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs. [000997] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human NTS1 R nucleotide (i.e., corresponding to the NTS1 R gene) sequence as set forth for example in GenBank Accession Nos. AY429106.1 and NM 002531 .2. In representative examples of this type, the polynucleotide comprises a NTS1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[000998] In some embodiments, a NTS1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian NTS1 R polynucleotide, or a fragment thereof. In some embodiments, the NTS1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian NTS1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.59 Orexin receptor 1 (0X1 R) polypeptides

[000999] The OX1 R has important roles in the regulation of feeding, reward, nociception and stress. It is believed that antagonism of the 0X1 R could be beneficial in treating sleep disorders, obesity, pain and addiction (Yin et al., 2016).

[0001000] A crystal structure of the 0X1 R has been published (Yin et al., 2016), providing insight into the ligand-binding and activation mechanisms of the receptor. The extracellular part of OX1 R consists of the N-terminal segment (Met1 -Tyr45 of the human OX1 R), ECL1 (Glu1 10-Phe1 14 of the human OX1 R) linking helices II and III, ECL2 (Glu184-Asp208 of the human OX1 R) linking helices IV and V, and ECL3 (Gly325-Asp332 of the human OX1 R) linking helices VI to VII.

[0001001 ] In specific embodiments of the present invention, the OX1 R polypeptide comprises a 0X1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an 0X1 R protein sequence.

[0001002] In some embodiments, the 0X1 R protein sequence corresponds to a mammalian 0X1 R protein sequence. Suitable 0X1 R sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3WN13), chimpanzee (UniProtKB Accession No. H2PYJ1 ), cow (UniProtKB Accession No. Q0GBZ5), dog (UniProtKB Accession No. E2R2A3), guinea pig (UniProtKB Accession No. H0V9F2), horse (UniProtKB Accession No. F6Y1 U3), human (UniProtKB Accession No. 043613), marmoset (UniProtKB Accession No. F7H2M7), mouse (UniProtKB Accession No. P58307), pig (UniProtKB Accession No. F1 SVA1 ), pig (UniProtKB Accession No. 097661 ), rabbit (UniProtKB Accession No. G1 SPJ3), rat (UniProtKB Accession No. P56718), sheep (UniProtKB Accession No. W5NTE0).

[0001003] In some preferred embodiments, the 0X1 R protein sequence corresponds to a human 0X1 R protein sequence. In some embodiments, the 0X1 R polypeptide comprises a human full-length wild-type 0X1 R protein sequence (UniProtKB Accession No. 043613), as set forth below, or a functional fragment of the wild-type 0X1 R protein sequence.

[0001004] MEPSATPGAQMGVPPGSREPSPVPPDYEDEFLRYLWRDYLYPKQYEWVLIAA

YVAVFVVALVGNTLVCLAVWRNHHMRTVTNYFIVNLSLADVLVTAICLPASLLVDIT ESWLFG

HALCKVIPYLQAVSVSVAVLTLSFIALDRWYAICHPLLFKSTARRARGSILGIWAVS LAIMVPQ

AAVMECSSVLPELANRTRLFSVCDERWADDLYPKIYHSCFFIVTYLAPLGLMAMAYF QIFRK

LWGRQIPGTTSALVRNWKRPSDQLGDLEQGLSGEPQPRARAFLAEVKQMRARRKTAK ML

MVVLLVFALCYLPISVLNVLKRVFGMFRQASDREAVYACFTFSHWLVYANSAANPII YNFLS

GKFREQFKAAFSCCLPGLGPCGSLKAPSPRSSASHKSLSLQSRCSISKISEHVVLTS VTTVL

P [SEQ ID NO: 44].

[0001005] In one form of the invention, the 0X1 R polypeptide comprises a truncated form of a mammalian wild-type 0X1 R protein sequence. For example, the 0X1 R polypeptide sequence may comprise the human wild-type 0X1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 374-425 may be truncated). Alternatively or in addition, the 0X1 R polypeptide sequence may comprise the wild-type 0X1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type 0X1 R protein sequence (e.g., amino acid residues 189-196 may be truncated). By way of a non-limiting illustrative example, a 0X1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-188 and 197-373 of the human wild-type 0X1 R protein sequence as set forth in SEQ ID NO: 44.

3.60 Constructs and nucleotide sequences encoding 0X1 R polypeptides.

[0001006] The present invention also encompasses isolated polynucleotide sequences and constructs encoding 0X1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs. [0001007] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human 0X1 R nucleotide (i.e., corresponding to the HCRTR1 gene) sequence as set forth for example in GenBank Accession Nos. AF041243.1 , NM 001525.2 and BC074796.2. In representative examples of this type, the polynucleotide comprises an 0X1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001008] In some embodiments, an 0X1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian 0X1 R polynucleotide, or a fragment thereof. In some embodiments, the 0X1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian 0X1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.61 Orexin receptor 2 ( 0X2 R) polypeptides

[0001009] Like the 0X1 R, it is believed that the OX2R could be beneficial in treating sleep disorders, obesity, pain and addiction. However the OX2R has distinct physiological functions from the 0X1 R, as it predominantly mediates the pro-arousal effects of the orexin peptides (Yin et al., 2016).

[0001010] A crystal structure of the OX2R has been published (Yin et al., 2015), providing insight into the ligand-binding and activation mechanisms of the receptor. The extracellular part of OX2R consists of the N-terminal segment (Met1 -Gu52 of the human OX2R), ECL1 (Glu1 18-Phe122 of the human OX2R) linking helices II and III, ECL2 (Glu192-Gly216 of the human OX2R) linking helices IV and V, and ECL3 (Gly331 -Asp338 of the human OX2R) linking helices VI to VII.

[000101 1 ] In specific embodiments of the present invention, the OX2R polypeptide comprises a OX2R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an OX2R protein sequence.

[0001012] In some embodiments, the OX2R protein sequence corresponds to a mammalian OX2R protein sequence. Suitable OX2R sequences may suitably be from mammal selected from the group comprising cat (UniProtKB Accession No. M3X961 ), chimpanzee (UniProtKB Accession No. H2QT82), cow (UniProtKB Accession No. F1 MG23), dog (UniProtKB Accession No. Q9TUP7), gorilla (UniProtKB Accession No. G3SII0), horse (UniProtKB Accession No. F6XS13), human (UniProtKB Accession No. 043614), marmoset (UniProtKB Accession No. F7D3X8), mouse (UniProtKB Accession No. P58308), panda (UniProtKB Accession No. G1 MGF7), pig (UniProtKB Accession No. 062809), rabbit (UniProtKB Accession No. G1 STQ8), rat (UniProtKB Accession No. P56719), sheep (UniProtKB Accession No. W5P9D8).

[0001013] In some preferred embodiments, the OX2R protein sequence corresponds to a human OX2R protein sequence. In some embodiments, the OX2R polypeptide comprises a human full-length wild-type OX2R protein sequence (UniProtKB Accession No. 043614), as set forth below, or a functional fragment of the wild-type OX2R protein sequence.

[0001014] MSGTKLEDSPPCRNWSSASELNETQEPFLNPTDYDDEEFLRYLWREYLHPKE

YEWVLIAGYIIVFVVALIGNVLVCVAVWKNHHMRTVTNYFIVNLSLADVLVTITCLP ATLVVDIT

ETWFFGQSLCKVIPYLQTVSVSVSVLTLSCIALDRWYAICHPLMFKSTAKRARNSIV IIWIVSC

IIMIPQAIVMECSTVFPGLANKTTLFTVCDERWGGEIYPKMYHICFFLVTYMAPLCL MVLAYL

QIFRKLWCRQIPGTSSVVQRKWKPLQPVSQPRGPGQPTKSRMSAVAAEIKQIRARRK TAR

MLMIVLLVFAICYLPISILNVLKRVFGMFAHTEDRETVYAWFTFSHWLVYANSAANP IIYNFLS

GKFREEFKAAFSCCCLGVHHRQEDRLTRGRTSTESRKSLTTQISNFDNISKLSEQVV LTSIS

TLPAANGAGPLQNW [SEQ ID NO: 45].

[0001015] In one form of the invention, the OX2R polypeptide comprises a truncated form of a mammalian wild-type OX2R protein sequence. For example, the OX2R polypeptide sequence may comprise the human wild-type OX2R protein sequence with a C-terminal truncation (e.g., amino acid residues 380-444 may be truncated). Alternatively or in addition, the OX2R polypeptide sequence may comprise the wild-type OX2R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type OX2R protein sequence (e.g., amino acid residues 195-204 may be truncated). By way of a non-limiting illustrative example, a OX2R polypeptide suitable for using with the present invention comprised amino acid residues 2-194 and 205-379 of the human wild-type OX2R protein sequence as set forth in SEQ ID NO: 45.

3.62 Constructs and nucleotide sequences encoding OX2R polypeptides.

[0001016] The present invention also encompasses isolated polynucleotide sequences and constructs encoding OX2R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs. [0001017] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human OX2R nucleotide (i.e., corresponding to the HCRTR2 gene) sequence as set forth for example in GenBank Accession Nos. AF041245.2, NM 001526.4, KC812500.1 , KC812499.1 , NG_012447.2 and AF283760.1 . In representative examples of this type, the polynucleotide comprises an OX2R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001018] In some embodiments, an OX2R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian OX2R polynucleotide, or a fragment thereof. In some embodiments, the OX2R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian OX2R protein, or a fragment thereof under low, medium or high stringency conditions.

3.63 Prostaglandin E 1 receptor (EP1R) polypeptides

[0001019] The EP1 R mediates stress responses (ACTH secretion and stress behaviour), promotes chemical carcinogenesis and mediates inflammatory thermal hyperalgesia (Sugimoto & Narumiya, 2007).

[0001020] To date, a crystal structure of the EP1 R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of EP1 R consists of the N-terminal segment (Met1 -Ser27 of the human EP1 R), ECL1 (Ala101 -Ala106 of the human EP1 R) linking helices II and III, ECL2 (Val176-Arg199 of the human EP1 R) linking helices IV and V, and ECL3 (Trp324-Ser328 of the human EP1 R) linking helices VI to VII.

[0001021 ] In specific embodiments of the present invention, the EP1 R polypeptide comprises a EP1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an EP1 R protein sequence.

[0001022] In some embodiments, the EP1 R protein sequence corresponds to a mammalian EP1 R protein sequence. Suitable EP1 R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P34995), bat (UniProtKB Accession No. G1 PBB3), cow (UniProtKB Accession No. F1 MUY4), dog (UniProtKB Accession No. Q9BGL8), frog (UniProtKB Accession No. F7C7N3), gorilla (UniProtKB Accession No. G3S6M0), macaque (UniProtKB Accession No. Q564H5), marmoset (UniProtKB Accession No. F7HTH9), monkey (UniProtKB Accession No. A0A0D9R1 Y9), mouse (UniProtKB Accession No. P35375), panda (UniProtKB Accession No. G1 M4C2), rat (UniProtKB Accession No. P70597), Tasmanian devil (UniProtKB Accession No. G3VPS1 ).

[0001023] In some preferred embodiments, the EP1 R protein sequence corresponds to a human EP1 R protein sequence. In some embodiments, the EP1 R polypeptide comprises a human full-length wild-type EP1 R protein sequence (UniProtKB Accession No. P34995), as set forth below, or a functional fragment of the wild-type EP1 R protein sequence.

[0001024] MSPCGPLNLSLAGEATTCAAPWVPNTSAVPPSGASPALPIFSMTLGAVSNLLAL

ALLAQAAGRLRRRRSAATFLLFVASLLATDLAGHVIPGALVLRLYTAGRAPAGGACH FLGGC

MVFFGLCPLLLGCGMAVERCVGVTRPLLHAARVSVARARLALAAVAAVALAVALLPL ARVG

RYELQYPGTWCFIGLGPPGGWRQALLAGLFASLGLVALLAALVCNTLSGLALLRARW RRRS

RRPPPASGPDSRRRWGAHGPRSASASSASSIASASTFFGGSRSSGSARRARAHDVEM VG

QLVGIMVVSCICWSPMLVLVALAVGGWSSTSLQRPLFLAVRLASWNQILDPWVYILL RQAVL

RQLLRLLPPRAGAKGGPAGLGLTPSAWEASSLRSSRHSGLSHF [SEQ ID NO: 46].

[0001025] In one form of the invention, the EP1 R polypeptide comprises a truncated form of a mammalian wild-type EP1 R protein sequence. For example, the EP1 R polypeptide sequence may comprise the human wild-type EP1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 378-402 may be truncated). Alternatively or in addition, the EP1 R polypeptide sequence may comprise the wild-type EP1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type EP1 R protein sequence (e.g., amino acid residues 185-189 may be truncated). By way of a non-limiting illustrative example, a EP1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-184 and 190-377 of the human wild-type EP1 R protein sequence as set forth in SEQ ID NO: 46.

3.64 Constructs and nucleotide sequences encoding EP 1 R polypeptides.

[0001026] The present invention also encompasses isolated polynucleotide sequences and constructs encoding EP1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001027] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human EP1 R nucleotide (i.e., corresponding to the PTGER1 gene) sequence as set forth for example in GenBank Accession Nos. AY275470.1 , NM 000955.2 and BC029768.1 . In representative examples of this type, the polynucleotide comprises an EP1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001028] In some embodiments, an EP1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian EP1 R polynucleotide, or a fragment thereof. In some embodiments, the EP1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian EP1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.65 Serotonin 5-HT1a receptor (5-HT1aR) polypeptides

[0001029] The 5-HT1 aR is involved in mediating anxiety, stress responses, depression, schizophrenia and addiction. It is also believed to have neuroprotective actions against ischemic brain damage (Nichols & Nichols, 2008).

[0001030] To date, a crystal structure of the 5-HT1 aR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of 5- HT1 aR consists of the N-terminal segment (Met1 -Thr32 of the human 5-HT1 aR), ECL1 (Asn100-Leu104 of the human 5-HT1 aR) linking helices II and III, ECL2 (Trp175-Lys191 of the human 5-HT1 aR) linking helices IV and V, and ECL3 (Cys371 -Met377 of the human 5- HT1 aR) linking helices VI to VII. It is believed to form a disulphide bridge between helices III and ECL2 (Nichols & Nichols, 2008).

[0001031 ] In specific embodiments of the present invention, the 5-HT1 aR polypeptide comprises a 5-HT1 aR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a 5-HT1 aR protein sequence.

[0001032] In some embodiments, the 5-HT1 aR protein sequence corresponds to a mammalian 5-HT1 aR protein sequence. Suitable 5-HT1 aR sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P08908), alligator (UniProtKB Accession No. A0A151 MXE8), chicken (UniProtKB Accession No. D2K8P9), chimpanzee (UniProtKB Accession No. Q9N298), dog (UniProtKB Accession No. Q6XXX9), fox (UniProtKB Accession No. Q6XXY0), frog (UniProtKB Accession No. Q98998), gorilla (UniProtKB Accession No. Q9N297), horse (UniProtKB Accession No. Q0EAB6), mouse (UniProtKB Accession No. Q64264), orangutan (UniProtKB Accession No. Q9N296), rat (UniProtKB Accession No. P19327).

[0001033] In some preferred embodiments, the 5-HT1 aR protein sequence corresponds to a human 5-HT1 aR protein sequence. In some embodiments, the 5-HT1 aR polypeptide comprises a human full-length wild-type 5-HT1 aR protein sequence (UniProtKB Accession No. P08908), as set forth below, or a functional fragment of the wild-type 5-HT1 aR protein sequence.

[0001034] MDVLSPGQGNNTTSPPAPFETGGNTTGISDVTVSYQVITSLLLGTLIFCAVLGNA

CVVAAIALERSLQNVANYLIGSLAVTDLMVSVLVLPMAALYQVLNKWTLGQVTCDLF IALDVL

CCTSSILHLCAIALDRYWAITDPIDYVNKRTPRRAAALISLTWLIGFLISIPPMLGW RTPEDRS

DPDACTISKDHGYTIYSTFGAFYIPLLLMLVLYGRIFRAARFRIRKTVKKVEKTGAD TRHGAS

PAPQPKKSVNGESGSRNWRLGVESKAGGALCANGAVRQGDDGAALEVIEVHRVGNSK EH

LPLPSEAGPTPCAPASFERKNERNAEAKRKMALARERKTVKTLGIIMGTFILCWLPF FIVALV

LPFCESSCHMPTLLGAIINWLGYSNSLLNPVIYAYFNKDFQNAFKKIIKCKFCRQ [SEQ ID NO:

47].

[0001035] In one form of the invention, the 5-HT1 aR polypeptide comprises a truncated form of a mammalian wild-type 5-HT1 aR protein sequence. For example, the 5-HT1 aR polypeptide sequence may comprise the human wild-type 5-HT1 aR protein sequence with a C-terminal truncation (e.g., amino acid residues 416-2 may be truncated). Alternatively or in addition, the 5-HT1 aR polypeptide sequence may comprise the wild-type 5-HT1 aR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N- terminal truncation, a truncation may be performed to remove an internal section of the wild- type 5-HT1 aR protein sequence (e.g., amino acid residues 181 -187 may be truncated). By way of a non-limiting illustrative example, a 5-HT1 aR polypeptide suitable for using with the present invention comprised amino acid residues 2-180 and 188-415 of the human wild-type 5-HT1 aR protein sequence as set forth in SEQ ID NO: 47.

3.66 Constructs and nucleotide sequences encoding 5-HT1aR polypeptides.

[0001036] The present invention also encompasses isolated polynucleotide sequences and constructs encoding 5-HT1 aR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs. [0001037] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human 5-HT1 aR nucleotide (i.e., corresponding to the HTR1 A gene) sequence as set forth for example in GenBank Accession Nos. NM 000524.3, AF498978.1 , NG_032816.1 , AB041403.1 , BC136263.1 and BC069159.1 . In representative examples of this type, the polynucleotide comprises a 5-HT1 aR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001038] In some embodiments, a 5-HT1 aR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian 5-HT1 aR polynucleotide, or a fragment thereof. In some embodiments, the 5-HT1 aR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian 5-HT1 aR protein, or a fragment thereof under low, medium or high stringency conditions.

3.67 Serotonin 5-HT2a receptor (5-HT2aR) polypeptides

[0001039] In the CNS, the 5-HT2aR is one of the key sites for hallucinogenic action. In the periphery, the 5-HT2aR has various other functions, including proliferation of arterial fibroblasts, migration of aortic smooth muscle cells, arterial vasoconstriction and analgesia (Nichols & Nichols, 2008).

[0001040] To date, a crystal structure of the 5-HT2aR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of 5- HT2aR consists of the N-terminal segment (Met1 -Glu73 of the human 5-HT2aR), ECL1 (Gly138-Leu143 of the human 5-HT2aR) linking helices II and III, ECL2 (Leu215-Ala-230of the human 5-HT2aR) linking helices IV and V, and ECL3 (Lys350-Asn354 of the human 5- HT2aR) linking helices VI to VII. It is believed to form a disulphide bridge between helices III and ECL2 (Nichols & Nichols, 2008).

[0001041 ] In specific embodiments of the present invention, the 5-HT2aR polypeptide comprises a 5-HT2aR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with a 5-HT2aR protein sequence.

[0001042] In some embodiments, the 5-HT2aR protein sequence corresponds to a mammalian 5-HT2aR protein sequence. Suitable 5-HT2aR sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P28223), chicken (UniProtKB Accession No. E1 BVI5), cow (UniProtKB Accession No. Q75Z89), dog (UniProtKB Accession No. 046635), hamster (UniProtKB Accession No. P18599), hedgehog (UniProtKB Accession No. A0A1 S2ZLM4), macaque (UniProtKB Accession No. P50128), mouse (UniProtKB Accession No. P35363), orangutan (UniProtKB Accession No. Q5R4Q6), pig (UniProtKB Accession No. P50129), rat (UniProtKB Accession No. P14842).

[0001043] In some preferred embodiments, the 5-HT2aR protein sequence corresponds to a human 5-HT2aR protein sequence. In some embodiments, the 5-HT2aR polypeptide comprises a human full-length wild-type 5-HT2aR protein sequence (UniProtKB Accession No. P28223), as set forth below, or a functional fragment of the wild-type 5-HT2aR protein sequence.

[0001044] MDILCEENTSLSSTTNSLMQLNDDTRLYSNDFNSGEANTSDAFNWTVDSENRT

NLSCEGCLSPSCLSLLHLQEKNWSALLTAVVIILTIAGNILVIMAVSLEKKLQNATN YFLMSLAI

ADMLLGFLVMPVSMLTILYGYRWPLPSKLCAVWIYLDVLFSTASIMHLCAISLDRYV AIQNPIH

HSRFNSRTKAFLKIIAVWTISVGISMPIPVFGLQDDSKVFKEGSCLLADDNFVLIGS FVSFFIPL

TIMVITYFLTIKSLQKEATLCVSDLGTRAKLASFSFLPQSSLSSEKLFQRSIHREPG SYTGRRT

MQSISNEQKACKVLGIVFFLFVVMWCPFFITNIMAVICKESCNEDVIGALLNVFVWI GYLSSA

VNPLVYTLFNKTYRSAFSRYIQCQYKENKKPLQLILVNTIPALAYKSSQLQMGQKKN SKQDA

KTTDNDCSMVALGKQHSEEASKDNSDGVNEKVSCV [SEQ ID NO: 48].

[0001045] In one form of the invention, the 5-HT2aR polypeptide comprises a truncated form of a mammalian wild-type 5-HT2aR protein sequence. For example, the 5-HT2aR polypeptide sequence may comprise the human wild-type 5-HT2aR protein sequence with a C-terminal truncation (e.g., amino acid residues 396-471 may be truncated). Alternatively or in addition, the 5-HT2aR polypeptide sequence may comprise the wild-type 5-HT2aR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N- terminal truncation, a truncation may be performed to remove an internal section of the wild- type 5-HT2aR protein sequence (e.g., amino acid residues 219-224 may be truncated). By way of a non-limiting illustrative example, a 5-HT2aR polypeptide suitable for using with the present invention comprised amino acid residues 2-218 and 225-397 of the human wild-type 5-HT2aR protein sequence as set forth in SEQ ID NO: 48.

3.68 Constructs and nucleotide sequences encoding 5-HT2aR polypeptides.

[0001046] The present invention also encompasses isolated polynucleotide sequences and constructs encoding 5-HT2aR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001047] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human 5-HT2aR nucleotide (i.e., corresponding to the HTR2A gene) sequence as set forth for example in GenBank Accession Nos. NM 000621 .4, NM_001 165947.2, NG_01301 1 .1 , AF498982.1 , EU796424.1 , EU796429.1 , EU796434.1 , KC603613.1 , BC074849.2, BC074848.2, BC069576.1 and BC069356.1 . In representative examples of this type, the polynucleotide comprises a 5-HT2aR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001048] In some embodiments, a 5-HT2aR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian 5-HT2aR polynucleotide, or a fragment thereof. In some embodiments, the 5-HT2aR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian 5-HT2aR protein, or a fragment thereof under low, medium or high stringency conditions.

3.69 Serotonin 5-HT2b receptor (5-HT2bR) polypeptides

[0001049] In the CNS, the 5-HT2bR appears to have a role in the auditory system, and is associated with vulnerability to drug abuse. However it is more highly expressed in the periphery where it is important during development, coordinating the proper formation of the heart and brain (Nichols & Nichols, 2008).

[0001050] To date, two studies have reported solving the crystal structure of the CXCR4, providing insight into how it binds its ligands (Liu et al., 2013; Wacker et al., 2013). The extracellular part of 5-HT2bR consists of the N-terminal segment (Met1 -Lys53 of the human 5-HT2bR), ECL1 (Glu1 18-Leu123 of the human 5-HT2bR) linking helices II and III, ECL2 (Ile195-Arg213 of the human 5-HT2bR) linking helices IV and V, and ECL3 (Asp351 -Asn354 of the human 5-HT2bR) linking helices VI to VII. It is believed to form a disulphide bridge between helix III and ECL2 (Nichols & Nichols, 2008).

[0001051 ] In specific embodiments of the present invention, the 5-HT2bR polypeptide comprises a 5-HT2bR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an 5-HT2bR protein sequence.

[0001052] In some embodiments, the 5-HT2bR protein sequence corresponds to a mammalian 5-HT2bR protein sequence. Suitable 5-HT2bR sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P41595), cat (UniProtKB Accession No. M3WRH8), catfish (UniProtKB Accession No. W5UAH8), chimpanzee (UniProtKB Accession No. H2QJL0), cow (UniProtKB Accession No. F6QI78), frog (UniProtKB Accession No. F6WGJ8), guinea pig (UniProtKB Accession No. A3RL34), hedgehig (UniProtKB Accession No. A0A1 S2ZHK9), horse (UniProtKB Accession No. F6V714), macaque (UniProtKB Accession No. F7HQF4), mouse (UniProtKB Accession No. Q02152), rat (UniProtKB Accession No. P30994), sheep (UniProtKB Accession No. W5QHN7), zebrafish (UniProtKB Accession No. Q0GH74).

[0001053] In some preferred embodiments, the 5-HT2bR protein sequence corresponds to a human 5-HT2bR protein sequence. In some embodiments, the 5-HT2bR polypeptide comprises a human full-length wild-type 5-HT2bR protein sequence (UniProtKB Accession No. P41595), as set forth below, or a functional fragment of the wild-type 5-HT2bR protein sequence.

[0001054] MALSYRVSELQSTIPEHILQSTFVHVISSNWSGLQTESIPEEMKQIVEEQGNKLH

WAALLILMVIIPTIGGNTLVILAVSLEKKLQYATNYFLMSLAVADLLVGLFVMPIAL LTIMFEAM

WPLPLVLCPAWLFLDVLFSTASIMHLCAISVDRYIAIKKPIQANQYNSRATAFIKIT VVWLISIGI

AIPVPIKGIETDVDNPNNITCVLTKERFGDFMLFGSLAAFFTPLAIMIVTYFLTIHA LQKKAYLV

KNKPPQRLTWLTVSTVFQRDETPCSSPEKVAMLDGSRKDKALPNSGDETLMRRTSTI GKK

SVQTISNEQRASKVLGIVFFLFLLMWCPFFITNITLVLCDSCNQTTLQMLLEIFVWI GYVSSGV

NPLVYTLFNKTFRDAFGRYITCNYRATKSVKTLRKRSSKIYFRNPMAENSKFFKKHG IRNGIN

PAMYQSPMRLRSSTIQSSSIILLDTLLLTENEGDKTEEQVSYV [SEQ ID NO: 49].

[0001055] In one form of the invention, the 5-HT2bR polypeptide comprises a truncated form of a mammalian wild-type 5-HT2bR protein sequence. For example, the 5-HT2bR polypeptide sequence may comprise the human wild-type 5-HT2bR protein sequence with a C-terminal truncation (e.g., amino acid residues 396-481 may be truncated). Alternatively or in addition, the 5-HT2bR polypeptide sequence may comprise the wild-type 5-HT2bR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N- terminal truncation, a truncation may be performed to remove an internal section of the wild- type 5-HT2bR protein sequence (e.g., amino acid residues 200-208 may be truncated). By way of a non-limiting illustrative example, a 5-HT2bR polypeptide suitable for using with the present invention comprised amino acid residues 2-199 and 209-395 of the human wild-type 5-HT2bR protein sequence as set forth in SEQ ID NO: 49.

3.70 Constructs and nucleotide sequences encoding 5-HT2bR polypeptides.

[0001056] The present invention also encompasses isolated polynucleotide sequences and constructs encoding 5-HT2bR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001057] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human 5-HT2bR nucleotide (i.e., corresponding to the HTR2B gene) sequence as set forth for example in GenBank Accession Nos. NM 001320758.1 , NM_000867.4, EU796439.1 , EU796444.1 , AY1 14103.1 , AH007819.3, AY136751 .1 and BC063123.1 . In representative examples of this type, the polynucleotide comprises an 5- HT2BR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001058] In some embodiments, an 5-HT2bR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian 5-HT2bR polynucleotide, or a fragment thereof. In some embodiments, the 5- HT2bR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian 5-HT2bR protein, or a fragment thereof under low, medium or high stringency conditions.

3.71 Serotonin 5-HT2c receptor (5-HT2cR) polypeptides

[0001059] The 5-HT2cR has been shown to modulate mesolimbic dopaminergic function. Additionally they are thought to play roles in mediation of anxiety, body weight regulation and psychostimulant abuse (Nichols & Nichols, 2008).

[0001060] To date, a crystal structure of the 5-HT2cR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of 5- HT2cR consists of the N-terminal segment (Met1 -Val52 of the human 5-HT2cR), ECL1 (Asp1 17-Leu122 of the human 5-HT2cR) linking helices II and III, ECL2 (Leu194-Asn210 of the human 5-HT2cR) linking helices IV and V, and ECL3 (Glu338-Asn342 of the human 5- HT2cR) linking helices VI to VII. It is believed to form a disulphide bridge between helix III and ECL2 (Nichols & Nichols, 2008).

[0001061 ] In specific embodiments of the present invention, the 5-HT2cR polypeptide comprises a 5-HT2cR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an 5-HT2cR protein sequence.

[0001062] In some embodiments, the 5-HT2cR protein sequence corresponds to a mammalian 5-HT2cR protein sequence. Suitable 5-HT2cR sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P28335), alligator (UniProtKB Accession No. A0A151 N7D9), baboon (UniProtKB Accession No. A0A096MPH1 , cat (UniProtKB Accession No. M3X5M0), chicken (UniProtKB Accession No. F1 N989), chimpanzee (UniProtKB Accession No. Q5IS66), dog (UniProtKB Accession No. Q60F97), frog (UniProtKB Accession No. F7AZS9), hedgehog (UniProtKB Accession No. A0A1 S3ADC2), horse (UniProtKB Accession No. F6PSU6), human (UniProtKB Accession No. P28335), macaque (UniProtKB Accession No. F7A578), mouse (UniProtKB Accession No. P34968), pig (UniProtKB Accession No. G8YY03), rat (UniProtKB Accession No. P08909).

[0001063] In some preferred embodiments, the 5-HT2cR protein sequence corresponds to a human 5-HT2cR protein sequence. In some embodiments, the 5-HT2cR polypeptide comprises a human full-length wild-type 5-HT2cR protein sequence (UniProtKB Accession No. P28335), as set forth below, or a functional fragment of the wild-type 5-HT2cR protein sequence.

[0001064] MVNLRNAVHSFLVHLIGLLVWQCDISVSPVAAIVTDIFNTSDGGRFKFPDGVQN

WPALSIVIIIIMTIGGNILVIMAVSMEKKLHNATNYFLMSLAIADMLVGLLVMPLSL LAILYDYVW

LPRYLCPVWISLDVLFSTASIMHLCAISLDRYVAIRNPIEHSRFNSRTKAIMKIAIV WAISIGVSV

PIPVIGLRDEEKVFVNNTTCVLNDPNFVLIGSFVAFFIPLTIMVITYCLTIYVLRRQ ALMLLHGH

TEEPPGLSLDFLKCCKRNTAEEENSANPNQDQNARRRKKKERRPRGTMQAINNERKA SKV

LGIVFFVFLIMWCPFFITNILSVLCEKSCNQKLMEKLLNVFVWIGYVCSGINPLVYT LFNKIYR

RAFSNYLRCNYKVEKKPPVRQIPRVAATALSGRELNVNIYRHTNEPVIEKASDNEPG IEMQV

ENLELPVNPSSVVSERISSV [SEQ ID NO: 50].

[0001065] In one form of the invention, the 5-HT2cR polypeptide comprises a truncated form of a mammalian wild-type 5-HT2cR protein sequence. For example, the 5-HT2cR polypeptide sequence may comprise the human wild-type 5-HT2cR protein sequence with a C-terminal truncation (e.g., amino acid residues 384-458 may be truncated). Alternatively or in addition, the 5-HT2cR polypeptide sequence may comprise the wild-type 5-HT2cR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N- terminal truncation, a truncation may be performed to remove an internal section of the wild- type 5-HT2cR protein sequence (e.g., amino acid residues 198-205 may be truncated). By way of a non-limiting illustrative example, a 5-HT2cR polypeptide suitable for using with the present invention comprised amino acid residues 2-197 and 206-383 of the human wild-type 5-HT2cR protein sequence as set forth in SEQ ID NO: 50.

3.72 Constructs and nucleotide sequences encoding 5-HT2cR polypeptides

[0001066] The present invention also encompasses isolated polynucleotide sequences and constructs encoding 5-HT2cR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001067] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human 5-HT2cR nucleotide (i.e., corresponding to the HTR2C gene) sequence as set forth for example in GenBank Accession Nos. NM 001256760.2, NM_000868.3, NM_001256761 .2, NG_012082.2, EU796454.1 , AF498983.1 and BC095543.1 . In representative examples of this type, the polynucleotide comprises an 5- HT2CR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001068] In some embodiments, an 5-HT2cR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian 5-HT2cR polynucleotide, or a fragment thereof. In some embodiments, the 5- HT2cR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian 5-HT2cR protein, or a fragment thereof under low, medium or high stringency conditions.

3.73 Serotonin 5-HT4 receptor (5-HT4R) polypeptides

[0001069] In the CNS the 5-HT4R is believed to be involved in learning and memory, and mediation of locomotor activity. In the periphery they play a role in gastrointestinal and cardiovascular functions (Nichols & Nichols, 2008). [0001070] To date, a crystal structure of the 5-HT4R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of 5-HT4R consists of the N-terminal segment (Met1 -Ser10 of the human 5-HT4R), ECL1 (Glu84-Tyr88 of the human 5-HT4R) linking helices I I and III, ECL2 (Trp161 -Val188 of the human 5-HT4R) linking helices IV and V, and ECL3 (Glu286-Val289 of the human 5-HT4R) linking helices VI to VII. It is believed to form a disulphide bridge between helix III and ECL2 (Nichols & Nichols, 2008).

[0001071 ] In specific embodiments of the present invention, the 5-HT4R polypeptide comprises a 5-HT4R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an 5-HT4R protein sequence.

[0001072] In some embodiments, the 5-HT4R protein sequence corresponds to a mammalian 5-HT4R protein sequence. Suitable 5-HT4R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. Q13639), alligator (UniProtKB Accession No. A0A151 M6L1 ), cow (UniProtKB Accession No. Q5E9Q5), frog (UniProtKB Accession No. F7BIA5), guinea pig (UniProtKB Accession No. 070528), hedgehog (UniProtKB Accession No. A0A1 S2ZAK9), horse (UniProtKB Accession No. D2Y0Z2), marmoset (UniProtKB Accession No. F7FE43), mouse (UniProtKB Accession No. P97288), pig (UniProtKB Accession No. F1 RLB0), rat (UniProtKB Accession No. Q62758).

[0001073] In some preferred embodiments, the 5-HT4R protein sequence corresponds to a human 5-HT4R protein sequence. In some embodiments, the 5-HT4R polypeptide comprises a human full-length wild-type 5-HT4R protein sequence (UniProtKB Accession No. Q13639), as set forth below, or a functional fragment of the wild-type 5-HT4R protein sequence.

[0001074] MDKLDANVSSEEGFGSVEKVVLLTFLSTVILMAILGNLLVMVAVCWDRQLRKIK

TNYFIVSLAFADLLVSVLVMPFGAIELVQDIWIYGEVFCLVRTSLDVLLTTASIFHL CCISLDRY

YAICCQPLVYRNKMTPLRIALMLGGCWVIPTFISFLPIMQGWNNIGIIDLIEKRKFN QNSNSTY

CVFMVNKPYAITCSVVAFYIPFLLMVLAYYRIYVTAKEHAHQIQMLQRAGASSESRP QSADQ

HSTHRMRTETKAAKTLCIIMGCFCLCWAPFFVTNIVDPFIDYTVPGQVWTAFLWLGY INSGL

NPFLYAFLNKSFRRAFLIILCCDDERYRRPSILGQTVPCSTTTINGSTHVLRDAVEC GGQWE

SQC H P PATS P L VAAQ PS DT [SEQ ID NO: 51 ]. [0001075] In one form of the invention, the 5-HT4R polypeptide comprises a truncated form of a mammalian wild-type 5-HT4R protein sequence. For example, the 5-HT4R polypeptide sequence may comprise the human wild-type 5-HT4R protein sequence with a C-terminal truncation (e.g., amino acid residues 329-388 may be truncated). Alternatively or in addition, the 5-HT4R polypeptide sequence may comprise the wild-type 5-HT4R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type 5- HT4R protein sequence (e.g., amino acid residues 166-183 may be truncated). By way of a non-limiting illustrative example, a 5-HT4R polypeptide suitable for using with the present invention comprised amino acid residues 2-165 and 184-328 of the human wild-type 5-HT4R protein sequence as set forth in SEQ ID NO: 51 .

3.74 Constructs and nucleotide sequences encoding 5-HT4R polypeptides.

[0001076] The present invention also encompasses isolated polynucleotide sequences and constructs encoding 5-HT4R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001077] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human 5-HT4R nucleotide (i.e., corresponding to the HTR4 gene) sequence as set forth for example in GenBank Accession Nos. NM 000870.6, NM_001286410.1 , NM_001040173.2, NM_001040172.2, NM_001040169.2, NM_199453.3, NR 104445.1 , NG_029052.1 , AM712912.1 , AJ633645.1 , AJ278982.1 , AJ278981 .1 , AJ278980.1 , AJ278979.1 , AB070620.2 and AB070621 .1 . In representative examples of this type, the polynucleotide comprises an 5-HT4R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001078] In some embodiments, an 5-HT4R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian 5-HT4R polynucleotide, or a fragment thereof. In some embodiments, the 5-HT4R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian 5-HT4R protein, or a fragment thereof under low, medium or high stringency conditions. 3.75 Somatostatin 2 receptor (SST2R) polypeptides

[0001079] The SST2R is widely expressed throughout the body, including the brain, lung, heart, stomach, kidney, liver and numerous type of immune cells (De Martino et al., 2010). It is also believed to mediate cell cycle regulation, apoptosis and transcriptional regulation (Theodoropoulou & Stalla, 2013).

[0001080] To date, a crystal structure of the SST2R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of SST2R consists of the N-terminal segment (Met1 -Glu39 of the human SST2R), ECL1 (Val106- Phe1 10 of the human SST2R) linking helices II and III, ECL2 (Gly182-Glu200 of the human SST2R) linking helices IV and V, and ECL3 (Ala283-Pro286 of the human SST2R) linking helices VI to VII. It is belived to form a disulphode bridge between helix III and ECL2 (Patel, 1999).

[0001081 ] In specific embodiments of the present invention, the SST2R polypeptide comprises a SST2R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an SST2R protein sequence.

[0001082] In some embodiments, the SST2R protein sequence corresponds to a mammalian SST2R protein sequence. Suitable SST2R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P30874), alligator (UniProtKB Accession No. A0A151 N260), carp (UniProtKB Accession No. B2D1T6), cat (UniProtKB Accession No. M3W2Q8), chicken (UniProtKB Accession No. Q58G84), chimpanzee (UniProtKB Accession No. G2HFH4), cow (UniProtKB Accession No. P34993), dog (UniProtKB Accession No. Q49LX6), frog (UniProtKB Accession No. F6T888), macaque (UniProtKB Accession No. G7NJD6), mouse (UniProtKB Accession No. P30875), pig (UniProtKB Accession No. P34994), rabbit (UniProtKB Accession No. G1 SX13), rat (UniProtKB Accession No. P30680), sheep (UniProtKB Accession No. W5NPT8).

[0001083] In some preferred embodiments, the SST2R protein sequence corresponds to a human SST2R protein sequence. In some embodiments, the SST2R polypeptide comprises a human full-length wild-type SST2R protein sequence (UniProtKB Accession No. P30874), as set forth below, or a functional fragment of the wild-type SST2R protein sequence.

[0001084] MDMADEPLNGSHTWLSIPFDLNGSVVSTNTSNQTEPYYDLTSNAVLTFIYFVVC IIGLCGNTLVIYVILRYAKMKTITNIYILNLAIADELFMLGLPFLAMQVALVHWPFGKAI CRVVMT VDGINQFTSIFCLTVMSIDRYLAVVHPIKSAKWRRPRTAKMITMAVWGVSLLVILPIMIY AGLR SNQWGRSSCTINWPGESGAWYTGFIIYTFILGFLVPLTIICLCYLFIIIKVKSSGIRVGS SKRKK SEKKVTRMVSIVVAVFIFCWLPFYIFNVSSVSMAISPTPALKGMFDFVVVLTYANSCANP ILY AFLSDNFKKSFQNVLCLVKVSGTDDGERSDSKQDKSRLNETTETQRTLLNGDLQTSI [SEQ ID NO: 52].

[0001085] In one form of the invention, the SST2R polypeptide comprises a truncated form of a mammalian wild-type SST2R protein sequence. For example, the SST2R polypeptide sequence may comprise the human wild-type SST2R protein sequence with a C-terminal truncation (e.g., amino acid residues 329-369 may be truncated). Alternatively or in addition, the SST2R polypeptide sequence may comprise the wild-type SST2R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type SST2R protein sequence (e.g., amino acid residues 183-193 may be truncated). By way of a non-limiting illustrative example, a SST2R polypeptide suitable for using with the present invention comprised amino acid residues 2-182 and 194-328 of the human wild-type SST2R protein sequence as set forth in SEQ ID NO: 52.

3.76 Constructs and nucleotide sequences encoding SST2R polypeptides.

[0001086] The present invention also encompasses isolated polynucleotide sequences and constructs encoding SST2R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001087] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human SST2R nucleotide (i.e., corresponding to the SSTR2 gene) sequence as set forth for example in GenBank Accession Nos. NM 001050.2, NG_029371 .1 , L34689.1 , AY236542.1 , M81830.1 , AF184174.1 , L13033.1 , BC095495.1 and BC019610.1 . In representative examples of this type, the polynucleotide comprises an SST2R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001088] In some embodiments, an SST2R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian SST2R polynucleotide, or a fragment thereof. In some embodiments, the SST2R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian SST2R protein, or a fragment thereof under low, medium or high stringency conditions.

3.77 Sphingosine 1 -phosphate receptor 1 (S1P1R) polypeptides

[0001089] The S1 P1 R mediates a variety of physiological functions, including astrocyte migration, B cell chemotaxis and inhibition of egress, T cell chemotaxis and inhibition of egress, increased cardiomyocyte positive inotropy, early vascular system development and migration of neural stem cells (Rosen et al., 2009).

[0001090] A crystal structure of the S1 P1 R has been published (Hanson et al., 2012), providing insight into the ligand-binding and activation mechanisms of the receptor. The extracellular part of S1 P1 R consists of the N-terminal segment (Met1 -Lys41 of the human S1 P1 R), ECL1 (Ser105-Leu1 12 of the human S1 P1 R) linking helices II and III, ECL2 (Trp182-Tyr198 of the human S1 P1 R) linking helices IV and V, and ECL3 (Lys283-Cys287 of the human S1 P1 R) linking helices VI to VII. Two disulphide bonds help to shape the extracellular side of S1 P1 R (Hanson et al., 2012).

[0001091 ] In specific embodiments of the present invention, the S1 P1 R polypeptide comprises a S1 P1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an S1 P1 R protein sequence.

[0001092] In some embodiments, the S1 P1 R protein sequence corresponds to a mammalian S1 P1 R protein sequence. Suitable S1 P1 R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P21453), aligator (UniProtKB Accession No. A0A151 M4S4), cat (UniProtKB Accession No. M3W6R4), chimpanzee (UniProtKB Accession No. H2PZI1 ), cow (UniProtKB Accession No. Q5E9P3), dog (UniProtKB Accession No. W5VNF7), guinea pig (UniProtKB Accession No. H0V7G9), hedgehog (UniProtKB Accession No. A0A1 S3A399), macaque (UniProtKB Accession No. H9EUW9), marmoset (UniProtKB Accession No. F7HBI1 ), mouse (UniProtKB Accession No. 008530), rabbit (UniProtKB Accession No. G1T9R2), rat (UniProtKB Accession No. P48303), Tasmanian devil (UniProtKB Accession No. G3WFF9), turtle (UniProtKB Accession No. K7F150).

[0001093] In some preferred embodiments, the S1 P1 R protein sequence corresponds to a human S1 P1 R protein sequence. In some embodiments, the S1 P1 R polypeptide comprises a human full-length wild-type S1 P1 R protein sequence (UniProtKB Accession No. P21453), as set forth below, or a functional fragment of the wild-type S1 P1 R protein sequence. [0001094] MGPTSVPLVKAHRSSVSDYVNYDIIVRHYNYTGKLNISADKENSIKLTSVVFILIC

CFIILENIFVLLTIWKTKKFHRPMYYFIGNLALSDLLAGVAYTANLLLSGATTYKLT PAQWFLR

EGSMFVALSASVFSLLAIAIERYITMLKMKLHNGSNNFRLFLLISACWVISLILGGL PIMGWNCI

SALSSCSTVLPLYHKHYILFCTTVFTLLLLSIVILYCRIYSLVRTRSRRLTFRKNIS KASRSSEK

SLALLKTVIIVLSVFIACWAPLFILLLLDVGCKVKTCDILFRAEYFLVLAVLNSGTN PIIYTLTNKE

MRRAFIRIMSCCKCPSGDSAGKFKRPIIAGMEFSRSKSDNSSHPQKDEGDNPETIMS SGNV

NSSS [SEQ ID NO: 53].

[0001095] In one form of the invention, the S1 P1 R polypeptide comprises a truncated form of a mammalian wild-type S1 P1 R protein sequence. For example, the S1 P1 R polypeptide sequence may comprise the human wild-type S1 P1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 328-382 may be truncated). Alternatively or in addition, the S1 P1 R polypeptide sequence may comprise the wild-type S1 P1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type S1 P1 R protein sequence (e.g., amino acid residues 187-193 may be truncated). By way of a non-limiting illustrative example, a S1 P1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-186 and 194-327 of the human wild-type S1 P1 R protein sequence as set forth in SEQ ID NO: 53.

3.78 Constructs and nucleotide sequences encoding S1P1R polypeptides.

[0001096] The present invention also encompasses isolated polynucleotide sequences and constructs encoding S1 P1 R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001097] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human S1 P1 R nucleotide (i.e., corresponding to the S1 PR1 gene) sequence as set forth for example in GenBank Accession Nos. NM 001320730.1 , NM_001400.4, NG_016181 .1 and BC018650.1 . In representative examples of this type, the polynucleotide comprises an S1 P1 R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001098] In some embodiments, an S1 P1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian S1 P1 R polynucleotide, or a fragment thereof. In some embodiments, the S1 P1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian S1 P1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.79 Sphingosine 1 -phosphate receptor 3 (S1 P3R) polypeptides

[0001099] The S1 P3R is found in the brain, heart, spleen, liver, lung, thymus, kidney, testis and skeletal muscle. It mediates a variety of physiological functions, including cardiomyocyte survival following ischemia-reperfusion and dendritic cell lethality/inflammation/coagulation (Rosen et al, 2009).

[0001 100] To date, a crystal structure of the S1 P3R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of S1 P3R consists of the N-terminal segment (Met1 -Ala35 of the human S1 P3R), ECL1 (Ser99-Leu106 of the human S1 P3R) linking helices II and III, ECL2 (Trp176-Tyr192 of the human S1 P3R) linking helices IV and V, and ECL3 (Arg270-Cys274 of the human S1 P3R) linking helices VI to VII.

[0001 101 ] In specific embodiments of the present invention, the S1 P3R polypeptide comprises a S1 P3R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an S1 P3R protein sequence.

[0001 102] In some embodiments, the S1 P3R protein sequence corresponds to a mammalian S1 P3R protein sequence. Suitable S1 P3R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. Q99500), chimpanzee (UniProtKB Accession No. H2RBL8), cow (UniProtKB Accession No. A6QR17), dog (UniProtKB Accession No. E2RF74), guinea pig (UniProtKB Accession No. H0WDH2), hedgehog (UniProtKB Accession No. A0A1 S3ASI7), macaque (UniProtKB Accession No. G7NEM6), marmoset (UniProtKB Accession No. F7F4H4), mouse (UniProtKB Accession No. Q9Z0U9), orangutan (UniProtKB Accession No. K7EVQ8), rabbit (UniProtKB Accession No. G1 T9A8), rat (UniProtKB Accession No. F1 M9D3), salmon (UniProtKB Accession No. A0A1 S3LU52), Tasmanian devil (UniProtKB Accession No. G3VPD8).

[0001 103] In some preferred embodiments, the S1 P3R protein sequence corresponds to a human S1 P3R protein sequence. In some embodiments, the S1 P3R polypeptide comprises a human full-length wild-type S1 P3R protein sequence (UniProtKB Accession No. Q99500), as set forth below, or a functional fragment of the wild-type S1 P3R protein sequence. [0001 104] MATALPPRLQPVRGNETLREHYQYVGKLAGRLKEASEGSTLTTVLFLVICSFIVL

ENLMVLIAIWKNNKFHNRMYFFIGNLALCDLLAGIAYKVNILMSGKKTFSLSPTVWF LREGSM

FVALGASTCSLLAIAIERHLTMIKMRPYDANKRHRVFLLIGMCWLIAFTLGALPILG WNCLHNL

PDCSTILPLYSKKYIAFCISIFTAILVTIVILYARIYFLVKSSSRKVANHNNSERSM ALLRTVVIVV

SVFIACWSPLFILFLIDVACRVQACPILFKAQWFIVLAVLNSAMNPVIYTLASKEMR RAFFRLV

CNCLVRGRGARASPIQPALDPSRSKSSSSNNSSHSPKVKEDLPHTAP

SSCIMDKNAALQNGIFCN [SEQ ID NO: 54].

[0001 105] In one form of the invention, the S1 P3R polypeptide comprises a truncated form of a mammalian wild-type S1 P3R protein sequence. For example, the S1 P3R polypeptide sequence may comprise the human wild-type S1 P3R protein sequence with a C-terminal truncation (e.g., amino acid residues 315-378 may be truncated). Alternatively or in addition, the S1 P3R polypeptide sequence may comprise the wild-type S1 P3R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type S1 P3R protein sequence (e.g., amino acid residues 181 -188 may be truncated). By way of a non-limiting illustrative example, a S1 P3R polypeptide suitable for using with the present invention comprised amino acid residues 2-180 and 189-314 of the human wild-type S1 P3R protein sequence as set forth in SEQ ID NO: 54.

3.80 Constructs and nucleotide sequences encoding S 1 P3R polypeptides.

[0001 106] The present invention also encompasses isolated polynucleotide sequences and constructs encoding S1 P3R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001 107] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human S1 P3R nucleotide (i.e., corresponding to the S1 PR3 gene) sequence as set forth for example in GenBank Accession Nos. NM 005226.3, BC060827.1 and BC069579.1 . In representative examples of this type, the polynucleotide comprises an S1 P3R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001 108] In some embodiments, an S1 P3R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian S1 P3R polynucleotide, or a fragment thereof. In some embodiments, the S1 P3R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian S1 P3R protein, or a fragment thereof under low, medium or high stringency conditions.

3.81 Thyrotrophin-releasing hormone receptor 1 (TRH1R) polypeptides

[0001 109] The TRH1 R stimulates the release of prolactin and thyrotropin (Perret et al., 1988) and the action of TRH on TRH1 R is essential for normal function of the thyroid axis (Hollenberg, 2008).

[0001 1 10] To date, a crystal structure of the TRH1 R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of TRH1 R consists of the N-terminal segment (Met1 -Ala21 of the human TRH1 R), ECL1 (Tyr88-Tyr93 of the human TRH1 R) linking helices II and III, ECL2 (Asp165-lle183 of the human TRH1 R) linking helices IV and V, and ECL3 (Ser293-Gln297 of the human TRH1 R) linking helices VI to VII.

[0001 1 1 1 ] In specific embodiments of the present invention, the TRH1 R polypeptide comprises a TRH1 R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an TRH1 R protein sequence.

[0001 1 12] In some embodiments, the TRH1 R protein sequence corresponds to a mammalian TRH1 R protein sequence. Suitable TRH1 R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P34981 ), alligator (UniProtKB Accession No. A0A151 P2B9), chicken (UniProtKB Accession No. 093603), cow (UniProtKB Accession No. 046639), frog (UniProtKB Accession No. F6X1 15), hedgehog (UniProtKB Accession No. A0A1 S2ZTF7), macaque (UniProtKB Accession No. F7DWM0), mouse (UniProtKB Accession No. P21761 ), pig (UniProtKB Accession No. D5FUH1 ), rat (UniProtKB Accession No. Q01717), sheep (UniProtKB Accession No. Q28596).

[0001 1 13] In some preferred embodiments, the TRH1 R protein sequence corresponds to a human TRH1 R protein sequence. In some embodiments, the TRH1 R polypeptide comprises a human full-length wild-type TRH1 R protein sequence (UniProtKB Accession No. P34981 ), as set forth below, or a functional fragment of the wild-type TRH1 R protein sequence. [0001 1 14] MENETVSELNQTQLQPRAVVALEYQVVTILLVLIICGLGIVGNIMVVLVVMRTKH

MRTPTNCYLVSLAVADLMVLVAAGLPNITDSIYGSWVYGYVGCLCITYLQYLGINAS SCSITA

FTIERYIAICHPIKAQFLCTFSRAKKIIIFVWAFTSLYCMLWFFLLDLNISTYKDAI VISCGYKISR

NYYSPIYLMDFGVFYVVPMILATVLYGFIARILFLNPIPSDPKENSKTWKNDSTHQN TNLNVN

TSNRCFNSTVSSRKQVTKMLAVVVILFALLWMPYRTLVVVNSFLSSPFQENWFLLFC RICIYL

NSAINPVIYNLMSQKFRAAFRKLCNCKQKPTEKPANYSVALNYSVIKESDHFSTELD DITVTD

TYLSATKVSFDDTCLASEVSFSQS [SEQ ID NO: 55].

[0001 1 15] In one form of the invention, the TRH1 R polypeptide comprises a truncated form of a mammalian wild-type TRH1 R protein sequence. For example, the TRH1 R polypeptide sequence may comprise the human wild-type TRH1 R protein sequence with a C-terminal truncation (e.g., amino acid residues 337-398 may be truncated). Alternatively or in addition, the TRH1 R polypeptide sequence may comprise the wild-type TRH1 R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type TRH1 R protein sequence (e.g., amino acid residues 167-175 may be truncated). By way of a non-limiting illustrative example, a TRH1 R polypeptide suitable for using with the present invention comprised amino acid residues 2-166 and 176-336 of the human wild-type TRH1 R protein sequence as set forth in SEQ ID NO: 55.

3.82 Constructs and nucleotide sequences encoding TRH1R polypeptides.

[0001 1 16] The present invention also encompasses isolated polynucleotide sequences and constructs encoding TRHR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001 1 17] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human TRHR nucleotide (i.e., corresponding to the TRHR gene) sequence as set forth for example in GenBank Accession Nos. NM_003301 .5, NG_017161 .1 , AJ01 1701 .1 , BC1 13360.1 , BC105045.1 and AY493373.1 . In representative examples of this type, the polynucleotide comprises an TRHR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001 1 18] In some embodiments, a TRH1 R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian TRH1 R polynucleotide, or a fragment thereof. In some embodiments, the TRH1 R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian TRH1 R protein, or a fragment thereof under low, medium or high stringency conditions.

3.83 Vasopressin receptor 1A (V1AR) polypeptides

[0001 1 19] The V1 AR is expressed in many tissues including the liver, vascular smooth muscle cells and the brain. In the vasculature the receptor mediates vasoconstriction, while in the brain it mediates anxiety- and aggression-producing responses (Manning et al., 2008).

[0001 120] To date, a crystal structure of the V1 AR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of V1 AR consists of the N-terminal segment (Met1 -Asn47 of the human V1 AR), ECL1 (Thr1 14- Phe1 17 of the human V1 AR) linking helices II and III, ECL2 (Ser190-lle208 of the human V1 AR) linking helices IV and V, and ECL3 (Pro318-Trp322 of the human V1 AR) linking helices VI to VII.

[0001 121 ] In specific embodiments of the present invention, the V1 AR polypeptide comprises a V1 AR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an V1 AR protein sequence.

[0001 122] In some embodiments, the V1 AR protein sequence corresponds to a mammalian V1 AR protein sequence. Suitable V1 AR sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P37288), cat (UniProtKB Accession No. M3WKK5), chimpanzee (UniProtKB Accession No. H2Q6D6), cow (UniProtKB Accession No. A2VDS9), dog (UniProtKB Accession No. E2R8D3), frog (UniProtKB Accession No. F7B836), horse (UniProtKB Accession No. F6QLP1 ), macaque (UniProtKB Accession No. F6ZXQ6), mouse (UniProtKB Accession No. Q62463), pig (UniProtKB Accession No. A0A1 D5NXS1 ), rabbit (UniProtKB Accession No. G1 TLM4), rat (UniProtKB Accession No. P30560), sheep (UniProtKB Accession No. P48043), turtle (UniProtKB Accession No. K7FSS7), vole (UniProtKB Accession No. Q9WTV8).

[0001 123] In some preferred embodiments, the V1 AR protein sequence corresponds to a human V1 AR protein sequence. In some embodiments, the V1 AR polypeptide comprises a human full-length wild-type V1 AR protein sequence (UniProtKB Accession No. P37288), as set forth below, or a functional fragment of the wild-type V1 AR protein sequence. [0001 124] MRLSAGPDAGPSGNSSPWWPLATGAGNTSREAEALGEGNGPPRDVRNEELA

KLEIAVLAVTFAVAVLGNSSVLLALHRTPRKTSRMHLFIRHLSLADLAVAFFQVLPQ MCWDIT

YRFRGPDWLCRVVKHLQVFGMFASAYMLVVMTADRYIAVCHPLKTLQQPARRSRLMI AAA

WVLSFVLSTPQYFVFSMIEVNNVTKARDCWATFIQPWGSRAYVTWMTGGIFVAPVVI LGTC

YGFICYNIWCNVRGKTASRQSKGAEQAGVAFQKGFLLAPCVSSVKSISRAKIRTVKM TFVIV

TAYIVCWAPFFIIQMWSVWDPMSVWTESENPTITITALLGSLNSCCNPWIYMFFSGH LLQDC

VQSFPCCQNMKEKFNKEDTDSMSRRQTFYSNNRSPTNSTGMWKDSPKSSKSIKFIPV ST

[SEQ ID NO: 56].

[0001 125] In one form of the invention, the V1 AR polypeptide comprises a truncated form of a mammalian wild-type T V1 AR protein sequence. For example, the V1 AR polypeptide sequence may comprise the human wild-type V1 AR protein sequence with a C-terminal truncation (e.g., amino acid residues 365-418 may be truncated). Alternatively or in addition, the V1 AR polypeptide sequence may comprise the wild-type V1 AR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type V1 AR protein sequence (e.g., amino acid residues 192-205 may be truncated). By way of a non-limiting illustrative example, a V1 AR polypeptide suitable for using with the present invention comprised amino acid residues 2-191 and 206-364 of the human wild-type V1 AR protein sequence as set forth in SEQ ID NO: 56.

3.84 Constructs and nucleotide sequences encoding V1AR polypeptides.

[0001 126] The present invention also encompasses isolated polynucleotide sequences and constructs encoding V1 AR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001 127] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human V1 AR nucleotide (i.e., corresponding to the AVPR1 A gene) sequence as set forth for example in GenBank Accession Nos. NM 000706.4, KJ534780.1 , KJ534779.1 , BC074803.2, BC074804.2, AF208541 .1 and AY322550.1 . In representative examples of this type, the polynucleotide comprises an V1 AR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences.

[0001 128] In some embodiments, a V1 AR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian V1 AR polynucleotide, or a fragment thereof. In some embodiments, the V1 AR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian V1 AR protein, or a fragment thereof under low, medium or high stringency conditions.

3.85 Vasopressin receptor 1B (V1BR) polypeptides

[0001 129] The V1 BR is expressed in many tissues including the brain, kidney and the adrenal medulla. In the anterior pituitary it stimulates the release of adrenocorticotropic hormone and it is also believed to mediate anxiety and stress responses (Manning et al., 2008).

[0001 130] To date, a crystal structure of the V1 BR has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of V1 BR consists of the N-terminal segment (Met1 -Glu30 of the human V1 BR), ECL1 (Thr97-Phe100 of the human V1 BR) linking helices II and III, ECL2 (Ser173-Gly191 of the human V1 BR) linking helices IV and V, and ECL3 (Lys308-Glu312 of the human V1 BR) linking helices VI to VII.

[0001 131 ] In specific embodiments of the present invention, the V1 BR polypeptide comprises a V1 BR protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an V1 BR protein sequence.

[0001 132] In some embodiments, the V1 BR protein sequence corresponds to a mammalian V1 BR protein sequence. Suitable V1 BR sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P47901 ), cat (UniProtKB Accession No. M3W6E7), chicken (UniProtKB Accession No. F1 NVW1 ), chimpanzee (UniProtKB Accession No. H2R1 Q0), cow (UniProtKB Accession No. F1 ME85), dog (UniProtKB Accession No. F1 P9D6), frog (UniProtKB Accession No. F7EHY9), guinea pig (UniProtKB Accession No. H0V8M7), horse (UniProtKB Accession No. F7BNU0), macaque (UniProtKB Accession No. F7BAU2), mouse (UniProtKB Accession No. Q9WU02), pig (UniProtKB Accession No. F1 SF06), rabbit (UniProtKB Accession No. G1 SSV5), rat (UniProtKB Accession No. P48974), Tasmanian devil (UniProtKB Accession No. G3WX95).

[0001 133] In some preferred embodiments, the V1 BR protein sequence corresponds to a human V1 BR protein sequence. In some embodiments, the V1 BR polypeptide comprises a human full-length wild-type V1 BR protein sequence (UniProtKB Accession No. P47901 ), as set forth below, or a functional fragment of the wild-type V1 BR protein sequence.

[0001 134] MDSGPLWDANPTPRGTLSAPNATTPWLGRDEELAKVEIGVLATVLVLATGGNL

AVLLTLGQLGRKRSRMHLFVLHLALTDLAVALFQVLPQLLWDITYRFQGPDLLCRAV KYLQV

LSMFASTYMLLAMTLDRYLAVCHPLRSLQQPGQSTYLLIAAPWLLAAIFSLPQVFIF SLREVI

QGSGVLDCWADFGFPWGPRAYLTWTTLAIFVLPVTMLTACYSLICHEICKNLKVKTQ AWRV

GGGGWRTWDRPSPSTLAATTRGLPSRVSSINTISRAKIRTVKMTFVIVLAYIACWAP FFSVQ

MWSVWDKNAPDEDSTNVAFTISMLLGNLNSCCNPWIYMGFNSHLLPRPLRHLACCGG PQ

PRMRRRLSDGSLSSRHTTLLTRSSCPATLSLSLSLTLSGRPRPEESPRDLELADGEG TAETII

F [SEQ ID NO: 57].

[0001 135] In one form of the invention, the V1 BR polypeptide comprises a truncated form of a mammalian wild-type T V1 BR protein sequence. For example, the V1 BR polypeptide sequence may comprise the human wild-type V1 BR protein sequence with a C-terminal truncation (e.g., amino acid residues 355-424 may be truncated). Alternatively or in addition, the V1 BR polypeptide sequence may comprise the wild-type V1 BR protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type V1 BR protein sequence (e.g., amino acid residues 175-183 may be truncated). By way of a non-limiting illustrative example, a V1 BR polypeptide suitable for using with the present invention comprised amino acid residues 2-174 and 184-354 of the human wild-type V1 BR protein sequence as set forth in SEQ ID NO: 57.

3.86 Constructs and nucleotide sequences encoding V1BR polypeptides.

[0001 136] The present invention also encompasses isolated polynucleotide sequences and constructs encoding V1 BR polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001 137] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human V1 BR nucleotide (i.e., corresponding to the AVPR1 B gene) sequence as set forth for example in GenBank Accession Nos. NM 000707.3, DQ194816.1 and EU4321 1 1 .1 . In representative examples of this type, the polynucleotide comprises a V1 BR nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences. [0001 138] In some embodiments, a V1 BR polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian V1 BR polynucleotide, or a fragment thereof. In some embodiments, the V1 BR polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian V1 BR protein, or a fragment thereof under low, medium or high stringency conditions.

3.87 Vasopressin receptor 2 (V2R) polypeptides

[0001 139] The V2R mediates anti-diuresis through receptors present in the collecting duct of the kidney. Additionally it appears to mediate the pain response induced by vasopressin (Manning et al, 2008).

[0001 140] To date, a crystal structure of the V2R has not yet been published, so the structural and ligand-binding features of the receptor have to be inferred through indirect approaches such as mutagenesis and homology modelling. The extracellular part of V2R consists of the N-terminal segment (Met1 -Glu33 of the human V2R), ECL1 (Thr102-Phe105 of the human V2R) linking helices II and III, ECL2 (Ala179-Ala197 of the human V2R) linking helices IV and V, and ECL3 (Pro298-Leu302 of the human V2R) linking helices VI to VII.

[0001 141 ] In specific embodiments of the present invention, the V2R polypeptide comprises a V2R protein sequence or shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with an V2R protein sequence.

[0001 142] In some embodiments, the V2R protein sequence corresponds to a mammalian V2R protein sequence. Suitable V2R sequences may suitably be from mammal selected from the group comprising human (UniProtKB Accession No. P30518), cat (UniProtKB Accession No. M3WF07), chicken (UniProtKB Accession No. F1 NV74), chimpanzee (UniProtKB Accession No. H2RCC1 ), cow (UniProtKB Accession No. P48044), dog (UniProtKB Accession No. 077808), horse (UniProtKB Accession No. F6UX08), mouse (UniProtKB Accession No. 088721 ), pig (UniProtKB Accession No. P32307), rat (UniProtKB Accession No. Q00788), sheep (UniProtKB Accession No. W5NTL0), turkey (UniProtKB Accession No. G1 NAI7), turtle (UniProtKB Accession No. K7FJ45).

[0001 143] In some preferred embodiments, the V2R protein sequence corresponds to a human V2R protein sequence. In some embodiments, the V2R polypeptide comprises a human full-length wild-type V2R protein sequence (UniProtKB Accession No. P30518), as set forth below, or a functional fragment of the wild-type V2R protein sequence.

[0001 144] MLMASTTSAVPGHPSLPSLPSNSSQERPLDTRDPLLARAELALLSI VFVAVALS

NGLVLAALARRGRRGHWAPIHVFIGHLCLADLAVALFQVLPQLAWKATDRFRGPDAL CRAV

KYLQMVGMYASSYMILAMTLDRHRAICRPMLAYRHGSGAHWNRPVLVAWAFSLLLSL PQL

FIFAQRNVEGGSGVTDCWACFAEPWGRRTYVTWIALMVFVAPTLGIAACQVLIFREI HASLV

PGPSERPGGRRRGRRTGSPGEGAHVSAAVAKTVRMTLVIVVVYVLCWAPFFLVQLWA AW

DPEAPLEGAPFVLLMLLASLNSCTNPWIYASFSSSVSSELRSLLCCARGRTPPSLGP QDES

CTTASSSLAKDTSS [SEQ ID NO: 58].

[0001 145] In one form of the invention, the V2R polypeptide comprises a truncated form of a mammalian wild-type V2R protein sequence. For example, the V2R polypeptide sequence may comprise the human wild-type V2R protein sequence with a C-terminal truncation (e.g., amino acid residues 342-371 may be truncated). Alternatively or in addition, the V2R polypeptide sequence may comprise the wild-type V2R protein sequence with a N-terminal truncation. Alternatively or in addition to a C-terminal or N-terminal truncation, a truncation may be performed to remove an internal section of the wild-type V2R protein sequence (e.g., amino acid residues 187-191 may be truncated). By way of a non-limiting illustrative example, a V2R polypeptide suitable for using with the present invention comprised amino acid residues 2-186 and 192-341 of the human wild-type V2R protein sequence as set forth in SEQ ID NO: 58.

3.88 Constructs and nucleotide sequences encoding V2R polypeptides.

[0001 146] The present invention also encompasses isolated polynucleotide sequences and constructs encoding V2R polypeptides as broadly described above and elsewhere herein. Also contemplated are host cells comprising those polynucleotide sequences or constructs.

[0001 147] In some embodiments, the polynucleotide sequences comprise a sequence that corresponds to a human V2R nucleotide (i.e., corresponding to the AVPR2 gene) sequence as set forth for example in GenBank Accession Nos. NM_000054.4, NM_001 146151 .1 , NR_027419.1 , U04357.1 , NG_008687.1 , BC1 12181 .1 and BC101484.1 . In representative examples of this type, the polynucleotide comprises an V2R nucleotide sequence that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity with any one of these sequences. [0001 148] In some embodiments, an V2R polynucleotide coding sequence comprises a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% sequence identity to a wild type mammalian V2R polynucleotide, or a fragment thereof. In some embodiments, the V2R polynucleotide comprises a nucleotide sequence that hybridises to an open reading frame for a wild type mammalian V2R protein, or a fragment thereof under low, medium or high stringency conditions.

4. Cells expressing a certain co- located GPCR and/or RAGE

[0001 149] Nucleic acid molecules (preferably in the form of constructs including vectors) encoding a certain co-located GPCR, such as an angiotensin receptor, such as AT ₁ R or a certain chemokine receptor, such as CCR2, and/or RAGE can be transiently or stably transfected or co-transfected into a host cell, suitably any cell line, including stable cell lines. In preferred embodiments, the cell line is a mammalian-derived cell line. Preferred cell lines are those derived from the same organism as from which RAGE and the certain co-located GPCR are derived. Thus, the cell line used can be optimised for each specific purpose, for example, if the polynucleotides to be expressed in said cells encode human protein (or a polypeptide that is derived from a human protein), then a cell line that is derived from human cells will likely be the most suitable. However, this does not exclude using polypeptides and cells that are derived from different organisms. An example is expressing rat or mouse- derived polypeptides on the surface of a human-derived cell. Another example is expressing rat or human-derived polypeptides on the surface of a hamster-derived cell, such as a Chinese hamster ovary (CHO) cell.

[0001 150] One example of a human-derived cell line that is suitable for performing the present invention is a HEK293 cell line (e.g., a HEK293 cell line, or a HEK293FT cell line). Other examples of suitable cell lines include but are not limited to a COS-1 cell line, or a COS-7 cell line, or a CHO cell line, or a HeLa cell line.

[0001 151 ] Accordingly, the cells of the present invention express RAGE and a certain co- located GPCR, such as an angiotensin receptor, such as AT ₁ R or a certain chemokine receptor, such as CCR2.

5. Candidate Agents

[0001 152] In certain aspects, the present invention provides methods of screening for agents that modulate signalling in a cell resulting from RAGE ligand-independent activation of RAGE by certain co-located activated GPCRs, including AT ₁ R and CCR2. The agents can bind to one or both of the certain co-located activated GPCR and RAGE and/or proteins complexed with either, provided that they modulate (e.g., inhibit or signal) RAGE ligand- independent activation of RAGE by the certain co-located activated GPCR. Representative agents can bind to any domain of the certain co-located activated GPCR and RAGE or other proteins complexed with RAGE and/or the certain co-located activated GPCR other than the ectodomain of RAGE. Without wishing to be bound by any one theory or mode of operation, the inventors hypothesise that the site mediating RAGE ligand-independent activation of RAGE by the certain co-located activated GPCR is present on the cytoplasmic (cytosolic) side of the membrane. As such, in some embodiments the candidate agents screened are determined as being cell penetrable (i.e., able to transport through the plasma membrane) or are modified to be cell penetrable. In one embodiment, the cells of the present invention express a RAGE polypeptide lacking the ectodomain and a certain co-located activated GPCR. In another embodiment, the cells of the present invention express the cytosolic tail of RAGE, or a fragment thereof, and a certain co-located activated GPCR. In another embodiment, the cells of the present invention express a certain co-located activated GPCR but do not express endogenous RAGE.

[0001 153] Any known type of screening assay is contemplated as being suitable to identify candidate agents as being modulators of RAGE ligand-independent activation of RAGE by the certain co-located activated GPCR. In one embodiment, a proximity screening assay is used. One example of a proximity screening assay to run is a bioluminescence resonance energy transfer (BRET) assay to investigate a change in proximity between the certain co- located activated GPCR and RAGE. In some embodiments, a screening assay using soluble recombinant polypeptides and performed in vitro is envisaged. Examples of such screening assays are conventional two-hybrid, surface plasmon resonance (SPR) or complementation systems. In another embodiment, a binding assay is utilised, for example using a fluorescently-labelled peptide or other agent that binds to the certain co-located activated GPCR and/or RAGE. This may or may not be used with NanoLuc conjugated to the certain co-located activated GPCR or RAGE in order to utilise BRET (Stoddart et al., 2015). The fusion of NanoLuc to the certain co-located activated GPCR or RAGE may occur at the N- terminus, at the C-terminus, or at another suitable site within the certain co-located activated GPCR or RAGE polypeptide.

[0001 154] Immunoassays can also be used to analyse specific binding and include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well-known in the art (see, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). In some embodiments, a RAGE polypeptide is assayed for binding to a certain co-located GPCR, such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, using ELISA. In illustrative examples of this type, either a recombinant RAGE polypeptide or a recombinant certain co-located GPCR polypeptide together with a candidate agent is contacted to a microtitre plate whose bottom surface has been coated with the other binding partner, e.g., a limiting amount of the target. The plate is washed with buffer to remove non- specifically bound polypeptide and excess candidate agent. Then the amount of the RAGE polypeptide or the certain co-located GPCR polypeptide that is bound to the target on the plate is determined by probing the plate with an antibody that can recognize the RAGE polypeptide or the certain co-located GPCR polypeptide, e.g., a tag or constant portion of the polypeptide. The antibody is linked to a detection system (e.g., an enzyme such as alkaline phosphatase or horse radish peroxidase (HRP) which produces a colorimetric product when appropriate substrates are provided).

[0001 155] In other embodiments, the ability of a candidate agent to modulate the binding of a RAGE polypeptide to a certain co-located GPCR polypeptide is analysed using a homogenous assay, i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, e.g., U.S. Pat. No. 5,631 ,169; and U.S. Pat. No. 4,868,103). A fluorophore label on the first molecule (e.g., the RAGE polypeptide) is selected such that resonance energy can be absorbed by a fluorescent acceptor on a second molecule (e.g., the certain co-located GPCR polypeptide) if the RAGE polypeptide is in proximity to the certain co-located GPCR polypeptide. The fluorescent acceptor on the certain co-located GPCR polypeptide fluoresces when it absorbs the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which the polypeptides are proximal, the fluorescence emission of the fluorescent acceptor in the assay should be detectable. The FRET can be conveniently measured through standard fluorometric detection means, e.g., using a fluorimeter.

[0001 156] In other embodiments, interaction between a RAGE polypeptide and a certain co-located GPCR polypeptide is analysed using Surface Plasmon Resonance (SPR). SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labelling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which is measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S. Pat. No. 5,641 ,640; Raether, 1988; Sjolander and Urbaniczky (1991 ), Szabo et al. (1995) and on-line resources provide by BIAcore International AB (Uppsala, Sweden).

[0001 157] Upon identification of a candidate agent's ability to modulate an interaction between a RAGE polypeptide and a certain co-located activated GPCR, such as angiotensin receptor such as AT ₁ R or certain chemokine receptor such as CCR2, toxicity and/or efficacy of the candidate agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapies that exhibit large therapeutic indices are preferred. While therapies that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0001 158] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in mammals, including humans. The dosage of such agents lies suitably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any therapy used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0001 159] Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Dalton. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, desirably at least two of the functional chemical groups. The candidate agent often comprises cyclical carbon or heterocyclic structures or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.

[0001 160] Small (non-peptide) molecule modulators are particularly advantageous. In this regard, small molecules are desirable because such molecules are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene (e.g. by interacting with the regulatory region or transcription factors involved in gene expression); or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules.

[0001 161 ] Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification or amidification, to produce structural analogues.

[0001 162] Screening may also be directed to known pharmacologically active compounds and chemical analogues thereof.

[0001 163] Compounds may be further tested in the animal models to identify those compounds having the most potent in vivo effects. These molecules may serve as "lead compounds" for the further development of pharmaceuticals by, for example, subjecting the compounds to sequential modifications, molecular modelling, and other routine procedures employed in rational drug design.

5. 1 Fragments, analogues and derivatives of RAGE polypeptide

[0001 164] In some embodiments the candidate agent is a fragment of the cytosolic tail of RAGE, wherein the candidate agent comprises a fragment of a RAGE polypeptide or DNA coding for a RAGE polypeptide from which the fragment is subsequently generated following gene delivery. Suitably, the candidate agent is a peptide that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with, or differs at no more than 1 , 2, 3, 5 or even 10, 15 or 20 amino acid residues from, a fragment of the cytosolic tail of RAGE polypeptide sequence. As described above, in some embodiments of the invention the RAGE fragment amino acid sequence corresponds to the cytosolic tail of of the wild-type RAGE polypeptide (for example, amino acid residues 362 to 404 of the wild-type RAGE protein (SEQ ID NO: 1 or a fragment thereof).

[0001 165] In some preferred examples, the fragment corresponds to the C terminal cytosolic tail of a wild-type RAGE protein (i.e., amino acid residues 362 to 404 of the amino acid sequence set forth in SEQ ID NO: 1 ). The peptide analogue may also comprise additional amino acid residues at its N-terminal or C-terminal end, or derivative thereof. Similarly, the analogue may comprise any 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42 amino acid fragment of the C-terminal cytosolic tail of a wild-type RAGE protein, or derivative thereof.

[0001 166] As such, the peptide may comprise the following amino acid sequence: [SEQ ID NO: 1 ].

[0001 167] Alternatively, the analogue can share at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with, or differs at no more than 1 , 2, 3, 5 or even 10 amino acid residues from, the sequence set forth in SEQ ID NO: 1 .

[0001 168] In some embodiments the candidate agent is an activator, an inhibitor, an allosteric modulator, or a non-functional substitute for the transmembrane domain of RAGE or part thereof, wherein the candidate agent comprises a fragment of a RAGE polypeptide or DNA coding for a RAGE polypeptide from which the fragment is subsequently generated following gene delivery. Suitably, the candidate agent is a peptide that shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with, or differs at no more than 1 , 2, 3, 5 or even 10, 15 or 20 amino acid residues from, a fragment of the RAGE polypeptide sequence that comprises the transmembrane domain of RAGE or a fragment thereof. A non-functional substitute is a candidate agent that takes the place of the transmembrane domain of RAGE in the presence of certain co-located GPCRs, is not able to be activated by them or induce downstream RAGE-dependent signalling, and inhibits signalling that normally occurs through activation of the cytosolic tail of RAGE and RAGE-dependent signalling resulting therefrom. In some embodiments, the candidate agent comprises a transmembrane domain of RAGE or a part thereof and a fragment of the RAGE ectodomain. In some embodiments, the candidate agent comprises a transmembrane domain of RAGE or a part thereof and a fragment of the cytosolic tail of RAGE. In some embodiments, the candidate agent comprises a transmembrane domain of RAGE or part thereof and a fragment of the RAGE ectodomain and/or a fragment of the cytosolic tail of RAGE.

[0001 169] In some embodiments, the analogue amino acid sequence is distinguished from a wild-type RAGE polypeptide sequence by the substitution, addition or deletion of at least one (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid residues, which results in modulation of activation of the cytosolic tail of RAGE by a certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2.

[0001 170] In some embodiments, the peptide, analogue, fragment or derivative thereof suitably inhibits signalling resulting from a cognate ligand activating the certain co-located GPCR.

[0001 171 ] Thus, in some embodiments the peptide, analogue, fragment, or derivative thereof corresponds to a RAGE polypeptide sequence with an impaired certain activated co- located GPCR-binding site. The at least one amino acid substitution, addition or deletion is suitably located at any position corresponding to a key residue in the interaction between RAGE and the certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2. In illustrative examples of this type, the peptide analogue amino acid sequence is distinguished from the wild-type RAGE polypeptide sequence by a substitution, addition or deletion at amino acid residue position S391 of the cytosolic tail of wild-type RAGE protein (as set forth in SEQ ID NO: 1 ) with another amino acid residue.

[0001 172] In some embodiments, the peptide, analogue, fragment or derivative thereof corresponds to a RAGE polypeptide sequence with an impaired site at which the certain activated co-located GPCR exerts its functional interaction. The at least one amino acid substitution, addition or deletion is suitably located at any position corresponding to a key residue in the functional interaction between RAGE and the certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2. [0001 173] The residue at amino acid position 391 is a site that the present inventors determined to be important in the RAGE ligand-independent activation of RAGE by a certain activated co-located GPCR, such as AT ₁ R and CCR2. Suitably, in these embodiments, the serine 391 is removed or substituted with another amino acid (such as alanine, aspartate, phenylalanine, histidine, lysine, arginine, tyrosine, asparagine, valine, glycine, cysteine or glutamate) in order to inhibit activation of the cytosolic tail of RAGE. In other embodiments, the serine 391 is retained or substituted with another amino acid (such as proline, glutamine, leucine, isoleucine methionine, threonine or tryptophan), in order for it to be activated following activation of a certain co-located GPCR, and thereby induce RAGE-dependent signalling, with or without requiring the expression of wild type RAGE. Without wishing to be bound by theory, the inventors believe the substitution or removal of S391 influences the conformation of the alpha-helical coil (e.g. RAGE379-390) proximal to this residue (i.e. RAGE379-390) and/or its affinity for binding partners.

[0001 174] In other examples of this type, the peptide, analogue, fragment or derivative thereof is distinguished from the wild-type RAGE polypeptide sequence by a substitution, addition or deletion of any amino acid that is critical to the conformation of the alpha helix proximal to the S391 site (e.g., an amino acid directly adjacent to amino acid position S391 ). For example, the glutamine residue at amino acid position 390 of the wild-type RAGE protein sequence may, when substituted with a polar amino acid (e.g., arginine or lysine), enhance the capping of the alpha helix at its C-terminus [e.g. SEQ ID NO: 9 & 10]. Such substitutions result in a peptide that is an inhibitor of RAGE ligand-independent activation of RAGE by certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2.

[0001 175] Alternatively, the peptide, analogue, fragment or derivative thereof shares at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or similarity with, or differs at no more than 1 , 2, 3, 5 or even 10 amino acid residues from, the sequence set forth in SEQ ID NO: 5. Any truncations of the above peptide, analogue, fragment or derivative thereof, at either or both of the C-terminal or N terminal end, are also contemplated. Thus fragments comprising 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42 amino acids, from the sequence set forth in SEQ ID NO: 5 are suitable as peptides, analogues, fragments or derivatives thereof, providing they comprise a substitution or deletion corresponding to position Ser391 of the wild-type RAGE amino acid sequence of the cytosolic tail (as set forth in SEQ ID NO: 1 ). [0001 176] In some embodiments, the RAGE peptide mimetics of the present invention are conjugated, fused or otherwise linked to a natural or synthetic protein transduction domain or mimetic or placed in a non-covalent carrier system (e.g. liposomes, PEP-1 ), which suitably targets the RAGE peptides into the intracellular component of a cell, and are well known in the art. Non-limiting examples of such molecules include fusions with the natural cell membrane penetrating peptides, including the HIV-TAT motif (SEQ ID NO:4: YGRKKRRQRRR, as reviewed in Schwarze et al., 2000), as well natural and/or synthetic cell membrane penetrating peptides such as those described for example in U.S. Pat. Appl. Nos. 2014/0213775, 2014/0141452, and 2013/0136742, which are incorporated herein by reference in their entirety.

6. Proximity Screening Assays

[0001 177] In certain embodiments, the screening method assesses whether a candidate agent can modulate the interaction between a RAGE polypeptide and a certain activated co- located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2 by assessing a change in proximity of the RAGE polypeptide to the AT ₁ R certain activated co-located GPCR. In illustrative examples of this type, the RAGE polypeptide is coupled (e.g., conjugated, fused or otherwise linked) to a first reporter component and the certain activated co-located GPCR is coupled (e.g., conjugated or otherwise linked) to a second reporter component. The first and second reporter components can be the same or different. For example, one reporter component may be a proximity signal or energy donor, and the other may be a proximity signal or energy acceptor. The first and second reporter components can be any known molecules (e.g., an organic molecule, an inorganic molecule, a proteinaceous molecule, a non-proteinaceous molecule, or a combination thereof) that are capable of emitting a detectable signal when in close proximity.

[0001 178] Screening assays suitable for assessing the proximity of interacting polypeptides are well established in the art, and any format of such assays is suitable for the methods of the present invention as described above and elsewhere herein. For example, U.S. Patent No. 8,283,127 describes one such suitable system for the detection of molecular associations, the contents of which is incorporated herein by reference in its entirety.

[0001 179] By way of a generic example, to determine whether a candidate agent is capable of modulating the interaction between a RAGE polypeptide and a certain activated co- located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, each of the three components (i.e., (i) the RAGE polypeptide coupled to the first reporter component, (ii) the certain activated co-located GPCR coupled to a second reporter component, and (iii) the candidate agent) are provided to the assay system. The proximity signal from the first and second reporter components is observed and compared to a reference proximity signal that is emitted from the interacting RAGE and certain activated co-located GPCR in the absence of a candidate agent (i.e. the proximity signal emitted by the first and second reporter components when the RAGE polypeptide and the certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2 are free to interact). When the candidate agent does not modulate the interaction between a RAGE polypeptide and a certain activated co- located GPCR, no change to the reference proximity signal is observed. However, when the candidate agent is capable of modulating the interaction between a RAGE polypeptide and a certain activated co-located GPCR, a change to the proximity signal emitted from the first and second reporter components is observed upon the presence of the candidate agent to the assay system. Typically, a decrease in the proximity signal when compared to the reference proximity signal is indicative of the candidate agent antagonising or otherwise inhibiting the interaction between a RAGE polypeptide and a certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2. Conversely, an increase in proximity signal when compared to the reference proximity signal is generally indicative of the candidate agent agonising or otherwise stimulating the interaction between a RAGE polypeptide and a certain activated co-located GPCR. Representative reporter components are suitably selected from bioluminescent donor molecules, and fluorescent acceptor molecules. In some illustrative embodiments, the bioluminescent donor molecules and the fluorescent acceptor molecules are proteins.

[0001 180] The coupling of the certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or certain chemokine receptor such as CCR2 and/or the RAGE polypeptide to a reporter component can be by direct or indirect coupling and may be by any known covalent or non-covalent means of coupling two molecules. Illustrative examples of coupling methods include chemical cross-linking, chemical modification of proteins, chemical modification of amino acids, chemical modification of nucleic acids, chemical modification of carbohydrates, chemical modification of lipids, chemical modification of any other organic or inorganic molecule, biotin-avidin interactions, antigen-antibody interaction and nucleic acid hybridisation. In some forms of the invention, the first and/or second reporter component is coupled indirectly to the RAGE and/or certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2 by a linker. In some embodiments, the linker comprises an enzyme cleavage site. [0001 181 ] Alternatively, the RAGE polypeptide and/or certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or certain chemokine receptor such as CCR2 may be directly coupled to a proteinaceous reporter component. Approaches for directly conjugating proteinaceous molecules are well established in the art, for example, by genetic fusion, wherein nucleic acids encoding the RAGE polypeptide and/or certain activated co- located GPCR and the first and/or second reporter components are fused to produce a nucleic acid that encodes a single polypeptide.

[0001 182] In a particularly preferred embodiment of the invention, the RAGE polypeptide and/or certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or certain chemokine receptor such as CCR2 and the first and/or second reporter components each respectively form part of single polypeptides. Additional functionality may form part of the same polypeptide. For example, the RAGE and/or certain activated co-located GPCR and the first and/or second reporter components respectively form part of a single polypeptide that additionally comprises any one or more of: an amino acid sequence for affinity purification; a amino acid sequence that directs the polypeptide to a subcellular compartment of a eukaryotic cell; an amino acid sequence that facilitates the penetration of a eukaryotic cell membrane; and an amino acid sequence enabling expression levels to be assessed by the use of antibodies or otherwise.

[0001 183] In some embodiments, the screening assay employs energy donor and energy acceptor molecules for assessing interaction between the certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2 and RAGE polypeptides. The principle of energy transfer between two molecules can be exploited as a means to provide information about relative changes in their proximity and orientation to one another. Resonance Energy Transfer (RET) is the transfer of excited state energy from a donor to an acceptor molecule. Forster or fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. This can only occur if the absorption spectrum of the acceptor molecule overlaps with the emission spectrum of the donor. Forster determined that the degree of resonance energy transfer between the energy donor and energy acceptor is inversely proportional to the distance between the two molecules to the sixth power. In the case of FRET, an external light source of specific wavelength is used to excite the donor molecule.

[0001 184] Bioluminescence Resonance Energy Transfer (BRET) is described in Pfleger and Eidne (2006), US Patent 8,283,127, Jaeger et al., 2014 and Stoddart et al. 2015. Luciferases that have been used in BRET include those from the firefly, Renilla reniformis and Gaussia princeps, as well as NanoLuc luciferase (Promega).

[0001 185] In specific embodiments, the screening assay uses a BRET technique or assay. In order to perform the BRET screening, three components are required: a bioluminescent donor, a modulator and a fluorescent acceptor. The fluorescent acceptor can accept energy from the bioluminescent donor and will generate luminescence when these components are in an appropriate special relationship and in the presence of the appropriate substrate (bioluminescence initiating compound). The modulator can either influence the proximity and/or orientation of the bioluminescent donor and the fluorescent acceptor and thereby modulate the energy transfer between the components, or it can play a different role in affecting the energy transfer between the bioluminescent donor-generated luminescence and the fluorescent acceptor.

[0001 186] One advantage of the BRET technique is that protein-protein interactions in living cells can be monitored and/or determined in real time following activation with ligands.

[0001 187] In some embodiments a certain activated co-located GPCR (such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2) or a RAGE polypeptide is fused or otherwise linked to a bioluminescent donor. In some embodiments a certain activated co-located GPCR or a RAGE polypeptide is fused or otherwise linked to a fluorescent acceptor. For example, in one embodiment a certain activated co-located GPCR is fused or otherwise linked to a bioluminescent donor and a RAGE polypeptide is fused or otherwise linked to a fluorescent acceptor.

6. 1 Bioluminescent donors

[0001 188] As mentioned above, the bioluminescent donor includes, but is not limited to, a first target protein (a RAGE polypeptide or a certain activated co-located GPCR, such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2) and a bioluminescent donor molecule. The bioluminescent donor molecule can include, but is not limited to, Renilla luciferases and fragments thereof; polypeptide variants of Renilla luciferase; and the like. In particular, the bioluminescent donor molecule can include, but is not limited to, any of the following Renilla luciferase protein sequences:

Wild-type Renilla luciferase:

MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASS YLW RHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHD WGA CLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVE TML PSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRA SD DLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLK NE

Q [SEQ ID NO: 59].

Cys124Ala/Met185Val variant Renilla luciferase:

MTSKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASS YLW

RHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFV GHDWGA

ALAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNF FVETVL

PSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAY LRASD

DLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVER VLKNE

Q [SEQ ID NO: 60].

RLuc8 variant Renilla luciferase:

MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNATSS YLW

RHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFV GHDWGA

ALAFHYAYEHQDRIKAIVHMESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNF FVETVL

PSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAY LRASD

DLPKLFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFLQEDAPDEMGKYIKSFVER VLKNE

Q [SEQ ID NO: 61 ].

[0001 189] Other suitable bioluminescent donor molecules include, but are not limited to, other luciferases or photo-proteins such as NanoLuc luciferase (Promega), Coleoptera luciferase, firefly (Photinus pyralis) luciferase (fLuc), Gaussia luciferase, Anachnocampa sp luciferase, click beetle red luciferase and aequorin photoprotein. Alternative, non-luciferase, bioluminescent donor molecules that can be employed in this invention are any enzymes which can act on suitable substrates to generate a luminescent signal. Specific examples of such enzymes are β-galactosidase, lactamase, horseradish peroxidase, alkaline phophatase, β-glucuronidase and β-glucosidase. Synthetic luminescent substrates for these enzymes are well known in the art and are commercially available from companies, such as Tropix Inc. (Bedford, Mass., USA).

[0001 190] In a preferred embodiment, a bioluminescent donor molecule with a small molecular weight is used to prevent or minimise an inhibition of the interaction due to steric hindrance. The bioluminescent donor preferably consists of a single polypeptide chain. Also the bioluminescent proteins preferably do not form oligomers or aggregates. The bioluminescent donor molecules Renilla luciferase, Gaussia luciferase, Firefly luciferase and NanoLuc meet ah or most of these criteria. In some preferred embodiments, the bioluminescent donor comprises a certain activated co-located GPCR (such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2), as broadly defined above.

[0001 191 ] In some embodiments, the bioluminescent donor comprises a luciferase variant. The luciferase variants retain luciferase activity (i.e., catalyse the conversion of a coelenterazine substrate into a luminescent product in the presence of molecular oxygen). The luciferase variants may have at least one of the following properties relative to their corresponding reference wild-type protein: modulated stability; enhanced light output; modulated emission wavelength maximum; and modulated substrate utilization. In certain embodiments, the luciferase variants suitable for the present invention include two or more of the above properties (e.g., modulated stability and enhanced brightness, enhanced light output and modulated emission maximum, modulated stability and modulated emission maximum, and the like), or include three or more of the above properties (e.g., modulated stability, enhanced light output and modulated emission maximum).

[0001 192] As mentioned above, one such variant Renilla luciferase protein that is specifically contemplated includes, but is not limited to, eight amino acid substitutions in comparison to the wild-type amino acid sequence (SEQ ID NO: 61 ). These amino acid substitutions include Ala55Thr, Cys124Ala, Ser130Ala, Lys136Arg, Ala143Met, Met185Val, Met253Leu, and Ser287Leu. In addition, variant Renilla luciferase proteins can include one or more additional conservative substitution as long as the conservatively modified variant retains the characteristics of the mutated Renilla luciferase protein.

6.2 The bioluminescent donor encoding vector

[0001 193] In some embodiments, a vector encoding the bioluminescent donor can include, but is not limited to, polynucleotides that encode the bioluminescent donor (e.g., encoding a certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2 fused or otherwise linked to RLuc8). Methods of producing vectors (e.g., viral and non-viral) and polynucleotides are well known in the art. It should be noted that the bioluminescent donors can be expressed using other expression systems and the bioluminescent donor vector is merely an illustrative embodiment.

6.3 Fluorescent acceptors

[0001 194] As mentioned above, the fluorescent acceptor includes, but is not limited to, a second target protein, wherein the second target protein consists of either a RAGE polypeptide or a certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, and a fluorescent acceptor molecule. In general, fluorophores absorb electromagnetic and/or resonance energy at one wavelength and emit electromagnetic energy at a second wavelength. In some preferred embodiments, the fluorescent acceptor comprises a RAGE polypeptide, as broadly defined above and elsewhere herein.

[0001 195] Representative fluorescent acceptor molecules (i.e., fluorophores) can include, but are not limited to, sgGFP, sgBFP, blue-shifted GFP (Y66H), Blue Fluorescent Protein (BFP), Cyan Fluorescent Protein (CFP), Cyan GFP, DsRed, DsRed2, monomeric RFP, enhanced BFP (EBFP), enhanced CFP (ECFP), enhanced GFP (EGFP), destabilised EGFP, destabilised ECFP, destabilised EYFP, GFP (S65T), red-shifted GFP (rsGFP), wild-type GFP (GFP), GFPuv, HcRed, t-HcRed, rsGFP, Sapphire GFP, sgBFP™, sgBFP™ (super glow BFP), sgGFP™, sgGFP™ (super glow GFP), wild-type GFP, yellow variant GFP, YFP, Venus, Emerald, Topaz, Citrine, YPet, t-dimer2, t dimer2(12), mRFP1 , pocilloporin, Renilla GFP, Monster GFP, paGFP, and Kaede protein. Favourably, Venus contains an amino acid substitution compared to the YFP sequence, which accelerates the oxidation of the chromophore at 37°C, the rate limiting step of maturation. Venus also has other substitutions (e.g., F63L, M153T, V163A, and S175G) that permit the fluorophore to fold well and improves the protein's tolerance to acidosis and CI ^" .

[0001 196] Other representative fluorescent acceptor molecules (i.e., fluorophores) can include, but are not limited to: Alexa Fluor derivatives, BODIPY derivatives, and those referenced in US Patent 8,283,127, 1 ,5 IAEDANS; 1 ,8-ANS; 4-Methylumbelliferone; 5- carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5- HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5- TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7- Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9- Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs— AutoFluorescent Protein— (Quantum Biotechnologies); ALEXA FLUOR 350™; ALEXA FLUOR 430™; ALEXA FLUOR 488™; ALEXA FLUOR 532™; ALEXA FLUOR 546™; ALEXA FLUOR 568™; ALEXA FLUOR 594™; ALEXA FLUOR 633™; ALEXA FLUOR 647™; ALEXA FLUOR 660™; ALEXA FLUOR 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide (Hoechst); bis-BTC; Blancophor FFG; BlancophorSV; BOBO™-1 ; BOBO™-3; Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581 /591 ; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl- Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1 ; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; CALCIUM CRIMSON™; Calcium Green; Calcium Green-1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5- ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coumarin Phalloidin; C- phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.18; Cy3.5™; Cy3™; Cy5.18; Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3 ' DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-DM 6-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1 ); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1 -43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; lndo-1 , high calcium; lndo-1 , low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1 ; JO-JO-1 ; JO-PRO-1 ; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1 ; LO-PRO-1 ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1 ; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1 ; POPO-3; PO-PRO-1 ; PO-PRO- 3; Primuline; Procion Yellow; Propidium lodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 1 10; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A; S65C; S65L; S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1 ; SNAFL-2; SNARF calcein; SNARF1 ; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine Extra; SYTO 1 1 ; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21 ; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41 ; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61 ; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81 ; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); TEXAS RED™; TEXAS RED-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1 ; TO-PRO-3; TO-PRO-5; TOTO-1 ; TOTO-3; Tricolor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; WW 781 ; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1 ; YO-PRO-3; YOYO-1 ; YOYO-3, Sybr Green, Thiazole orange (interchelating dyes), or combinations thereof.

[0001 197] The fluorescent acceptor molecule may be a fluorophore conjugated to a molecule that enables attachment another molecule. An example of a molecule that enables attachment to another molecule is HaloTag. Examples of fluorophores that may or may not be used attached to HaloTag and may or may not be described as HaloTag ligands as a result are Oregon Green, tetramethylrhodamine (TMR) and nonchloro TOM (NCT).

6.4 Fluorescent acceptor vectors

[0001 198] The fluorescent acceptor vector can include, but is not limited to, a polynucleotide that encodes the fluorescent acceptor fusion protein (for example, a RAGE polypeptide and the green fluorescent protein variant, Venus) and degenerate nucleotide sequences thereof. Methods of producing polynucleotides and vectors (e.g., viral and non- viral) are well known in the art. It should be noted that the fluorescent fusion protein can be expressed using other expression systems and the fluorescent vector is merely an illustrative embodiment.

6.5 Bioluminescence initiating compound

[0001 199] The choice of the bioluminescence initiating compound can impact on the wavelength and the intensity of the light generated by the bioluminescent donor.

[0001200] An exemplary bioluminescence initiating compound that is known to be suitable for BRET techniques is coelenterazine. Coelenterazine occurs in cnidarians, copepods, chaetgnaths, ctenophores, decapod shrimps, mysid shrimps, radiolarians and some fish taxa (Greer and Szalay, 2002). For Renilla luciferase and variants of Renilla luciferase, coelenterazine analogues and functional derivatives thereof are available that result in light emission between 418 and 512 nm (Inouye et al., 1997). Some coelenterazine analogues and functional derivatives thereof may result in light emission at wavelengths below 418 nm. Bisdeoxycoelenterazine (also known as DeepBlueC, also known as colelenterazine 400a) and derivatives thereof are examples of substrates with emission spectra that include light emission below 418 nm as well as light emission above 418 nm.

[0001201 ] The bioluminescence initiating compound can include, but is not limited to, coelenterazine and analogues, and functional derivatives thereof. Exemplary derivatives of coelenterazine include, but are not limited to, coelenterazine 400a, coelenterazine cp, coelenterazine f, coelenterazine fcp, coelenterazine h, coelenterazine hep; coelenterazine ip, coelenterazine n, coelenterazine O, coelenterazine c, coelenterazine c, coelenterazine i, coelenterazine icp, coelenterazine 2-methyl, benzyl-coelenterazine bisdeoxycoelenterazine, EnduRen, ViviRen, deep blue coelenterazine (DBC; DeepBlueC) (described in more detail in U.S. Pat. Nos. 6,020,192; 5,968,750 and 5,874,304), furimazine and variants thereof.

[0001202] In general, coelenterazines are known to luminesce when acted upon by a wide variety of bioluminescent proteins, specifically Luciferases. Useful, but non-limiting, coelenterazines are disclosed in U.S. patent application Ser. No. 10/053,482, the disclosure which is hereby incorporated by reference in its entirety. Coelenterazines are available from Promega Corporation (U.S.A.). and from Molecular Probes, Inc. (U.S.A.). Coelenterazines may also be synthesized as described for example in Shimomura et al., Biochem. J. 261 : 913-20, 1989; Inouye et al., Biochem. Biophys. Res. Comm. 233: 349-53, 1997; and Teranishi et al., Anal. Biochem. 249: 37-43, 1997.

6.6 Methods of performing the BRET assay

[0001203] As mentioned above, the present invention includes methods for determining (i.e., detecting, localizing, or quantifying) the proximity between a RAGE polypeptide and an AT ₁ R polypeptide inside a living cell, tissue, or organ, or a living organism using the BRET system. For example, a living animal can be imaged using the BRET system to measure the proximity of a RAGE polypeptide and an AT ₁ R polypeptide and the degree of that proximity. This approach facilitates the study of protein-protein interactions to understand fundamental cell biology and will enable the in vivo testing of candidate agents that have been identified as being a modulator of the RAGE-AT ₁ R complex.

[0001204] For example, the BRET system can be used to measure and quantify the proximity between RAGE and AT ₁ R in living cells to determine the ability of candidate agents administered to modulate (i.e., increase or reduce) the proximity. Further, the BRET system can be used to measure the effects of candidate agents targeting the RAGE-AT ₁ R complex.

[0001205] In some embodiments, a cell line transfected with vector constructs as described above and elsewhere herein that is developed utilizing coding regions for the RAGE polypeptides and the AT ₁ R polypeptides, followed by measurement in a suitable microplate reader or optical imaging to quantitate the proximity between RAGE and AT ₁ R in the presence and absence of candidate agents to identify whether said candidate agent modulates (i.e., stimulate or inhibit) proximity between RAGE and AT ₁ R. As will be appreciated by the skilled practitioner, this technique will significantly accelerate candidate agent validation by allowing testing in vivo. [0001206] As will be appreciated by one of skill in the art, such screening may also be done in cell culture. Preferably, the compounds screened are suitable for administering to mammals. Even more preferably, the compounds screened are suitable for administering to humans.

6.7 BRET Kits

[0001207] The present invention encompasses kits which may include two or more, three or more, four or more, or all of the following: a bioluminescent donor (that comprises, for example, an AT ₁ R polypeptide fused or otherwise linked to the bioluminescent donor molecule RLuc8); a vector comprising a bioluminescent donor; a fluorescent acceptor (that comprises, for example, a RAGE polypeptide fused or otherwise linked to the fluorescent acceptor molecule Venus); a vector comprising a fluorescent acceptor; a bioluminescence initiating compound; a candidate agent; and directions (written instructions for their use). Accordingly, in some embodiments, the bioluminescent donor will comprises an AT ₁ R polypeptide and the fluorescent acceptor comprises a RAGE polypeptide. The kit can also include appropriate buffers and reagents known in the art for administering various combinations of the components listed above to a host cell or a host organism.

[0001208] By way of a specific illustrative example, the kit may comprise a bioluminescent donor comprising an AT ₁ R polypeptide fused or otherwise linked to the bioluminescent donor molecule Rluc8; and a fluorescent acceptor comprising a RAGE polypeptide fused or otherwise linked to the fluorescent acceptor molecule Venus.

[0001209] By way of a specific illustrative example, to perform the BRET assay the bioluminescent donor and the fluorescent acceptor are co-expressed in cells as broadly described above. The generation of a strong and saturable BRET signal indicates close proximity (for example, less than 10 nm) between the AT ₁ R polypeptide and the RAGE polypeptide, allowing energy resulting from the oxidation of a cell-permeable coelenterazine substrate by the donor to transfer to the acceptor, which in turn fluoresces at a longer characteristic wavelength. Accordingly, identification of a candidate agent as a modulator of the AT ₁ R polypeptide and the RAGE polypeptide proximity is made on the basis of a change (e.g., increase or reduction) of the BRET signal generated by a cell co-expressing labelled AT ₁ R polypeptide and labelled RAGE polypeptide, as a result of the cell being exposed to the candidate agent. Specifically, a reduction in the BRET signal generated by the cell in response to exposure to a candidate agent identifies the agent as an inhibitor of the proximity between RAGE and AT ₁ R. Conversely, an increase in the BRET signal generated by the cell in response to exposure to a candidate agent identifies the agent as an inducer of the proximity between RAGE and AT ₁ R. Construct Systems

[0001210] In accordance with the present invention, a construct system is provided for identifying modulators of proximity between a RAGE polypeptide and an AT ₁ R polypeptide. In one broad form, the system comprises a first synthetic construct encoding a RAGE polypeptide and an energy donor molecule (e.g., a bioluminescent donor molecule); and a second synthetic construct encoding an AT ₁ R polypeptide and an energy acceptor molecule (e.g., a fluorescent acceptor molecule). In another broad form, the system comprises a first synthetic construct encoding an AT ₁ R polypeptide and an energy donor molecule (e.g., a bioluminescent donor molecule); and a second synthetic construct encoding a RAGE polypeptide and an energy acceptor molecule (e.g., a fluorescent acceptor molecule).

[000121 1 ] The synthetic constructs of the invention each comprise a regulatory sequence that is operably connected to the bioluminescent donor coding sequence or the fluorescent acceptor coding sequence. The regulatory sequence suitably comprises transcriptional and/or translational control sequences, which will be compatible for expression in the cell or organism of interest. Typically, the transcriptional and translational regulatory control sequences include, but are not limited to, a promoter sequence, a 5' non-coding region, a cis-regulatory region such as a functional binding site for transcriptional regulatory protein or translational regulatory protein, an upstream open reading frame, ribosomal-binding sequences, transcriptional start site, translational start site, and/or nucleotide sequence which encodes a leader sequence, termination codon, translational stop site and a 3' non- translated region. Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. Promoter sequences contemplated by the present invention may be native to the organism of interest or may be derived from an alternative source, where the region is functional in the chosen organism. The choice of promoter will differ depending on the intended host. For example, promoters which could be used for expression in plants include plant promoters such as: constitutive plant promoters examples of which include CaMV35S plant promoter, CaMV19S plant promoter, FMV34S plant promoter, sugarcane bacilliform badnavirus plant promoter, CsVMV plant promoter, Arabidopsis ACT2/ACT8 actin plant promoter, Arabidopsis ubiquitin UBQ1 plant promoter, barley leaf thionin BTH6 plant promoter, and rice actin plant promoter; tissue specific plant promoters examples of which include bean phaseolin storage protein plant promoter, DLEC plant promoter, PHSD plant promoter, zein storage protein plant promoter, conglutin gamma plant promoter from soybean, AT2S1 gene plant promoter, ACT1 1 actin plant promoter from Arabidopsis, napA plant promoter from Brassica napus and potato patatin gene plant promoter; and inducible plant promoters examples of which include a light-inducible plant promoter derived from the pea rbcS gene, a plant promoter from the alfalfa rbcS gene, DRE, MYC and MYB plant promoters which are active in drought; INT, INPS, prxEa, Ha hsp17.7G4 and RD21 plant promoters active in high salinity and osmotic stress, and hsr203J and str246C plant promoters active in pathogenic stress. Alternatively, promoters which could be used for expression in mammals include the metallothionein promoter, which can be induced in response to heavy metals such as cadmium, the β-actin promoter as well as viral promoters such as the SV40 large T antigen promoter, human cytomegalovirus (CMV) immediate early (IE) promoter, Rous sarcoma virus LTR promoter, adenovirus promoter, or a HPV promoter, particularly the HPV upstream regulatory region (URR) may also be used. All these promoters are well described and readily available in the art.

[0001212] The synthetic constructs of the present invention may also comprise a 3' non- translated sequence. A 3' non-translated sequence refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is characterised by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognised by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. The 3' non-translated regulatory DNA sequence preferably includes from about 50 to 1 ,000 nucleotide base pairs and may contain transcriptional and translational termination sequences in addition to a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.

[0001213] In specific embodiments, the synthetic constructs further contain a selectable marker gene to permit selection of an organism or a precursor thereof that contains a synthetic construct. Selection genes are well known in the art and will be compatible for expression in cell or organism of interest, or a progenitor or precursor thereof.

[0001214] In some embodiments, the synthetic constructs of the invention are in the form of viral vectors, such as simian virus 40 (SV40) or bovine papilloma virus (BPV), which has the ability to replicate as extra-chromosomal elements (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982; Sarver et al., 1981 , Mol. Cell. Biol. 1 :486). Viral vectors include retroviral (lentivirus), adeno-associated virus (see, e.g., Okada, 1996, Gene Ther. 3:957-964; Muzyczka, 1994, J. Clin. Invst. 94:1351 ; U.S. Pat. Nos. 6,156,303; 6,143,548 5,952,221 , describing AAV vectors; see also U.S. Pat. Nos. 6,004,799; 5,833,993), adenovirus (see, e.g., U.S. Pat. Nos. 6,140,087; 6,136,594; 6,133,028; 6,1 20,764), reovirus, herpesvirus or rotavirus genomes, modified for introducing and directing expression of a polynucleotide or transgene in cells. Retroviral vectors can include those based upon murine leukemia virus (see, e.g., U.S. Pat. No. 6,132,731 ), gibbon ape leukemia virus (see, e.g., U.S. Pat. No. 6,033,905), simian immuno-deficiency virus, human immuno-deficiency virus (see, e.g., U.S. Pat. No. 5,985,641 ), and combinations thereof.

[0001215] Vectors also include those that efficiently deliver genes to animal cells in vivo (e.g., stem cells) (see, e.g., U.S. Pat. Nos. 5,821 ,235 and 5,786,340; Croyle et al, 1998, Gene Ther. 5:645; Croyle et al., 1998, Pharm. Res. 15:1348; Croyle et al., 1998, Hum. Gene Ther. 9:561 ; Foreman et al., 1998, Hum. Gene Ther. 9:1313 ; Wirtz et al., 1999, Gut 44:800). Adenoviral and adeno-associated viral vectors suitable for in vivo delivery are described, for example, in U.S. Pat. Nos. 5,700,470, 5,731 ,172 and 5,604,090. Additional vectors suitable for in vivo delivery include herpes simplex virus vectors (see, e.g., U.S. Pat. No. 5,501 ,979), retroviral vectors (see, e.g., U.S. Pat. Nos. 5,624,820, 5,693,508 and 5,674,703; and WO92/05266 and W092/14829), bovine papilloma virus (BPV) vectors (see, e.g., U.S. Pat. No. 5,719,054), CMV-based vectors (see, e.g., U.S. Pat. No. 5,561 ,063) and parvovirus, rotavirus and Norwalk virus vectors. Lentiviral vectors are useful for infecting dividing as well as non-dividing cells (see, e.g., U.S. Pat. No. 6,013,516).

[0001216] Vectors for insect cell expression commonly use recombinant variations of baculoviruses and other nucleopolyhedrovirus, e.g., Bombyx mori nucleopolyhedrovirus vectors (see, e.g., Choi, 2000, Arch. Virol. 145:171 -177). For example, Lepidopteran and Coleopteran cells are used to replicate baculoviruses to promote expression of foreign genes carried by baculoviruses, e.g., Spodoptera frugiperda cells are infected with recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV) carrying a heterologous, e.g., a human, coding sequence (see, e.g., Lee, 2000, J. Virol. 74:1 1873- 1 1880; Wu, 2000, J. Biotechnol. 80:75-83). See, e.g., U.S. Pat. No. 6,143,565, describing use of the polydnavirus of the parasitic wasp Glyptapanteles indiensis to stably integrate nucleic acid into the genome of Lepidopteran and Coleopteran insect cell lines. See also, U.S. Pat. Nos. 6,130,074; 5,858,353; 5,004,687.

[0001217] The invention further contemplates cells containing therein the synthetic constructs of the invention. In this regard, it will be appreciated that the construct system of the present invention is applicable to prokaryotic as well as eukaryotic host cells and includes for example unicellular organisms and cells derived from multicellular organisms, such as but not limited to yeast, plants and animals including vertebrate animals such as mammals, reptiles, fish and birds, as well as invertebrate animals such as metazoa, sponges, worms, molluscs, nematodes, crustaceans and echinoderms. In certain embodiments, the construct system is used to determine the translational efficiency of different synonymous codons in plant cells or animal cells or to determine the ability of a candidate agent to modulate proximity between a RAGE polypeptide and a AT ₁ R polypeptide.

[0001218] Illustrative examples of eukaryotic organisms include, but are not limited to, fungi such as yeast and filamentous fungi, including species of Aspergillus, Trichoderma, and Neurospora; animal hosts including vertebrate animals illustrative examples of which include fish (e.g., salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod and zebrafish), birds (e.g., chickens, ducks, quail, pheasants and turkeys, and other jungle foul or game birds) and mammals (e.g., dogs, cats, horses, cows, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, marine mammals including dolphins and whales, as well as cell lines, such as human or other mammalian cell lines of any tissue or stem cell type (e.g., COS, NIH 3T3 CHO, BHK, HEK293, or HeLa cells), and stem cells, including pluripotent and non-pluripotent and embryonic stem cells, and non- human zygotes), as well as invertebrate animals illustrative examples of which include nematodes (representative generae of which include those that infect animals such as but not limited to Ancylostoma, Ascaridia, Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus, Haernonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia, Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris, Trichostrongylus, Tflichonema, Toxocara, Uncinaria, and those that infect plants such as but not limited to Bursaphalenchus, Criconerriella, Diiylenchus, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Melodoigyne, Nacobbus, Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus, and Xiphinerna) and other worms, drosophila, and other insects (such as from the families Apidae, Curculionidae, Scarabaeidae, Tephritidae, Tortricidae, representative orders of which include Coleoptera, Diptera, Lepidoptera, and Homoptera).

[0001219] The synthetic constructs of the present invention may be introduced directly ex vivo or in cell culture into a cell of interest The synthetic constructs of the present invention may be introduced into a cell of interest using any suitable method, and the kind of method employed will differ depending on the intended cell type of interest. For example, four general classes of methods for delivering nucleic acid molecules into cells have been described: (1 ) chemical methods such as calcium phosphate precipitation, polyethylene glycol (PEG)-mediate precipitation and lipofection; (2) physical methods such as microinjection, electroporation, acceleration methods and vacuum infiltration; (3) vector based methods such as bacterial and viral vector-mediated transformation; and (4) receptor- mediated. Transformation techniques that fall within these and other classes are well known to workers in the art, and new techniques are continually becoming known. The particular choice of a transformation technology will be determined by its efficiency to transform certain host species as well as the experience and preference of the person practicing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce a synthetic construct of the invention into cells is not essential to or a limitation of the invention, provided it achieves an acceptable level of nucleic acid transfer. Thus, the synthetic constructs are introduced into tissues or host cells by any number of routes, including viral infection, phage infection, microinjection, electroporation, or fusion of vesicles, lipofection, infection by Agrobacterium tumefaciens or A. rhizogenes, or protoplast fusion. Jet injection may also be used for intramuscular administration (as described for example by Furth et al., 1992, Anal Biochem 205:365-368). The synthetic constructs may be coated onto microprojectiles, and delivered into a host cell or into tissue by a particle bombardment device, or "gene gun" (see, for example, Tang et al., 1992, Nature 356:152-154). Alternatively, the synthetic constructs can be fed directly to, or injected into, a host organism or it may be introduced into a cell (i.e., intracellular^) or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, or introduced orally. Methods for oral introduction include direct mixing of the synthetic constructs with food of the organism. In certain embodiments, a hydrodynamic nucleic acid administration protocol is employed (e.g., see Chang et al., 2001 , J. Virol. 75:3469-3473; Liu et al., 1999, Gene Ther. 6:1258-1266; Wolff et al., 1990, Science 247:1465-1468; Zhang et al., 1999, Hum. Gene Ther. 10:1735-1737; and Zhang et al., 1999, Gene Ther. 7:1344-1349). Other methods of nucleic acid delivery include, but are not limited to, liposome-mediated transfer, naked DNA delivery (direct injection) and receptor-mediated transfer (ligand-DNA complex).

8. Assays that detect downstream signalling mediated by activation of RAGE

[0001220] Activation of RAGE is known to trigger many intracellular signalling pathways involved in specific aspects of cellular function, depending on the specific cell type, its state and the duration of stimulation. These signalling pathways are activated through production of ROS via NADPH oxidase and activation of various kinases, including mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 /2 (ERK1 /2), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38-MAP kinase; Rho kinase (Rho), AMP kinase (AMPK), phosphoinositide 3 kinases/Akt (PI3K/Akt); Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT); glycogen synthase kinase 3 beta (Θ5Κ3β) (Batkulwar KB et a/ 2015). These kinases phosphorylate downstream signalling molecules and cause specific cellular responses often via activation of transcription factors nuclear factor kappa B (NF-κΒ), egrl and specificity protein 1 (SP1 ), thereby modulating specific cellular processes, including inflammation, cell motility, adhesion, and structure.

[0001221 ] The generation of ROS following the activation of RAGE can be measured by assays detecting ROS themselves (through modification of probes by ROS and detection of their adducts), measurement of modifications induced by ROS on endogenous or synthetic substrates, or the induction of signalling pathways resulting from them.

[0001222] These specific downstream signals following the activation of RAGE can be measured by assays detecting the activation of these kinases (i.e. kinase activity assays), phosphorylation of synthetic or endogenous targets of kinase activity, or downstream induction of transcription factors (e.g. N FKB, AP-1 , EGR-1 ), changes in gene expression, protein expression or changes in cellular function/phenotype resulting from them.

9. Pharmaceutical compositions

[0001223] Generally, upon identifying a candidate agent as a modulator of RAGE ligand- independent activation of RAGE by certain activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2, and thus selected as having therapeutic potential, the agents are manufactured in the form of pharmaceutical compositions that optionally comprise a pharmaceutically acceptable carrier, excipient and/or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). These compositions are generally in the form of lyophilized formulations or aqueous solutions. Antibody crystals are also contemplated (see, U.S. Pat. Appl. 2002/0136719). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Lyophilized antibody formulations are described in WO 97/04801 .

[0001224] The pharmaceutical compositions contain the active compounds as necessary for the particular indication being treated, desirably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0001225] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0001226] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0001227] In some embodiments, and dependent on the intended mode of administration, the compositions will generally contain about 0.000001 % to 90%, about 0.0001 % to 50%, or about 0.01 % to about 25%, by weight of the modulator, the remainder being suitable pharmaceutical carriers, diluents, or excipients. The dosage of the modulator can depend on a variety of factors, such as mode of administration, the species of the affected subject, age, sex, weight and general health condition, and can be easily determined by a person of skill in the art using standard protocols. The dosages will also take into consideration the binding affinity of the modulator to its binding partner (i.e., co-located GPCR such as AT ₁ R and/or RAGE), its bioavailability and its in vivo and pharmacokinetic properties. In this regard, precise amounts of the agents for administration can also depend on the judgment of the practitioner. In determining the effective amount of the agents to be administered in the treatment or prevention of a cardiometabolic disease and/or condition, the physician or veterinarian may evaluate the progression of the disease or condition over time. In any event, those of skill in the art may readily determine suitable dosages of the agents of the invention without undue experimentation. The dosage of the actives administered to a patient should be sufficient to effect a beneficial response in the patient over time, and/or in the treatment and/or prevention of a cardiometabolic disease and/or condition). The dosages may be administered at suitable intervals to ameliorate the symptoms of the cardiometabolic disease and/or condition. Such intervals can be ascertained using routine procedures known to persons of skill in the art and can vary depending on the type of active agent employed and its formulation. For example, the interval may be daily, every other day, weekly, fortnightly, monthly, bimonthly, quarterly, half-yearly or yearly.

[0001228] For any modulator identified using the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (e.g., the concentration of a modulator which achieves a half-maximal inhibition of RAGE ligand-independent activation of RAGE by activated co-located GPCR such as an angiotensin receptor such as AT ₁ R or a certain chemokine receptor such as CCR2). Such information can be used to more accurately determine useful doses in mammals, including humans.

[0001229] Dosage amount and interval may be adjusted individually to provide plasma levels of the active agent, which are sufficient to maintain therapeutic effects. Usual patient dosages for systemic administration range from 1 -2000 mg/day, commonly from 1 -250 mg/day, and typically from 10-150 mg/day. Stated in terms of patient body weight, usual dosages range from 0.02-25 mg/kg/day, commonly from 0.02-3 mg/kg/day, typically from 0.2-1 .5 mg/kg/day. Stated in terms of patient body surface areas, usual dosages range from 0.5-1200 mg/m ² /day, commonly from 0.5-150 mg/m2/day, typically from 5-100 mg/m ² /day.

[0001230] The modulator may be administered concurrently with at least one ancillary therapy that treats or ameliorates the symptoms or reverses or inhibits the development or progression of a cardio-metabolic disease and/or condition in a subject. The modulator may be used therapeutically after the ancillary therapy or may be used, before the therapy is administered, or together with the therapy. Accordingly, the present invention contemplates combination therapies, which employ a modulator and concurrent administration of an ancillary therapy (e.g., medical treatment), non-limiting examples of which include diuretics, beta blockers, alpha inhibitors, ACE inhibitors and angiotensin receptor blockers. [0001231 ] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

In each of the following examples 1-21 independently, the following general materials and Methods apply, unless the context requires otherwise.

Animal Models

[0001232] C57bl6 mice, AGER knockout (KO) mice, apoE KO mice, AGERIapoE double knockout (DKO), Ace2lapoE DKO and AGER/Ace2/apoE triple knockout (TKO) mice were sourced and generated in-house at the AMPREP animal house. All mice were bred on a C57bl6 background. For experimental studies, male mice aged 6-8 weeks and weighing between 20-25g were used, with at least 8 animals per group for in vivo studies and n=6/group for ex vivo studies. Throughout the study animals were given access to mouse chow and water ad libitum. All experiments were approved by the animal ethics committee of the Alfred Medical Research Precinct and conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Aortic Plaque Area Quantitation method

[0001233] Aortic arches were removed from mice, cleaned of excess fat, placed in 10% neutral buffered formalin and subsequently stained with Sudan IV-Herxheimer's solution (0.5% w/vol; Gurr, BDH Limited, Poole, UK). Aortae were then dissected longitudinally, and pinned flat onto a wax pad. Plaque accumulation across the aortic arch was quantitated as the percentage area stained red.

Aortic Expression of Pro-inflammatory Mediators by Quantitative Real-Time PCR

[0001234] At the end of each study period, thoracic and abdominal aortae were placed in TRIZOL, snap frozen and stored at -80°C. RNA extraction and cDNA synthesis was performed in aortic homogenates using the Trizol method. Gene expression of pro- atherogenic mediators including adhesion molecules, cytokines, inflammatory, oxidative stress and macrophage markers, were estimated in aortic homogenates by quantitative realtime RT-PCR, performed using the TaqMan system based on real-time detection of accumulated fluorescence (ABI Prism 7700, Perkin-Elmer Inc, PE Biosystems, Foster City, CA, USA). Gene expression was normalized to 18S mRNA and reported as fold change compared to the level of expression in untreated control mice/cells, which were given an arbitrary value of 1 .

Induction of Oxidative Stress

[0001235] The induction of oxidative stress in each animal model was estimated by the measurement of plasma levels of 8-hydroxydeoxyguanosine using the OxiSelect Oxidative DNA Damage ELISA Kit (8-OHdG Quantitation; Cell Biolabs, Inc), and performed as per manufacturer's instructions. In addition, gene expression of the superoxide-producing NADPH oxidase subunits, NOX-1 and NOX-4 were quantitated in the aortae of mice using real time RT-PCR, as detailed above.

Circulating concentration of RAGE ligands

[0001236] Changes in circulating levels of RAGE ligands were assessed in animal models. S100A8/A9 levels were estimated by ELISA (Immundiagnostik, Germany) performed according to the manufacturer's instructions. Plasma AGEs were measured by in-house ELISA. Plasma methylglyoxal, a reactive precursor of AGEs, was measured by HPLC.

Systolic BP measurement

[0001237] Systolic blood pressure was measured by tail-cuff plethysmography in conscious, pre-warmed mice using a computerized, non-invasive, tail-cuff system (Kent Scientific, USA). Animals were habituated to the device before measuring the pressures to ensure accurate measurements.

In vitro studies

Cell culture

[0001238] Primary aortic endothelial cells (PMAEC) were isolated from the aortae of (wild- type) C57bl6 mice and AGER KO mice and cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 endothelial cell growth supplement (ECGS) supplemented media. Chinese Hamster Ovary (CHO) cells were cultured using F12 media (10% FCS with 2mM glutamine). Human microvascular endothelial cells (HMEC) were cultured in MCDB 131 medium (10% FCS with 10mM glutamine, EGF and hydrocortisone). Generation of transgenic Chinese Hamster Ovary Cells

[0001239] 100ng of AT ₁ R-Rluc8 construct was transfected into CHO cells using Lipofectamine 2000 (Thermo). Stable transfectants were selected using G418. AT1 R-CHO were then transiently transfected with RAGE constructs using Lipofectamine 2000 (Invitrogen) and incubated for 16h. The Homo sapiens AGER transcript variant 1 coding sequence (NM 001 136) was synthesised and cloned between the Nhel and Xbal sites of pClneo (Promega) to generate the vector pClneo-RAGE. This plasmid was then used to generate a library of mutant AGER clones (NP_001 127). Primers 1 -RAGE-SP-5' and mCherry-Xhol-Notl-3' were used to amplify the coding sequence of mCherry from the vector pmCherry-C1 . The resultant PCR product was then used as template for a second PCR using primers 2-RAGE-SP-5' and mCherry-Xhol-Notl-3'. The PCR product generated was digested with Nhel and Notl and inserted between the Nhel and Notl sites of pClneo to generate pClneo-SP-mCherry. The sequence of the insert was confirmed by DNA sequencing (Micromon, Monash University). For truncated RAGE constructs, primers 1 - RAGE-SP-5' and mCherry-Xhol-Notl-3' were used to amplify the coding sequence of mCherry from the vector pmCherry-C1 . The resultant PCR product was then used as template for a second PCR using primers 2-RAGE-SP-5' and mCherry-Xhol-Notl-3'. The PCR product generated was digested with Nhel and Notl and inserted between the Nhel and Notl sites of pClneo to generate pClneo-SP-mCherry. The sequence of the insert was confirmed by DNA sequencing (Micromon, Monash University).

Cellular expression of pro-inflammatory markers and mediators by quantitative real-time PCR

[0001240] After 2 hours of exposure to Ang II (1 μΜ) or the RAGE ligand, S100A8/A9 (5ng/ml_) cells were placed in Trizol, mRNA extracted and cDNA synthesized. Changes in the gene expression of the N FKB subunit, p65 (RelA) or NFKB-activated target genes (e.g ICAM- 1, VCAM- 1) were estimated by quantitative real-time RT-PCR, performed using the TaqMan system based on real-time detection of accumulated fluorescence (ABI Prism 7700, Perkin-Elmer Inc, PE Biosystems, Foster City, CA, USA). Gene expression was normalized to 18S mRNA and reported as fold change compared to the level of expression in untreated control mice/cells, which were given an arbitrary value of 1 .

Selective silencing of gene expression using siRNA

[0001241 ] PMAEC from C57bl6 mice were also transfected with siRNA to RAGE (2nM) Ambion, ID:s621 19), p65 (RelA) (10nM; Ambion, ID:s72857), MyD88 (10nM; Ambion. ID:s201719), ΡΚCζ (10nM; Ambion, ID:s71716), IQGAP-1 , Ambion, ID:s781 19), Diaphl (10nM; sense5'-UACAGAGGAAGCUGAUAUUGAAGCC, antisense 3'-

GGCUUCAAUAUCAGCUUCCUCUGUA; Invitrogen) or scrambled control #1 (2nM or 10nM; Ambion) using Lipofectamine RNAiMAX (Invitrogen) as per manufacturer's instructions. Cells were allowed to recover in media containing 10% FBS for 16 hours prior to treatment with 1 μΜ Ang II or 5ng/ml of the RAGE ligand, SiooA8/A9, as a signalling control.

Generation of peptides

[0001242] Oligopeptides (Cherry-TAT (control), mCherry-TAT-RAGE ₃₆ 2-404 and mCherry- S391 A-TAT-RAGE ₃ 62-404 were generated by transformation of the Escherichia coli strain ClearColi BL21 (DE3; Lucigen). A single transformed colony was inoculated into 50 ml 2YT medium containing 100 μg/ml ampicillin and the culture was grown overnight at 37°C with shaking at 250 rev/min. 10 ml overnight culture was used to inoculate 1 litre 2YT medium containing 100 μg/ml ampicillin and the culture was grown at 37°C with shaking at 250 rev/min. When the cultures reached an OD600 of 0.8, the temperature was shifted to 15°C for 30 min after which protein expression was induced by the addition of 1 mM isopropyl β- D-1 -thiogalacto-pyranoside (IPTG). Following the addition of IPTG the cultures were grown for -16 h at 15°C with shaking at 250 rev/min. Cells were harvested by centrifugation (3500g, 4°C, 20 min) and resuspended in ice-cold lysis buffer (50 mM Tris pH 7.4, 300 mM NaCI, 10 mM imidazole and 5mM beta-mercaptoethanol). The resuspended cells were sonicated and insoluble material was pelleted by centrifugation (13 OOOg, 4°C, 30 min). The supernatant was filtered (0.2 μηι) and applied onto a gravity-flow column with a 1 ml Ni-NTA Agarose resin bed volume (Qiagen) pre-equilibrated with lysis buffer. After the supernatant was passed over the resin, the column was washed with 100 ml of wash buffer (20 mM imidazole, 50 mM Tris pH 7.4, 150 mM NaCI, 5mM beta-mercaptoethanol). His-tagged TAT-mCherry and TAT-mCherry-RAGE peptides were eluted from the resin using elution buffer (250 mM imidazole, 50 mM Tris pH 7.4, 150 mM NaCI, 5mM beta-mercaptoethanol). The purity of the purified proteins was assessed by SDS-PAGE. Typically, 10 mg protein was produced per litre of culture. The eluted fractions containing protein were pooled and quantified by Nanodrop Spectrophotometry (Thermo Scientific) and BCA Protein Assay (Pierce).

Bioluminescence resonance energy transfer (BRET)

[0001243] BRET is an established technology for studying protein-protein proximity in live cells, particularly involving GPCRs (Pfleger and Eidne, 2006). One protein of interest was linked to a bioluminescent donor enzyme, Rluc8, a variant of Renilla luciferase, and a second linked to an acceptor fluorophore, Venus, a variant of green fluorescent protein. If in close proximity (<10 nm), energy resulting from the rapid oxidation of a cell-permeable coelenterazine substrate by the donor can transfer to the acceptor, which in turn fluoresces at a longer characteristic wavelength.

[0001244] Plasmids were transiently co-expressed in human embryonic kidney (HEK) 293FT cells and BRET measurements taken at 37°C using a POLARstar Omega or LUMIstar plate reader (BMG Labtech, Mornington, Victoria, Australia) with 460-490 nm ('donor emission') and 520-550 nm ('acceptor emission') filters, or a VICTOR Light plate reader (Perkin Elmer, Glen Waverley, Victoria, Australia) with 400-475 nm ('donor emission') and 520-540 nm ('acceptor emission') filters, or a CLARIOstar plate reader (BMG Labtech, Mornington, Victoria, Australia) with 420-480 nm ('donor emission') and 520-620 nm ('acceptor emission') filters.

[0001245] The BRET ratio was calculated by subtracting the ratio of 'acceptor emission' over 'donor emission' for a cell sample expressing Rluc8-tagged protein alone from the same ratio for a cell sample expressing both Rluc8 and Venus-tagged proteins. Alternatively, the ligand-induced BRET signal was calculated by subtracting the ratio of 'acceptor emission' over 'donor emission' for a vehicle-treated cell sample from the same ratio for a second aliquot of the same cells treated with agonist.

[0001246] For BRET kinetic assays, the final pre-treatment reading is presented at the zero time point (time of ligand/vehicle addition). For the saturation assay, fluorescence after light excitation was measured on an EnVision 2102 multi-label plate reader (PerkinElmer, Glen Waverley, Victoria, Australia) using a 485/14 excitation filter, 535/25 emission filter and D505 mirror. The fluorescence/luminescence ratio was generated by dividing the fluorescence values in arbitrary units (obtained with the EnVision) by the luminescence values also in arbitrary units (obtained as part of the BRET assay).

[0001247] For Receptor-HIT assays with RAGE/Rluc8 and β-arrestin2/Venus, GPCRs untagged with respect to the BRET system were co-expressed in the HEK293FT cells. These cells were then treated with an appropriate cognate agonist selective for the co- expressed GPCR, in order to promote recruitment of β-arrestin2/Venus to that GPCR. A ligand-induced BRET signal was indicative of recruitment of the β-arrestin2/Venus proximal to RAGE/Rluc8, thereby indicating close proximity between RAGE and the activated GPCR.

[0001248] The BRET measurements in Figure 21 B,C,D were taken at 37°C using a CLARIOstar plate reader (BMG Labtech, Mornington, Victoria, Australia) with 420-480 nm ('donor emission') and 520-580 nm ('acceptor emission') filters. The luminescence measurements in Figure 21 E were taken as part of the BRET measurements ('donor emission'). The fluoresence measurements in Figure 21 E were taken using a CLARIOstar plate reader with a 497/15 nm excitation filter, 540/20 nm emission filter and 517.2 nm dichroic mirror for Venus, and a 580/8 nm excitation filter, 615/10 nm emission filter and 597 nm dichroic mirror for mCherry.

Statistics

[0001249] Continuous data are expressed as mean ± SEM. Differences in the mean among groups were compared using 2-way ANOVA. Pair-wise multiple comparisons were made with Student-Newman-Keuls post-hoc analysis to detect significant differences between groups. P<0.05 was considered statistically significant.

EXAMPLE 1. ATHEROGENESIS ASSOCIATED WITH AN INFUSION OF ANG II IS REDUCED IN AGER/APOE DKO MICE

[0001250] This example demonstrates that the expression of RAGE is required for an infusion of angiotensin II (Ang II) to increase atherogenesis in atherosclerosis-susceptible apoE KO mice.

[0001251 ] A four-week infusion of Ang II (1μg/kg/min) increases atherogenesis in atherosclerosis-susceptible apoE KO mice when compared to untreated apoE KO mice, as demonstrated by an increased percentage of the aortic arch surface area staining positive to Sudan IV denoting the presence of atherosclerotic plaque (Figure 1 A).

[0001252] A four-week infusion of Ang II (1μg/kg/min) also increases the aortic expression of genes coding for pro-atherogenic mediators (Figure 1 B) and markers of oxidative stress (Figure 1 C).

[0001253] In apoE KO mice, a four-week infusion of Ang II (1μg/kg/min) also increases circulating levels of RAGE ligands, including S100A8/A9, AGEs and their reactive dicarbonyl precursors (Figure 1 D), as well as increasing the gene expression of RAGE ligand Mac1/CD1 1 b in the aorta of Ang ll-treated mice (Figure 1 B).

[0001254] Genetic deletion of RAGE prevents the increase in atherogenesis observed following a four-week infusion of Ang II (1μg/kg/min) in AGERIapoE DKO mice (Figure 1 A), as well as preventing Ang ll-induced aortic expression of pro-atherogenic mediators (Figure 1 B), Ang ll-induced oxidative stress (Figure 1 C) and Ang ll-induced circulating levels of RAGE ligands that are observed in apoE KO mice following an infusion of Ang II (Figure 1 D). [0001255] Systolic blood pressure levels are increased to a similar extent following a four- week infusion of Ang II (1 μg/kg/min) in both apoE KO and AGER/apoE DKO mice (Figure 1 E) showing that RAGE deficiency has no impact on hemodynamic signalling induced by Ang II via the AT ₁ R.

Additional materials and methods Angiotensin Infusion model

[0001256] Male apoE KO and AGER/apoE DKO mice (n=8/group), aged 6-8 weeks were randomized to receive Ang II (1μg/kg/min; Sigma-Aldrich, Castle Hill, Australia) or vehicle via subcutaneous infusion for 4-weeks using an osmotic mini-pump (Model 2004, ALZET, BioScientific, Gymea, Australia) placed into the subcutaneous space along the dorsal midline of each mouse under general anaesthetic via intraperitoneal injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). At the end of each study period in each model, mice were humanely killed using C0 ₂ asphyxiation followed by exsanguination via cardiac puncture.

EXAMPLE 2. ATHEROGENESIS ASSOCIATED WITH A LOW SODIUM DIET THAT ACTIVATES THE RAAS IS REDUCED IN AGER/APOE DKO MICE

[0001257] This example shows that the expression of RAGE is required for RAAS activation associated with a low sodium diet to induce atherogenesis in atherosclerosis- susceptible apoE KO mice.

[0001258] A low sodium diet is an accepted experimental model of physiological renin- angiotensin-aldosterone system (RAAS) activation and is a test of the capacity of the RAAS to appropriately retain salt as part of its homeostatic functions.

[0001259] Six-weeks of exposure to a 0.05% (low) sodium diet results in RAAS-dependent increase in plaque accumulation in apoE KO mice (Figure 2A), as demonstrated by an increased percentage of the aortic arch surface area staining positive to Sudan IV denoting the presence of atherosclerotic plaque.

[0001260] Six-weeks of exposure to a 0.05% (low) sodium diet also results in increased aortic expression of pro-atherogenic mediators (Figure 2B), increased markers of oxidative stress (Figure 2C) and increased circulating concentrations of soluble MCP-1 and ICAM-1 (Figure 2D). [0001261 ] More leukocytes adhere to aortae taken from apoE KO mice after 1 -week of exposure to a low sodium diet when compared to aortae from apoE KO mice fed with normal chow (Figure 2E).

[0001262] In apoE KO mice, a low salt diet also increases circulating levels of RAGE ligands, including S100A8/A9, AGEs and their reactive dicarbonyl precursors (Figure 2F), as well as the gene expression of RAGE ligand Mac1/CD1 1 b in the aorta (Figure 2B).

[0001263] Genetic deletion of RAGE prevents the induction of atherogenesis in AGER/apoE DKO mice following four weeks of a low sodium diet when compared to apoE KO mice fed the same diet (Figure 2A), and also prevents the increase in the aortic expression of markers of vascular inflammation, increases in markers of oxidative stress or increases in circulating inflammatory markers (Figures 2B, 2C & 2D).

[0001264] Aortae taken from AGER/apoE DKO fed a low sodium diet for one week do not manifest the increased adhesiveness of labelled leucocytes to the aortic endothelium ex vivo when compared to apoE KO mice fed the same diet (Figure 2E).

[0001265] Increased circulating levels of the RAGE ligands, S100A8/A9 and AGEs in response to feeding with a low sodium diet seen in apoE KO mice are not observed in AGER/apoE KO mice treated with the same low sodium diet for four weeks (Figures 2F).

[0001266] Blood pressure levels are not modified by a low sodium diet in either apoE KO or AGER/apoE DKO mice (Figure 2G).

[0001267] The physiological induction of markers of RAAS activation (e.g. plasma aldosterone, plasma renin) and sodium retention associated with a low sodium diet are not significantly different between apoE KO and AGER/apoE DKO mice (Figure 2H).

Additional materials and methods

Exposure of mice to a 0.05% (low) sodium diet

[0001268] Exposure to a low sodium diet (0.05% sodium) is an accepted experimental model of physiological RAAS activation (Tikellis et al. 2012). In this study, apoE KO and AGER/apoE DKO mice (n=8/group) were randomized to receive an isocaloric diet with low- salt content (0.05% sodium) or normal salt-content (0.3% sodium) both containing 6% fat (Specialty Feeds, Perth, Australia) for 1 -week or 6-weeks.

Plasma renin and aldosterone [0001269] Activation of the RAAS is associated with increased circulating levels of aldosterone and (pro)-renin. These RAAS components were estimated in each group by commercial radioimmunoassay (ProSearch International, Malvern, Australia).

Circulating soluble MCP-1 and ICAM- 1

[0001270] Circulating soluble MCP-1 and ICAM-1 were estimated by ELISA (R&D Biosystems) each performed according to the manufacturer's instructions.

Urinary sodium excretion

[0001271 ] After 6-weeks of a normal or low sodium diet, mice were placed into individual metabolic cages (Iffa Credo, L'Arbresele, France) for 24 hours and their weight, water and food (sodium) intake and urine output documented. The sodium concentration was measured in diluted urine with a (COBAS INTEGRA 400 auto-analyser; (Roche Diagnostics, Indianapolis, USA) using an ion-sensitive electrode and the total sodium excretion (μΓΤΐοΙ/day) was estimated by multiplying with total daily urinary output.

Adherence of labelled leukocytes to aortae

[0001272] After 1 week of a low sodium or normal sodium diet, apoE KO and AGER/apoE DKO animals were humanely killed and aortae were isolated, cleaned of fat and mounted in a vessel chamber primed with Krebs buffer and maintained at physiological pH by infusing carbogen gas (95% 0 ₂ ; 5% C0 ₂ ) through the buffer at 37°C, as previously described. Whole blood labelled with DNC18 (1 :1000) was then perfused through the aorta at 0.12ml/min. As a positive control, vessels (n=6/group) were pre-treated with TNFa (4ng/ml) for 4hrs at 37°C or Ang II (1 μΜ). Images and videos of vessel wall-cell interactions were acquired using a fluorescence microscope (Zeiss Discovery. V20), coupled to a digital camera (HAMAMATSU ORCA-ER) and analysed with AxioVison software. Two to three frames were taken at each time point and the number of adherent cells per frame recorded.

EXAMPLE 3. ATHEROGENESIS IN ACE2/APOE DOUBLE KO MICE IS REDUCED IN ACE2, 'AGER/APOE TRIPLE KO MICE.

[0001273] This example shows that the expression of RAGE is required for genetic Ace2 deficiency to induce increased atherogenesis in atherosclerosis-susceptible Ace2/apoE DKO mice.

[0001274] ACE2 is an enzyme that metabolises Ang II, the major effector of the RAAS. Genetic deficiency of Ace2 in Ace2lapoE DKO mice results in constitutive activation of the RAAS associated with increased circulating levels of Ang II (Figure 3A), increased systolic blood pressure (Figure 3B) and augmented plaque accumulation in the aortic arch when compared to apoE KO mice constitutively replete in Ace2 (Figure 3C).

[0001275] The aortae of Ace2/apoE DKO mice also show increased expression of pro- atherogenic mediators, when compared to apoE KO mice (Figure 3D).

[0001276] Ace2/apoE KO mice have increased circulating markers of oxidative stress (Figure 3E) and elevated circulating levels of RAGE ligands, including S100A8/A9 and AGEs, when compared to apoE KO mice (Figures 3F).

[0001277] Genetic deletion of the AGER gene coding for RAGE in ce IA GERIapoE TKO mice prevents the increase in atherogenesis (Figure 3C) and the induction of proinflammatory markers (Figure 3D), markers of oxidative stress (Figure 3E) and RAGE ligands (Figure 3F) observed in Ace2lapoE DKO mice, restoring these markers to levels similar to those observed in apoE KO mice.

[0001278] Blood pressure levels and circulating concentrations of Ang II are not significantly different between Ace2lapoE DKO or Ace2l AGERI apoEI O (Figure 3A & 3B).

Additional materials and methods

Genetic deficiency of Ace2 in apoE KO mice

[0001279] Ace2 is the major enzyme that metabolizes Ang II in the vasculature. Genetic deficiency of Ace2 in atherosclerosis-susceptible apoE KO mice results in increased circulating levels of Ang II and increased plaque accumulation when compared to wild-type apoE KO mice (Thomas et al. 2012). In these studies, male Ace2lapoE DKO and AGERI Ace2l apoE TKO mice were followed to 16-18 weeks of age, a time point associated with increased plaque accumulation in Ace2lapoE DKO mice.

Plasma angiotensin II

[0001280] Plasma angiotensin II levels were estimated by commercial radioimmunoassay (ProSearch International, Malvern, Australia).

EXAMPLE 4. THE INDUCTION OF PRO-INFLAMMATORY SIGNALLING IN THE AORTA AND ENDOTHELIUM BY EXPOSURE TO ANGIOTENSIN II IS DEPENDENT ON RAGE EXPRESSION [0001281 ] This example shows that the expression of RAGE is required for exposure to Ang II to induce pro-inflammatory signalling in aortae and aortic endothelial cells taken from them.

[0001282] Ex vivo exposure of isolated aortae from apoE KO mice to Ang II (1 μΜ) increases the expression of pro-atherogenic mediators (Figure 4A) and results in an increased number of adherent leukocytes when compared to untreated aortae (Figure 4B).

[0001283] Genetic deletion of RAGE in AGER/apoE DKO mice prevents the induction of these atherogenic mediators (Figure 4A) or increased endothelial adhesiveness of primed leucocytes (Figure 4B) to the aorta following exposure to Ang II (1 μΜ) when compared to aortae from apoE KO mice.

[0001284] In primary murine aortic endothelial cells (PMAEC) isolated from the aortae of C57bl6 mice, exposure to Ang II (1 μΜ) also results in the induction of pro-inflammatory signalling. This is associated with increased adhesion of labelled THP-1 (monocyte-type) cells to a monolayer of Ang ll-treated PMAEC (Figure 4C) and increased expression of key pro-inflammatory genes (Figure 4D), as well as a rapid increase in the production of ROS and other markers of oxidative stress, activated rac-1 and oxidised glutathione (Figure 4E).

[0001285] In PMAEC isolated from the aortae of C57bl6 mice, exposure to Ang II also results in the induction of both canonical and non-canonical signalling downstream of NFKB, as demonstrated by the induction of classical markers CXCL2 and CXCL 12 (Figure 4F). TNFα is shown as a positive control for its ability to selectively induce canonical signalling downstream of N FKB activation. The expression of VCAM-1 is shown as an additional target response gene, replicating the data in Figure 4D.

[0001286] In PMAEC isolated from the aortae of C57bl6 mice, exposure to the RAGE ligand, S100A8/A9, also results in the induction of target NFKB-dependent pro-inflammatory genes, including ICAM-1, VCAM- 1, TNFa and MCP-1 (Figure 4G).

[0001287] In PMAEC isolated from AGER KO mice, treatment with Ang II does not lead to the induction of key pro-inflammatory genes (Figure 4D), markers of canonical or non- canonical activation of N FKB (Figure 4F), an increase in ROS production and oxidative stress (Figure 4E) or a functional increase in endothelial adhesiveness (Figure 4C).

[0001288] In PMAEC isolated from AGER KO mice, treatment with the RAGE ligand, S100A8/A9, does not lead to the induction of pro-inflammatory genes, including ICAM- 1, VCAM-1 and MCPA (Figure 4G). [0001289] Silencing of AGER expression in PMAEC using siRNA achieves a similar phenotype to genetic RAGE deletion, preventing the NFKB-dependent induction of VCAM-1 gene expression by Ang II or the RAGE ligand, S100A8/A9 (Figure 4H). Silencing of p65 expression is shown as a positive control for NFKB-dependence of this signalling pathway.

[0001290] Taken together, the findings in endothelial cells from AGER KO mice and PMAEC in which RAGE expression has been suppressed with siRNA, both demonstrate that the actions of S100A8/A9 in these cells are specifically mediated via RAGE, and that other receptors through which S100A8/A9 also has the potential to activate N FKB and downstream signalling pathways (e.g. Toll-Like Receptors) which are present in other cell systems (e.g. in leucocytes and glial cells) do not play a significant role in this phenomenon in PMAECs.

[0001291 ] The observed attenuation in the effects of Ang II in PMAECs from AGER KO mice is not due to a loss of RAAS-signalling in the absence of RAGE, as exposure of RAGE- deficient PMAECs to Ang II leads to a similar increase in inositol phosphate synthesis as estimated by an increase in IP-1 and downstream induction of the early growth response gene (EGR1 ), both considered to be markers of competent Ang ll-mediated signalling (Figure 4I).

Additional materials and methods Ex vivo Studies in Isolated Aortae

[0001292] Aortae were isolated from apoE KO and AGERIapoE DKO mice, divided and placed in Krebs buffer and maintained at physiological pH by infusing carbogen gas (95% 0 ₂ ; 5% C0 ₂ ) through the buffer at 37°C. Vessels (n=6/group) were then treated with Ang II (1 μΜ) for 4 hours. After which time, whole blood labelled with DNC18 (1 :1000) was then perfused through the aorta at 0.12ml/min. Images and videos of vessel wall-cell interactions were observed using a fluorescence microscope (Zeiss Discovery. V20), coupled to a digital camera (HAMAMATSU ORCA-ER) and analysed with AxioVison software.

Static adhesion assay

[0001293] Functional responsiveness to Ang II was determined in PMAEC using a static adhesion assay, in which PMAEC were seeded at 50,000 cells per well into six-well plates and allowed to grow to 70% confluency before treatment with 1 μΜ Ang II for 24 hours. Human THP-1 cells, which mimic monocytes and macrophages in the vasculature, were stained using CellVue Burgundy Fluorescent Cell labelling Kit (LICOR) as per manufacturer's instructions before seeding them onto the endothelial cell monolayers at 3x10 ⁵ viable cells per well and incubated for 20 minutes at 37°C. The cells that had not adhered to the endothelial cell monolayer were removed and the wells washed with PBS before fixing with 4% formalin in PBS for 30 min. The adhesion of cells was then quantitated using the ODYSSEY infra-red imager (Licor). Additionally adhered cells were photographed at x20 using light microscopy (Olympus CKX41 ).

Generation of superoxide and additional markers of oxidative stress

[0001294] To quantitate superoxide production induced by Ang II, a vessel chamber assay was used to measure total cytoplasmic and mitochondrial superoxide production in real time in PMAEC from AGER KO and C57BI6 aortae. PMAEC were grown on collagen coated cover slips and mounted into a sealed glass chamber when they were 80% confluent. The chambers were perfused with Krebs buffer for an hour with fluorescent readings taken every 10 minutes to establish baseline readings. The cells were then perfused with 1 μΜ Ang II for 60 minutes and fluorescent readings were taken every 1 minute for a further 10 minutes. Fluorescence readings were expressed as change (Δ) in fluorescence arbitrary units from baseline. Levels of the activated NADPH oxidase subunit, Rac-1 activation was also measured in cell lysates after exposure to Ang II using a Rac-1 G-LISA activation assay (Cytoskeleton), as per manufacturers' instructions. Additional markers of oxidative stress were also assayed including levels of oxidized glutathione, (Cayman Chemical Company, US), performed as per manufacturer's instructions.

Integrity of signalling via the ATiR

[0001295] To confirm that the AT ₁ R signalling cascade was functional in PMAEC from AGER KO, we measured myo-lnositol phosphate (IP1 ), a stable downstream metabolite of inositol phosphate (IP ₃ ), which accumulates in cells following Gq receptor activation using an IP-one HTRF assay (CisBio bioassays, Bagnols-sur-Ceze Cedex, France) using the manufacturer's instructions and changes in the gene expression of EGR1 , and AT ₁ R were then determined 2 hours after exposure to 1 μΜ Ang II, as assessed by real-time RT-PCR.

EXAMPLE 5. THE MODULATORY EFFECTS OF FULL LENGTH AND TRUNCATED HUMAN RAGE CONSTRUCTS ON PRO-INFLAMMATORY SIGNALLING IN ATiR-CHO CELLS

[0001296] This example shows that for the exposure to Ang II to induce expression of the key pro-inflammatory transcription factor, p65-NFKB and increase N FKB activity in Chinese Hamster Ovary (CHO) cells, the expression of both AT ₁ R and RAGE are also required. [0001297] CHO cells express few cell surface receptors, and specifically do not express endogenous AT ₁ R or RAGE on their surface making them an ideal system to explore the role of the AT ₁ R-RAGE interaction. In addition, CHO cells do not express TLRs that potentially have the capacity to bind RAGE ligands and be activated by them, resulting in activation of N FKB.

[0001298] Stable transfection of CHO cells with the human AT ₁ R gene generates AT ₁ R- CHO cells, and confers classical responsiveness to exogenous Ang II (1 μΜ), as demonstrated by the induction of inositol phosphate synthesis (Figure 5A) and the induction of early growth response gene, EGR1 (Figure 5B).

[0001299] By contrast, exogenous Ang II (1 μΜ) is unable to induce expression of the key pro-inflammatory transcription factor, p65-NFKB or increase N FKB activation in AT ₁ R-CHO cells (Figure 5C).

[0001300] Exogenous Ang II (1 μΜ) is able to induce activation of the key pro-inflammatory transcription factor, p65-NFKB only when AT ₁ R-CHO cells are also transfected with and expressing full-length human RAGE polypeptide (Figure 5C). The RAGE ligand, S100A8/A9 (5ng/ml_) is also able to induce activation of the key pro-inflammatory transcription factor, P65-N FKB, only when cells are also expressing the RAGE polypeptide (Figure 5C), which serves as a positive control for the transgenic expression of RAGE and the integrity of its signalling pathways in transfected CHO cells.

[0001301 ] Transfection of AT1 R-CHO cells with N-truncated mCherry-RAGE constructs that are (i) lacking the ectodomain of RAGE (i.e. mCherry-RAGE ₃ 42-404) , (ii) lacking the ectodomain and the transmembrane domain of RAGE (i.e. m Cherry- RAG E362-404) or (iii) lacking the ectodomain, the transmembrane domain and the Diaphanous-1 binding domain (366-367) of RAGE (i.e. mCherry-RAGE ₃ 7o-404) retain the ability of full length RAGE ₂₂ - ₄ o4 to mediate pro-inflammatory signalling in response to Ang II when compared to empty plasmid alone (vector), provided that Q379 is retained (Figure 5D). N-Truncation beyond Q379 (e.g. mCherry-RAGE ₃ 8o-404) results in an inactive mutant RAGE peptide that is unable to mediate pro-inflammatory signalling in response to Ang II.

[0001302] Transfection of AT1 R-CHO cells with C-truncated RAGE constructs retain the ability to mediate pro-inflammatory signalling in response to Ang II, provided that at least Ser391 of the C-terminal of wild-type RAGE remains intact (i.e. RAGE22-391 ; Figure 5E). [0001303] The smallest construct retaining the ability to mediate pro-inflammatory signalling in response to Ang II in AT1 R-CHO cells is the 13-mer construct mCherry-RAGE ₃ 79-39i (Figure 5E).

[0001304] N-truncated mCherry-RAGE constructs lacking the RAGE ligand-binding ectodomain are not able to facilitate signalling when transfected into AT1 R-CHO cells in response to the RAGE ligand S100A8/A9 (Figure 5F) as RAGE ligand-dependent activation of RAGE requires this ligand-binding ectodomain to bind the ligand, in addition to other parts of RAGE to subsequently transfer its signal, recruiting and activating its intracellular signalling cascade.

[0001305] C-truncated RAGE constructs are able retain the ability to mediate proinflammatory signalling when transfected into AT1 R-CHO cells in response to S100A8/A9, provided that at least Ser391 of the C-terminal of wild-type RAGE remains intact (i.e. RAGE22-391 ; Figure 5G).

[0001306] The additional mCherry protein provides a practical means to ensure expression of transgene constructs in cell models. However, it plays no role in the pro-inflammatory signalling actions of N-truncated RAGE as (i) the expression of mCherry on its own has no effect on signalling pathways (i.e. mCherry control in Figure 5F) and (ii) N-truncated RAGE constructs in which the mCherry protein has been omitted (Figure 5H) achieve the same levels of signalling function in response to Ang II as those expressing the mCherry-RAGE fusion protein (Figure 5D, 5F). Similarly N-truncation beyond Q379 destabilises the alpha helix and results in a loss of signalling function in contructs with and without mCherry-RAGE fusion (Figure 5F and 5H respectively).

Additional materials and methods

Classical GPCR signalling

[0001307] Classical GPCR signalling was confirmed in cells treated with 1 μΜ Ang II, by the induction of myo-lnositol phosphate (IP1 ), a stable downstream metabolite of IP ₃ , which accumulates in cells following Gq receptor activation using an IP-one HTRF assay (CisBio bioassays, Bagnols-sur-Ceze Cedex, France) using the manufacturer's instructions.

NFKB activity assay

[0001308] To measure the induction of N FKB activity by 1 μΜ Ang II, or 5ng/ml of the RAGE-ligand S100A8/A9 as a signalling control, AT1 R-CHO cells were transfected with 0.4 g of plasmids encoding N FKB-SEAP or β-galactosidase (β-Gal) using Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions. After 24 hours, cells transfected with each plasmid combination were then treated with Ang I I (1 μΜ) or left untreated and incubated for 4 hours before cell supernatants and cell lysates were collected. Cell supernatants were analysed using a chemiluminescent SEAP reporter gene assay (Roche Applied Science) to detect SEAP production after treatment. To control for transfection efficiency, cell lysates were assayed for β-Gal using a commercially available kit (Promega).

EXAMPLE 6. RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE FOLLOWING ACTIVATION OF THE ATiR WITH ANG II

[0001 309] This example shows that activation of RAGE following binding of Ang II to AT ₁ R occurs via a RAGE ligand-independent pathway and not via liberation of a RAGE ligand.

[0001 31 0] Activation of the AT ₁ R is known to trigger activation of tyrosine-kinase receptors due to activation of pathways leading to protease-mediated extracellular "shedding" of membrane-tethered ligands, which are then freed to bind to and activate these tyrosine kinase receptors.

[0001 31 1 ] Pre-treatment with a neutralizing antibody targeting the ligand-binding ectodomain of RAGE (RAGEab) or a soluble RAGE decoy with ligand-binding affinity (SRAGE22-331 ) both block RAGE ligand-dependent induction of pro-inflammatory signalling by the RAGE ligand S100A8/A9 in RAGE-AT1 R-CHO cells expressing both full length RAGE and AT ₁ R (Figure 6A).

[0001 31 2] Pre-treatment with a neutralizing antibody targeting the ligand-binding ectodomain of RAGE (RAGEab) or a soluble RAGE decoy with ligand-binding affinity (SRAGE22-331 ) do not affect RAGE ligand-independent induction of pro-inflammatory signalling by Ang I I in RAGE-AT ₁ R-CHO (Figure 6A).

[0001 31 3] Pre-treatment with a neutralizing antibody targeting the ligand-binding ectodomain of RAGE (RAGEab) or a soluble RAGE decoy with ligand-binding affinity (SRAGE22-331 ) also does not attenuate Ang II-AT1 R -dependent induction of pro-inflammatory signalling in PMAEC, endogenously replete in both AT1 R and RAGE (Figure 6B). PMAEC from AGER KO mice are shown as a negative control.

[0001 314] By contrast, pre-treatment with a neutralizing antibody targeting the ligand- binding ectodomain of RAGE (RAGEab) or a soluble RAGE decoy with ligand-binding affinity (SRAGE22-331 ) is able to attenuate pro-inflammatory signalling induced following exposure to the RAGE ligand S100A8/A9 in PMAEC, endogenously replete in both AT ₁ R and RAGE (Figure 6C).

[0001315] Taken together with the findings using N-truncated RAGE constructs detailed in Example 5, these data confirm that activation of the RAGE receptor following exposure to Ang II is independent of the liberation of RAGE ligands or the activation of the ectodomain of RAGE by such liberated ligands.

Additional materials and methods

[0001316] Additional experiments were conducted in the presence and absence of a 1 hour pre-treatment with a neutralizing antibody to RAGE (RAGEab; R&D systems; 1 μg/mL ) or soluble RAGE22-331 (sRAGE; 1 μg/mL) prior to exposure to 1 μΜ Ang II or 5ng/ml of the RAGE ligand, S100A8/A9.

EXAMPLE 7 RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR DOES NOT REQUIRE PHOSPHORYLATION OF THE CYTOSOLIC TAIL TO INDUCE PRO-INFLAMMATORY SIGNALLING

[0001317] This example shows that RAGE ligand-independent activation of RAGE by a certain co-located GPCR does not require phosphorylation of RAGE to induce proinflammatory signalling.

[0001318] Phosphorylation of Ser391 has been previously thought to be important for ligand-dependent signalling via RAGE, possibly by facilitating the recruitment of adaptor proteins to the RAGE cytosolic tail, which then transduce pro-inflammatory signalling.

[0001319] However, many other mammals have another amino acid other than serine at the 391 position of RAGE that cannot be phosphorylated. For example, camels have a glutamine at 391 , while a proline is naturally found in the same position in cattle, sheep, goats, pigs, deer as well as some other mammals. These residues are unable to sustain phosphorylation, and yet these animals have intact RAGE signalling pathways.

[0001320] Transfection of AT1 R-CHO cells with full length RAGE constructs in which S391 is mutated to glutamine (S391 Q-RAGE) or proline (S391 P-RAGE), thereby blocking potential phosphorylation at this site, continue to mediate Ang ll-dependent induction of p65-NFKB gene expression. Transfection with full length RAGE constructs in which the putative phosphorylation site at S391 is mutated to glutamine (S391 Q-RAGE) or proline (S391 P- RAGE) also continue to mediate S100A8/A9-dependent induction of N FKB expression in AT1 R-CHO cells (Figure 7A).

[0001321 ] Blockade of all other potential phosphorylation sites on the human RAGE cytosolic tail (i.e. S399, S400 and T401 ) was achieved by replacing the last eleven C- terminal residues of RAGE 394-404 with those residues found in the murine RAGE cytosolic tail that naturally do not contain potential phosphorylation sites (i.e. hRAGE ₃ 94-404; P394EAGESSTGGP is replaced with mRAGE ₃₉ 2-404; A ₃₉₄ EMPENGAGGP, generating a chimeric RAGE (cRAGE) with no potential sites for phosphorylation (S391 Q-cRAGE); Notably this construct was able to signal the same as wild-type RAGE in AT1 R-CHO cells in response to Ang II (Figure 7B), confirming that signalling-induced phosphorylation of RAGE is not necessary for ligand-independent pro-inflammatory signalling via RAGE, and that other sites do not convey redundancy for RAGE phosphorylation when S391 is mutated.

[0001322] S391 Q-CRAGE was also shown to mediate S100 A8/A9- induced signalling in AT1 R-CHO cells (Figure 7B). These data confirm that signalling-induced phosphorylation of RAGE is also not necessary for ligand-dependent pro-inflammatory signalling via RAGE or that other phosphorylation sites in the cytosolic tail can convey redundancy for RAGE phosphorylation when S391 is mutated.

[0001323] RAGE constructs in which S391 has been deleted (e.g. RAGE22-390) have no ability to mediate Ang ll-dependent induction of N FKB activation or p65 expression in AT ₁ R- CHO cells (see Example 5, Figure 5G).

[0001324] Full length RAGE constructs in which only S391 is mutated to alanine (S391 A- RAGE) have no ability to mediate Ang ll-dependent or S100A8/A9-dependent induction of N FKB expression or activity (as determined by p65 expression) in AT ₁ R-CHO cells (Figure 7A), despite the presence of other potential phosphorylation sites at S399, S400 and T401 in human RAGE.

[0001325] Full length and N-truncated RAGE constructs in which only the S391 is selectively mutated to alanine (S391 A-RAGE) also have no ability to mediate Ang ll- dependent induction of N FKB expression or activity (as determined by p65 expression) in AT1 R-CHO cells (Figure 7C).

[0001326] RAGE constructs in which only the S391 is mutated to cysteine (S391 C-RAGE) also have no ability to mediate Ang ll-dependent or S100A8/A9 dependent induction of N FKB expression or activity (as determined by p65 expression) in AT ₁ R-CHO cells (Figure 7A). [0001327] Full length RAGE constructs in which S391 is mutated to leucine (S391 L-RAGE) retain the ability to mediate Ang ll-dependent induction of N FKB expression in AT ₁ R-CHO cells, but lose signalling induced by the RAGE ligand S100A8/A9 (Figure 7A).

[0001328] By contrast, full length RAGE constructs in which S391 is mutated to glutamic acid (S391 E-RAGE) lose the ability to mediate Ang ll-dependent induction of N FKB expression in AT1 R-CHO cells (Figure 7A), but retain the ability to signal following exposure to the RAGE ligand, S100A8/A9 (Figure 7A).

[0001329] Transfection of AT1 R-CHO cells with N-truncated mCherry-RAGE ₃₆ 2-404 constructs in which the S391 is mutated to isoleucine, methionine, threonine, or tryptophan retain the ability of transfection with wild type RAGE362-404 to mediate Ang ll-dependent induction of N FKB expression (as determined by p65 expression) (Figure 7D). By contrast, transfection with N-truncated RAGE362-404 constructs in which the S391 is mutated to alanine, aspartate, glutamate, phenylalanine, glycine, histidine, lysine, asparagine, arginine, valine or tyrosine fail to restore Ang ll-dependent induction of NFKB expression or activity (as determined by p65 expression) in AT1 R-CHO cells when compared to wild type RAGE (Figure 7D).

EXAMPLE 8. THE COMMON ROLE OF MYD88 IN RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR AND RAGE LIGAND-DEPENDENT ACTIVATION OF RAGE.

[0001330] This example describes the common role of myeloid differentiation factor 88 (myD88) in RAGE ligand-independent activation of RAGE by a certain co-located GPCR as well as RAGE ligand-dependent activation of RAGE.

[0001331 ] The common adaptor protein, myD88, has been previously shown to be important for downstream pro-inflammatory signalling following activation of RAGE as well as many other receptors including all Toll-like receptors (TLRs) except TLR-3. Ligand- dependent activation of RAGE is thought to facilitate the recruitment of MyD88 and other common adaptor proteins including, Toll lnterleukin-1 receptor domain containing adaptor protein (TIRAP) to the cytosolic tail of RAGE, which then recruit and transduce signalling through IRAK4, ultimately leading to N FKB activation.

[0001332] Silencing of myD88 expression using siRNA inhibits the NFKB-dependent induction of the key adhesion molecule, ICAM-1 , induced by the RAGE ligand S100A8/A9 in PMAECs (Figure 8A). This effect is comparable to silencing of p65, its downstream target. [0001333] Silencing of MyD88 expression using siRNA also inhibits signalling induced by RAGE ligand-independent activation of RAGE by Ang II in PMAECs, including the N FKB- dependent activation of gene expression of the key adhesion molecule, ICAM- 1 (Figure 8B).

[0001334] Silencing of MyD88 expression using siRNA also inhibits NFKB-dependent activation of gene expression of the key adhesion molecule, MCP-1 induced by RAGE ligand-independent activation of RAGE by Ang II in HMECs (Figure 8C). This inhibition is rescued by RAGE362-404 (Figure 8C), confirming the functional substitute of RAGE identified by the inventors, is functioning independent of MyD88.

EXAMPLE 9. THE COMMON ROLE OF PKC-ZETA IN RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR AND RAGE LIGAND-DEPENDENT ACTIVATION OF RAGE.

[0001335] This example describes the common role of ΡΚC iζn RAGE ligand-independent activation of RAGE by a certain co-located GPCR and ligand-dependent activation of RAGE.

[0001336] The atypical PKC, ΡΚCζ has been previously shown to be important for RAGE ligand-dependent activation of RAGE and subsequent activation of downstream pathways. ΡΚCζ has been shown to bind to the cytosolic tail of RAGE as well as Diaphl , another RAGE binding partner.

[0001337] ΡΚCζ is a pleiotropic kinase involved in many signalling pathways including MAP kinase signalling, NFKB-activation, cell polarity, insulin-mediated glucose uptake and long- term memory potentiation. ΡΚC iζs also involved in many other signalling pathways including TLR2/4-dependent induction of pro-inflammatory signalling. ΡΚCζ also modulates the phosphorylation status of activated GPCRs, a key event in receptor downstream signalling and functioning.

[0001338] Silencing of ΡΚCζ expression using siRNA or inhibition of ΡΚ wCζith a pseudo- substrate (ίΡΚCζ) inhibits signalling induced by the RAGE ligand S100A8/A9 in PMAEC (Figure 9A), with an equivalent effect to silencing of RAGE expression. This further confirms that silencing or inhibition of ΡΚC iζs not acting by silencing signalling of S100A8/A9 through TLR2/4, but rather specifically through altering signalling specifically through full length RAGE.

[0001339] Silencing of ΡΚCζ expression using siRNA or inhibition of ΡΚC wζith a pseudo- substrate (ίΡΚCζ) also inhibits signalling induced by RAGE ligand-independent activation of full length RAGE by Ang II, including the NFKB-dependent activation of the key adhesion molecule, ICAM- 1 (Figure 9B).

[0001340] The inhibition of ΡΚCζ with a pseudo-substrate (ΡΚCζί) also inhibits signalling induced by RAGE ligand-independent activation of full-length S391 Q-cRAGE ₂ 2-404 (which contains no serines or threonines in the cytosolic tail capable of being phosphorylated), as assessed by the NFKB-dependent activation of p65 and PCNA following exposure to Ang II in AT1 R-CHO cells (Figure 9C), confirming that ΡΚC isζ not acting in the RAGE signalling pathway by inducing phosphorylation of RAGE.

[0001341 ] By contrast, silencing of ΡΚCζ expression using siRNA does not inhibit NFKB- dependent activation of gene expression of the key adhesion molecule, MCP-1 induced by RAGE ligand-independent activation of RAGE362-404 by Ang II in HMECs (Figure 9D), confirming that the functional substitute of RAGE identified by the inventors, is functioning independent of ΡΚCζ.

Additional materials and methods

[0001342] Additional experiments were conducted in the presence and absence of a 1 hour pre-treatment with a pseudo-substrate for ΡΚC (ίζΡΚCζ, 5μΜ, Tocris).

EXAMPLE 10. THE DIFFERENTIAL ROLE OF DIAPHANOUS-1 IN RAGE LIGAND- INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR COMPARED TO RAGE LIGAND-DEPENDENT ACTIVATION OF RAGE.

[0001343] This example describes the differential role of Diaphanous-1 (Diaphl ) in RAGE ligand-independent activation of RAGE by a certain co-located GPCR compared to RAGE ligand-dependent activation of RAGE, specifically its dependence on the charged patch (R366-Q367) of RAGE and the alpha-turn it stabilises that are known to complex with Diaphl , indicating a key point of difference between RAGE ligand-dependent and RAGE ligand-independent (transactivation) induced signalling via RAGE.

[0001344] The formin-type protein, Diaphanous-1 (Diaphl ) is regarded as a key element for pro-inflammatory signalling induced by RAGE ligands.

[0001345] Silencing of Diaphl expression attenuates ligand-dependent signalling via full length RAGE induced by the RAGE ligand, S100A8/A9 (5ng/ml_) in PMAEC (Figure 10A), with an effect equivalent to silencing of RAGE, which serves as a negative control. [0001346] The charged patch (R366-Q367) of RAGE is thought to form an alpha-turn that stabilises its complex with Diaphi (Rai, et al. 2016). Mutation of R366-Q367 that selectively disrupts the charged patch through which Diaphi and RAGE are thought to interact, is able to prevent S100A8/A9-induced signalling in AT ₁ R CHO cells expressing full length R366A- Q367A-RAGE (Figure 10B).

[0001347] Silencing of Diaphi expression also attenuates RAGE ligand-independent signalling via full-length RAGE induced by Ang II including the induction of ICAM-1 and VCAM-1 expression in PMAEC (Figure 10C) and inhibits the functional induction of leukocyte adhesion to an endothelial monolayer following exposure to Ang II (Figure 10D).

[0001348] By contrast, mutated full-length R366A-Q367A-RAGE ₂₂ - ₄ 04 and N-truncated mCherry-R366A-Q367A-RAGE ₃ 62-404 in which the charged patch through which Diaphi and RAGE are thought to interact is disrupted, still mediate RAGE ligand-independent signalling induced by Ang II in AT1 R-CHO cells (Figure 10B).

[0001349] Furthermore, deletion of the charged patch through which Diaphi and RAGE are thought to interact (e.g. in RAGE370-404) does not prevent RAGE ligand-independent activation of RAGE and pro-inflammatory signalling induced by Ang II in AT ₁ R-CHO cells (Figure 10E).

[0001350] In addition, silencing of Diaphi expression using siRNA does not inhibit N FKB- dependent activation of MCP-1 gene expression induced by RAGE ligand-independent activation of RAGE362-404 by Ang II in HMECs (Figure 10F), confirming that the functional substitute of RAGE identified by the inventors, is functioning independent of Diaphi .

[0001351 ] Taken together, these data imply that methods to disrupt the interaction between the charged patch of RAGE and Diaphi will have no direct effect on RAGE ligand- independent signalling via RAGE by a certain co-located GPCR.

[0001352] Without wishing to be bound by theory, the inventors believe that mutation or deletion of the charged patch through which Diaphi and full length RAGE are thought to interact alleviates binding constraints that occur through this interaction, meaning that N- truncated RAGE peptides without this binding domain (e.g. RAGE370-404) or mutated peptides in which this domain is altered (e.g. R366A-Q367A-RAGE ₃ 62-404) are able to overcome the inhibition of transactivation conferred by pre-treatment with mCherry-S391 A-RAGE362-404 (0.4ng/ml), a construct in which the charged patch remains present (Figure 10G). EXAMPLE 11. THE COMMON ROLE OF IQGAP- 1 IN RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR AND RAGE LIGAND-DEPENDENT ACTIVATION OF RAGE.

[0001353] This example describes the role of IQGAP-1 in the activation of RAGE.

[0001354] IQGAP-1 is a scaffold protein involved in regulating various cellular processes ranging from organization of the actin cytoskeleton, transcription, and cellular adhesion to regulating the cell cycle. IQGAP-1 is known to bind to RAGE effector molecules including active forms of Cdc42, Rac-1 , calmodulin-1 , and ERK 1 /2.

[0001355] Silencing of IQGAP- 1 expression attenuates RAGE ligand-independent signalling via RAGE induced by Ang II in PMAEC (Figure 1 1 A) and RAGE ligand-dependent signalling induced by S100A8/A9 in PMAECs (Figure 1 1 B).

[0001356] The mutant form of the RAGE cytosolic tail, S391 A-RAGE _362-404 , preferentially associates with IQGAP-1 as indicated by its ability to be differentially purified from other cytosolic components using a column coated with S391 A-RAGE _362-404 (Figure 1 1 C). This strategy also pulled down the IQGAP-1 associated proteins, ezrin/radixin/moesin.

[0001357] Silencing of IQGAP- 1 expression using siRNA also inhibits NFKB-dependent activation of MCP-1 gene expression induced by RAGE ligand-independent activation of RAGE ₃ 62-404 by Ang II in HMECs (Figure 1 1 D), confirming that the functional substitute of RAGE identified by the inventors, is functionally dependent on IQGAP-1 .

Additional materials and methods

Methods for pull-down experiments

[0001358] The TAT-Cherry-S391 A-RAGE ₃₆ 2-404 peptide and TAT-mCherry control peptide were bound to a gravity flow NiNTA column, and equilibrated using lysis buffer (50mM Tris pH 7.5, 300mM NaCI, 5mM βΒ-mercaptoethanol, 10mM imidazole. 5 x 10 ⁷ CHO cells stably transfected with Hs-ATiR were lysed using RIPA buffer (1 x PBS supplemented with 1 % NP- 40, 0.5% Na-deoxycholate with protease and phosphatase inhibitors), diluted to 50ml in lysis buffer and passed over the Ni-NTA column twice by gravity flow. The 1 ml column was washed extensively (200ml wash buffer - 50mM Tris pH 7.4, 300mM NaCI, 5mM β- mercaptoethanol, 20mM imidazole). After washing, 2ml of elution buffer (50mM Tris pH 7.4, 300mM NaCI, 5mM β-mercaptoethanol, 250mM imidazole) was applied to the column, and used to elute proteins that had bound to either the A peptide or TAT-mCherry control peptide. 1 ml of sample was delivered for proteomic analysis by Mass Spectroscopy. Venny 2.1 .0 was used to generate lists of the proteins differentially bound to the two proteins.

EXAMPLE 12. INHIBITION OF RAGE-LIGAND INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR IN MURINE SVEC

[0001359] This example describes using specific components of the RAGE cytosolic tail to inhibit RAGE ligand-independent signalling induced via RAGE in murine SVEC following exposure to Ang II.

[0001360] Transfection of murine SVEC with full length S391 A-RAGE _22-404 and N-truncated mCherry-S391 A-RAGE ₃ 62-404 is able to prevent the induction of ICAM-1 gene expression by Ang II in murine SVEC that are endogenously replete in RAGE expression (Figure 12A).

[0001361 ] Transfection of murine SVEC with full length (RAGE ₂₂ - ₄₀₄ ) and N-truncated mCherry-RAGE constructs in which the wild-type conformation (S391 ) is retained does not affect the induction of ICAM-1 gene expression following exposure to Ang II (Figure 12A).

[0001362] Transfection of murine SVEC with C-truncated S391 X-RAGE mutants (i.e. RAGE ₂₂ -39o and m-Cherry-RAGE ₃ 7o-39o) also prevents the induction of ICAM- 1 by Ang II in murine SVEC that are endogenously replete in RAGE expression (Figure 12A).

[0001363] The smallest sequence demonstrated to be inhibitory for RAGE signalling in SVEC is mCherry- RAG E379-390 (Figure 12B). Further N-or C-terminal truncations (e.g. mCherry-RAGE ₃ 8o-39o) or peptides comprising other components of the cytosolic tail but not containing the RAGE379-390 dodecapeptide show no inhibitory effect on the pro-inflammatory actions of Ang II via RAGE activation.

[0001364] The additional mCherry protein provides a practical means to ensure expression of transgene constructs in cell models. However, it plays no role in the functional inhibitory actions of N-truncated RAGE as (i) the expression of mCherry (control) on its own has no inhibitory effect on signalling pathways (Figure 12B) and (ii) N-truncated RAGE constructs in which the mCherry protein has been omitted achieve the same inhibition of Ang ll-dependent signalling in RAGE-AT1 R-CHO cells (Figure 12C), as constructs expressing the mCherry- RAGE fusion proteins, detailed above (Figure 12B).

[0001365] The peptide resulting from this RAGE sequence is predicted to be an alpha helix. As alpha helices can be stabilised or disrupted by changes to capping amino acids, additional selective mutation was performed. Notably, loss of either glutamine 379 or glutamine 390 by alanine substitution destabilised the helix and resulted in a loss of inhibition, while glutamine-to-lysine mutants Q390K-RAGE ₃₇ o- ₃ 9o and Q379K-RAGE ₃₇ o- ₃ 9o appeared to retain the greatest inhibitory effect, consistent with the known art that terminal lysine or arginine possess a greater effect in stabilising dodecapeptides in an alpha helix conformation (Figure 12D).

[0001366] This sequence of the dodecapeptide RAGE379-390 has homology to sequences in immunomodulatory bacterial proteins from commensal organisms including Streptomyces (Figure 12E). Although only mammals possess the gene for RAGE, the presence of RAGE inhibitory sequences in pathogens is consistent with a survival mechanism to avoid/attenuate mammalian host defences including inflammation.

[0001367] Although the capacity for RAGE370-390 to inhibit the induction of ICAM-1 expression following activation of the AT1 R with Ang II in SVEC cells is lost with the following glutamine-to-alanine mutations: Q390A-RAGE ₃₇₀ - ₃ 9o or Q379A-RAGE ₃₇₀ - ₃ 9o (Figure 12D), L388A-RAGE ₃ 7o-39o, E384A-RAGE ₃₇₀ - ₃ 9o, E382A-RAGE ₃₇₀ - ₃ 9o and E380A-RAGE ₃₇₀ - ₃ 9o, continue to inhibit signalling in SVEC cells as wild type RAGE ₃₇ o- ₃ go.

Additional materials and methods

[0001368] To explore the potential for RAGE constructs to inhibit transactivation of endogenous RAGE, additional experiments were conducted in immortalized mouse endothelial cells (Svec4-10, ATCC) cultured in 10% FBS/DMEM with 25mM glucose were used. SVEC were transfected with truncated RAGE constructs using Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions, or treated with RAGE oligopeptides (280pM). Cells were allowed to recover in media containing 10% FBS for 16 hours prior to treatment with 1 μΜ Ang II.

EXAMPLE 13. TARGETING RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR IN VITRO

[0001369] In this example, we demonstrate that treatment of cells with a cell-penetrating peptide modulator of RAGE ligand-independent activation of RAGE by a certain co-located GPCR is able to modulate the induction of pro-inflammatory signalling via RAGE ligand- independent RAGE activation in cell culture.

[0001370] To validate the therapeutic potential of targeting AT ₁ R-dependent signalling via activation of the cytosolic tail of RAGE, oligopeptides comprising the C-terminal 43 amino acids of RAGE tagged with mCherry-fluorescent protein (to quantify expression and delivery) and an HIV-TAT motif (to facilitate cellular penetration) were generated to act as substitutes. In one version, the serine residue at 391 was changed to alanine (S391 A-RAGE _362-404 ; SEQ ID NO: 1 ) to neutralise its ability to signal and enhance its ability to inhibit activation of endogenous RAGE.

[0001371 ] Treatment of AT ₁ R-CHO cells (not expressing RAGE) with the TAT-mCherry- S391 A-RAGE ₃ 62-404 fusion protein; 0.4ng/ml), different at only one amino acid residue from the activating wild type-construct, does not facilitate the induction of expression of p65-NFKB by Ang II (Figure 13A).

[0001372] In AT1 R-CHO cells pre-treated with the wild-type TAT-mCherry-RAGE ₃₆₂ - ₄ o4 fusion peptide (1 ng/ml) subsequent treatment with TAT-mCherry-S391 A-RAGE _362-404 fusion protein (1 ng/ml), is able to prevent the restoration of Ang ll-induced pro-inflammatory signalling by the wild-type peptide (Figure 13B).

[0001373] In AT1 R-CHO cells treated with TAT-mCherry-S391 A-RAGE _362-404 fusion protein (1 ng/ml), subsequent treatment with wild-type TAT-mCherry-RAGE _362-404 fusion peptide, even in 1000-fold excess, is not able to restore responsiveness to Ang ll-induced expression of p65 when compared to untreated cells (Figure 13C).

[0001374] In AT1 R-CHO cells expressing S391 Q-cRAGE ₂₂ - ₄ 04 (i.e. a construct with no phosphorylatable elements) subsequent treatment with TAT-mCherry-S391 A-RAGE _362-404 fusion protein (0.4ng/ml), is able to prevent Ang ll-induced induction of p65-NFKB and the proliferation marker PCNA, confirming that modulation is achieved independent of the phosphorylation of RAGE (Figure 13D).

[0001375] Treatment of PMAEC from AGER KO mice (deficient in RAGE) with the wild-type TAT-mCherry-RAGE ₃ 62-404 fusion peptide (0.4ng/ml) is able to restore the Ang ll-mediated induction of expression of p65-NFKB and NFKB-dependent pro-inflammatory genes, ICAM- 1, VCAM-1 and MCP-1 (Figure 13E).

[0001376] Treatment of RAGE-replete PMAEC obtained from c57bl6 mice with TAT- mCherry-S391 A-RAGE ₃₆ 2-404 fusion protein (0.4ng/ml), blocks Ang ll-induced induction of downstream signalling leading to activation of canonical and non-canonical pathways of N FKB activation (as detected by induction of the expression of CXCL 12 and CXCL2 respectively) and the downstream NFKB-dependent induction of VCAM-1 gene expression (Figure 13F). TNFa is shown as a positive control for canonical NFKB-signalling and is unaffected by TAT-mCherry-S391 A-RAGE _362-404 fusion protein. [0001377] Treatment of RAGE-replete human AEC with TAT-mCherry-S391 A-RAGE _362-404 fusion protein (0.4ng/ml) also blocks Ang ll-induced downstream signalling leading to the NFKB-dependent induction of pro-inflammatory genes, ICAM- 1, VCAM- 1, MCP- 1, TNFa and IL-6 (Figure 13G). The inhibitory effect is comparable to that achieved with the AT ₁ R blocker, irbesartan in HAECs.

[0001378] Treatment with TAT-mCherry-S391 A-RAGE _362-404 fusion protein (0.4ng/ml) also inhibits subsequent induction of pro-inflammatory gene expression by the RAGE ligand, S100A8/A9 in (i) RAGE-AT1 R-CHO cells and (ii) PMAECs replete in RAGE (Figure 13H).

Additional material and methods

[0001379] To confirm the applicability of inhibition strategies to human tissues, additional experiments were undertaken in primary aortic endothelial cells from humans (Lonza). HAECs were grown in EGM-2 media. In each case cells were treated with mCherry-TAT (control), TAT-m Cherry- RAG E _362-404 (wild type), or TAT-mCherry-S391 A-RAGE _362-404 at a dose of 0.4ng/ml for 30min prior to treatment with either Ang II (1 μΜ), or S100A8/A9 (2 μg/ml; R&D systems) for 2h. As a further control, the inhibitory effect of the AT ₁ R blocker irbesartan (1 μΜ; 30 minutes before dosing with Ang II) was also explored in HAECs.

EXAMPLE 14. TARGETING RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR IN THE AORTA EX VIVO

[0001380] In this example, we demonstrate that treatment of aortae with a cell-penetrating peptide inhibitor of RAGE ligand-independent activation of RAGE (TAT-mCherry-S391 A- RAGE ₃₆ 2-404 fusion protein, 8ng/ml) is able to prevent the induction of RAAS-mediated inflammation ex vivo.

[0001381 ] As also detailed in Example 4, ex vivo exposure to Ang II increases the expression of pro-atherogenic mediators (Figure 14A) in isolated aortae from apoE KO mice. By contrast, ex vivo treatment of aortae taken from AGER/apoE DKO mice with Ang II does not lead to the induction of these atherogenic mediators (Figure 14B).

[0001382] Pre-treatment of aortae from apoE KO mice with a cell-penetrating peptide inhibitor of RAGE ligand-independent activation of RAGE (TAT-mCherry-S391 A-RAGE ₃₆₂ - ₄ o4 fusion protein, 8ng/ml) prevents Ang ll-dependent induction of adhesion molecules, as detected by gene expression measured by real time RT-PCR (Figure 14A). [0001383] Pre-treatment of aortae from AGERIapoE DKO mice with a cell-penetrating wild- type RAGE peptide (TAT-mCherry-RAGE ₃ 62-404 fusion protein, 8μg/ml) restores Ang ll- dependent induction of adhesion molecules and inflammatory markers, as detected by gene expression measured by real time RT-PCR (Figure 14B).

Additional materials and methods

[0001384] Aortae were isolated from apoE KO and AGERIapoE DKO mice, divided and placed in Krebs buffer and maintained at physiological pH by infusing carbogen gas (95% 0 ₂ ; 5% C0 ₂ ) through the buffer at 37°C. Aortae were randomised to receive pre-treatment with mCherry-TAT (control), mCherry-TAT-RAGE ₃₆ 2- ₄ 04 (wild type), or mCherry-TAT-S391 A- RAGE ₃ 62-404 oligopeptide (all 8ng/ml_). Thirty minutes later, aortae were exposed to Ang II (1 μΜ) for 4 hours. At the end of the incubation, aortae were placed in Trizol, mRNA extracted and cDNA synthesized. Gene expression of pro-atherogenic mediators was then estimated by quantitative real-time RT-PCR, as detailed above.

EXAMPLE 15. TARGETING RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CO-LOCATED GPCR (TRANSACTIVATION) IN LIVE MICE

[0001385] In this example, we demonstrate that treatment with a cell-penetrating peptide inhibitor of RAGE ligand-independent activation of RAGE is able to prevent the induction of RAAS-mediated atherosclerosis in apoE KO mice.

[0001386] As detailed in Example 3, genetic deficiency of Ace2 in Ace2/apoE DKO mice results in augmented plaque accumulation at 16 weeks of age when compared to apoE KO mice replete in Ace2. Ace2/AGER/apoE triple KO mice are protected against this increase in atherosclerosis associated with genetic Ace2 deficiency (Figure 15A).

[0001387] Treatment of Ace2/apoE DKO mice with a cell-penetrating peptide inhibitor of RAGE ligand-independent activation of RAGE (TAT-mCherry-S391 A-RAGE _362-404 fusion protein (S391 A); 10 μg/kg/IP every second day for ten weeks) attenuates Ang ll-dependent atherosclerosis in this model (Figure 15A).

[0001388] Treatment of Ace2/AGER/apoE triple KO mice with a cell-penetrating peptide transducer of RAGE ligand-independent activation of RAGE (TAT-mCherry-RAGE _362-404 fusion protein (WT); 10 μg/kg/IP every second day for ten weeks) restores atherosclerosis to that observed in Ace2/apoE DKO mice replete in RAGE (Figure 15A) despite only restoring the signalling capable RAGE cytosolic tail and the ongoing absence of full-length RAGE and its associated ligand-binding domain. [0001389] Treatment of Ace2/apoE DKO mice with a cell-penetrating peptide inhibitor of RAGE ligand-independent activation of RAGE (TAT-mCherry-S391 A-RAGE _362-404 fusion protein; 10 μg/kg/IP every second day for ten weeks) does not affect blood pressure levels (Figure 15C).

[0001390] Streptozotocin-induced diabetes is also a model associated with RAAS activation and increased atherosclerosis, which is prevented by RAAS inhibition, without lowering blood pressure or altering glucose control (Candido et al., 2002).

[0001391 ] AGER/apoE DKO mice are protected against an increase in atherosclerosis following the induction of diabetes (Soro-Paavonen et al., 2008).

[0001392] Treatment of diabetic apoE KO mice with a cell-penetrating peptide inhibitor of RAGE ligand-independent activation of RAGE (TAT-mCherry-S391 A-RAGE _362-404 fusion protein (S391 A); 10 μg/kg/IP every second day for ten weeks) attenuates atherosclerosis in this model (Figure 15B) without affecting blood pressure levels (Figure 15C).

[0001393] Treatment of diabetic apoE KO mice with a cell-penetrating peptide transducer of RAGE ligand-independent activation of RAGE (TAT-mCherry-RAGE ₃ 62-404 fusion protein; 10 μg/kg/IP every second day for ten weeks) increases atherosclerosis beyond that seen with diabetes alone (Figure 15B) despite only restoring the signalling capable RAGE cytosolic tail and the ongoing absence of full-length RAGE and its associated ligand-binding domain.

[0001394] Treatment of diabetic AGER/apoE DKO mice with a cell-penetrating peptide transducer of RAGE ligand-independent activation of RAGE (TAT-mCherry-RAGE ₃ 62-404 fusion protein, 10 μg/kg/IP every second day for ten weeks) reverses the protection afforded by genetic RAGE deletion and increases atherosclerosis (Figure 15B) to a level observed in RAGE replete mice, despite only restoring the signalling capable RAGE cytosolic tail and the ongoing absence of full-length RAGE and its associated ligand-binding domain.

Additional materials and Methods

[0001395] Male apoE KO mice, Ace2/apoE DKO mice and Ace2/ AGER/apoE TKO mice were randomized to receive the TAT-Cherry-S391 A-RAGE ₃₆ 2- ₄ 04 (inhibitor) peptide (10 μg/kg/IP every second day), TAT-Cherry-RAGE _362-404 (wild-type; 10 μg/kg/IP every second day) oligopeptide or mCherry-TAT (control; 10 μg/kg/IP every second day) for 10 weeks, at which time they were humanely killed. [0001396] In a second model of Ang ll-dependent atherosclerosis, apoE KO mice and AGER/apoE DKO were randomly allocated to receive streptozotocin (55mg/kg, Sigma Chemical Co, St. Louis, MO, USA) or buffer (sodium citrate buffer pH 4.5) delivered intraperitoneal^ in five consecutive daily doses (Soro-Pavinonen et al. 2008). This regimen induces an insulinopenic form of diabetes associated with hyperglycaemia (blood glucose -30 mM) but with sufficient beta-cell function to prevent ketosis or require insulin supplementation. The presence of diabetes was confirmed by a fasting blood glucose level > 15 mM, one week after the first dose of streptozotocin. All diabetic mice also achieved an HbA1 c>8% (median 1 1 .2%) after ten weeks. After one week of diabetes, mice were further randomized to receive the mCherry-TAT-S391 A-RAGE ₃ 62-404 (inhibitor) peptide (100μg/kg/IP every second day), mCherry-TAT-RAGE ₃ 62-404 (wild-type; 100μg/kg/IP every second day) oligopeptide or mCherry-TAT (control; 100μg/kg/IP every second day) for 10 weeks, at which time they were humanely killed.

EXAMPLE 16. BRET INDICATES CLOSE PROXIMITY OF ATiR AND RAGE WHEN CO- EXPRESSED IN LIVE CELLS

[0001397] In this example we demonstrate co-expression of Rluc8-labeled AT ₁ R (AT ₁ R /Rluc8) and Venus labelled-RAGE (RAGE/Venus) in HEK293FT cells results in the generation of a strong and saturable BRET signal consistent with their close proximity, which is reduced upon treatment with Ang II (Figure 16A). The presence of soluble RAGE22-331 (sRAGE) does not alter these results (Figure 16A).

[0001398] Treatment of cells co-expressing AT ₁ R/Rluc8 and β-arrestin2/Venus with Ang II results in the induction of a robust and stable ligand-induced BRET signal consistent with recruitment of -arrestin2 to the AT1 R (Figure 16B). Treatment of cells co-expressing Chemokine (C-C Motif) Receptor 4 (CCR4)/Rluc8 and β-arrestin2/Venus with its cognate ligand CCL22 also results in the induction of a robust ligand-induced BRET signal consistent with recruitment of β-arrestin2 to the CCR4 (Figure 16B).

[0001399] Treatment of cells co-expressing RAGE/Rluc8 and β-arrestin2/Venus with Ang II induces a BRET signal in the presence of untagged AT1 R (Figure 16C), indicative of close proximity between RAGE and AT1 R.

[0001400] By way of a control, this is not observed in the presence of untagged CCR4 when treated with its cognate ligand CCL22 (Figure 16C). [0001401 ] As an additional control to demonstrate the expression/functioning of untagged CCR4, a CCL22-induced BRET signal is observed when Gai/Nluc and Gv2/Venus are co- expressed (Figure 16D).

Additional materials and Methods

[0001402] An alternative BRET donor to Rluc8 is Nanoluc (Nluc) and this can be used in combination with Venus with a furimazine substrate, as described in Tiulpakov et al., 2016. This approach was used when monitoring the proximity of Gai/Nluc and GY2/Venus to assess the functionality of CCR4.

EXAMPLE 17. BRET INDICATES CLOSE PROXIMITY OF RAGE TO CERTAIN ACTIVATED GPCRS IN ADDITION TO ATiR WHEN CO-EXPRESSED IN LIVE CELLS

[0001403] In this example, we demonstrate that close proximity between RAGE and AT ₁ R in live cells, described in Example 16 above, is also observed between RAGE and certain other activated co-located GPCRs.

[0001404] Treatment of cells co-expressing an Rluc8-labelled GPCR and β-arrestin2 Venus with an appropriate cognate agonist for that GPCR resulted in the induction of a robust ligand-induced BRET signal consistent with recruitment of β-arrestin2 to the activated Rluc8- labelled GPCR. This was observed for AT ₁ R with Ang II (Figure 17A), TRHR1 with TRH (Figure 17B), OxR1 with OxA (Figure 17C), bradykinin receptor 2 (BDKR) with bradykinin (Figure 17D), V2R with AVP (Figure 17E), CCR2 with MCP1 (Figure 17F) and CCR5 with ΜΙΡ1 β (Figure 17G).

[0001405] Receptor-HIT: Treatment of cells co-expressing Rluc8-labelled RAGE (RAGE/Rluc8) and β-arrestin2/Venus in the presence of AT ₁ R (Figure 17A), OxR1 (Figure 17C), V2R (Figure 17E) or CCR2 (Figure 17F) with the appropriate cognate agonist for that GPCR resulted in the induction of a clear ligand-induced BRET signal consistent with recruitment of β-arrestin2 to the GPCR. This in turn is indicative of RAGE proximity to the activated GPCR. No ligand-induced BRET signal was observed in the absence of the co- expressed GPCR.

[0001406] Treatment of cells co-expressing Rluc8-labelled RAGE (RAGE/Rluc8) and β- arrestin2/Venus in the presence of TRHR1 (Figure 17B) or bradykinin receptor 2 (BDKR; Figure 17D) with the appropriate cognate agonist for that GPCR resulted in the induction of a weak ligand-induced BRET signal discernible from the lack of ligand-induced BRET signal observed in the absence of the co-expressed GPCR and consistent with recruitment of β- arrestin2 to the GPCR. This in turn may be indicative of RAGE proximity to the activated GPCR, but this is not as clear as observed for AT ₁ R, OxR1 , V2R or CCR2.

[0001407] Treatment of cells co-expressing Rluc8-labelled RAGE (RAGE/Rluc8) and β- arrestin2/Venus in the presence of CCR5 (Figure 17G) with ΜΙΡ1 β resulted in the induction of a weak ligand-induced BRET signal that was difficult to discern from that observed in the absence of the co-expressed GPCR.

EXAMPLE 18. BRET INDICATES CLOSE PROXIMITY OF RAGE TO CERTAIN ACTIVATED CHEMOKINE RECEPTORS WHEN CO-EXPRESSED IN LIVE CELLS

[0001408] In this example, we demonstrate that close proximity between RAGE and certain chemokine receptors is observed in live cells using the Receptor-HIT assay configuration.

[0001409] Treatment of cells co-expressing Rluc8-labelled RAGE (RAGE/Rluc8) and β- arrestin2/Venus in the presence of CCR1 (Figure 18A), CCR2 (Figure 18B), CCR6 (Figure 18E), CCR7 (Figure 18F), CXCR2 (Figure 181) or CXCR6 (Figure 17F) with the appropriate cognate agonist for that GPCR (as labelled) resulted in the induction of a clear ligand- induced increase in BRET signal consistent with recruitment of β-arrestin2 to the GPCR. This in turn is indicative of RAGE proximity to the activated GPCR.

[0001410] Treatment of cells co-expressing Rluc8-labelled RAGE (RAGE/Rluc8) and β- arrestin2/Venus in the presence of CXCR1 (Figure 18H) with CXCL8 resulted in the induction of a discernible ligand-induced increase in BRET signal consistent with recruitment of β-arrestin2 to the GPCR. This in turn is indicative of RAGE proximity to the activated GPCR, but this is not as clear as for the aforementioned receptors.

[000141 1 ] Treatment of cells co-expressing Rluc8-labelled RAGE (RAGE/Rluc8) and β- arrestin2/Venus in the presence of CXCR4 (Figure 18K) with CXCL12 resulted in the induction of a clear ligand-induced decrease in BRET signal consistent with a reduction in proximity between β-3ΠΌ5ίίη2 and RAGE, or a conformational change resulting in less resonance energy transfer between Rluc8 and Venus. This in turn is indicative of a change in proximity of RAGE to the activated GPCR to which the is recruited.

[0001412] Treatment of cells co-expressing Rluc8-labelled RAGE (RAGE/Rluc8) and β- arrestin2/Venus in the presence of CCR4 (Figure 18C), CCR5 (Figure 18D), CCR10 (Figure 18G) or CXCR3 (Figure 17J) with the appropriate cognate agonist for that GPCR (as labelled) did not result in the induction of a discernible ligand-induced change in BRET signal. This is despite very strong ligand-induced BRET signals being observed for CCL22-induced recruitment of β-arrestin2/Venus to CCR4/Rluc8 (Figure 16B) and CCL4-induced recruitment of β-arrestin2/Venus to CCR5/Rluc8 (n=3; data not shown). Weaker, but still clear ligand- induced BRET signals were also observed for CCL27-induced recruitment of β- arrestin2/Venus to CCR10/Rluc8 and CXCL1 1 -induced recruitment of β-arrestin2/Venus to CXCR3/Rluc8 (n=3; data not shown).

EXAMPLE 19. BRET INDICATES THAT CO-LOCATED GPCR ACTIVATION RESULTS IN A CHANGE IN RAGE SUBCELLULAR LOCALISATION IN LIVE CELLS

[0001413] In this example, we demonstrate that proximity between RAGE and subcellular compartment markers changes upon activation of certain co-located GPCRs, indicating an effect of GPCR activation on RAGE function for a number of GPCRs.

[0001414] Venus-tagged subcellular compartment markers used were the plasma membrane marker KRAS, as well as RabGTPases Rab1 (endoplasmic reticulum trafficking to the cis-Golgi), Rab4 (early endosome recycling), Rab5 (early endosomes), Rab7 (late endosomes/lysosomes), Rab8 (trans- Golgi network to the plasma membrane), Rab9 (late endosome trafficking to the trans-Golgi network) and Rab1 1 (recycling endosomes). Proximity of Rluc8-tagged RAGE to Venus-tagged subcellular compartment markers was assessed in real-time before and after addition of an appropriate cognate agonist for the GPCR at time zero and at the concentration indicated.

[0001415] For adrenergic α1A receptor, adrenergic α1 B receptor, angiotensin receptor AT ₁ R, bradykinin receptor B2, CCR2, CCR6, CCR9, CXCR4, CXCR5, dopamine D1 receptor, endothelin receptor type B, histamine H3 receptor, muscarinic M2 receptor, neuropeptide Y1 receptor, orexin receptor 1 , orexin receptor 2, prostaglandin E1 receptor, serotonin 5-HT2c receptor, serotonin 5-HT4b receptor, somatostatin 2 receptor, sphingosine 1 -phosphate receptor S1 P3, vasopressin receptor 1 A and vasopressin receptor 1 B, a particularly clear change in the ligand-induced BRET signal indicative of a change in proximity to at least one of the subcellular compartment markers was observed as a consequence of ligand addition.

[0001416] For CCR3, CCR4, neurotensin 1 receptor and serotonin 5-HT2b receptor, a clear change in the ligand-induced BRET signal indicative of a change in proximity to at least one of the subcellular compartment markers was observed as a consequence of ligand addition.

[0001417] For adenosine A1 receptor, adrenergic a2B receptor, CCR1 , CCR5, CCR7, CXCR2, endothelin receptor type A, muscarinic M1 receptor, muscarinic M3 receptor, serotonin 5-HT1 a receptor, serotonin 5-HT2a receptor, sphingosine 1 -phosphate receptor S1 P1 , thyrotropin-releasing hormone receptor 1 and vasopressin receptor 2, a small yet discernible change in the ligand-induced BRET signal indicative of a change in proximity to at least one of the subcellular compartment markers was observed as a consequence of ligand addition.

For adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic a2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1 , CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1 , neurotensin 2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor, little discernible change in the ligand-induced BRET signal for any of the subcellular compartment markers was observed as a consequence of ligand addition (data not shown).

EXAMPLE 20. RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CERTAIN ACTIVATED CO-LOCATED GPCRS OTHER THAN AT ₁ RECEPTOR

[0001418] In this example, we demonstrate that the activation of certain chemokine receptors (CCR2 and CXCR2) by their cognate ligands (MCP-1 and CXCL8, respectively) to induce the expression of the key pro-inflammatory transcription factor, p65-NFKB, also requires the co-expression of RAGE.

[0001419] In CHO cells stably transfected with a plasmid coding for CCR2 and expressing CCR2 on their surface (CCR2-CHO cells), transfection with RAGE and exposure to MCP-1 (10 ^~7 ) the cognate ligand for CCR2, results in the activation of N FKB and induction of the expression of p65 (Figure 20A). In the absence of RAGE co-expression, MCP-1 does not induce the activation of N FKB to induce the expression of p65 in CCR2-CHO cells.

[0001420] In CHO cells stably transfected with a plasmid coding for CXCR2 and expressing CXCR2 on their surface (CXCR2-CHO cells), transfection with RAGE and exposure to the cognate ligand, IL-8 (CXCL8), results in the increased expression of p65 in the presence of co-expressed RAGE (Figure 20B). In the absence of RAGE, IL-8 does not induce the activation of N FKB to induce the expression of p65 in CXCR2-CHO cells.

[0001421 ] In bone marrow-derived primary murine macrophages, exposure to MCP-1 (10 ^~7 M) the cognate ligand for CCR2, results in the activation of N FKB and induction of the expression of p65 (Figure 20C). The induction of expression of p65-NFKB by MCP-1 in primary macrophages is blocked by pre-treatment with the TAT-mCherry-S391 A-RAGE ₃ 62-404 fusion protein (0.4ng/ml). [0001422] In HMEC exposure to MCP-1 (10 ^-7 M) the cognate ligand for CCR2, results in the activation of N FKB and auto-induction of the expression of MCP-1 (Figure 20D). The auto- induction of MCP-1 expression of HMEC is blocked by pre-treatment with the TAT-mCherry- S391 A-RAGE ₃ 62-404 fusion protein (0.4ng/ml). Taken together these data confirm the role of co-located endogenous RAGE in the MCP-1/CCR2 signalling process.

[0001423] In HMEC expressing CXCR2 exposure to IL-8 (10 ⁷ M), the cognate ligand for CXCR2, results in the activation of N FKB and induction of the expression of MCP-1 (Figure 20E). The induction of MCP-1 expression of HMEC is blocked by pre-treatment with the TAT-mCherry-S391 A-RAGE ₃ 62-404 fusion protein (0.4ng/ml). Taken together these data confirm the role of co-located endogenous RAGE in the IL-8/CXCR2 signalling process.

Additional materials and methods

Generation of transgenic Chinese Hamster Ovary Cells

[0001424] 10Ong of CCR2-Rluc8 construct or CXCR2-Rluc8 was transfected into CHO cells using Lipofectamine 2000 (Thermo). Stable transfectants were selected using G418. CCR2- CHO and CXCR2-CHO cells were then transiently transfected with full length RAGE constructs using Lipofectamine 2000 (Invitrogen) and incubated for 16h. CCR2-CHO cells were then exposed to MCP-1 (10 ⁷ ) the cognate ligand for CCR2 for two hours. CXCR2- CHO cells were exposed to the cognate ligand, IL-8 (CXCL8) for two hours. Cells were then placed in Trizol, mRNA extracted and cDNA synthesized. Changes in the gene expression of the N FKB subunit, p65 (RelA) were determined by quantitative real-time RT-PCR, performed using the TaqMan system based on real-time detection of accumulated fluorescence (ΑΒΙ Prism 7700, Perkin-Elmer Inc, PE Biosystems, Foster City, CA, USA), as detailed in the general methods.

Primary bone-marrow derived macrophages

[0001425] Primary bone marrow-derived macrophages were pre-treated with RAGE362-404, the mutant RAGE peptide, S391 A-RAGE362-404 or TAT-cherry control for 1 hour. Cells were then exposed to MCP-1 (10 ^~7 M) the cognate ligand for CCR2 for two hours. Cells were then placed in Trizol, mRNA extracted and cDNA synthesized. Changes in the gene expression of the N FKB subunit, p65 (RelA) were determined by quantitative real-time RT-PCR, performed using the TaqMan system based on real-time detection of accumulated fluorescence (ABI Prism 7700, Perkin-Elmer Inc, PE Biosystems, Foster City, CA, USA), as detailed in the general methods. EXAMPLE 21. RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE BY CERTAIN CO- LOCATED GPCRS IS INHIBITED BY TARGETING THE TRANSMEMBRANE DOMAIN OF RAGE

[0001426] This example shows that RAGE ligand-independent activation of RAGE by a certain co-located GPCR and activation of RAGE by a RAGE ligand are inhibited following modulation of the transmembrane domain (TMD) of RAGE.

[0001427] RAGE is a type 1 transmembrane protein that comprises a single transmembrane spanning helix (residues 343-361 of SEQ ID NO: 14). The RAGE TMD is highly conserved across all mammalian species.

[0001428] The RAGE TMD is important for membrane targeting and anchoring, as RAGE isoforms lacking this hydrophobic domain (e.g. soluble RAGE; sRAGE) are secreted and not retained in the plasma membrane.

[0001429] Homodimerization of RAGE on the cell surface is essential for RAGE-mediated signal transduction. The V, C1 and C2 domains of the extracellular portion of RAGE contain elements that promote RAGE dimerization (e.g. RAGE22-331 is able to homodimerize) and dimerization of the RAGE can be inhibited by overexpression of sRAGE. The TMD of RAGE also potentially plays an active role in oligomerization, and the TMD has been previously shown to homodimerize, independent of the RAGE ectodomain (Su et al. 2013).

[0001430] The RAGE TMD is also required for transmission of the RAGE ligand-induced signal from the ectodomain to signalling elements in the cytosolic domain. Without wishing to be bound by theory, the inventors believe that this occurs by the transduction of conformational changes (such as scissor-like motion to increase the distance between the C-termini to 100 A) following ligand binding (Xue et al. 2017).

[0001431 ] RAGE ligand binding is known to induce ectodomain shedding of RAGE22-331 by the secretases ADAM10/17 and MMP9 following which the remaining membrane-bound C- terminal fragment of RAGE is processed by γ-secretase, an intramembrane cleaving protease. Inhibition of γ-secretase results in increased retention of the C-terminal fragment of RAGE in the membrane after RAGE ligand-binding as well as inhibiting RAGE ligand- induced signalling, suggesting that intramembrane proteolysis of RAGE transmembrane domain is a critical step for RAGE ligand-dependent signaling (Braley et al. 2016). [0001432] Taken together, these data provide a rationale for targeting the RAGE TMD as a means to modulate RAGE ligand-independent activation of RAGE following activation of certain co-located GPCRs.

[0001433] We demonstrate that a BRET signal is observed between N-terminally tagged mCherry-RAGE _338-361 (the RAGE TMD with an extracellular juxta-membrane fragment linked to mCherry) and an extracellularly luciferase-tagged ATi receptor (Nluc-ATi ; Fig. 21 A), confirming that the TMD is also involved in the interaction between RAGE and certain GPCRs.

[0001434] Overexpression of mCherry-RAGE _338-361 modulates BRET between full length RAGE and ATi in a dose dependent fashion, demonstrated with BRET saturation assays (Fig. 21 B) and kinetic assays (Fig. 21 C), confirming that BRET between full length RAGE and ATi is inhibited by a non-functional substitute of the RAGE TMD with a juxta-membrane portion of the ectodomain. Notably, sRAGE (i.e. the extracellular ligand-binding V-C1 -C2 domains of RAGE; RAGE22-331 ) does not inhibit BRET between AT1 R and RAGE (Fig 16A).

[0001435] Control experiments are shown in Fig. 21 D showing that there is no ligand- induced BRET signal between AT1 /Rluc8 and mCherry/RAGE _338-361 , even with the appropriate filter set. The mCherry tag is used to separately assess the relative expression level of the mCherry/RAGE _338-361 fusion protein, as shown in Fig. 21 E, following excitation by an external light source. Also shown in Fig. 21 E, similar expression levels of AT1/Rluc8 across the various transfections are demonstrated by the similar relative luminescence emissions from the luciferase. Furthermore, similar expression levels of RAGE/Venus across the various transfections are demonstrated by the similar relative fluorescence emissions at the emission wavelength of Venus following excitation by an external light source.

[0001436] For adrenergic a2B receptor, angiotensin receptor ATi (AT1 R), bradykinin receptor 2 (B2R), CCR1 , CCR2, CCR4, CCR5, CCR6, CCR9, CXCR2, CXCR4, neuropeptide Y1 receptor (NPY1 R), orexin receptor 2, sphingosine 1 -phosphate receptor 1 (S1 PR1 ), thyrotropin-releasing hormone receptor 1 (TRHR1 ), vasopressin receptor 1 A (V1 aR), vasopressin receptor 1 B (V1 bR) and vasopressin receptor 2 (V2R), a particularly clear inhibition of the change in the ligand-induced BRET signal between the Rluc8-tagged GPCR and RAGE/Venus was observed when mCherry-RAGE _338-361 was co-expressed (Fig. 21 F), confirming that BRET between full length RAGE and these GPCRs is inhibited by a non-functional substitute of the RAGE TMD with a juxta-membrane portion of the ectodomain. [0001437] For adenosine A1 receptor (ADORA1 ), CCR7 and CXCR5, a small yet discernible inhibition of the change in the ligand-induced BRET signal between the Rluc8- tagged GPCR and RAGE/Venus was observed when m Cherry- RAG E338-361 was co- expressed (Fig. 21 F), confirming that BRET between full length RAGE and these GPCRs is inhibited by a non-functional substitute of the RAGE TMD with a juxta-membrane portion of the ectodomain.

[0001438] For adrenergic α1A receptor, CCR3, muscarinic acetylcholine receptor 2 (CHRM2) and orexin receptor 1 , little discernible change in ligand-induced BRET signal was observed between the Rluc8-tagged GPCR and RAGE/Venus meaning that the effect of mCherry-RAGE _338-361 co-expression could not be determined (Fig. 21 F).

[0001439] As detailed above, transactivation of RAGE requires expression of the cytosolic domain (RAGE379-391 ), consequently RAGE338-361 cannot be activated. However, Ang II- mediated pro-inflammatory signaling, as indicated by ICAM- 1 gene expression, induced following transactivation of full-length RAGE in human microvascular endothelial cells (HMEC) is inhibited by transfection with RAGE338-361 . This inhibition is rescued by additional expression of mCherry-RAGE ₃ 62-404 (i.e. the cytosolic domain of RAGE; Figure 21 G). This suggests that the TMD and the cytosolic domain are able to function independently, but are co-dependent in full length RAGE constructs. Notably, sRAGE (i.e. the RAGE ectodomain) does not inhibit RAGE ligand-independent activation of RAGE following activation of certain co-located GPCRs.

[0001440] To validate the hypothesis that the TMD of RAGE alone can act as an inhibitor of RAGE ligand-independent activation of RAGE by certain co-located GPCRs, HMEC were transfected with only the TMD of RAGE (RAGE343-361 ) or mCherry-TMD fusion. Expression of either construct was able to prevent the induction of both MCP1 and ICAM1 gene expression by Ang II in HMEC that are endogenously replete in RAGE expression (Figure 21 H).

[0001441 ] To validate the hypothesis that the TMD of RAGE alone can also act as an inhibitor of RAGE ligand-dependent activation of RAGE, CHO cells expressing full length human RAGE were treated with the RAGE ligand S100A8/A9 in the presence and absence of transfection of a construct expressing only the TMD of RAGE (RAGE343-361 ). Expression of this construct was able to prevent the induction of p65 gene expression by S100A8/A9, similar to RAGE370-390 (Figure 21 1). [0001442] The actions of mDiaphI and ΡΚCζ in RAGE transactivation are mediated by their actions at the RAGE TMD, as silencing of mDiaphI and ΡΚCζ using siRNA inhibits signalling induced by RAGE ₃ 43-404 but not RAGE362-404. (Figure 21 J).

[0001443] Taken together these data confirm that specific modulation of the RAGE TMD is able to inhibit the transactivation of RAGE by certain co-located GPCRs as well as RAGE ligand-dependent signalling following activation of the RAGE ectodomain.

EXAMPLE 22. PROPHETIC EXAMPLES OF SCREENING FOR POTENTIAL INHIBITORS OF RAGE LIGAND-INDEPENDENT ACTIVATION OF RAGE

[0001444] The following assays are prophetic examples of how the invention described herein would be logically applied to identify a potential inhibitor of RAGE ligand-independent RAGE signalling via activation of a certain co-located GPCR (e.g. AT ₁ R-induced or CCR2- mediated transactivation of RAGE).

[0001445] Assay 1 : Competition assays to measure inhibition of binding between the cytosolic tail of RAGE (full length or truncated, wild type or inhibitory mutants) and IQGAP-1 or fragments thereof.

[0001446] Methods: Sandwich assay has either IQGAP or cytosolic tail of RAGE bound to plate or column (full length or truncated). Add binding partner (i.e. either IQGAP or cytosolic tail of RAGE) in presence of serial dilution of test compound/modulator. Presence of binding partner measured by intrinsic label (fluorescent label, enzyme fusion) or by way of detection probe (e.g. antibody to binding partner brings label). Absence of binding partner indicates added test compound is a competitive inhibitor of binding between cytosolic RAGE and IQGAP-1 or fragments thereof.

[0001447] Assay 2. Yeast-two hybrid used to measure binding between the cytosolic tail of RAGE and IQGAP-1 .

[0001448] Methods: Yeast two hybrid assay expresses IQGAP-1 and cytosolic RAGE (full length or truncated, wild type or inhibitory mutants). Presence of binding partner measured by intrinsic label (e.g. fluorescent label, enzyme fusion, reporter system (e.g. KISS). Absence of binding partner indicates added test compound is a competitive inhibitor of binding between cytosolic tail of RAGE and IQGAP-1 or fragments thereof.

[0001449] Assay 3: BRET used to measure binding between the cytosolic tail of RAGE (full length or truncated, wild type or inhibitory mutants) and IQGAP-1 . [0001450] Methods: BRET assay expresses labelled IQGAP and cytosolic RAGE and measures the transfer of resonance energy between them, indicating their proximity. Inhibition of the BRET signal upon addition of the test compound/modulator indicates that the test compound/modulator is a competitive inhibitor of interaction between cytosolic RAGE and IQGAP or fragments thereof.

[0001451 ] Assay 4: Inhibition of RAGE ligand-independent activation of RAGE by certain activated co-located GPCR (e.g. AT ₁ R-induced) by detecting the induction of downstream signalling via RAGE.

[0001452] An inhibitor of RAGE ligand-independent (e.g. AT ₁ R-induced transactivation) RAGE signalling is indicated by a test compound that reduces RAGE-dependent downstream N FKB activation in CHO cells expressing both AT ₁ R and RAGE exposed to Ang II (or another GPCR exposed to its cognate ligand), but not RAGE signalling induced by S100A8/A9 or downstream N FKB activation induced by S100A8/A9 or other RAGE ligands, or RAGE-independent Gq signalling induced following activation of the AT ₁ R by Ang II (e.g. calcium influx, inositol phosphate levels, expression markers such as EGR).

[0001453] Assay 5. Binding partners of RAGE379-390.

[0001454] Methods: Having demonstrated that RAGE379-390 is an important signalling element for modulation of RAGE ligand independent activation of RAGE, identification of high-affinity specific binding partners to RAGE379-390 is undertaken in silico or in vitro of screening peptide and non-peptide libraries (i.e. chemicals) or structure based drug design.

CONCLUSIONS

[0001455] Activation of certain co-located GPCRs by their cognate ligands, such as activation of AT ₁ R by Ang II and CCR2 by MCP-1 , triggers inflammation through pathways distinct from classical canonical signalling via GPCRs that induce, for example, calcium influx, inositol phosphate synthesis and activation of PKA. Here, the inventors show that RAGE ligand-independent activation of the cytosolic tail of RAGE by certain co-located GPCRs is a key determinant of this division. The inventors demonstrate that activation of N FKB, which is critical for the pro-inflammatory signalling downstream of many GPCRs, is mediated by RAGE ligand-independent activation of co-located RAGE. Consequently, many of the adverse vascular changes induced, for example, by Ang II-AT1 R activation are attenuated when RAGE is deleted or when RAGE activation is inhibited. By contrast, AT1 R- dependent signalling via classic Gq pathways is unaffected by RAGE expression and RAGE deletion has no deleterious effect on vascular homeostasis in salt deficiency or blood pressure responsiveness to Ang I I.

[0001456] The inventors provide herein specific evidence that RAGE and GPCRs, including the AT ₁ R or CCR2, form a heteromeric complex, with the generation of a clear Receptor-HIT BRET signal. This is further supported by the specific evidence provided by the inventors herein that the heteromeric complex formation and transactivation is inhibited by expression of a non-functional substitute of the transmembrane domain of RAGE with or without an additional juxta-membrane fragment of the ectodomain. Inactive state preassembly of GPCRs into transient complexes with signalling components is thought to enable rapid and augmented responsiveness. Without wishing to be bound by theory, the inventors believe that preassembly of RAGE with certain co-located GPCRs serves a similar role, enabling rapid RAGE ligand-independent activation of the cytosolic tail of RAGE and downstream proinflammatory signalling following activation of GPCRs.

[0001457] The inventors also show that the conformation of residue 391 of RAGE is important for GPCR-dependent pro-inflammatory signalling, as S391 A-RAGE and 390X- RAGE mutants do not induce pro-inflammatory signalling following activation of certain co- located GPCRs, such as the AT ₁ R by Ang I I, unlike wild-type RAGE which is different only at one amino acid (serine391 ). This is not because Ser391 is phosphorylated following RAGE activation, as previously suggested, because RAGE containing different amino acids at this position, namely Q391 and P391 (naturally found in camels and cattle, respectively) can also be activated by RAGE ligands as well as by RAGE ligand-independent activation by certain activated co-located GPCRs (see Example 7). Although ΡΚCζ inhibition is able to prevent activation of full-length RAGE, it is not acting by preventing phosphorylation of RAGE, as it also inhibits signalling via a full-length S391 Q chimeric RAGE mutant that contains no amino acids capable of sustaining phosphorylation (i.e. no serines, threonines or tyrosines).

[0001458] The interaction of the cytosolic tail of RAGE with Diaph i is known to be important for pro-inflammatory signalling, possibly as it facilitates recruitment and activation of ΡΚCζ, with which Diaphi also binds, and subsequent "scissor-like" changes in structure of RAGE mediated via interaction with the transmembrane domain . In the inventors' experiments, silencing of Diaphi or Ρ^ζ inhibits both Ang II and s1 00-induced proinflammatory signalling via full-length RAGE and signalling with Ang I I mediated by N- truncated RAGE constructs retaining the RAGE TMD. However, N-truncated RAGE constructs missing the RAGE TMD are still able to be activated by Ang I I following silencing of Diaphi or ΡΚCζ. Diaphi is thought to interact with RAGE in the cytosol via a small charged patch. Alanine-substitution of this charged patch (R366A-Q367A) prevents signalling mediated by the RAGE ligand, S1 00A8/A9. However, mutation of this charged patch or deletion of the alpha-loop it stabilises (e.g. RAGE370-404) has no effect on RAGE ligand-independent activation of the cytosolic tail of RAGE following activation of certain co- located GPCRs, such as the AT1 R by Ang I I. Consequently, proposed methods to disrupt the binding of Diaph-1 to RAGE using small molecules or other inhibitors (Ramasamy et al. 201 6) will have no effect on RAGE ligand-independent activation of RAGE by certain activated co- located GPCRs. Moreover, the ability of N-trucated RAGE ₃ 62-404 to be activated by certain co- located GPCRs, such as the AT1 R is not inhibited by inhibition of ΡΚC oζr Diaph-1 using siRNA, confirming the mechanism of action of this domain is independent of both these RAGE binding partners.

[0001459] Even though the RAGE ectodomain has historically been considered to be essential for its functions, without wishing to be bound by theory, the inventors believe the RAGE ligand-independent activation of the cytosolic tail of RAGE by certain activated co- located GPCRs appears to be the dominant mechanism inducing downstream effector activation and signalling. Consistent with this hypothesis, treatment with the wild-type RAGE ₃ 62-404 oligopeptide restores atherogenesis in diabetic AGER/apoE DKO mice, suggesting that the relative vasculo-protection observed in AGER KO mice is mediated through inhibition of this RAGE ligand-independent transactivation pathway, further highlighting its pathophysiological importance and rationale for inhibition of RAGE ligand- independent activation of the cytosolic tail of RAGE by certain activated co-located GPCRs.

[0001460] Only some cells (e.g. endothelial cells, leucocytes) express RAGE under basal conditions, meaning that the RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs described by the inventors is limited to these cell types under basal conditions. Most other cell types do not express RAGE under basal conditions. Over and above findings that RAGE expression has no effect on classic Gq signalling induced following AT ₁ R activation, this limited distribution of expression may partly explain why RAGE deletion/inhibition does not impact on blood pressure, natriuresis or other homeostatic functions of the systemic RAAS. However, following injury, inflammation, stress or hypoxia, the de novo or enhanced expression of RAGE may provide a conduit for the proinflammatory effects of local GPCR activation.

[0001461 ] Although systemic antagonists of GPCR-mediated signalling currently exist (e.g . AT ₁ R antagonists, CCR2 antagonists), the inhibition achieved by this strategy removes all signalling arising from respective blocked GPCRs. This is likely to be suboptimal in some circumstances as it may induce feedback escape from inhibition or result in unwanted effects arising from inhibition of physiological signalling. For example, inhibition of the AT ₁ R results in blood pressure lowering which may be undesirable in some settings (e.g. following a myocardial infarction, stroke or in severe heart failure), as well as inducing feedback 'escape' activation of the RAAS. The potential advantage of targeting only key cells in which this RAGE ligand-independent activation of RAGE is in operation, and then only the proinflammatory signals arising from GPCR activation, is that the limitations of systemic inhibition do not apply.

[0001462] As a proof of principle, the inventors show that in endothelial cells replete in both RAGE and AT ₁ R, over-expression of S391 A-RAGE or treatment with a TAT-S391 A- RAGE ₃ 62-404 oligopeptide is able to inhibit Ang II-AT1 R dependent pro-inflammatory signalling, as effectively as an AT1 R blocker, but without modifying canonical IP-1 -dependent signalling. The same oligopeptide applied to the aorta ex vivo is also able to block the induction of proinflammatory molecules following exposure to Ang II. In addition, when delivered to Ace2/apoE DKO mice or diabetic apoE KO mice for ten weeks, the S391 A-RAGE362-404 peptide was able to attenuate RAAS-dependent atherosclerosis, without affecting blood pressure levels. Furthermore, the inventors have shown that RAGE338-361 or RAGE343-361 can also inhibit Ang II-AT1 R dependent pro-inflammatory signalling in endothelial cells replete in both RAGE and AT1 R. These data underline the therapeutic potential of specifically targeting this novel pro-inflammatory signalling pathway.

[0001463] The inhibition achieved with the S391 A-RAGE362-404 oligopeptide is not overcome by excess wild-type RAGE362-404, while low concentrations of S391 A-RAGE362-404 oligopeptide overcomes existing wild-type RAGE, suggesting its actions are not simply competitive. Moreover, that equivalent inhibition can be achieved using the dodecapeptide RAGE379-390 shows that changes over a small region of an alpha helix, and specifically key residues over this region, alters the affinity of such RAGE mutants for signalling mediators, including IQGAP-1 , which are then rendered blocked to endogenous signalling by wild type RAGE.

[0001464] Notably, the efficacy of S391 A-RAGE362-404 in inhibiting pro-inflammatory signalling induced following activation of co-located GPCRs (e.g. CCR2 by MCP-1 ), and the actions of RAGE in mediating activation of NFKB following activation of GPCRs by their cognate ligands (e.g. activation of CCR2-CHO cells by MCP or CXCR2-CHO cells by IL-8), confirms that the effect of an activated co-located GPCR resulting in RAGE ligand- independent activation of RAGE is not exclusive to AT1 R, and generalizable to proinflammatory signalling induced following activation of certain other co-located GPCRs. Without wishing to be bound by theory, the inventors believe that the key RAGE pharmacophore identified herein interacts with signalling elements common to many GPCRs with pro-inflammatory effects, or by interaction with wild-type RAGE.

[0001465] The inventors anticipate that mechanistic understanding of RAGE-GPCR heteromers will lead directly to the development of novel therapeutics that are able to specifically target the adverse effects of RAGE ligand-independent activation of RAGE by certain activated co-located GPCRs without compromising physiological signalling or induce feedback 'escape' from their inhibition. These data may be applicable across a broad range of conditions in which RAGE-mediated signalling has been implicated including atherosclerosis, neurodegenerative diseases, malignancy, diabetic complications and other important inflammatory and pro-proliferative conditions.

[0001466] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0001467] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0001468] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

[0001469] As used herein, "isolated" when describing a peptide modulator of the invention means a peptide described herein that is not in a natural state (e.g. it is disassociated from a larger protein molecule or cellular debris in which it naturally occurs or is normally associated with), or is a non-naturally occurring fragment of a naturally occurring protein (e.g. the peptide comprises less than 25%, preferably less than 10% and most preferably less than 5% of the naturally occurring protein). Isolated also may mean that the amino acid sequence of the peptide does not occur in nature, for example, because the sequence is modified from a naturally occurring sequence (e.g. by alteration of certain amino acids, including basic (i.e. cationic) amino acids such as arginine, tryptophan, or lysine), or because the sequence does not contain flanking amino acids which are present in nature. The term "isolated" may mean that the peptide or amino acid sequence is a man-made sequence or polypeptide and may be non-naturally occurring. [0001470] Likewise, "isolated" as used in connection with nucleic acids which encode peptides embraces all of the foregoing, e.g. the isolated nucleic acids are disassociated from adjacent nucleotides with which they are associated in nature, and can be produced recombinantly, synthetically, by purification from biological extracts, and the like. Isolated nucleic acids can contain a portion that encodes one of the foregoing peptides and another portion that codes for another peptide or protein. The isolated nucleic acids also can be labeled. The nucleic acids include codons that are preferred for animal, bacterial, plant, or fungal usage. In certain embodiments, the isolated nucleic acid is a vector, such as an expression vector, which includes a nucleic acid that encodes one of the foregoing isolated peptides. A general method for the construction of any desired DNA sequence is provided, e.g., in Brown J. et al. (1979), Methods in Enzymology, 68:109; Sambrook J, Maniatis T (1989), supra.

[0001471 ] The term "amino acid" or "residue" as used herein includes any one of the twenty naturally-occurring amino acids, the D-form of any one of the naturally-occurring amino acids, non-naturally occurring amino acids, and derivatives, analogues and mimetics thereof. Any amino acid, including naturally occurring amino acids, may be purchased commercially or synthesized by methods known in the art. Examples of non-naturally-occurring amino acids include norleucine ("Nle"), norvaline ("Nva"), β-Alanine, L- or D-naphthalanine, ornithine ("Orn"), homoarginine (homoArg) and others well known in the peptide art, including those described in M. Bodanzsky, "Principles of Peptide Synthesis," 1 st and 2nd revised ed., Springer-Verlag, New York, N.Y., 1984 and 1993, and Stewart and Young, "Solid Phase Peptide Synthesis," 2nd ed., Pierce Chemical Co., Rockford, III., 1984, both of which are incorporated herein by reference.

[0001472] Common amino acids may be referred to by their full name, standard single- letter notation (lUPAC), or standard three-letter notation for example: A, Ala, alanine; C, Cys, cysteine; D, Asp, aspartic acid (aspartate); E, Glu, glutamic acid (glutamate); F, Phe, phenylalanine; G, Gly, glycine; H, His, histidine; I, lie isoleucine; K, Lys, lysine; L, Leu, leucine; M, Met, methionine; N, Asn, asparagine; P, Pro, proline; Q, Gin, glutamine; R, Arg, arginine; S, Ser, serine; T, Thr, threonine; V, Val, valine; W, Trp, tryptophan; X, Hyp, hydroxyproline; Y, Tyr, tyrosine. Any and all of the amino acids in the compositions herein can be naturally occurring, synthetic, and derivatives or mimetics thereof.

[0001473] Non-peptide analogues of peptides, e.g., those that provide a stabilized structure or lessened biodegradation, are also contemplated. Peptide mimetic analogues can be prepared based on a selected peptide by replacement of one or more residues by non- peptide moieties. Preferably, the non-peptide moieties permit the peptide to retain its natural conformation, or stabilize a preferred, e.g., bioactive, conformation. One example of methods for preparation of non-peptide mimetic analogues from peptides is described in Nachman et al., Regul. Pept. 57:359-370 (1995). The term "peptide" as used herein embraces all of the foregoing.

[0001474] As mentioned above, the peptide of the present invention may be composed either of naturally occurring amino acids, i.e. L-amino acids, or of D-amino acids, i.e. of an amino acid sequence comprising D-amino acids in retro-inverso order as compared to the native sequence. The term "retro-inverso" refers to an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted. Thus, any sequence herein, being present in L-form is also inherently disclosed herein as a D-enantiomeric (retro-inverso) peptide sequence. D-enantiomeric (retro-inverso) peptide sequences according to the invention can be constructed, e.g. by synthesizing a reverse of the amino acid sequence for the corresponding native L-amino acid sequence. In D-retro- inverso enantiomeric peptides, e.g. a component of the isolated peptide, the positions of carbonyl and amino groups in each single amide bond are exchanged, while the position of the side-chain groups at each alpha carbon is preserved.

[0001475] Preparation of a component of the isolated peptide modulators of embodiments of the invention as defined above having D-enantiomeric amino acids can be achieved by chemically synthesizing a reverse amino acid sequence of the corresponding naturally occurring L-form amino acid sequence or by any other suitable method known to a skilled person. Alternatively, the D-retro-inverso-enantiomeric form of a peptide or a component thereof may be prepared using chemical synthesis as disclosed above utilizing an L-form of an peptide or a component thereof as a matrix for chemical synthesis of the D-retro-inverso- enantiomeric form.

[0001476] Various changes may be made including the addition of various side groups that do not affect the manner in which a peptide modulator of embodiments of the invention functions, or which favourably affect the manner in which a peptide modulator of embodiments of the invention functions. Such changes may involve adding or subtracting charge groups, substituting amino acids, adding lipophilic moieties that do not affect binding but that affect the overall charge characteristics of the peptide modulator of embodiments of the invention facilitating delivery across the blood-brain barrier, etc. For each such change, no more than routine experimentation is required to test whether the molecule functions according to the invention. One simply makes the desired change or selects the desired peptide and applies it in a fashion as described in detail in the examples. [0001477] In one form of the invention, the term "sequence identity" as defined herein means that the sequences are compared as follows. To determine the percent identity of two amino acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid sequence). The amino acids at corresponding amino acid positions can then be compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. For example, where a particular peptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide, which is 50% identical to the reference polypeptide over its entire length. Such a determination of percent identity of two sequences can be accomplished using a mathematical algorithm.

[0001478] A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873- 5877. Such an algorithm is incorporated into the NBLAST program, which can be used to identify sequences having the desired identity to the amino acid sequence of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997), Nucleic Acids Res, 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) can be used. The sequences further may be aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default (BLOSUM62) matrix (values-4 to +1 1) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (per each additional consecutive null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the claimed sequence. The described methods of determination of the percent identity of two amino acid sequences can be applied correspondingly to nucleic acid sequences.

[0001479] In one embodiment a peptide modulator of embodiments of the invention may be linked directly or via a linker. A "linker" in the present context is usually a peptide, oligopeptide or polypeptide and may be used to link multiples of the peptides to one another. The peptides of the invention selected to be linked to one another can be identical sequences, or are selected from any of the peptides of the invention. A linker can have a length of 1 -10 amino acids, more preferably a length of 1 to 5 amino acids and most preferably a length of 1 to 3 amino acids. In certain embodiments, the linker is not required to have any secondary structure forming properties, i.e. does not require a a-helix or β-sheet structure forming tendency, e.g. if the linker is composed of at least 35% of glycine residues. As mentioned hereinbefore, a linker can be a cleavable peptide such as an MMP peptide which can be cleaved intracellularly by normal cellular processes, effectively raising the intracellular dose of the previously linked peptides, while keeping the extracellular dose low enough to not be considered toxic. The use of a(n) intracellularly/endogenously cleavable peptide, oligopeptide, or polypeptide sequence as a linker permits the peptides to separate from one another after delivery into the target cell. Cleavable oligo- or polypeptide sequences in this context also include protease cleavable oligo- or polypeptide sequences, wherein the protease cleavage site is typically selected dependent on the protease endogenously expressed by the treated cell. The linker as defined above, if present as an oligo- or polypeptide sequence, can be composed either of D-amino acids or of naturally occurring amino acids, i.e. L-amino acids. As an alternative to the above, coupling or fusion of the peptides can be accomplished via a coupling or conjugating agent, e.g. a cross-linking reagent.

[0001480] There are several intermolecular cross-linking reagents which can be utilized, see for example, Means and Feeney, Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43. Among these reagents are, for example, N-succinimidyl 3-(2- pyridyldithio)propionate (SPDP) or N,N'-(1 ,3-phenylene)bismaleimide; N,N'-ethylene-bis- (iodoacetamide) or other such reagent having 6 to 1 1 carbon methylene bridges; and 1 ,5- difluoro-2,4-dinitrobenzene. Other cross-linking reagents useful for this purpose include: ρ,ρ'- difluoro-m,m'-dinitrodiphenylsulfone; dimethyl adipimidate; phenol-1 ,4-disulfonylchloride; hexamethylenediisocyanate or diisothiocyanate, or azophenyl-p-diisocyanate; glutaraldehyde and disdiazobenzidine. Cross-linking reagents may be homobifunctional, i.e., having two functional groups that undergo the same reaction. A preferred homobifunctional cross-linking reagent is bismaleimidohexane (BMH). BMH contains two maleimide functional groups, which react specifically with sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). The two maleimide groups are connected by a hydrocarbon chain. Therefore, BMH is useful for irreversible cross-linking of proteins (or polypeptides) that contain cysteine residues. Cross-linking reagents may also be heterobifunctional. Heterobifunctional cross- linking reagents have two different functional groups, for example an amine- reactive group and a thiol-reactive group, that will cross-link two proteins having free amines and thiols, respectively. Examples of heterobifunctional cross-linking reagents are succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS), and succinimide 4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analogue of MBS. The succinimidyl group of these cross-linking reagents with a primary amine, and the thiol-reactive maleimide forms a covalent bond with the thiol of a cysteine residue. Because cross-linking reagents often have low solubility in water, a hydrophilic moiety, such as a sulfonate group, may be added to the cross-linking reagent to improve its water solubility. Sulfo-MBS and sulfo-SMCC are examples of cross-linking reagents modified for water solubility. Many cross-linking reagents yield a conjugate that is essentially non-cleavable under cellular conditions. Therefore, some cross-linking reagents contain a covalent bond, such as a disulfide, that is cleavable under cellular conditions. For example, Traut's reagent, dithiobis (succinimidylpropionate) (DSP), and N-succinimidyl 3-(2- pyridyldithio)propionate (SPDP) are well-known cleavable cross-linkers. The use of a cleavable cross-linking reagent permits the peptides to be separated after delivery into the target cell, if desired, provided the cell is capable of cleaving a particular sequence of the crosslinker reagent. For this purpose, direct disulfide linkage may also be useful. Chemical cross-linking may also include the use of spacer arms. Spacer arms provide intramolecular flexibility or adjust intramolecular distances between conjugated moieties and thereby may help preserve biological activity. A spacer arm may be in the form of a protein (or polypeptide) moiety that includes spacer amino acids, e.g. proline. Alternatively, a spacer arm may be part of the cross-linking reagent, such as in "long-chain SPDP" (Pierce Chem. Co., Rockford, III., cat. No. 21651 H). Numerous cross-linking reagents, including the ones discussed above, are commercially available. Detailed instructions for their use are readily available from the commercial suppliers. A general reference on protein cross-linking and conjugate preparation is: Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press (1991 ).

[0001481 ] In one embodiment, peptide modulators may also contain a "derivative", "variant", or "functional fragment", i.e. a sequence of a peptide that is derived from the naturally occurring (L-amino-acid) sequence of a peptide of the invention as defined above by way of substitution(s) of one or more amino acids at one or more sites of the amino acid sequence, by way of deletion(s) of one or more amino acids at any site of the naturally occurring sequence, and/or by way of insertion(s) of one or more amino acids at one or more sites of the naturally occurring peptide sequence. "Derivatives" shall retain their biological activity if used as peptides of the invention. Derivatives in the context of the present invention may also occur in the form of their L- or D-amino-acid sequences as defined above, or both. [0001482] If substitution(s) of amino acid(s) are carried out for the preparation of a derivative of the peptides of the invention, conservative (amino acid) substitutions are preferred. Conservative (amino acid) substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid (aspartate) and glutamic acid (glutamate); asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Thus, preferred conservative substitution groups are aspartate-glutamate; asparagine-glutamine; valine-leucine-isoleucine; alanine-valine; and phenylalanine-tyrosine. By such mutations e.g. stability and/or effectiveness of a peptide may be enhanced. If mutations are introduced into the peptide, the peptide remains (functionally) homologous, e.g. in sequence, in function, and in antigenic character or other function. Such mutated components of the peptide can possess altered properties that may be advantageous over the non-altered sequences of the peptides of the invention for certain applications (e.g. increased pH optimum, increased temperature stability etc.).

[0001483] In one embodiment, a derivative of the peptide of the invention is defined as having substantial identity with the non-modified sequences of the peptide of the invention. Particularly preferred are amino acid sequences which have at least 30% sequence identity, preferably at least 50% sequence identity, even preferably at least 60% sequence identity, even preferably at least 75% sequence identity, even more preferably at least 80%, yet more preferably 90% sequence identity and most preferably at least 95% or even 99% sequence identity to the naturally occurring analogue. Appropriate methods for synthesis or isolation of a functional derivative of the peptides of the invention as well as for determination of percent identity of two amino acid sequences are described above. Additionally, methods for production of derivatives of the peptides as disclosed above are well known and can be carried out following standard methods which are well known by a person skilled in the art (see e.g., Sambrook J, Maniatis T (1989)).

[0001484] As a further embodiment, the invention provides pharmaceutical compositions or medicaments comprising the modulators as defined herein. In certain embodiments, such pharmaceutical compositions or medicaments comprise the modulators as well as an optional linker, as defined herein.

[0001485] Additionally, such a pharmaceutical composition or medicament can comprise a pharmaceutically acceptable carrier, adjuvant, or vehicle. A "pharmaceutically acceptable carrier, adjuvant, or vehicle" according to the invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity or physiological targeting of the modulator with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the pharmaceutical compositions of this invention include, but are not limited to those that can be applied cranially or intracranial^, or that can cross the blood-brain barrier (BBB). Notwithstanding this, the pharmaceutical compositions of the invention can include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0001486] The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, cerebrally, or via an implanted reservoir.

[0001487] The term "fragment" as used herein with reference to any domain of RAGE, or with reference to any other polypeptide sequence, is to be understood as meaning one or more amino acid residues less than the domain of RAGE or any other polypeptide sequence to which it refers.

[0001488] The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the pharmaceutical compositions of this invention may be aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0001489] As such, any bland fixed oil may be employed including synthetic mono- or di- glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0001490] The pharmaceutically acceptable compositions herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavouring or colouring agents may also be added.

[0001491 ] Alternatively, the pharmaceutical composition as defined herein may be administered in the form of suppository for rectal administration. Such a suppository can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and, therefore, will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0001492] The pharmaceutical composition as defined herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the brain, other intra-cranial tissues, the eye, or the skin. Suitable formulations are readily prepared for each of these areas or organs.

[0001493] For topical applications, the pharmaceutical composition as defined herein may be formulated in a suitable ointment containing modulators as identified herein, suspended or dissolved in one or more carriers. Carriers for topical administration of the peptide include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition as defined herein can be formulated in a suitable lotion or cream containing the peptide suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. [0001494] The pharmaceutical composition as defined herein may also be administered by nasal aerosol or inhalation. Such a composition may be prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. The pharmaceutically acceptable composition or medicament herein is formulated for oral or parenteral administration, e.g. by injection.

[0001495] For treatment purposes, a non-toxic, effective amount of the modulator may be used for preparation of a pharmaceutical composition as defined above. Therefore, an amount of the modulator may be combined with the carrier material(s) to produce a composition as defined above.

[0001496] The pharmaceutical composition is typically prepared in a single (or multiple) dosage form, which will vary depending upon the host treated and the particular mode of administration. Usually, the pharmaceutical composition is formulated so that a dosage range per dose of 0.0001 to 100 mg/kg body weight/day of the peptide can be administered to a patient receiving the pharmaceutical composition. Preferred dosage ranges per dose vary from 0.01 mg/kg body weight/day to 50 mg/kg body weight/day, even further preferred dosage ranges per dose range from 0.1 mg/kg body weight/day to 10 mg/kg body weight/day.

[0001497] However, dosage ranges and treatment regimens as mentioned above may be adapted suitably for any particular patient dependent upon a variety of factors, including the activity of the specific modulator employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician and the severity of the particular disease being treated. In this context, administration may be carried with in an initial dosage range, which may be varied over the time of treatment, e.g. by increasing or decreasing the initial dosage range within the range as set forth above. Alternatively, administration may be carried out in a continuous manner by administering a specific dosage range, thereby maintaining the initial dosage range over the entire time of treatment. Both administration forms may furthermore be combined, e.g. if the dosage range is to be adapted (increased or decreased) between various sessions of the treatment but kept constant within the single session so that dosage ranges of the various sessions differ from each other.

[0001498] When used therapeutically, the modulators of the invention are administered in therapeutically effective amounts. In general, a therapeutically effective amount means an amount necessary to delay the onset of, inhibit the progression of, or halt altogether the particular condition being treated. Generally, a therapeutically effective amount will vary with the subject's age and condition, as well as the nature and extent of the disease in the subject, all of which can be determined by one of ordinary skill in the art. The dosage may be adjusted by the individual physician, particularly in the event of any complications being experienced.

[0001499] As mentioned above, one aspect of the invention relates to nucleic acid sequences and their derivatives which code for an isolated peptide modulator or variant thereof and other nucleic acid sequences which hybridize to a nucleic acid molecule consisting of the above described nucleotide sequences, under stringent conditions. The term "stringent conditions" as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically, stringent conditions, as used herein, refers to hybridization at 65° C. in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% Polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 25 mMNaH ₂ P0 ₄ (pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M Sodium Chloride/0.15 M Sodium Citrate, pH 7; SDS is Sodium Dodecyl Sulphate; and EDTA is Ethylene diaminetetraacetic acid. After hybridization, the membrane upon which the DNA is transferred is washed at 2xSSC at room temperature and then at 0.1 xSSC/0.1 xSDS at 65 °C.

[0001500] The present invention furthermore provides kits comprising the abovementioned pharmaceutical composition (in one or more containers) in at least one of the above formulations and an instruction manual or information brochure regarding instructions and/or information with respect to application of the pharmaceutical composition.

[0001501 ] Those skilled in the field of the invention will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such functional variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein. Furthermore, the present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, neurobiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology, or techniques cited herein.

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