NEUROIMMUNOLOGICAL RESPONSE TO STRESS

TECHNICAL FIELD

[0001] The invention relates to the field of medical diagnostics and therapeutics, and more particularly to methods for recognizing and treating biological phenomena that significantly exarcebate disease via neuroimmunological responses to stress and for identifying molecules that may be selectively active on stress-related complications of human trauma and disease. The invention also relates to specific reagents and procedures of particular utility in the generation of therapeutic agents that regulate the binding of Rictor to its binding partners, thereby affecting the neurogenic complications of traumatic,

autoimmune and other disease processes.

BACKGROUND ART

[0002] Traumatic stress, such as from physical trauma, open-heart surgery or severe burns, is associated with secondary complications leading to morbidity and death in a subset of subjects that receive the traumatic insult. The mechanisms by which such serious consequences develop in a subset of the population are incompletely understood but are believed to involve neuroimmunological processes that may, for example, result in altered responses to infection, leading to sepsis and organ failure. In the intensive care unit (ICU) trauma and sepsis are leading causes of mortality. The rate of this type of ICU mortality has remained largely unchanged for several decades and new approaches for early detection of high-risk individuals and effective interventions that reduce neuroimmunological

complications, organ failure and death are desperately needed.

[0003] A neuroimmunological stress response (NSR) is characterized by a traumatic, xenobiotic, or physiological stressor followed by release of peptides such as Substance P, calcitonin-related peptide (CGRP) from sensory nerves into the tissues and the triggering of complicated downstream events such as acute inflammation, followed by extended suppression of inflammation. This sequence may sometimes result in sepsis, multiple organ failure, and death. During the NSR, changes in protein kinase C beta (PKC-beta) and TRPV family signaling in the dorsal root ganglia typically result in activation of neurokinin receptor activation. No effective intervention for the NRS has been developed to date. Over 200,000 people die of sepsis in the ICU each year. [0004] The NSR may also explain the underlying biology of many autoimmune processes. The so-called diseases of western civilization (chronic conditions such as arthritis, lupus, psoriasis, asthma, painful bladder syndrome, colitis, neuropathic pain, fibromyalgia and other immune-mediated diseases, osteoporosis, autism, atherosclerosis and other cardiovascular diseases, cancers and metastases of the breast, prostate and colon, metabolic syndrome -related conditions such as cardiovascular dysfunctions, diabetes, pulmonary arterial hypertension and polycystic ovary syndrome, neurodegenerative conditions such as Parkinson's and Alzheimer's, and ophthalmic diseases such as macular degeneration) are now increasingly viewed as associated with chronic relap sing-remitting inflammation cycles which, in turn, relate to the neuroimmunological mechanisms. One common feature of nearly all of the emerging diseases in the Western world is the complexity of the underlying neuroimmunological dysfunction. New methodology and reagents for identifying the best points for mechanistic intervention in such disease conditions is needed. Such methodology would provide a first step to the development of predictive diagnostics and adequately targeted interventions.

[0005] The inventor has recently shown that a central regulator of neuroimmunological responses to metabolic and xenobiotic stress is the highly conserved Rictor-containing complex in mammalian cells known as mTORC2. Neurogenic inflammation pursuant to stress is associated with the release of neuropeptides such as substance P from nerve fibers in organ tissues. In kidney, a key early-warning organ, mTORC2 controls substance P levels in tissue following xenobiotic stress, as well as downstream events such as macrophage activity, plasma IL-6 and TNF-alpha, tissue phosphorylation of p66shc, gene expression and other markers of oxidative damage and inflammation (Mascarenhas D et al [2012] Inflamm. Res. 61: 1395-1404).

[0006] TOR (target of rapamycin) proteins are conserved Ser/Thr kinases found in diverse eukaryotes ranging from yeast to mammals. The TOR kinase is found in two biochemically and functionally distinct complexes, termed TORC1 and TORC2. Aided by the compound rapamycin, which specifically inhibits TORC1, the role of TORC1 in regulating translation and cellular growth has been extensively studied. mTORC2 is largely rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton (Jacinto E, et al [2004] Nat Cell Biol. 6: 1122-1128). The physiological roles of TORC2 have remained largely elusive due to the lack of pharmacological inhibitors and its genetic lethality in mammals. PRR5/PROTOR and its related family of proteins are a new class of molecules found in association to mTORC2 complex, and may be required cofactors for the function of this central regulator of neuroimmunological responses to stress. The PRR5/PROTOR gene encodes a conserved proline-rich protein predominant in kidney (Johnstone CN et al [2005] Genomics 85: 338-351). The PRR5/PROTOR class of proteins is believed to physically associate with mTORC2 and regulate aspects of growth factor signaling and apoptosis (Woo SY et al [2007] /. Biol. Chem. 282: 25604-25612; Pearce LR et al [2007] Biochem J. 405: 513-522; Thedieck K et al [2007] PLoS ONE 2: el217). In this invention, the importance of a particular domain within PRR5/PROTOR comprising the sequence HESRGVTEDYLRLETLVQKVVSPYLGTYGL (SEQ ID NO: 3) is demonstrated. This sequence is conserved in human PRR5/PROTOR isoforms as well as in rat and mouse. Other obligate partners of Rictor, a central defining protein component of the mTORC2 complex, include Sinl (also known as MIP1). Sinl is an essential component of TORC2 but not of TORC1, and functions similarly to Rictor, the defining member of TORC2, in complex formation and kinase activity. Knockdown of Sinldecreases Akt phosphorylation in both Drosophila and mammalian cells and diminishes Akt function in vivo. It also disrupts the interaction between Rictor and mTOR. Furthermore, Sinl is required for TORC2 kinase activity in vitro (Yang Q et al [2006] Genes Dev. 20: 2820-2832). mTOR, SIN1 and Rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt, SGK1 (serum- and glucocorticoid- induced protein kinase 1), and conventional protein kinase C (PKC). This TORC2 function is growth factor independent and conserved from yeast to mammals.

[0007] mTORC2 activity was elevated in glioma cell lines as well as in primary tumor cells as compared with normal brain tissue (Masri J et al [2007] Cancer Res. 67: 11712- 11720). In these lines Rictor protein and mRNA levels were also elevated and correlated with increased mTORC2 activity. Xenograft studies using these cell lines also supported a role for increased mTORC2 activity in tumorigenesis and enhanced tumor growth. These data suggest that mTORC2 is hyperactivated in gliomas and functions in promoting tumor cell

proliferation and invasive potential. mTORC2 and its activation of downstream AGC kinases such as PKC-alpha, SGK1 and Akt have also been implicated in cancers of the prostate and breast (Guertin DA et al [2009] Cancer Cell. 15: 148-159; Sahoo S et al [2005] Eur J Cancer. 41: 2754-2759; Guo J, et al [2008] Cancer Res. 68: 8473-8481).

[0008] IRS-1 and IRS-2 are master traffic regulators in intracellular signal transduction pathways associated with growth and metabolism, playing key roles in the docking of accessory proteins to phosphorylated insulin and IGF receptors. Although similar in function, activated IRS-1 and IRS-2 proteins generate subtly different cellular outcomes, at least in part through the phosphorylation of different Akt (especially Akt 1 and Akt 2) and MAP kinase isoforms.

[0009] In diabetic humans and db/db mice the receptor for advanced glycated end products (RAGE) is activated by systemic ligands such as amphoterin, S100A9 and glycated hemoglobin (Goldin A et al [2006] Circulation 114: 597-605) and affects urinary albumin and/or NGAL (lipocalin-2). RAGE has been implicated in the development of kidney dysfunction consequent to elevated blood sugar (Tan AL et al [2007] Semin. Nephrol.

27: 130-143). The inventor has recently shown that inhibition of mTORC2 and PKC reduces urinary albumin and NGAL in diabetic models (Singh BK and Mascarenhas D [2008] Am J Nephrol 28:890-899; Singh BK et al [2010] Metab Syn Relat Dis. 8(4): 1-10).

[0010] Variability within patient populations creates numerous problems for medical treatment. Without reliable means for determining which individuals will respond to a given treatment, physicians are forced to resort to trial and error. Because not all patients will respond to a given therapy, the trial and error approach means that some portion of the patients must suffer the side effects (as well as the economic costs) of a treatment that is not effective in that patient. It is therefore desirable to develop diagnostic methods and reagents to facilitate the identification of patients most likely to benefit from treatment for the NSR.

[0011] For some therapeutics targeted to specific locations within the body, screening to determine eligibility for the treatment can be performed. For example, the estrogen antagonist tamoxifen targets the estrogen receptor, so it is normal practice to only administer tamoxifen to those patients whose tumors express the estrogen receptor. Likewise, the antitumor agent trastuzumab (HERCEPTIN®) acts by binding to a cell surface molecule known as HER2/neu; patients with HER2/neu negative tumors are not normally eligible for treatment with trastuzumab. Methods for predicting whether a patient will respond to treatment with IGF-I/IGFBP-3 complex have also been disclosed (U.S. Patent No.

5,824,467), as well as methods for creating predictive models of responsiveness to a particular treatment (U.S. Patent No. 6,087,090).

[0012] The inventor has previously disclosed certain IGFBP-derived peptides known as "MBD" peptides (U.S. Patent Application Publication Nos. 2003/0059430, 2003/0161829, and 2003/0224990). These peptides have a number of properties, which are distinct from the IGF-binding properties of IGFBPs, that make them useful as therapeutic agents. MBD peptides are internalized some cells, and the peptides can be used as cell internalization signals to direct the uptake of molecules joined to the MBD peptides (such as proteins fused to the MBD peptide). Therapeutic peptides are provided by U.S. Patent Nos. 7,618,816; 7,611,893; 7,662,624; and U.S. Patent Application Publication No. 2008/003,9393 Al;

2010/0152113 Al; the contents of each are hereby incorporated by reference is its entirety.

[0013] Combination treatments are increasingly being viewed as appropriate strategic options for designed interventions in complex disease conditions such as cancer, metabolic diseases, vascular diseases and neurodegenerative conditions. For example, the use of combination pills containing two different agents to treat the same condition (e.g. metformin plus a thiazolidinedione to treat diabetes, a statin plus a fibrate to treat hypercholesterolemia) is on the rise. It is therefore appropriate to envisage combination treatments that include moieties such as MBD in combination with other agents such as other peptides, antibodies, nucleic acids, chemo therapeutic agents and dietary supplements. Combinations may take the form of covalent extensions to the MBD peptide sequence, other types of conjugates, or coadministration of agents simultaneously or by staggering the treatments i.e. administration at alternating times.

[0014] We have shown that MBD peptide-mediated delivery of bioactive molecules in vivo can be applied to disease processes such as cancer (Huq A, et al [2009] Anti-Cancer Drugs 20: 21-31) and diabetes, as described above. Nephrilin, a peptide containing the MBD scaffold, is bioactive in reducing albuminuria in diabetic mice. Nephrilin was designed to interfere with mTORC2 complex and has been shown to disrupt the association of IRS proteins with Rictor (Singh BK et al [2010] Metab Syn Relat Dis. 8(4): 1-10; US Patent No. 7,662,624). Similar approaches may be used to disrupt mTORC2 and IRS protein activity in human disease by competing the physical interaction of Rictor with obligate cofactors such as PRR5/PROTOR or Sinl/MIPl. The competing molecule may be a cell-penetrating peptide, protein, antibody or nucleic acid, or a small chemical molecule. In this work we describe in vitro assay systems that facilitate rapid screening of candidate molecules for such a purpose. Any metabolic, systemic, degenerative, or inflammatory disease process may be a candidate for interventions using such molecules.

[0015] The central role played by mTORC2 in regulating diseases of stress and aging has not been well documented. Nephrilin, an inhibitor of the binding of Rictor— the canonical component of mTORC2 complex— to its binding partners or cofactors such as

PRR5/PROTOR, Sinl and IRS proteins, is the only specific inhibitor of its class described to date. The inventor has shown that nephrilin can reverse immunological dysfunctions and disease processes relating to complications of diabetes and hypertension; acute kidney injury from rhabdomyolysis or xenotoxic stress with platinum compounds or aminoglycoside antibiotics; cancer metastasis; the neuroimmunological sequelae of burn trauma; and mortality from sepsis. These results implicate mTORC2 as a central regulator of diseases of aging. Fundamental common mechanisms suggested for the gamut of diseases of aging— the so-called diseases of western civilization— include oxidative stress [Pinton P and Rizzuto R

(2008) Cell Cycle. 7(3): 304-308], loss of circadian circuitry [Uchida Y et al (2010) Biol. Pharm. Bull. 33(4) 535— 544], loss of selective protein turnover mechanisms [Hussain S et al

(2009) Cell Cycle 8: 11, 1688-1697], and the epithelial-mesenchymal transition, EMT

[Slattery C et al (2005) American Journal of Pathology, 167(2): 395-407]. The inventor has shown that biochemical signatures associated with each of these pathways can be reversed by treatment with nephrilin and has demonstrated, for the first time, that specific inhibition of mTORC2 may be the key to controlling diseases of stress and aging. Thus, therapeutic agents that disrupt binding of Rictor (the canonical component mTORC2) to its binding partners are of particular interest in the treatment of metabolic and cardiovascular diseases, especially those characterized by some underlying combination of insulin resistance, hyperglycemia, hypertension and hyperlipidemia; cancer progression and metastasis; acute kidney injury (AKI) in critical care settings, also including sepsis, systemic inflammatory conditions such as shock, post-operative stress such as after cardiopulmonary bypass or transplant, burns, pancreatitis, rhabdomyolysis, xenobiotic stresses caused by cocaine, alcohol, aminoglycoside antibiotics, antiviral compounds or platinum compounds; neurodegenerative diseases such as Parkinson's Alzheimer's, Huntington's and ALS/Lou Gehrig's disease; ototoxicities;

autoimmune conditions such as lupus erythematosus and multiple sclerosis; genetic diseases such as cystinosis, Fanconi's and other conditions affecting mitochondrial respiration;

pulmonary diseases, especially COPD and asthma and pulmonary arterial hypertension;

migraine; ocular diseases such as cataracts and retinopathies, especially diabetic

complications; and liver diseases, including chronic viral infections such as hepatitis. These disease states are now increasingly viewed as secondary to chronic inflammatory conditions that may, in turn, relate to neurogenic signaling and oxidative stress. A correlation between oxidative stress and processes of aging may explain the rising incidence of these diseases as a direct consequence of an aging population.

[0016] A key regulator of oxidative damage and aging is the adapter protein p66shc. Activation of this molecule by phosphorylation at serine 36 leads to mitochodrial

translocation and increased production of free oxygen radicals. P66shc gene knockout mice live significantly longer and are protected from many of the diseases of aging listed above [Pinton P and Rizzuto R (2008) Cell Cycle. 7(3): 304-308]. The inventor has shown for the first time, that mTORC2 regulates the activation of protein kinase C beta- II (PKC-beta-II) by phosphorylation at threonine 641. PKC-beta was shown to be the activator of p66shc by phosphorylation of S36 [Pinton P, et al. (2007) Science 315: 659-663]. Nephrilin reverses both PKC-beta-II-T641 and p66shc-S36 phosphorylation events [Mascarenhas D et al, 2012].

[0017] A recently recognized histological consequence of cellular stress is the formation of microscopically visible punctate structures in or around the nuclei of stressed cells [Bart J. et al (2008) Cytometry 73A: 816-824]. The inventor has shown, in diseased hypertensive animals, the presence of such structures in kidney cells by immunohistochemical staining. The incidence of such structures is much reduced in animals treated with the mTORC2 inhibitor, nephrilin.

[0018] Epithelial cells of renal proximal tubules (PTECs) are known to be exquisitely sensitive to p66shc-mediated oxidative stress [Sun L et al (2010) Am J Physiol Renal Physiol. 299(5): F1014-F1025]. Damage to PTECs can be monitored by measuring albumin or lipocalin-2/NGAL in urine by using commercially available kits. In many of the

proinflammatory disease conditions listed above, elevated levels of NGAL or albumin have been documented. This is especially true of AKI settings such as those encountered in patients with burns, hypoperfusion, pancreatitis and sepsis [Cruz D et al (2010) Intensive Care Med 36:444-451]. In AKI, moreover, a proinflammatory condition reminiscent of human systemic inflammatory states encountered in critical care settings, as enumerated above, can be generated in experimental animals by placing artificial stress on kidneys, such as in rhabdomyolysis and gentamycin models [Zager R et al (2006) Am J Physiol Renal Physiol 291:F546-F556]. The inventor has shown, for the first time, that this type of proinflammatory state is regulated by mTORC2 and can be successfully treated by an inhibitor of mTORC2, nephrilin.

[0019] Adaptive biochemical signatures are provided by U.S. Patent Application

Publications Nos: 2011/0202281 Al and 2011/0212079 Al.

[0020] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0021] The present invention provides methods of understanding, diagnosing and treating disease conditions in a mammal, especially neuroimmunological dysfunctions. In some aspects, the invention provides method for treating a neuroimmunological stress response in a mammal characterized by significant change in one or more of a physiological attribute selected from Substance P release, CGRP release, elevated salivary alpha-amylase and expression of a RAGE ligand gene; said method comprising treating the mammal with a therapeutic agent that disrupts the binding of Rictor to one or more binding partners selected from PRR5/PROTOR/Protor, Sinl, IRS1 and IRS2; thereby reducing or reversing the neuroimmunological stress response in the mammal. In some embodiments, the

physiological attribute further includes one or more of urinary albumin, urinary lipocalin- 2/NGAL, phosphorylated p66shc, PKC-beta, plasma IL-6, plasma TNF-alpha and tissue expression of UCHL1 gene. In some embodiments, the mammal is a human. In some embodiments, the neuroimmunological stress response is associated with traumatic stress, xenobiotic stress, metabolic stress or autoimmune stress. In some embodiments, the neuroimmunological stress response is associated with traumatic stress selected from the group consisting of blunt trauma, severe burn, surgery, sepsis, pancreatitis, rhabdomyolysis and psychological stress. In some embodiments, the neuroimmunological stress response is associated with one or more forms of autoimmune stress selected from the group consisting of systemic lupus erythematosis, arthritis, colitis, Sjogren's syndrome, painful bladder syndrome, fibromyalgia, psoriasis, asthma and diabetes.

[0022] In some embodiments of the invention, the therapeutic agent is a peptide, a protein, an antibody, a nucleic acid, or a small chemical molecule. In some embodiments, the therapeutic agent is nephrilin or a functional variant thereof. Nephrilin is a 40-amino acid peptide of the sequence RGVTEDYLRLETLVQKVVSKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 1).

[0023] In some aspects, the invention provides a method of identifying a therapeutic agent for treating a neuroimmunological stress response in a mammal, wherein the therapeutic agent disrupts the binding of nephrilin to its binding target site on Rictor. In some embodiments, the therapeutic agent binds

ACRLYATKHLRVLLRANVEFFNNWGIELLVTQLHDKNKTISSEALDILDEACE (SEQ ID NO:2). In some embodiments, the therapeutic agent competes with nephrilin for binding to ACRLYATKHLRVLLRANVEFFNNWGIELLVTQLHDKNKTISSEALDILDEACE (SEQ ID NO:2).

[0024] In some aspects, the invention provides methods to identify a therapeutic agent for treating an immunological stress response as described above, said method comprising i) exposing a cell simultaneously to stress and to either a control or the therapeutic agent (e.g., a candidate therapeutic agent), ii) measuring expression of one or more promoter-reporter gene constructs selected from Substance P, CGRP, PKC-beta, osteopontin, S100A9 and their respective orthologs and paralogs, and Hi) comparing expression of the promoter-reporter gene construct in the presence of the therapeutic agent with expression of the promoter-gene construct in the absence of the therapeutic agent; wherein a therapeutic agent is identified by regulation of expression of the promoter gene construct.

[0025] In some embodiments, the therapeutic agent is selected by random search. For example, the therapeutic agent is selected for random search for binding to

ACRLYATKHLRVLLRANVEFFNNWGIELLVTQLHDKNKTISSEALDILDEACE (SEQ ID NO:2) or for modulating expression of a promoter-gene construct.

[0026] In some embodiments, the therapeutic agent is selected by rational design. For example, the therapeutic agent is selected for rational design for binding to

ACRLYATKHLRVLLRANVEFFNNWGIELLVTQLHDKNKTISSEALDILDEACE (SEQ ID NO:2) or for modulating expression of a promoter-gene construct.

[0027] In some aspects, the invention provides methods for treating a

neuroimmunological stress response in a mammal characterized by significant change in one or more of a physiological attribute selected from the group consisting of Substance P release, CGRP release, PKC-beta, elevated salivary alpha-amylase, elevated plasma IL-6, and expression of a RAGE ligand gene; said method comprising treating the mammal with a therapeutic agent identified by the method of any one of claims 9- 14; thereby reducing or reversing the neuroimmunological stress response in the mammal (e.g. a human).

[0028] In some embodiments, the invention provides a diagnostic method for identifying mammal subsets for treatment according to the methods of the invention, wherein the physiological attribute measured is one or more of tissue Substance P release, tissue CGRP, tissue phosphorylation of p66shc, urinary lipocalin-2/NGAL, urinary albumin, salivary alpha- amylase, plasma IL-6, plasma TNF-alpha, and tissue expression of a RAGE ligand gene or UCHL1 gene. In some embodiments, the physiological attribute measured is one or more of tissue Substance P release, tissue CGRP release, tissue phosphorylation of p66shc, elevated urinary lipocalin-2/NGAL, elevated urinary albumin, elevated salivary alpha-amylase, elevated plasma IL-6, elevated plasma TNF-alpha, and tissue expression of a RAGE ligand gene or downregulated UCHL1 gene. In some embodiments, the physiological attribute measured one or more of tissue Substance P release, tissue CGRP release, tissue

phosphorylation of p66shc, elevated salivary alpha-amylase, elevated plasma IL-6, and tissue expression of a RAGE ligand gene, and further comprises elevated urinary lipocalin- 2/NGAL, elevated urinary albumin, elevated plasma TNF-alpha, and/or downregulated UCHL1. In some embodiments, the RAGE ligand gene is S 100A9. [0029] The invention provides compositions of inhibitors of mTORC2 and methods for finding other such molecules and their use in clinical settings. Neuroimmunological stress response and disease conditions include but are not limited to diabetes, gastrointestinal disease, obesity, metabolic and cardiovascular diseases (especially those characterized by some underlying combination of insulin resistance, hyperglycemia, hypertension and hyperlipidemia); cancer progression and metastasis; acute kidney injury (AKI) in critical care settings, also including sepsis, systemic inflammatory conditions such as shock, postoperative stress such as after cardiopulmonary bypass or transplant, burns, blunt trauma, pancreatitis, rhabdomyolysis, xenobiotic stresses caused by cocaine, alcohol, aminoglycoside antibiotics, antiviral compounds or platinum compounds; neuropathic pain and migraine; neurodegenerative diseases such as Parkinson's Alzheimer's, Huntington's and ALS/Lou Gehrig's disease; ototoxicities; autoimmune conditions such as lupus erythematosus, arthritis, psoriasis, colitis, painful bladder syndrome, fibromyalgia, and multiple sclerosis; genetic diseases such as cystinosis, Fanconi's and other conditions affecting mitochondrial respiration; pulmonary diseases, especially COPD, PAH and asthma; ocular diseases such as cataracts and retinopathies, especially diabetic complications; and liver diseases, including chronic viral infections such as hepatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows the effect of treatment with nephrilin in a rat burn model. Plasma cytokines. A=sham, B=burn+saline, C=burn+nephrilin 4mg/kg; * # p<0.05; ** ## p<0.01;

[0031] Figure 2 shows the effect of treatment with nephrilin in a rat burn model. PKC and CGRP in dorsal root ganglia. A=sham, B=burn+saline, C=burn+nephrilin 4mg/kg; * # p<0.05; ** ## p<0.01;

[0032] Figure 3 shows the effect of treatment with nephrilin in a rat burn model.

Substance P release and p66shc phosphorylation in kidney tissue. Right panel shows immunohistochemistry of kidney using anti-p66shc antibody. A=sham, B=burn+saline, C=burn+nephrilin 4mg/kg; * # p<0.05; ** ## p<0.01;

[0033] Figure 4 shows the effect on survival of daily treatment with nephrilin in a mouse CLP sepsis model.

[0034] Figure 5 shows S100A9 gene expression profiling of tissue samples from burn and sepsis models.

[0035] Figure 6 shows plasma TNF-alpha and Substance P-CGRP ratio in kidney tissue extracts from the mouse CLP sepsis model. [0036] Figure 7 shows peptide mapping of the Rictor-Protor binding site.

DISCLOSURE OF THE INVENTION

[0037] The present invention provides methods for recognizing and treating biological phenomena that significantly exarcebate disease via neuroimmunological responses to stress and for identifying molecules that may be selectively active on stress-related complications of human trauma and disease.

[0038] The invention also relates to specific reagents and procedures of particular utility in the generation of therapeutic agents that regulate the binding of Rictor to its binding partners, thereby affecting the neurogenic complications of traumatic, autoimmune and other disease processes. In some aspects, the invention provides an agent capable of disrupting the physical association of Rictor protein with one of its obligate cofactors, thereby reversing the effects of a biochemical signature. Examples of agents include but are not limited to peptides, proteins, antibodies, nucleic acid and small chemical molecules.

[0039] In some aspects, the invention provides methods for random search or rational design of a therapeutic agent, said method comprising either (a) in vitro or in vivo inhibition of binding between Rictor and its binding partners selected from a group comprising

PRR5/PROTOR, Sinl, IRS1 and IRS2; or (b) counter-regulation of a promoter-reporter gene construct in a cell line or live organism wherein the promoter is selected from a group of genes comprising S100A9, osteopontin, PKC-beta, B4galNT4, UCHLl, PER2, and their respective orthologs and paralogs.

[0040] In some aspects, the invention provides methods for treating an inflammatory condition in a mammal exhibiting a biochemical signature characterized by (a) a

physiological attribute selected from the group comprising elevated substance P, urinary albumin, urinary lipocalin-2/NGAL, plasma IL-6 or TNF-alpha, and tissue expression of S100A9 gene in response to neuroimmunological stimulus; and (b) a tissue phosphorylation attribute selected from the group comprising elevated PKC-beta-II-T641 and elevated p66shc-S36; and (c) an altered gene transcript level attribute selected from the group comprising elevated S100A9, B4galNT4, down-regulated UCHLl, and downregulated PER2; said method comprising treating the mammal with a therapeutic agent that disrupts the binding of Rictor to a binding partner selected from the group comprising PRR5/PROTOR, Sinl, IRS1 and IRS2; thereby reducing or reversing the neuroimmunological dysfunction or condition. [0041] In embodiments of the invention, the therapeutic agent is capable of disrupting the physical association of Rictor protein with one of its binding partners selected from the group comprising PRR5/PROTOR, Sinl, IRS1 and IRS2, thereby reversing detrimental effects of the neuroimmunological stress response.

[0042] In embodiments of the invention, the composition can be administered via any route including but not limited to intravenous, oral, subcutaneous, intraarterial, intramuscular, intracardial, intraspinal, intrathoracic, intraperitoneal, intraventricular, sublingual, transdermal, and inhalation.

[0043] In an embodiment of the invention, nucleic acids encoding fusion proteins are used in methods of diagnosing or treating an inflammatory disease condition. Inflammatory disease conditions include but are not limited to trauma, sepsis, cancer, diabetes,

cardiovascular disease, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, autoimmune disease, rheumatological disease and infectious disease.

[0044] In another aspect the invention provides methods of diagnosing or treating an inflammatory disease condition comprising administering an effective amount of a polypeptide of the invention to a mammal. Neuroimmunological dysfunctions or conditions include but are not limited to psychological or psychosocial stress, trauma, sepsis, cancer, diabetes, cardiovascular disease, chronic kidney disease, acute kidney injury, retinopathy, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, autoimmune disease, rheumatological disease, infectious disease, genetic disease, and xenotoxicity.

[0045] The compositions of the invention may be administered by means which include but are not limited to intravenous, oral, subcutaneous, intraarterial, intramuscular,

intracardial, intraspinal, intrathoracic, intraperitoneal, intraventricular, sublingual, transdermal, and inhalation. In some embodiments, the composition is administered to a mammal at less than about 20 mg/kg/day.

[0046] The invention includes methods to diagnose or treat neuroimmunological stress response, dysfunction or condition by administering nucleic acids and/or vectors encoding polypeptides of the invention to a mammal.

[0047] In one aspect, the invention provides methods for treating a neuroimmunological stress response in a mammal characterized by one or more of a physiological attribute selected from the group consisting of elevated Substance P, CGRP, PKC-beta, urinary albumin, urinary lipocalin-2/NGAL, plasma IL-6 or TNF-alpha, and tissue expression of a RAGE ligand gene; said method comprising treating the mammal with a therapeutic agent that disrupts the binding of Rictor to a binding partner selected from the group consisting of PRR5/PROTOR/Protor, Sinl, IRSl and IRS2; thereby reducing or reversing the neuroimmunological stress response in the mammal. In some embodiments, the

neuroimmunological dysfunction or condition is selected from a group consisting of trauma, acute kidney injury, neurodegenerative disease, autoimmune disease, infectious disease, metabolic disease, cancer, genetic disease, and xenotoxicity.

[0048] In some aspects, the invention provides a therapeutic agent capable of disrupting the physical association of Rictor protein with one of its binding partners selected from the group consisting of PRR5/PROTOR, Sinl, IRSl and IRS2, thereby reversing the effects of a biochemical signature characteristic of an neuroimmunological dysfunction in a mammal exhibiting a biochemical signature characterized by one or more of a physiological attribute selected from the group consisting of substance P, CGRP, PKC-beta, phosphorylated p66shc, urinary albumin, urinary lipocalin-2/NGAL, plasma IL-6 or TNF-alpha, and expression of a RAGE-ligand gene such as S100A9 in tissues, in response to traumatic, xenobiotic, metabolic or autoimmune stress; wherein the therapeutic agent disrupts the binding of Rictor to a binding partner selected from the group consisting of PRR5/PROTOR, Sinl, IRSl and IRS2; thereby reducing or reversing the neuroimmunological dysfunction or condition. In some embodiments the agent is a peptide, a protein, an antibody, a nucleic acid, or a small chemical molecule. In some embodiments, the therapeutic agent is nephrilin or a sequence variant thereof. For example, the variant may have 80%, 90%, 95% or 99% identity to nephrilin in its effector domain.

[0049] In another aspect, the invention provides methods to identify a therapeutic agent according to the invention, wherein the method comprising either (a) exposing rictor and one of its binding partners selected from the group consisting of PRR5/PROTOR, Sinl, IRSl and IRS2 to a candidate therapeutic agent in vitro or in vivo; measuring binding of rictor to the binding partner, and comparing binding of Rictor and its partner in the presence of the candidate therapeutic agent with binding in the absence of the candidate therapeutic agent; or (b) exposing a cell to the candidate therapeutic agent, measuring expression of a promoter- reporter gene construct selected from the group consisting of S100A9, PKC-beta,

osteopontin, B4galNT4, UCHL1, PER2, and their respective orthologs and paralogs, and comparing expression of the promoter-reporter gene construct in the presence of the candidate therapeutic agent with expression of the promoter-gene construct in the absence of the candidate therapeutic agent. As such, a therapeutic agent is identified by inhibition of binding of Rictor to its binding partner or by counter regulation of the promoter gene construct. In some embodiments, the cell is in vitro; for example, in a cell culture. In some embodiments, the cell is in vivo; for example, in an organism such as a mammal. In some embodiments, therapeutic agents are selected by random search prior to the identification steps outlined above. In some embodiments, the candidate therapeutic agent is first selected by rational design prior to the identification steps outlined above. In some embodiments, the invention provides a therapeutic agent identified by the methods outlined above.

[0050] In another aspect, the invention provides methods to identify a therapeutic agent according to the invention, wherein the therapeutic agent is nephrilin or functional variants thereof. Nephrilin is a 40-amino acid peptide of the sequence

RGVTEDYLRLETLVQKVVSKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 1) that has been previously described (Singh BK et al [2010] Metab Syn Relat Dis. 8(4): 1-10;

Mascarenhas D et al [2012] Inflamm. Res. 61: 1395-1404). A functional variant of nephrilin has one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions of the nephrilin sequence and maintains the ability to disrupt the binding of Rictor to a binding partner. Alternatively, a functional variant of nephrilin has at least any one of 95%, 90%, 85%, 80%, or 75% sequence identity to nephrilin and maintains the ability to disrupt the binding of Rictor to a binding partner.

[0051] In yet another aspect the invention provides methods to identify a therapeutic agent wherein the therapeutic agent competes with PRR5/Protor or nephrilin in binding to its binding site on Rictor or subsets of that binding site. Preferably the therapeutic agent competes with binding of PRR5/Protor or nephrilin to the sequence

ACRLYATKHLRVLLRANVEFFNNWGIELLVTQLHDKNKTISSEALDILDEACE (SEQ ID NO:2) or a subset thereof, which is part of the Rictor binding site. To measure binding, any of several available protocols, well-known to practitioners in the art, may be used. For example, to measure the binding of nephrilin to various candidate peptides carrying subsets of the Rictor binding domain previously located by deletion mapping [Pearce L et al (2007) Biochem J. 405(3): 513-522] one may use the following method: Bind 100 ug of nephrilin to a Ni-NTA column (bed volume 0.2 ml) and pass candidate biotinylated agent (l-50ug) plus 1 ug of streptavidin-HRP conjugate through the column. After washing the column with 4-5 column volumes of binding buffer (PBS), sample is eluted in 2 column volumes of PBS containing 100 mM imidazole and the eluate assayed for HRP using commerically available reagent TMB (read at 655nm). Therapeutic agents, selected from the group consisting of a peptide, a protein, an antibody, a nucleic acid, and a small chemical molecule, may be identified using this assay, either as binding directly to nephrilin, as described above, or as competitors of binding of nephrilin to subsets of its binding site, as described above. [0052] The invention discloses a method for treating a neuroimmunological stress response in a mammal characterized by significant change in one or more of a physiological attribute selected from the group consisting of Substance P, CGRP, PKC-beta, alpha- amylase, IL-6, and expression of a RAGE ligand gene; said method comprising treating the mammal with a therapeutic agent that disrupts the binding of Rictor to a binding partner selected from the group consisting of PRR5/PROTOR/Protor, Sinl, IRS1 and IRS2; thereby reducing or reversing the neuroimmunological stress response in the mammal. The physiological attribute may also be selected from the group consisting of urinary albumin, urinary lipocalin-2/NGAL, plasma TNF-alpha and tissue expression of UCHL1 gene.

[0053] The neuroimmunological stress response is associated with traumatic, xenobiotic, metabolic or autoimmune stress. It may be associated with traumatic stress selected from the group consisting of blunt trauma, severe burn, surgery, sepsis, pancreatitis, rhabdomyolysis and psychological stress. It may also be associated with one or more forms of autoimmune stress selected from the group consisting of systemic lupus erythematosis, arthritis, colitis, Sjogren's syndrome, painful bladder syndrome, fibromyalgia, psoriasis, asthma and diabetes.

[0054] In another aspect the present invention describes diagnostic methods for identifying patient subsets for treatment according to the invention wherein the physiological attribute measured is selected from the group consisting of tissue Substance P, tissue CGRP, urinary lipocalin-2/NGAL, urinary albumin, salivary alpha-amylase, plasma IL-6, plasma TNF-alpha, and tissue expression of RAGE ligand gene or UCHL1 gene.

MODES FOR CARRYING OUT THE INVENTION

Methods of identifying candidates for treatment

[0055] The invention provides methods for identifying candidates for treatment therapies.

[0056] As will be understood by those of skill in the art, the mode of detection of the signal will depend on the exact detection system utilized in the assay. For example, if a radiolabeled detection reagent is utilized, the signal will be measured using a technology capable of quantitating the signal from the biological sample or of comparing the signal from the biological sample with the signal from a reference sample, such as scintillation counting, autoradiography (typically combined with scanning densitometry), and the like. If a chemiluminescent detection system is used, then the signal will typically be detected using a luminometer. Methods for detecting signal from detection systems are well known in the art and need not be further described here. [0057] When more than one biochemical readout is measured (i.e., measured values for two or more readouts are obtained), the sample may be divided into a number of aliquots, with separate aliquots used to measure different readouts (although division of the biological sample into multiple aliquots to allow multiple determinations of the levels of the readouts in a particular sample are also contemplated). Alternately the sample (or an aliquot therefrom) may be tested to determine the levels of multiple readouts in a single reaction using an assay capable of measuring the individual levels of different readouts in a single assay, such as an array-type assay or assay utilizing multiplexed detection technology (e.g., an assay utilizing detection reagents labeled with different fluorescent dye markers).

[0058] As will be understood by those in the art, the exact identity of a reference value will depend on the tissue that is the target of treatment and the particular measuring technology used. In some embodiments, the comparison determines whether the measured value is above or below the reference value. In some embodiments, the comparison is performed by finding the "fold difference" between the reference value and the measured value (i.e., dividing the measured value by the reference value).

[0059] Although some assay formats will allow testing of biological samples without prior processing of the sample, it is expected that most biological samples will be processed prior to testing. Processing generally takes the form of elimination of cells (nucleated and non-nucleated), such as erythrocytes, leukocytes, and platelets in blood samples, and may also include the elimination of certain proteins, such as certain clotting cascade proteins from blood.

[0060] Commonly, readouts will be measured using an affinity-based measurement technology. Affinity-based measurement technology utilizes a molecule that specifically binds to the readout protein being measured (an "affinity reagent," such as an antibody or aptamer), although other technologies, such as spectroscopy-based technologies (e.g., matrix- assisted laser desorption ionization-time of flight, or MALDI-TOF, spectroscopy) or assays measuring bioactivity (e.g., assays measuring mitogenicity of growth factors) may be used.

[0061] Affinity-based technologies include antibody-based assays (immunoassays) and assays utilizing aptamers (nucleic acid molecules which specifically bind to other molecules), such as ELONA. Additionally, assays utilizing both antibodies and aptamers are also contemplated (e.g., a sandwich format assay utilizing an antibody for capture and an aptamer for detection).

[0062] If immunoassay technology is employed, any immunoassay technology which can quantitatively or qualitatively measure the readout in a biological sample may be used. Suitable immunoassay technology includes radioimmunoassay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, ELISA, immuno-PCR, and western blot assay.

[0063] Likewise, aptamer-based assays that can quantitatively or qualitatively measure the level of a relevant readout in a biological sample may be used in the methods of the invention. Generally, aptamers may be substituted for antibodies in nearly all formats of immunoassay, although aptamers allow additional assay formats (such as amplification of bound aptamers using nucleic acid amplification technology such as PCR (U.S. Patent No. 4,683,202) or isothermal amplification with composite primers (U.S. Patents Nos. 6,251,639 and 6,692,918).

[0064] A wide variety of affinity-based assays are known in the art. Affinity-based assays will utilize at least one epitope derived from the readout protein of interest, and many affinity-based assay formats utilize more than one epitope (e.g., two or more epitopes are involved in "sandwich" format assays; at least one epitope is used to capture the marker, and at least one different epitope is used to detect the marker).

[0065] Affinity-based assays may be in competition or direct reaction formats, utilize sandwich-type formats, and may further be heterogeneous (e.g., utilize solid supports) or homogenous (e.g., take place in a single phase) and/or utilize or immunoprecipitation. Most assays involve the use of labeled affinity reagent (e.g., antibody, polypeptide, or aptamer); the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA and ELONA assays.

[0066] In a heterogeneous format, the assay utilizes two phases (typically aqueous liquid and solid). Typically readout protein- specific affinity reagent is bound to a solid support to facilitate separation of the readout indicator protein from the bulk of the biological sample. After reaction for a time sufficient to allow for formation of affinity reagent/readout indicator protein complexes, the solid support containing the antibody is typically washed prior to detection of bound polypeptides. The affinity reagent in the assay for measurement of readout proteins may be provided on a support (e.g., solid or semi-solid); alternatively, the polypeptides in the sample can be immobilized on a support. Examples of supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates), polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, and Protein A beads. Both standard and competitive formats for these assays are known in the art.

[0067] Array-type heterogeneous assays are suitable for measuring levels of readout proteins when the methods of the invention are practiced utilizing multiple readout proteins. Array-type assays used in the practice of the methods of the invention will commonly utilize a solid substrate with two or more capture reagents specific for different readout proteins bound to the substrate a predetermined pattern (e.g., a grid). The biological sample is applied to the substrate and readout proteins in the sample are bound by the capture reagents. After removal of the sample (and appropriate washing), the bound readout proteins are detected using a mixture of appropriate detection reagents that specifically bind the various readout proteins. Binding of the detection reagent is commonly accomplished using a visual system, such as a fluorescent dye -based system. Because the capture reagents are arranged on the substrate in a predetermined pattern, array-type assays provide the advantage of detection of multiple readout proteins without the need for a multiplexed detection system.

[0068] In a homogeneous format the assay takes place in single phase (e.g., aqueous liquid phase). Typically, the biological sample is incubated with an affinity reagent specific for the readout protein in solution. For example, it may be under conditions that will precipitate any affinity reagent/antibody complexes that are formed. Both standard and competitive formats for these assays are known in the art.

[0069] In a standard (direct reaction) format, the level of readout protein/affinity reagent complex is directly monitored. This may be accomplished by, for example, determining the amount of a labeled detection reagent that forms is bound to readout protein/affinity reagent complexes. In a competitive format, the amount of readout protein in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled readout protein (or other competing ligand) in the complex. Amounts of binding or complex formation can be determined either qualitatively or quantitatively.

[0070] Complexes formed comprising readout protein and an affinity reagent are detected by any of a number of known techniques known in the art, depending on the format of the assay and the preference of the user. For example, unlabelled affinity reagents may be detected with DNA amplification technology (e.g., for aptamers and DNA-labeled antibodies) or labeled "secondary" antibodieswhich bind the affinity reagent. Alternately, the affinity reagent may be labeled, and the amount of complex may be determined directly (as for dye- (fluorescent or visible), bead-, or enzyme-labeled affinity reagent) or indirectly (as for affinity reagents "tagged" with biotin, expression tags, and the like). [0071] As will be understood by those of skill in the art, the mode of detection of the signal will depend on the exact detection system utilized in the assay. For example, if a radiolabeled detection reagent is utilized, the signal will be measured using a technology capable of quantitating the signal from the biological sample or of comparing the signal from the biological sample with the signal from a reference sample, such as scintillation counting, autoradiography (typically combined with scanning densitometry), and the like. If a chemiluminescent detection system is used, then the signal will typically be detected using a luminometer. Methods for detecting signal from detection systems are well known in the art and need not be further described here.

[0072] When more than one readout protein is measured, the biological sample may be divided into a number of aliquots, with separate aliquots used to measure different readout proteins (although division of the biological sample into multiple aliquots to allow multiple determinations of the levels of the readout protein in a particular sample are also

contemplated). Alternately the biological sample (or an aliquot therefrom) may be tested to determine the levels of multiple readout proteins in a single reaction using an assay capable of measuring the individual levels of different readout proteins in a single assay, such as an array-type assay or assay utilizing multiplexed detection technology (e.g., an assay utilizing detection reagents labeled with different fluorescent dye markers).

[0073] It is common in the art to perform 'replicate' measurements when measuring readout proteins. Replicate measurements are ordinarily obtained by splitting a sample into multiple aliquots, and separately measuring the readout protein (s) in separate reactions of the same assay system. Replicate measurements are not necessary to the methods of the invention, but many embodiments of the invention will utilize replicate testing, particularly duplicate and triplicate testing.

[0074] In some aspects of the invention, the following readout proteins or markers include, but are not limited to: S100A9 (SI 00 calcium binding protein A9 [Homo sapiens] NM_002965.3), IRS-1 (insulin receptor substrate- 1; IRS-1 [Homo sapiens]

gil386257lgblAAB27175.1), IRS-2 (insulin receptor substrate-2 [Homo sapiens]

gill8652857ldbjlBAB84688.1), mTORCl and mTORC2 complexes which are traditionally defined by the protein components Raptor and Rictor respectively, Rictor (RICTOR protein [Homo sapiens] gil30704352lgblAAH51729.1) » mTORC2, Raptor (raptor [Homo sapiens] gil21979456lgblAAM09075.1) » mTORCl; AKT SUB-FAMILY OF AGC KINASES: AKT1 protein [Homo sapiens] gill8027298lgblAAL55732.1, AKT2 protein [Homo sapiens] gill l l309392lgblAAI20996.1, AKT3 protein [Homo sapiens] gil62089468lgblAAH20479.1 (NOTE:AKT is also known as Protein Kinase B, or PKB); SGK SUB-FAMILY OF AGC KINASES: SGK1 Serum/glucocorticoid regulated kinase 1 [Homo sapiens]

gill2654839lgblAAH01263.1, SGK2 protein [Homo sapiens] gil41351348lgblAAH65511.1, SGK3 Serum/glucocorticoid regulated kinase 3 [Homo sapiens]

gil 15929810lgblAAH15326.1; PKC SUB-FAMILY OF AGC KINASES: PKC-alpha; Protein kinase C, alpha [Homo sapiens] gil80479084lgblAAI09275.1, PKC-beta; Protein kinase C, beta [Homo sapiens] gil22209072lgblAAH36472.1, PKC-beta; protein kinase C, beta isoform 1 [Homo sapiens] gil47157322lreflNP_997700.1, PKC-beta; protein kinase C, beta isoform 2 [Homo sapiens] gil20127450lreflNP_002729.2, PKC-delta; protein kinase C, delta [Homo sapiens] gil47157325lreflNP_997704.1, PKC-gamma; Protein kinase C, gamma [Homo sapiens] gil28839171lgblAAH47876.1, PKC-zeta 1; protein kinase C, zeta isoform 1 [Homo sapiens] gil52486327lreflNP_002735.3, PKC-zeta 2; protein kinase C, zeta isoform 2 [Homo sapiens] gil75709226lreflNP_001028753.1, PKC-epsilon; protein kinase C, epsilon [Homo sapiens] gil4885563lreflNP_005391.1, PKC-theta; protein kinase C, theta [Homo sapiens] gil5453976lreflNP_006248.1, PKC-iota; Homo sapiens protein kinase C, iota

gblNM_002740. In some aspects of the invention, the readout proteins or markers are from a non-human mammal.

Kits

[0075] The invention provides kits for carrying out the methods of the invention. Kits of the invention comprise at least one probe specific for an readout gene (and/or at least one affinity reagent specific for an readout protein) and instructions for carrying out a method of the invention. More commonly, kits of the invention comprise at least two different readout gene probes (or at least two affinity reagents specific for readout proteins), where each probe/reagent is specific for a different readout gene.

[0076] Kits comprising a single probe for a readout gene (or affinity reagent specific for a readout protein) will generally have the probe/reagent enclosed in a container (e.g., a vial, ampoule, or other suitable storage container), although kits including the probe/reagent bound to a substrate (e.g., an inner surface of an assay reaction vessel) are also contemplated.

Likewise, kits including more than one probe/reagent may also have the probes/reagents in containers (separately or in a mixture) or may have the probes/affinity reagents bound to a substrate (e.g., such as an array or microarray). [0077] A modified substrate or other system for capture of readout gene transcripts or readout proteins may also be included in the kits of the invention, particularly when the kit is designed for use in an array format assay.

[0078] In certain embodiments, kits according to the invention include the

probes/reagents in the form of an array. The array includes at least two different

probes/reagents specific for a readout gene/protein (each probe/reagent specific for a different readout gene/protein) bound to a substrate in a predetermined pattern (e.g., a grid). The localization of the different probes/reagents allows measurement of levels of a number of different readout genes/ proteins in the same reaction.

[0079] The instructions relating to the use of the kit for carrying out the invention generally describe how the contents of the kit are used to carry out the methods of the invention. Instructions may include information as sample requirements (e.g., form, pre- assay processing, and size), steps necessary to measure the readout gene(s), and interpretation of results.

[0080] Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. In certain embodiments, machine-readable instructions comprise software for a programmable digital computer for comparing the measured values obtained using the reagents included in the kit.

[0081] Sequence "identity" and "homology", as referred to herein, can be determined using BLAST (Altschul, et al, 1990, J. Mol. Biol. 215(3): 403-410), particularly BLASTP 2 as implemented by the National Center for Biotechnology Information (NCBI), using default parameters (e.g., Matrix 0 BLOSUM62, gap open and extension penalties of 11 and 1, respectively, gap x_dropoff 50 and wordsize 3). Unless referred to as "consecutive" amino acids, a sequence optionally can contain a reasonable number of gaps or insertions that improve alignment.

[0082] For testing efficacy of an agent believed to alter a stress response, an effective amount of therapeutic agent is administered to a subject having a disease. In some embodiments, the agent is administered at about 0.001 to about 40 milligrams per kilogram total body weight per day (mg/kg/day). In some embodiments the agent is administered at about 0.001 to about 40 mg/kg/day.

[0083] The terms "subject" and "individual", as used herein, refer to a vertebrate individual, including avian and mammalian individuals, and more particularly to sport animals (e.g., dogs, cats, and the like), agricultural animals (e.g., cows, horses, sheep, and the like), and primates (e.g., humans).

[0084] The term "treatment" is used herein as equivalent to the term "alleviating", which, as used herein, refers to an improvement, lessening, stabilization, or diminution of a symptom of a disease. "Alleviating" also includes slowing or halting progression of a symptom.

[0085] For the purposes of this invention, a "clinically useful outcome" refers to a therapeutic or diagnostic outcome that leads to amelioration of the disease condition.

"Neuroimmunological stress response" means a disease condition that is characterized by sporadic or episodic elevation of substance P and or calcitonin-related peptide (CGRP) release from sensory nerves into the tissue followed by activation of both inflammatory and anti-inflammatory mechanisms, as well as oxidative damage.

[0086] As used herein, "in conjunction with", "concurrent", or "concurrently", as used interchangeably herein, refers to administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to administration of one treatment modality before, during or after delivery of the other treatment modality to the subject.

[0087] Techniques for the manipulation of recombinant DNA are well known in the art, as are techniques for recombinant production of proteins (see, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3 (Cold Spring Harbor Laboratory Press, 2 ed., (1989); or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley- Interscience: New York, 1987) and periodic updates). Derivative peptides or small molecules of known composition may also be produced by chemical synthesis using methods well known in the art.

[0088] Homologous sequences are orthologous if they were separated by a speciation event: when a species diverges into two separate species, the divergent copies of a single gene in the resulting species are said to be orthologous. Orthologs, or orthologous genes, are genes in different species that are similar to each other because they originated from a common ancestor.

[0089] Homologous sequences are paralogous if they were separated by a gene duplication event: if a gene in an organism is duplicated to occupy two different positions in the same genome, then the two copies are paralogous. A set of sequences that are paralogous are called paralogs of each other. Paralogs typically have the same or similar function, but sometimes do not: due to lack of the original selective pressure upon one copy of the duplicated gene, this copy is free to mutate and acquire new functions. [0090] Accordingly, the invention provides methods of treatment with fusions and/or conjugates of therapeutic or diagnostic molecules (such as agents) which are desired to be internalized into cells. The fusion partner molecules may be polypeptides, nucleic acids, or small molecules which are not normally internalized (e.g., because of large size,

hydrophilicity, etc.). The fusion partner can also be an antibody or a fragment of an antibody. As will be apparent to one of skill in the art, such fusions/conjugates will be useful in a number of different areas, including pharmaceuticals (to promote internalization of therapeutic molecules which do not normally become internalized), gene therapy (to promote internalization of gene therapy constructs), and research (allowing 'marking' of cells with an internalized marker protein).

[0091] Therapeutic agents are preferably administered via oral or parenteral

administration, including but not limited to intravenous (IV), intra- arterial (IA),

intraperitoneal (IP), intramuscular (IM), intracardial, subcutaneous (SC), intrathoracic, intraspinal, intradermal (ID), transdermal, oral, sublingual, inhaled, and intranasal routes. IV, IP, IM, and ID administration may be by bolus or infusion administration. For SC

administration, administration may be by bolus, infusion, or by implantable device, such as an implantable minipump (e.g., osmotic or mechanical minipump) or slow release implant. The agent may also be delivered in a slow release formulation adapted for IV, IP, IM, ID or SC administration. Inhaled agent is preferably delivered in discrete doses (e.g., via a metered dose inhaler adapted for protein delivery). Administration of a molecule comprising an agent via the transdermal route may be continuous or pulsatile. Administration of agents may also occur orally.

[0092] For parenteral administration, compositions comprising a therapeutic agent may be in dry powder, semi- solid or liquid formulations. For parenteral administration by routes other than inhalation, the composition comprising an agent is preferably administered in a liquid formulation. Compositions comprising an agent formulation may contain additional components such as salts, buffers, bulking agents, osmolytes, antioxidants, detergents, surfactants, and other pharmaceutical excipients as are known in the art.

[0093] A composition comprising an agent is administered to subjects at a dose of about 0.001 to about 40 mg/kg/day, more preferably about 0.01 to about 10 mg/kg/day, more preferably 0.05 to about 4 mg/kg/day, even more preferably about 0.1 to about 1 mg/kg/day.

[0094] As will be understood by those of skill in the art, the symptoms of disease alleviated by the instant methods, as well as the methods used to measure the symptom(s) will vary, depending on the particular disease and the individual patient. [0095] Patients treated in accordance with the methods of the instant invention may experience alleviation of any of the symptoms of their disease.

Definitions

[0096] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated, singular terms shall include pluralities and plural terms shall include the singular.

[0097] It is understood that aspect and embodiments of the invention described herein include "consisting" and/or "consisting essentially of" aspects and embodiments. As used herein, the singular form "a", "an", and "the" includes plural references unless indicated otherwise.

[0098] In this application, the use of "or" means "and/or" unless expressly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim.

[0099] As is understood by one skilled in the art, reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0100] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.

[0101] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0102] As used herein, "treatment" is an approach for obtaining beneficial or desired clinical results. "Treatment" as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a human. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, any one or more of:

alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (e.g., neuroinflammation) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). "Alleviating" also includes slowing or halting progression of a symptom.

[0103] The terms "subject" and "individual", as used herein, refer to a vertebrate individual, including avian and mammalian individuals, and more particularly to sport animals (e.g., dogs, cats, and the like), agricultural animals (e.g., cows, horses, sheep, and the like), and primates (e.g., humans).

[0104] For the purposes of this invention, a "clinically useful outcome" refers to a therapeutic or diagnostic outcome that leads to amelioration of the disease condition.

"Neuroimmunological stress response" means a disease condition that is characterized by sporadic or episodic elevation of substance P and or calcitonin-related peptide (CGRP) release from sensory nerves into the tissue followed by activation of both inflammatory and anti-inflammatory mechanisms, as well as oxidative damage.

[0105] As used herein, "in conjunction with", "concurrent", or "concurrently", as used interchangeably herein, refers to administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to administration of one treatment modality before, during or after delivery of the other treatment modality to the subject.

[0106] The terms "inhibition" or "inhibit" refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To "reduce" or "inhibit" is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. In certain embodiments, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

[0107] To "elevate" is to increase an activity, function, and/or amount as compared to a reference. In certain embodiments, by "elevate" is meant the ability to cause an overall increase of 20% or greater. In another embodiment, by "elevate" is meant the ability to cause an overall increase of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or greater.

[0108] A "reference" as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy and/or non-diseased sample. In some examples, a reference may be obtained from an untreated sample. In some examples, a reference is obtained from a non-diseased on non-treated sample of a subject individual. In some examples, a reference is obtained from one or more healthy individuals who are not the subject or patient.

[0109] A "therapeutically effective amount" of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations.

[0110] An "article of manufacture" is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

EXAMPLES

[0111] The examples discussed below are intended to be purely exemplary of the invention and should not be considered to limit the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Rat Burn Model.

[0112] The objective of the rat burn experiments was to test the effect of treatment with nephrilin on the neuroimmunological sequelae of burn trauma. The protocol was as follows: Table 1. Drugs and Treatment

nep r n sc ; a y

[0113] Burn model protocol: The rat scald burn model (Herndon DN, 1978) induces inflammation and hypermetabolism similar to what is experienced by severely burned people. The rat scald burn model that induces inflammation and hypermetabolism in line with what patients experience is a modified Walker-Mason model. Dr. David Herndon refined this model and has >30 years of experience using this model. This injury, when performed correctly, has a mortality rate of < 5%. All animals continue to receive Ensure and water ad libitum throughout the study. Prophylactic analgesia (0.05 mg/kg body weight Buprenorphin) is administered followed by general anesthesia (50 mg/kg body weight Pentobarbital). The dorsum of the trunk and the abdomen are shaved, and a 60% of total body surface area (TBSA) burn administered by placing the animals in a mold exposing defined areas of the skin of the back and abdomen. The mold is placed in 97-99°C water, the back scalded for 10 seconds and the abdomen for 2 seconds; this method delivers a full-thickness cutaneous burn as confirmed by histologic examination. Burned rats will be resuscitated with 40 cc/kg Ringer's Lactate injected intraperitoneally. Animals are monitored and receive oxygen during the post-burn period until they wake up, at which point they are transferred to their own cages and supervised for another 3-5 hours. If the animals demonstrate discomfort (mobility, loss of appetite, lethargic, fur changes) they are given an analgesic (0.05 mg of

buprenorphine). Rounds are conducted twice daily by the investigators and twice daily by the veterinary staff to ensure that animals are comfortable. If the animals demonstrate discomfort (mobility, loss of appetite, lethargic, fur changes) they are given an analgesic (0.05 mg of buprenorphine). Combined observations of movement, eating, drinking, and response to stimuli are used to evaluate the rats, and significant decrease in or cessation of these behaviors are considered evidence of morbidity. Animals meeting these criteria are humanely euthanized. At the end of the study period the animals are first anesthetized with an overdose of Pentobarbital injected intraperitoneally. Once the rats are anesthetized cervical dislocation is performed in a manner consistent with the recommendations of the IACUC, the NIH's Office of Laboratory Animal Welfare (OLAW), and the AVMA. This animal protocol has been approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (see Appendix). The effects of a burn injury on nephrilin were evaluated using ANOVA. Statistical significance was accepted at p<0.05. Plasma TNF-alpha and IL-6, as well as tissue substance P were measured using kits from R&D Systems

(Minneapolis, MN). Frozen kidney slices were used for RNA extraction and qPCR for GAPDH and actin and S100A9 genes. Differences between treatment groups were evaluated using Student's t-test (p<0.05) for comparing effects on plasma and tissue markers.

[0114] Results are shown in Figures 1, 2 and 3. Nephrilin treatment significantly reduces neurogenic, alarmin, oxidative damage and inflammation markers in response to burn trauma.

Example 2. Cecal Ligation Mouse Model of Sepsis (CLP).

[0115] The objective of the 7-day CLP model experiment was to determine mortality in untreated and nephrilin-treated animals.

Table 2. Drugs and Treatment:

# - Control Group

[0116] Male C57BL/6 mice used for this study were commercially obtained (Taconic Labs) and were between 8-12 weeks of age. Mice were acclimated to housing suite for one week prior to experiment and had access to food and water ad lib and were on a 12: 12 L/D lighting cycle. Mice were anesthetized using 5% vaporized isoflurane. The abdomen was shaved and disinfected prior to surgery. A lateral incision was made through two layers and the cecum exposed and ligated at 0.75 cm from the most distal region of the cecum. Two perforations were made side by side, with a 21.5 gauge needle. After perforation, the ligated portion of the cecum was gently squeezed to expose a small amount of fecal matter. Cecum was returned to the abdominal cavity and abdomen was closed in two layers, using 4-0 braided silk sutures to close the inner peritoneal cavity and 7mm stainless steel wound clips to close the outer skin layer. Two sham mice were operated on in the same manner, exposing the cecum but no ligation or perforation was made. Before the animal was fully aroused post surgery, lmL of 37°C pre-warmed sterile saline solutions was injected subcutaneously on the animals' dorsal side. Upon arousal, each animal was given a subcutaneous injection of the analgesic, buprenorphine, at 0.05mg/kg of body weight. Mice were randomly selected to be given either of two treatments, Nephrilin given at a dose of 4mg/kg of body weight vs. sterile saline solution. Treatments were delivered to mice subcutaneously daily by user blind to treatment identity. 24 hours post-op, blood was collected from each animal. Collection was made from the tail vein into a capillary blood collection tube, coated with EDTA. Blood was also collected at the time of death via cardiac puncture, with the exception of mice found dead over night, where blood collection was not possible. Plasma separation was done in a centrifuge at 700rpm for 25 mins and immediately stored at -80°C. One kidney was collected per animal at the time of death and immediately stored at -80°C.

[0117] Tissues were extracted and assayed by ELISA as previously described [Singh BK et al (2010) Metab Syndr Relat Disord. 8(4): 355-363]. The results are shown in Figure 4. They show that nephrilin significantly inhibits mortality in this model. Figure 6 shows biochemical correlates from the same experiment. The nephrilin-treated group has significantly reduced plasma TNF-alpha and SP-CGRP ratio in kidney tissue extracts.

Example 3. Gene array and qPCR analysis of gene expression from burn and sepsis models.

[0118] The objective of this experiment was to identify genes that are controlled by disease processes regulated by mTORC2 in these models. Such genes are expected to be counter-regulated by treatment of the animals with nephrilin. The preliminary gene array comparisons performed suggested that the best marker at 24-hours post-insult in the burn model was S100A9, a well-known RAGE ligand. Figure 5 shows regulation of S100A9 in the preliminary gene array (4 tissues) as well as confirmation by qPCR (kidney tissue). Only signals that met statistical significance and minimum intensity criteria were considered in the analysis. These results show that S100A9 is a useful reporter for neuroimmunological disease states regulated by mTORC2.

Example 4. Screening inhibitors and agonists of mTORC2-Protor binding.

[0119] This experiment was designed to measure the binding of nephrilin, which carries the Protor sequence involved in binding to Rictor, and various candidate peptides carrying subsets of the Rictor binding domain previously located by deletion mapping [Pearce L et al (2007) Biochem J. 405(3): 513-522], We have used peptide mapping to narrow down the Rictor binding site for PRR5/PROTOR to the sequence

ACRLYATKHLRVLLRANVEFF ^' NNWGiELLVTQLHD KTISSEALDiLDEACE (PEPTIDE RICb) (SEQ ID NO:2) by comparison to RICa:

ERSLQ NGLLl SQeYn.FIGTLSCHPHGV MLE CSVFQCLL LCSL NQD (SEQ ID NO:4)

[0120] Additional fine mapping may, for example, involve the peptides RICc and RICd: RICc: ACRANVEFFNNWGIELLVTQLHDKNKTISSEALDILDEACE (SEQ ID NO:5) RICd: ACRLYATKHLRVLLRANVEALDILDEACE (SEQ ID NO:6)

[0121] Briefly, the method involved binding 100 ug of nephrilin to a Ni-NTA column (bed volume 0.2 ml) and passing candidate biotinylated peptide (50ug) plus 1 ug of streptavidin-HRP conjugate through the column. After washing the column with 4-5 column volumes of binding buffer (PBS), sample was eluted in 2 column volumes of PBS containing 100 mM imidazole and the eluate was assayed for HRP using commerically available reagent TMB (read at 655nm). As an example, the results of a comparison between two candidate peptides RICa and RICb are shown in Figure 7.

[0122] Using an in vitro binding assay, candidate molecules may be screened for inhibition of binding of Rictor (the canonical component of mTORC2) to its binding partners, such as PRR5/PROTOR, Sinl, IRS1 and IRS2. The interaction region of Rictor and Protor has been mapped, as described above. For example, candidate inhibitor molecules may be used in a competitive binding assay to disrupt binding interactions between a peptide like RICb and nephrilin, which carries the Protor-binding domain. Briefly, the method may, for example, involve binding 100 ug of nephrilin to a Ni-NTA column (bed volume 0.2 ml) and passing candidate biotinylated peptide (50ug) plus 1 ug of streptavidin-HRP conjugate plus or minus the test agent through the column. After washing the column with 4-5 column volumes of binding buffer (PBS), sample may be eluted in 2 column volumes of PBS containing 100 mM imidazole and the eluate was assayed for HRP using commerically available reagent TMB (read at 655nm).

[0123] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.