PREVENTING CARDIOVASCULAR DISEASE

Technical Field of the Invention

Aspects and embodiments of the present invention relate to the treatment and/or prevention of cardiovascular disease, for example thromboembolic conditions, such as atherothrombotic disease. Particularly although not exclusively, the present invention relates at least in part to an arginine methylation inhibitor ('ArgMe' inhibitor) for use in the treatment and/or prevention of cardiovascular disease such as thromboembolic conditions (for example atherothrombotic disease). The invention also provides inter alia pharmaceutical compositions comprising an arginine methylation inhibitor, together with methods of treating and/or preventing such conditions, diseases and/or disorders as well as other subject matter.

Background to the Invention

Thromboembolic conditions account for 1 in 4 deaths worldwide with atherothrombosis being a leading cause of death. There are many causal risk factors associated with these conditions but the combination of atherosclerosis, inflammation and thrombosis forms a central part of the pathobiological mechanisms behind such conditions. Platelets are key players in these processes. Atherosclerosis is characterised by the hardening and narrowing of arteries due to the deposition of fatty deposits (known as atheromas or atherosclerotic plaques) on their walls. In an advanced state, atherosclerosis increases the risk of thrombi that can potentially block the blood flow to organs like the heart or brain, leading to myocardial infarction or stroke, respectively. Every year, about 500,000 people in the UK suffer serious health complications, or death, from such conditions (Herrington et a/., Circ. Res. 2016, 118, 535-546). The biological basis of atherothrombosis is extremely complex and involves many different components, including the vessel wall and, crucially, platelets. Vascular injury leads to exposure of prothrombotic extracellular matrix ('ECM') proteins like von Willebrand factor ('vWF') and collagen, which trap and activate platelets. These interactions contribute to stable adhesion of platelets by generating intracellular signals that lead to shape change, secretion of granules and activation of integrins. Activation of integrins facilitates the binding of the plasma protein fibrinogen, which subsequently supports platelet aggregation and consolidation of the clot. Aggregation is initially a reversible process but is consolidated by the release of soluble agonists such as adenosine diphosphate ('ADP') and thromboxane A2 (TXA2 ), ensuring rapid entrapment of platelets (Jurk et al., Semin. Thromb. Hemost. 2005, 31, 381-392). While this process is critical to haemostatic protection of the vasculature after injury, the rupture of atherosclerotic plaques drives uncontrolled platelet activation that leads to arterial thrombosis and the clinical events mentioned above. Current therapeutic treatments, particularly for atherothrombosis-related cardiac and brain diseases, involve the use of antiplatelet agents, sometimes in combination with anticoagulation or thrombolytic agents. The use of antiplatelet agents in the treatment of atherothrombotic disease is well established (see for example Ungerer et a/., Thromobsis and Haemostasis 2013, 110, 868-875). Antiplatelet agents such as aspirin and clopidogrel block the actions of TXA2 and ADP, respectively and thus prevent platelet aggregation. However, a considerable number of patients under treatment still experience atherothrombotic events and, conversely, these and other antiplatelet agents significantly increase the risk of major bleeding (Franchi et al., Prog. Cardiovasc. Dis. 2015, 58, 267-277). Therefore, there is currently a clear need for novel antiplatelet and/or antithrombotic agents for the acute and long-term treatment of thromboembolic conditions, particularly in high-risk patients, that display high efficacy while maintaining a low risk of bleeding (Depta et a/., Annu. Rev. Pharmacol. Toxicol. 2015, 55, 373- 397). However, such a need can only be met when there is a greater understanding of the molecular processes regulating specific aspects of platelet function. The inventors have developed such an understanding and, in particular, have determined that the post-translational modification of arginine residues in proteins by methylation is very likely to play a critical role in the signalling pathways that regulate platelet physiology. The inventors have found that inhibition of arginine methylation significantly delays platelet aggregation in response to agonists.

In circulating platelets, activatory signalling pathways triggered by agonists are counterbalanced by inhibitory signalling pathways triggered by prostacyclin ('PGI2') and nitric oxide ('NO') released by the endothelium. Post-translational modifications ('PTMs') of proteins, most notably phosphorylation, play a paramount role in these pathways. PGI2 and NO, for example, trigger the formation of cyclic nucleotides, which in turn activate protein kinases A and G, respectively, which phosphorylate numerous substrates. Post-translational arginine methylation is thought to be as ubiquitous as phosphorylation in human cells (Larson et al., Sci Signal 2016, 9, rs9) and there are hundreds of proteins undergoing arginine methylation in brain and other tissues (Guo et al. Mol. Cell Proteomics 2014, 13, 372-387; Onwuli et al., Proteomics Clin Appl 2017, 11 , 1-9). Arginine methylation consists of the introduction of one ('monoArgMe') or two ('diArgMe') methyl groups on the Ω-nitrogen of arginine residues. Arginine methylation has been implicated in a number of cellular processes including signal transduction, RNA processing, and protein- protein interactions (Bedford etal., Mol. Cell. 2009, 33, 1-13). The effect of arginine methylation in the cardiovascular system, in particular of cardiac ion channels in the context of sudden cardiac death and heart failure has already been investigated (Beltran-Alvarez et al., Proteome Res. 2011, 10, 3712-3719; Beltran-Alvarez et al., FEBS Lett. 2013, 587, 3159-3165; Beltran-Alvarez et al., J. Mol. Cell. Cardiol. 2014, 76, 126-129; Beltran-Alvarez et al., Amino Acids 2015, 47, 429-434; Onwuli et al., Proteomics Clin. Applic. 2017, 11, 1-9). However, the extent, roles and relevance of arginine methylation have not yet been determined in platelets. Arginine methylation is catalysed by protein arginine methyl transferases ('PRMTs'). There are at least 9 enzymes encoded in the human genome ('PRMT1-9'), and proteomics analysis indicates the presence in platelets of at least PRMT1 and -5 (Kim et al., Nature 2014, 509, 575-581). Cell permeable arginine methylation inhibitors have been developed over the last decade, and are currently being investigated as possible therapeutic measures for the treatment of a range of cancers in animal models (Chan-Penebre et al. , Nat. Chem. Biol. 2015, 11, 432-437; Zhang et al., J. Trans. Med. 2015 13, 349; Zhang et al., Oncotarget 2015, 6, 22799-22811 ; Li et al., Oncotarget 2016, 7, 20236-20248; Moretttin et al., Mutagenesis 2015, 30, 177-189). Surprisingly, the inventors have found that the treatment of human platelets with an arginine methylation inhibitor (for example, a PRMT inhibitor such as AMI-1 (Cheng et al., J. Biol. Chem. 2004, 279, 23892-23899)) significantly delays platelet aggregation in response to thrombin and collagen in a dose dependent manner.

Most available antiplatelet therapies target one of four activatory pathways, namely the TXA2 pathway (e.g. aspirin), the ADP pathway (e.g. clopidogrel), the glycoprotein receptor GPIIb/llla (e.g. abciximab), and the thrombin pathway (e.g. vorapaxar). Whilst not wishing to be bound by theory, the inventors believe that arginine methylation inhibitors are effective as antiplatelet agents by a mechanism that targets the inhibitory pathways, and at least the cAMP pathway. It is therefore believed that arginine methylation inhibitors are able to produce potent antiplatelet effects without compromising the ability of thrombin to generate fibrin, and without completely inhibiting platelet activation. This could result in antiplatelet activity in the event of a blood clot, while maintaining protective haemostatic platelet functions, thus minimising risk of bleeding.

The present invention therefore provides for the use of arginine methylation inhibitor(s) as antiplatelet agent(s) for the treatment of cardiovascular disease and, in particular, thromboembolic conditions (for example atherothrombotic disease). Surprisingly, the inventors have also identified that VASP (vasodilator stimulated phosphoprotein) is modified by arginine methylation in human platelets. This, coupled with the inventors' determination of the importance of post-translational arginine methylation in the signalling pathways that regulate platelet physiology, has led to the realisation that inhibition of arginine methylation of VASP may also be useful in the treatment of cardiovascular disease.

VASP (vasodilator stimulated phosphoprotein) is a member of the Ena-VASP protein family. Ena-VASP family members contain an EHV1 N-terminal domain that binds proteins containing E/DFPPPPXD/E motifs and targets Ena-VASP proteins to focal adhesions. The NCBI accession number of VASP (homo sapiens) is NP_003361 (version 1). VASP proteins are actin-associated proteins involved in a range of processes dependent on cytoskeleton remodelling and cell polarity such as axon guidance, lamellipodial and filopodial dynamics, platelet activation and cell migration. VASP promotes actin filament elongation. It protects the barbed end of growing actin filaments against capping and increases the rate of actin polymerisation in the presence of capping protein. VASP stimulates actin filament elongation by promoting the transfer of profilin-bound actin monomers onto the barbed end of growing actin filaments. VASP thus regulates actin dynamics in platelets and plays an important role in regulating platelet aggregation. In platelets, VASP is modified by phosphorylation e.g. at Serine 157 and platelet reactivity inversely correlates with VASP S157 phosphorylation levels (Horstrup K, et al. 1994 Eur J Biochem 225, 21-7). A VASP phosphorylation index has been developed and kits to measure VASP phosphorylation index are commercially available (Diagnostica Stago, Asnieres, France) and used in research studies of cardiovascular disease (Laine M, et al. 2013 Thromb Res. 132(1):e15-8.

Summary of the Invention

It is an aim of certain embodiments of the present invention to at least partly mitigate the problems associated with the prior art.

It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use in the treatment and/or prevention of cardiovascular disease.

It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use in the treatment and/or prevention of thromboembolic condition(s). It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use in the treatment and/or prevention of atherothrombotic disease.

It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use in the treatment and/or prevention of atherothrombotic disease wherein the use of the arginine methylation inhibitor has a reduced risk of bleeding.

It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use as an antiplatelet agent and/or antithrombotic agent.

It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use as an antiplatelet agent in the event of a blood clot wherein the use of the arginine methylation inhibitor has a reduced risk of bleeding. It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use in the treatment and/or prevention of atherothrombotic disease wherein the use of the arginine methylation inhibitor maintains protective haemostatic platelet functions whilst reducing risk of bleeding. It is an aim of certain embodiments of the present invention to provide an arginine methylation inhibitor for use in the treatment and/or prevention of thrombosis (for example the prevention of thrombosis) in a subject undergoing a surgical procedure, for example an angioplasty procedure. It is an aim of certain embodiments of the present invention to identify target proteins important to platelet physiology wherein arginine methylation may be inhibited. It is envisaged that such inhibition may result in the provision of an arginine methylation inhibitor for use in the treatment and/or prevention of cardiovascular disease and, in particular, thromboembolic conditions, such as atherothrombotic disease.

In one aspect of the present invention, there is provided an arginine methylation inhibitor for use in the treatment and/or prevention of cardiovascular disease.

Aptly, the arginine methylation inhibitor is for use in the treatment and/or prevention of a thromboembolic condition. Aptly, the arginine methylation inhibitor is for use in the treatment and/or prevention of atherothrombotic disease.

Aptly, the arginine methylation inhibitor is for use in the treatment and/or prevention of a disease selected from coronary artery disease ('CAD'), ischemic heart disease ('IHD' also known as coronary heart disease ('CHD')), acute coronary syndrome ('ACS') (for example myocardial infarction and/or angina), left ventricular thrombus ('LVT'), cerebral ischemia, critical limb ischemia, peripheral artery disease ('PAD'), deep vein thrombosis ('DVT') and pulmonary embolism.

Aptly, use of the arginine methylation inhibitor as described herein has a reduced risk of bleeding.

Aptly, the arginine methylation inhibitor is for use as an antiplatelet agent and/or antithrombotic agent.

Aptly, use of the arginine methylation inhibitor as described herein maintains protective haemostatic platelet function whilst reducing the risk of bleeding. Aptly, the arginine methylation inhibitor is for use in the treatment and/or prevention of thrombosis (for example the prevention of thrombosis) in a subject undergoing a surgical procedure, for example an angioplasty procedure.

In one aspect of the invention, there is provided a pharmaceutical composition for use in the treatment and/or prevention of cardiovascular disease wherein the pharmaceutical composition comprises an arginine methylation inhibitor and a pharmaceutically acceptable carrier.

Aptly, the pharmaceutical composition is for use in the treatment and/or prevention of a thromboembolic condition.

Aptly, the pharmaceutical composition is for use in the treatment and/or prevention of atherothrombotic disease. Aptly, the pharmaceutical composition is for use in the treatment and/or prevention of a disease selected from coronary artery disease ('CAD'), ischemic heart disease ('IHD' also known as coronary heart disease ('CHD')), acute coronary syndrome ('ACS') (for example myocardial infarction and/or angina), left ventricular thrombus ('LVT), cerebral ischemia, critical limb ischemia, peripheral artery disease ('PAD'), deep vein thrombosis ('DVT) and pulmonary embolism. Aptly, use of the pharmaceutical composition as described herein has a reduced risk of bleeding.

Aptly, the pharmaceutical composition is for use as an antiplatelet agent and/or antithrombotic agent.

Aptly, use of the pharmaceutical composition as described herein maintains protective haemostatic platelet function whilst reducing the risk of bleeding.

Aptly, the pharmaceutical composition is for use in the treatment and/or prevention of thrombosis (for example the prevention of thrombosis) in a subject undergoing a surgical procedure, for example an angioplasty procedure.

Also provided is a kit including such a pharmaceutical composition. In one aspect of the invention, there is provided a method of treating and/or preventing cardiovascular disease, the method comprising administering a pharmaceutically effective amount of an arginine methylation inhibitor as described herein to a subject in need thereof.

Aptly the method is for treating and/or preventing a thromboembolic condition.

Aptly the method is for treating and/or preventing an atherothrombotic disease.

Aptly, the method is for treating and/or preventing a disease selected from coronary artery disease ('CAD'), ischemic heart disease ('IHD' also known as coronary heart disease ('CHD')), acute coronary syndrome ('ACS') (for example myocardial infarction and/or angina), left ventricular thrombus ('LVT'), cerebral ischemia, critical limb ischemia, peripheral artery disease ('PAD'), deep vein thrombosis ('DVT') and pulmonary embolism.

Aptly, administration of the arginine methylation inhibitor as described herein has a reduced risk of bleeding. Aptly, administration of the arginine methylation inhibitor is for use as an antiplatelet agent and/or antithrombotic agent.

Aptly, administration of the arginine methylation inhibitor as described herein maintains protective haemostatic platelet function whilst reducing the risk of bleeding.

Aptly, the method is for treating and/or preventing thrombosis (for example preventing of thrombosis) in a subject undergoing a surgical procedure, for example an angioplasty procedure.

In one aspect of the invention, there is provided a use of an arginine methylation inhibitor in the treatment and/or prevention of cardiovascular disease.

Aptly the use is for treating and/or preventing a thromboembolic condition.

Aptly the use is for treating and/or preventing an atherothrombotic disease.

Aptly, the use is for treating and/or preventing a disease selected from coronary artery disease ('CAD'), ischemic heart disease ('IHD' also known as coronary heart disease ('CHD')), acute coronary syndrome ('ACS') (for example myocardial infarction and/or angina), left ventricular thrombus ('LVT'), cerebral ischemia, critical limb ischemia, peripheral artery disease ('PAD'), deep vein thrombosis ('DVT') and pulmonary embolism.

Aptly, use of the arginine methylation inhibitor as described herein has a reduced risk of bleeding.

Aptly, use of the arginine methylation inhibitor is as an antiplatelet agent and/or antithrombotic agent. Aptly, use of the arginine methylation inhibitor as described herein maintains protective haemostatic platelet function whilst reducing the risk of bleeding.

Aptly, use of the arginine methylation inhibitor is for use in the treatment and/or prevention of thrombosis (for example the prevention of thrombosis) in a subject undergoing a surgical procedure, for example an angioplasty procedure. In one aspect of the invention, there is provided a use of an arginine methylation inhibitor as a research tool for the discovery, development and/or commercialisation of treatments and/or preventative methods for cardiovascular disease such as thromboembolic conditions (for example atherothrombotic disease).

In one aspect of the invention, there is provided an arginine methylation inhibitor which inhibits arginine methylation of VASP for use in the treatment and/or prevention of cardiovascular disease as described herein such as thromboembolic conditions (for example atherothrombotic disease). In certain embodiments, the arginine methylation inhibitor is a nucleic acid molecule e.g. an aptamer or an antibody or fragment thereof.

Further details of embodiments of the invention are provided below.

Brief Description of Drawings

Certain embodiments of the present invention are described in more detail below with reference to the following drawings and/or figures:

Figure 1.

A. Immunoblot detection of enzymes key to ArgMe including MAT2A (43 kDa), PRMT1 (42 kDa) and PRMT5 (72 kDa) in platelet lysates.

B. Pattern of arginine monomethylated proteins in platelet lysates and effect of AM 1-1. Washed human platelets were incubated with 1 mM AM 1-1 for 2 or 4 hours and lysed. Treatment of platelets for 4 h with AMI-1 reduced the levels of arginine methylation. The blot was probed with an α-monoArgMe antibody (#8711 , Cell Signalling Technologies), as well as an a-GAPDH antibody as loading control.

C. Washed platelets were incubated with PGI2 (100 nM) or thrombin (0.1 U/mL) for 1 min, then lysed. The observed decrease in the signal for ArgMe (#8015 Cell Signalling Technologies) after thrombin activation suggests that proteins modified by ArgMe participate in the response of platelets to thrombin. The blot was re-probed for actin as loading control.

D. Lane 1: Control platelets. Lane 2: Platelets activated with thrombin (0.1 U/mL, 5 min). The same samples were loaded 3 times on the same gel. The membrane was blotted with 3 different antibodies. Decreased ArgMe signal (red stars) after thrombin activation was only observed in protein(s) recognised by the #8015 α-monoArgMe antibody, supporting the specificity of this finding. Figure 2. VASP is modified by ArgMe in human platelets.

VASP was immunoprecipitated from washed, untreated platelet lysates. A. Immunoprecipitated VASP was recognised by an a-ArgMe antibody at the expected MW (ca. 45 kDa).

B. Available algorithms for prediction of ArgMe identified possible VASP ArgMe sites including R10.

C. Mass spectrum of the N-terminal VASP peptide SETVICSSRmeATVMLYDDGNK. (SEQ. ID. No. 2) including methylated RIO. LC-MS/MS analysis of the immunoprecipitated, Coomassie-stained, digested (LysC) VASP band was done using a Thermo Orbitrap Fusion. Protein identification was done using Mascot and Scaffold against the reference Swissprot human FASTA database (version 2017_03) to require a FDR of 1%. Note the identification of b9 to 615 diagnostic ions (in red). Mascot score: 48.5. Scaffold probability: 100%.

D. Human (PDB file: 1EGX) and murine (1QC6) VASP EVH1 domains highlighting hydrogen bonds between R10 and D98 (human numbering), note the 2.4-3.2 A distance between donors and acceptors. These interactions by hydrogen bonding between R and D (or E) at equivalent positions are conserved in other proteins containing EVH1 domains including Mena (2IYB), Spred2 (2JP2) and Falafel (4WSF). R10 methylation blocks a possible hydrogen bond and could thus impair the close packing of the EVH1 C-terminal a-helix against the β-barrel. Genetic variants at VASP R10 have been reported (rs 190763638). Mutation of the acidic partner of R10 leads to reduced expression of EVH1 -containing proteins.

E. provides data demonstrating that ArgMe regulates cAMP signalling in platelets. Washed human platelets were incubated with various concentrations of AMI-1 for4h and lysed. Lysates were probed with a phosphor-specific anti-VASP antibody that recognizes Ser ¹⁵⁷ , a target for PKA. As a positive control, platelets were stimulated with 100 μΜ prostaglandin (PGI2) for 5 min in the absence of AMI-1. The blot was re-probed for actin as a loading control. Data from three independent experiments were quantitated and presented as average ± SD fold increase relative to basal. All differences are statistically significant (ANOVA) relative to basal. Figure 2E shows a Western blot showing 5-fold increase in VASP S157 phosphorylation, a hallmark for platelet quiescence, upon treatment with AMI-1 Figure 3. Human platelets incubated ex vivo with ArgMe inhibitors aggregate much more slowly in response to platelet agonists.

Washed platelets were incubated with various concentrations of AMI-1 (A), GSK591 (B) or furamidine, effective in the μΜ range (C) for 4 h prior to platelet aggregation experiments. The bar diagrams show percentage aggregation (average ± SEM, 3 biological replicates) after 4 min relative to basal (no inhibitor, assigned 100%). All ArgMe inhibitors and concentrations caused a significant reduction of aggregation response (ANOVA).

Figure 4 illustrates the amino acid sequence of human VASP protein.

Detailed Description of Embodiments of the Invention

The practice of embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, pharmaceutical formulation, pharmacology and medicine, which are within the skill of those working in the art.

Most general chemistry techniques can be found in Comprehensive Heterocyclic Chemistry IF (Katritzky et a/., 1996, published by Pergamon Press); Comprehensive Organic Functional Group Transformations (Katritzky et a/., 1995, published by Pergamon Press); Comprehensive Organic Synthesis (Trost ef a/,. 1991, published by Pergamon); Heterocyclic Chemistry (Joule et al. published by Chapman & Hall); Protective Groups in Organic Synthesis (Greene et al., 1999, published by Wiley-lnterscience); and Protecting Groups (Kocienski et al., 1994).

Most general molecular biology techniques can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual (2001) Cold Harbor- Laboratory Press, Cold Spring Harbor, N.Y. or Ausubel et al., Current Protocols in Molecular Biology (1990) published by John Wiley and Sons, N.Y.

Most general pharmaceutical formulation techniques can be found in Pharmaceutical Preformulation and Formulation (2 ^nd Edition edited by Mark Gibson) and Pharmaceutical Excipients: Properties, Functionality and Applications in Research and Industry (edited by Otilia M Y Koo, published by Wiley).

Most general pharmacological techniques can be found in A Textbook of Clinical Pharmacology and Therapeutics (5 ^th Edition published by Arnold Hodder). Most general techniques on the prescribing, dispensing and administering of medicines can be found in the British National Formulary 72 (published jointly by BMJ Publishing Group Ltd and Royal Pharmaceutical Society). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2 ^nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3 ^rd ed., Academic Press; and the Oxford University Press, provide a person skilled in the art with a general dictionary of many of the terms used in this disclosure. For chemical terms, the skilled person may refer to the International Union of Pure and Applied Chemistry (lUPAC).

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to" and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and/or steps are mutually exclusive. The invention is not restricted to any details of any embodiments disclosed herein. The invention extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. Units, prefixes and symbols are denoted in their Systeme International d'Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.

The invention provides that in some embodiments, the arginine methylation inhibitor is a PRMT inhibitor.

Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor.

Aptly, the arginine methylation inhibitor is a PRMT2 inhibitor.

Aptly, the arginine methylation inhibitor is a PRMT3 inhibitor.

Aptly, the arginine methylation inhibitor is a PRMT4 inhibitor (also known as a CARM1). Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor. Aptly, the arginine methylation inhibitor is a PRMT6 inhibitor. Aptly, the arginine methylation inhibitor is a PRMT7 inhibitor.

Aptly, the arginine methylation inhibitor is a PRMT 8 inhibitor. Aptly, the arginine methylation inhibitor is a PRMT9 inhibitor. Aptly, the arginine methylation inhibitor is an inhibitor of arginine methylation of VASP. VASP (vasodilator stimulated phosphoprotein) is a member of the Ena-VASP protein family. Ena- VASP family members contain an EHV1 N-terminal domain that binds proteins containing E/DFPPPPXD/E motifs and targets Ena-VASP proteins to focal adhesions. The NCBI accession number of VASP (homo sapiens) is NP_003361 (version 1). The amino acid sequence of human VASP is shown in Figure 4 (SEQ. ID. No. 1).

In certain embodiments, the arginine methylation inhibitor inhibits methylation of an arginine residue at a position selected from 10, 35, 52, 236 and 308 of the VASP protein. In certain embodiments, the arginine inhibitor inhibits methylation of an arginine residue at position 10 of the VASP protein. In certain embodiments, the arginine inhibitor inhibits methylation of an arginine residue at position 35 of the VASP protein. In certain embodiments, the arginine inhibitor inhibits methylation of an arginine residue at position 52 of the VASP protein. In certain embodiments, the arginine methylation inhibitor inhibits methylation of an arginine residue at position 236 of the VASP protein. In certain embodiments, the arginine methylation inhibitor inhibits methylation of an arginine residue at position 308 of the VASP protein. The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Aptly, the arginine methylation inhibitor is a compound referred to herein as AMI-1, or a pharmaceutically acceptable salt or solvate thereof.

Aptly, the arginine methylation inhibitor is a compound referred to herein as AIM-5, or a pharmaceutically acceptable salt or solvate thereof.

Aptly, the arginine methylation inhibitor is a compound referred to herein as AIM-6, or a pharmaceutically acceptable salt or solvate thereof. Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound referred to herein as AM 1-1 , AMI-5 and AMI-6, or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular can be found in Cheng et al., J. Biol. Chem. 2004, 279, 23892-23899, the content of which are incorporated herein by reference.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from stilbamide and allantodapsone or pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in:

Spannhoff et a/., J. Med. Chem. 2007, 50, 2319-2325) (for stilbamidine and allantodapsone); Spannhoff et al., Bioorg. Med. Chem. Lett. 2007, 17, 4150-4153 (for compound RM65); Bonham et a/., FEBS J. 2010, 277, 2096-2108 (for compound 40);

Feng et al., J. Med. Chem. 2010, 53, 6028-6039 (for compound NS-1 (41));

Dowden et al., Org. Biomol. Chem. 2011, 9, 7814-7821 (for compound 42);

Dillion et al., ACS Chem. Biol. 2012, 7, 1198-1204 (for compounds CID 5380390 and CID 2818500);

Wang et al., J. Med. Chem. 2012, 55, 7978-7987 (for compound A36 (43)); and

Yan et al., J. Med. Chem. 2014, 57, 2611-2622 (for compound 44);

the content of which are incorporated herein by reference. The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular can be found in:

Siarheyeva et a/., Structure 2012, 20, 1425-1435 (for compound 45); and

Liu et al., J. Med. Chem. 2013, 56, 2110-2124 (for compounds 46 and 47);

the content of which are incorporated herein by reference.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular can be found in:

Huynh et al., Bioorg. Med. Chem. Lett. 2009, 19, 2924-2927 (for compound 48)

Selvi et al., J. Biol. Chem. 2010, 285, 7143-7152 (for compound 49);

the content of which are incorporated herein by reference.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

This compound is commercially available from Cayman, Tocris and Sigma. Methods for the preparation of such a compound will be known to the skilled person and, in particular can be found in Chan-Penebre et a/., Nat. Chem. Biol. 2015, 11, 432-437; the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein as GSK591 , or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, particular can be found in:

Drew ef a/., Sci Rep. 2017;7;17993 (www.ncbi.nlm.nih.gov/pubmed/29269946)

WO2014/144455; and/or

WO2014/144169;

the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT4 inhibitor selected from a compound referred to herein as 111, 112, EPZ025654, EZM2303 and 113, or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof. These compounds are commercially available from ChemBridge Corporation (San Diego, CA, USA). Methods for the preparation of such compounds will be known to the skilled person and, in particular, further information on these compounds can be found in Kong et a/., PLoS ONE 12(8): e0181601 (www.doi.org/10.1371/journal.pone.0181601); the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein as C1, C2, C3, C4, C5, C6, C7, C9 and C11, or a pharmaceutically acceptable salt or solvate thereof.

Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein as C2, C5, C6, C9 and C11 , or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in:

Chan-Penebre et al., Nat. Chem. Biol. 2015, 11 , 432-437 (for compound EPZ015666) Alinari et a/., Blood, 2015, 125, 2530-2543 (for compound CMP5)

WO2016/178870;

US20160244475;

WO2017/032840; and

Pugh et al., J. Biol. Chem. 1978, 253, 4075-4077;

the content of which are incorporated herein by reference. Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein as EPZ015666, CMP5, 3, 4, and 5(A9145C), or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in Mao et ai, J. Med. Chem. 2017, 60, 6298-6304; the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein as 6 (DC_P04), 7 (DC_P05), 8 (DC_P63), 9 (DC_P88), DC_P65) and 11 (DC_P08), or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

methyl 2-(2-((4-chloro-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (12);

methyl 2-(2-((4-methyl-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (13);

methyl 2-(2-((4-methoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (14);

methyl 2-(2-((5-methyl-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (15);

methyl-2-(2-((5-hydroxy-1H-benzo[d]imidazol-2-yl)-thio)aceta mido)-benzoate (16);

methyl 2-(2-((5-methoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (17);

methyl 2-(2-((5-ethoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (18);

methyl 2-(2-((5-propoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (19); methyl 2-(2-((5-isopropoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (20);

methyl 2-(2-((5-fluoro-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (21);

methyl 2-(2-((5-chloro-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (22);

methyl 2-(2-((5-bromo-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (23);

methyl 2-(2-((5-nitro-1 H-benzo[d]imidazol-2-yl)thio)acetamido)-benzoate (24);

methyl 2-(2-((5-amino-1 H-benzo[d]imidazol-2-yl)-thio)acetamido)benzoate (25);

methyl 2-(2-((5-(methylamino)-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (26); methyl 2-(2-((5-(dimethylamino)-1 H-benzo[d]imidazol-2-yl)-thio)acetamido)benzoate (27); methyl 2-(2-((5-methoxy-3H-imidazo[4,5-b]pyridin-2-yl)thio)-acetami do)benzoate (28);

methyl 2-(2-((3H-imidazo[4,5-c]pyridin-2-yl)thio)acetamido)-benzoat e (29);

2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-phenylacet amide(30);

2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(o-tolyl)- acetamide (31);

2-((5-methoxy-1H-benzo[d] imidazol-2- yl)thio) -N-(2-chlorophenyl)acetamide (32);

2-((5-methoxy-1H-benzo[d]imidazol-2- y I ) thio)-N-(2-methoxyphenyl)acetamide (33);

2-((5-methoxy-1H-benzo[d]imidazol-2- yl) thio)-N-(2-(dimethylamino)phenyl)acetamide (34); 2-((5-methoxy-1H-benzo[d]imidazol-2- yl) thio)-N-(2-acetylphenyl)acetamide (35);

2-(2-((5-methoxy-1 H-benzo[d]imidazol-2-yl)thio)acetamido)-benzoic acid (36);

ethyl 2-(2-((5-methoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (37);

2-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido) -Nmethylbenzamide(38);

2-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)acetamido) -N,N-dimethylbenzam-ide (39); methyl 3-(2-((5-methoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)benzoate (40);

methyl 4-(2-((5-methoxy-1H-benzo[d]imidazol-2-yl)thio)-acetamido)be nzoate (41); and methyl 3-(2-((5-methoxy-1 H-benzo[d]imidazol-2-yl)thio)-acetamido)isonicotinate (42);

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in Mao et a/., J. Med. Chem. 2017, 60, 6298-6304; the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein as any compound selected from compounds 12 to 42, or a pharmaceutically acceptable salt or solvate thereof.

Aptly, the arginine methylation inhibitor is methyl 2-(2-((5-methoxy-1H-benzo[d]imidazol-2- yl)thio)-acetamido)benzoate (17); or a pharmaceutically acceptable salt or solvate thereof. The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from formula (I):

wherein R is selected from 3-COOCH ₃ , 4-COOCH ₃ , 3-CF ₃ , 4-CF ₃ , 3-OCF ₃ , 4-OCF ₄ , 3-OCH ₃ , 4-OCH ₃ , 3-CN, and 4-CN;

or a pharmaceutically acceptable salt or solvate thereof.

Aptly R is selected from 3-COOCH ₃ , 3-CF ₃ , 4-CF ₃ , 3-OCF ₃ and 4-OCF ₄ .

Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in Yang et a/., Bioorganic & Medicinal Chemistry Letter 27 (2017) 4635-4642; the content of which are incorporated herein by reference. Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound wherein R is selected from 3-COOCH ₃ , 3-CF ₃ , 4-CF ₃ , 3-OCF ₃ and 4-OCF ₄ , or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from formula (II):

wherein R is selected from 3-COOCH ₃ , 4-COOCH ₃ , 3-CF ₃ , 4-CF ₃ , 3-OCF ₃ , 4-OCF ₄ , 3-OCH ₃ , 4-OCH ₃ , 3-CN, and 4-CN;

or a pharmaceutically acceptable salt or solvate thereof.

Aptly R is 3-OCF ₃ .

Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in Yang et a/., Bioorganic & Medicinal Chemistry Letter 27 (2017) 4635-4642; the content of which are incorporated herein by reference. Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound wherein R is 3-OCF3 or a pharmaceutically acceptable salt or solvate thereof. The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, particular, can be found in:

Xie et al., Org. Biomol. Chem. 2014, 12, 9665 (for compounds DCLX069 and DCLX078); Yan et al., J. Med. Chem. 2014, 57, 2611 (for compound DB75); and

Eram et al., ACS Chem. Biol. 2016, 11, 772 (for compound MS023).

the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound referred to herein as DCLC069, DCLX078, DB75 or MS023; or a pharmaceutically acceptable salt or solvate thereof.

Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound referred to herein as DB75; or a pharmaceutically acceptable salt or solvate thereof. DB75 is also known as furamidine. The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular, further information can be found in Dowden et a/., Bioorg. Med. Chem Lett. 2010, 20, 2103-2105; the content of which are incorporated herein by reference. Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound referred to herein as Ligand 1, Ligand 2 and Ligand 3 (hereinabove); or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

4,4-(methylenebis(oxy))dibenzimidamide (methamidine);

4,4-(ethane-1 ,2-diylbis(oxy))dibenzimidamide (ethamidine);

4,4-(propane-1 ,3-diylbis(oxy))dibenzimidamide (propamidine);

4,4-(butane-1 ,4-diylbis(oxy))dibenzimidamide (butamidine);

4,4-(pentane-1,5-diylbis(oxy))dibenzimidamide (pentamidine); and

4,4-(hexane-1 ,6-diylbis(oxy))dibenzimidamide (hexamidine);

or a pharmaceutically acceptable salt or solvate thereof.

Some of these compounds are commercially available from J&K Scientific (pentamidine and hexamidine). Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in Tidwell et a/., J. Med. Chem. 1990, 33, 1252; the content of which are incorporated herein by reference. Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound referred to herein as methamidine, ethamidine, propamidine, butamidine, pentamidine or hexamidine; or a pharmaceutically acceptable salt or solvate thereof. The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

wherein the linker is selected from -O- (compound 2a), -OCH2O- (compound 2b), -0(CH2)20- (compound 2c), -0(CH2)30- (compound 2d), -0(CH2)40- (compound 2e), - OCH ₂ CH=CHCH ₂ 0- (compound 2f), -0(CH ₂ )3CH(CH ₃ )0- (compound 2g), -0(CH ₂ ) ₅ 0- (compound 2h, pentamidine), -0(CH2)eO- (compound 2i, hexamidine) and -0(CH2)ioO- (compound 2j, decamidine),

or a pharmaceutically acceptable salt or solvate thereof.

Aptly the linker may be selected from -0(CH2) _m O- wherein m is an integer selected 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

Methods for the preparation of such compounds will be known to the skilled person and, in particular, further information can be found in Zhang et a/., Med. Chem. Commun., 2017, 8, 440-444; the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT1 inhibitor selected from a compound referred to herein as compounds 2a to 2j (hereinabove); or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound selected from:

2-(((2-(benzyloxy)naphthalen-1-yl)methyl)amino)-1-phenyletha n-1-ol (DC_C01);

1- phenyl-2-(((2-(pyridin-4-ylmethoxy)naphthalen-1-yl)methyl)am ino)ethan-1-ol (DC_C02);

2- (((2-phenethoxynaphthalen- 1 -yl)methyl)amino)-1 -phenylethan- 1 -ol (DC_C03) ;

1-(((2-hydroxy-2-phenylethyl)amino)methyl)naphthalen-2-yl benzoate (DC_C04); 2-(((2-((4-fluorobenzyl)oxy)naphthalen-1-yl)methyl)amino)-1- phenylethan-1-ol (DC_C05); 2-(((2-((4-chlorobenzyl)oxy)naphthalen-1-yl)methyl)amino)-1- phenylethan-1-ol (DC_C06); 4-(((1-(((2-hydroxy-2-phenylethyl)amino)methyl)naphthalen-2- yl)oxy)methyl) benzonitrile (DC_C07);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-phenylethan-1-ami ne (DC_C08);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(pyridin-2-yl) ethan-1-amine (DC_C09);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(1H-indol-3-yl )ethan-1-amine (DC_C10);

N-((2-(benzyloxy)naphthalen- 1-yl)methyl)-2-(piperidin- 1 -yl)ethan- 1 -amine (DC_C 11);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-morpholinoetha n-1-amine (DC_C12);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(4-fluoropheny l)ethan-1-amine (DC_C13);

4-(2-(((2-(benzyloxy)naphthalen-1-yl)methyl)amino)ethyl)p henol (DC_C14);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(p-tolyl)ethan -1-amine (DC_C15);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(4-chloropheny l)ethan-1-amine (DC_C16); N-((2-(benzyloxy)naphthalen-1-yl)methyl)-2-(3-fluorophenyl)e than-1-amine (DC_C17);

N-((2-(benzyloxy)naphthalen-1-yl)methyl)-N-methyl-2-pheny lethan-1-amine (DC_C18); and 2-((2-(benzyloxy) benzyl)am ino)- 1 -phenylethan- 1 -ol (DC_C 19) .

or a pharmaceutically acceptable salt or solvate thereof.

Methods for the preparation of such compounds will be known to the skilled person and, in particular, can be found in Ye et a/., Org. Biomol. Chem., 2017, 15, 3648-3661 (www.ncbi.nlm.nih.gov/pubmed/28397890); the content of which are incorporated herein by reference.

Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein a DC_C01 to DC_C19 (hereinabove); or a pharmaceutically acceptable salt or solvate thereof.

The invention provides that in some embodiments, the arginine methylation inhibitor is a compound of formula (III):

wherein

or a pharmaceutically acceptable salt or solvate thereof. These compounds are commercially available from SPECS. Methods for the preparation of such compounds will be known to the skilled person and, in particular, further information can be found in Ye et al., Org. Biomol. Chem., 2017, 15, 3648-3661

(www.ncbi.nlm.nih.gov/pubmed/28397890); the content of which are incorporated herein by reference. Aptly, the arginine methylation inhibitor is a PRMT5 inhibitor selected from a compound referred to herein as DC_P33 and DC_S01 to DC_S14 (hereinabove); or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the arginine methylation inhibitor is a nucleic acid molecule. The nucleic acid molecule may be single-stranded or double-stranded. In certain embodiments, the nucleic acid is an inhibitor of arginine methylation of VASP.

As used herein, the term "nucleic acid molecule" refers to deoxyribonucleotide molecules, ribonucleotide molecules, or modified nucleotides, and polymers thereof. The nucleic acid molecule may be in a single- or double-stranded form. The term encompasses a nucleic acid molecule which contains known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid molecule, and which are metabolized in a similar manner. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides and peptide-nucleic acids (PNAs).

Aptly, a nucleic acid molecule comprises a plurality of nucleotides. As used herein, the term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide, or a modified form thereof. Nucleotides include species that include purines (e.g., adenine, hypoxanthine, guanine, and the like) as well as pyrimidines (e.g., cytosine, uracil, thymine, and the like). When a base is indicated as "A", "C", "G", "U", or "T", it is intended to encompass both ribonucleotides and deoxyribonucleotides, and modified forms thereof.

The nucleic acid molecule may be synthetic or naturally-occurring. The term "naturally occurring" may refer to ssomething found in an organism without any intervention by a person; it could refer to a naturally-occurring wildtype or mutant molecule. A synthetic nucleic acid molecule may be an analogue of a naturally-occurring nucleic acid molecule or may be different.

In certain embodiments, the nucleic acid molecule is selected from a miRNA, an RNA aptamer and a DNA aptamer.

In one embodiment the nucleic acid molecule may be a miRNA. The term "miRNA" is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington et al., 2003, which is hereby incorporated by reference. As used herein, the term is used to refer to the single-stranded RNA molecule processed from a precursor. In certain embodiments, the nucleic acid molecule is an aptamer. Aptly, the aptamer is an RNA aptamer or a DNA aptamer.

The term "aptamer", as used herein, refers to a non-naturally occurring nucleic acid that has a desirable action on a target molecule. Desirable actions include, but are not limited to, binding of the target, inhibiting the activity of the target, enhancing the activity of the target, altering the binding properties of the target (such as, for example, increasing or decreasing affinity of the target for a ligand, receptor, cofactor, etc.), inhibiting processing of the target (such as inhibiting protease cleavage of a protein target), enhancing processing of the target (such as increasing the rate or extent of protease cleavage of a protein target), and inhibiting or facilitating the reaction between the target and another molecule. An aptamer may also be referred to as a "nucleic acid ligand."

In some embodiments, an aptamer specifically binds a target molecule, wherein the target molecule is a three-dimensional chemical structure other than a polynucleotide that binds to the aptamer through a mechanism which is independent of Watson/Crick base pairing or triple helix formation, and wherein the aptamer is not a nucleic acid having the known physiological function of being bound by the target molecule. In some embodiments, aptamers to a given target include nucleic acids that are identified from a candidate mixture of nucleic acids, by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield a ligand- enriched mixture of nucleic acids, whereby aptamers to the target molecule are identified.

An aptamer can include any suitable number of nucleotides. Aptamers may comprise DNA, RNA, both DNA and RNA, and modified versions of either or both, and may be single stranded, double stranded, or contain double stranded or triple stranded regions, or any other three- dimensional structures. In some embodiments, aptamers may be obtained by a technique called the systematic evolution of ligands by exponential enrichment (SELEX) process (Tuerk et al., Science 249:505-10 (1990), U.S. Patent Number 5,270,163, and U.S. Patent Number 5,637,459, each of which is incorporated herein by reference in their entirety). In certain embodiments, the arginine methylation inhibitor is a double-stranded nucleic acid molecule. Optionally, the double-stranded nucleic acid molecule is selected from siRNA, pDNA, a gene, e.g. a synthetic gene (linear, 5' and 3' end-hairpin ligated expression cassette), mRNA e.g. synthetic messenger RNA (mRNA).

The term "siRNA" (short interfering RNA) is a term used in the art and refers to a short double stranded RNA complex, typically 19-28 base pairs in length and which operates in the RNAi pathway where it interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription. Aptly, siRNA is a is double- stranded nucleic acid molecule comprising two nucleotide strands, each strand having about 19 to about 28 nucleotides (i.e. about 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). The complex often includes a 3'-overhang. SiRNA can be made using techniques known to one skilled in the art and a wide variety of siRNA is commercially available.

In certain embodiments, the nucleic acid molecule is a PRMT inhibitor. Aptly, the nucleic acid molecule is a PRMT1 inhibitor. Aptly, the nucleic acid molecule is a PRMT2 inhibitor. Aptly, the nucleic acid molecule is a PRMT3 inhibitor.

Aptly, the nucleic acid molecule is a PRMT4 inhibitor (also known as a CARM1).

Aptly, the nucleic acid molecule is a PRMT5 inhibitor. Aptly, the nucleic acid molecule is a PRMT6 inhibitor. Aptly, the nucleic acid molecule is a PRMT7 inhibitor. Aptly, the nucleic acid molecule is a PRMT 8 inhibitor. Aptly, the nucleic acid molecule is a PRMT9 inhibitor.

In certain embodiments, the arginine methylation inhibitor may be an antibody or fragment thereof. By "antibody" herein is meant a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (κ), lambda (λ), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (u), delta (δ), gamma (γ), sigma (a), and alpha (a) which encode the IgM, IgD, IgG (lgG1 , lgG2, lgG3, and lgG4), IgE, and IgA (lgA1 and lgA2) isotypes respectively. Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes. The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

In certain embodiments, the arginine methylation inhibitor may be an antibody or fragment thereof which inhibits the methylation of one or more than one arginine residue of a VASP protein. Arginine methylation inhibitors are compounds and other molecules e.g. nucleic acid molecules and antibodies that inhibit the post translational methylation of arginine. In particular, an arginine methylation inhibitor may be a compound or other molecule e.g. nucleic acid molecules and antibodies that partially or completely inhibits the activity of one or more protein arginine N-methyltransferase (PRMT). Methods and assays to determine if a compound or other molecules e.g. nucleic acid molecules and antibodies is an arginine methylation inhibitor are known and, for example, can be found in Cheng et a/., J. Biol. Chem. 2004, 279, 23892-23899, the content of which are incorporated herein by reference.

The arginine methylation inhibitors and/or pharmaceutical compositions of the invention may be used for the treatment and/or prevention of cardiovascular disease as described herein as a sole therapy or may be used in combination with other conventional surgery or therapies. In particular, the arginine methylation inhibitors and/or pharmaceutical compositions of the invention may be used in combination with other antiplatelet agents and/or anticoagulant agents and/or thrombolytic agents. For example, the arginine methylation inhibitors and/or pharmaceutical compositions of the invention may be used in combination with aspirin. For example, the arginine methylation inhibitors and/or pharmaceutical compositions of the invention may be used in combination with clopidogrel. For example, the arginine methylation inhibitors and/or pharmaceutical compositions of the invention may be used in combination with vorapaxar. For example, the arginine methylation inhibitors and/or pharmaceutical compositions of the invention may be used in combination with warfarin. For example, the arginine methylation inhibitors and/or pharmaceutical compositions of the invention may be used in combination with streptokinase. Other antiplatelet and/or anticoagulant and/or thrombolytic agents for use in combination with the arginine methylation inhibitors and/or pharmaceutical compositions of the invention can be found in Antiplatelet Therapy - A Summary for the General Physicians (Thachil J. Clin Med, 2016, 152-160), the contents of which are incorporated herein by reference.

The arginine methylation inhibitors of the present disclosure may be formulated as pharmaceutical compositions prepared for storage or administration for use in the treatment and/or prevention of cardiovascular disease (for example thromboembolic conditions) as described herein. Such a composition typically comprises a therapeutically effective amount of an arginine methylation inhibitor, in the appropriate form, and a pharmaceutically acceptable carrier.

The therapeutically effective amount of an arginine methylation inhibitor as described herein will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The arginine methylation inhibitors of the present disclosure may be particularly useful for treatment of humans.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the person skilled in the art. The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.Gennaro edit. 1985). For example, sterile saline and phosphate- buffered saline at slightly acidic or physiological pH may be used. pH buffering agents may be phosphate, citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS), N- Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine, or acetate or mixtures thereof. The term further encompasses any agents listed in the US Pharmacopeia for use in animals, including humans.

The term "pharmaceutically acceptable salt" refers to a salt of any one of the arginine methylation inhibitor of embodiments of the invention. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts include hydrochloride salts, citrate salts and acetate salts. Examples of basis salts include salts where the cation is selected from alkali metals, such as sodium and potassium, alkaline earth metals, such as calcium, and ammonium ions ⁺ N(R ³ )3(R ⁴ ), where R ³ and R ⁴ independently designates optionally substituted C -e-alkyl, optionally substituted C2-e-alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17 ^th edition. Ed. Alfonoso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S.A., 1985 and more recent editions, and in the Encyclopaedia of Pharmaceutical Technology.

The term "solvate" in the context of the present disclosure refers to a complex of defined stoichiometry formed between a solute (e.g., an arginine methylation inhibitor or pharmaceutically acceptable salt thereof according to the present disclosure) and a solvent. The solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate.

"Treatment" is an approach for obtaining beneficial or desired clinical results. For the purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treatment' is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures in certain embodiments. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. By treatment is meant inhibiting or reducing an increase in pathology or symptoms when compared to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant condition.

The pharmaceutical compositions for use in the treatment of a cardiovascular disease (for example a thromboembolic condition) can be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component, the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen. In certain embodiments, packaged forms include a label or insert with instructions for use. Compositions may be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

As used herein an "effective" amount or a "therapeutically effective amount" of an inhibitor or agent refers to a nontoxic but sufficient amount of the inhibitor or agent to provide the desired effect. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The terms "patient", "subject" and "individual" may be used interchangeably and refer to either a humans or non-human mammal. Aptly, the subject is a human.

The arginine methylation inhibitors described herein may be used to treat and/or prevent a cardiovascular disease (for example a thromboembolic condition) e.g. an atherothrombotic disease. In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of a disease selected from coronary artery disease ('CAD'), ischemic heart disease ('IHD' also known as coronary heart disease ('CHD')), acute coronary syndrome ('ACS') (for example myocardial infarction and/or angina), left ventricular thrombus ('LVT'), cerebral ischemia, critical limb ischemia, peripheral artery disease ('PAD'), deep vein thrombosis ('DVT') and pulmonary embolism.

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of coronary artery disease ('CAD').

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of ischemic heart disease ('IHD').

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of coronary heart disease ('CHD'). In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of acute coronary syndrome ('ACS') (for example myocardial infarction and/or angina).

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of left ventricular thrombus ('LVT').

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of cerebral ischemia. In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of critical limb ischemia.

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of peripheral artery disease ('PAD').

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of deep vein thrombosis ('DVT').

In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of pulmonary embolism. In one embodiment, the arginine methylation inhibitor is for use in the treatment and/or prevention of thrombosis (for example the prevention of thrombosis) in a subject undergoing a surgical procedure, for example an angioplasty procedure. A thromboembolic condition occurs when a blood clot (a thrombus) occurs in a blood vessel. The thrombus can break loose and be carried to by the blood stream to partially or fully block another vessel. The blockage is referred to as an embolism.

A thromboembolism which occurs in an artery is known as an arterial thromboembolism and results from atherothrombotic disease. An arterial thromboembolism can in principle affect any organ in the body resulting in tissue damage and/or death.

Coronary artery disease ('CAD') results when one or more artery that supplies blood to heart muscle becomes hardened and narrowed, usually as a result of atherosclerosis. Such a blockage may result in an ischemia.

Ischemia is a restriction in blood supply to tissues, causing a shortage of oxygen and glucose needed for cellular metabolism (to keep tissue alive). Ischemia is generally caused by problems with blood vessels, with resultant damage to or dysfunction of tissue. It also means local anemia in a given part of a body sometimes resulting from congestion (such as vasoconstriction, thrombosis or embolism). Ischemia comprises not only insufficiency of oxygen, but also reduced availability of nutrients and inadequate removal of metabolites.

Ischemic heart disease ('IHD') is a disease characterized by reduced blood supply to the heart. Ischemic heart disease is also known as coronary heart disease ('CHD').

Acute coronary syndrome (ACS) is a syndrome (set of signs and symptoms) resulting from decreased blood flow in the coronary arteries such that part of the heart muscle is unable to function properly or dies. Examples of ACS include myocardial infarction and angina. ACS is often associated with rupture of an atherosclerotic plaque and partial or complete thrombosis of the infarct-related artery. The signs and symptoms of ACS, which usually begin abruptly, may include one or more of:

• Chest pain (angina) or discomfort, often described as aching, pressure, tightness or burning

· Pain radiating from the chest to the shoulders, arms, upper abdomen, back, neck or jaw

• Nausea or vomiting Indigestion

Shortness of breath (dyspnea)

Sudden, heavy sweating (diaphoresis)

• Lightheadedness, dizziness or fainting

• Unusual or unexplained fatigue

• Feeling restless or apprehensive

Left ventricular thrombus ('LVT) is a thrombus in the left ventricle of the heart, typically on the wall (mural). It is a common complication of acute myocardial infarction. The primary risk of LVT is the occurrence of cardiac embolism when the thrombus detaches, causing for example cerebral ischemia.

Cerebral ischemia is an ischemia occurring in the brain and can be acute or chronic. Acute ischemic stroke is a neurologic emergency that may be reversible if treated rapidly. Chronic ischemia of the brain may result in a form of dementia called vascular dementia. A brief episode of ischemia affecting the brain is called a transient ischemic attack (TIA).

Peripheral vascular disease ('PVD') refers to a narrowing of blood vessels other than those that supply the heart and brain. When the blood vessel is an artery, the disease is known as peripheral artery disease ('PAD') although the terms are often used interchangeably. PAD most commonly affects the legs but other arteries may also be involved. In the context of this invention, PAD can be caused by arteriosclerosis (hardening and/or stiffening of the artery), which is most commonly due to the formation of atheromatous plaques. The classic symptom is leg pain when walking which resolves with rest, known as intermittent claudication. Other symptoms including skin ulcers, bluish skin, cold skin, or poor nail and hair growth may occur in the affected leg. Complications may include an infection or tissue death which may require amputation; coronary artery disease, or stroke. Up to 50% of cases of PAD are without symptoms. Complications of PAD include critical limb ischaemia (CLI) where blood flow to the limbs is severely restricted.

Critical limb ischemia (CLI) is an ischemia occurring in the arteries of the lower extremities. It is a serious form of PAD. It is a chronic condition that results in severe pain in the feet or toes, even while resting. Complications of poor circulation can include sores and wounds that won't heal in the legs and feet. Left untreated, the complications of CLI will result in amputation of the affected limb. The most prominent features of critical limb ischemia (CLI) are called ischemic rest pain— severe pain in the legs and feet while a person is not moving, or nonhealing sores on the feet or legs. Symptoms may include one or more of: • Pain or numbness in the feet

• Shiny, smooth, dry skin of the legs or feet

• Thickening of the toenails

• Absent or diminished pulse in the legs or feet

· Open sores, skin infections or ulcers that will not heal

• Dry gangrene (dry, black skin) of the legs or feet.

A pulmonary embolism refers to a blockage in an artery to the lungs. Signs and symptoms of a pulmonary embolism include chest pain, shortness of breath and coughing. In particular, symptoms can include:

• chest or upper back pain - a sharp, stabbing pain that may be worse when breathing in;

• shortness of breath - which can come on suddenly or develop gradually;

• coughing - this is usually dry, but may include coughing up blood or mucus that contains blood;

• feeling lightheaded or dizzy; and/or

• fainting.

Many pulmonary embolisms are caused by deep vein thrombosis (DVT). Some people with a pulmonary embolism therefore also have symptoms of DVT, such as pain, redness and swelling in one leg (usually the calf).

Blood clots that develop in a vein are known as venous thrombosis. Deep vein thrombosis (DVT) is characterized by the development of a blood clot within a deep vein in the body, usually in the leg. DVT usually occurs in a deep leg vein, a larger vein that runs through the muscles of the calf and the thigh. It can cause pain and swelling in the leg and may lead to complications such as pulmonary embolism. This is a serious condition that occurs when a piece of blood clot breaks off into the bloodstream and blocks one of the blood vessels in the lungs (see below).

Angioplasty (non-invasive or percutaneous) is a surgical procedure by which a catheter with a balloon is inserted into a vessel and directed to a position where there is a stenosis (narrowing). The balloon is inflated in order to widen a narrowed or obstructed artery or vein. A stent may or may not be placed to maintain the vessel dilated. The procedure is mainly (but not exclusively) used to treat stenosis caused by atherosclerosis. The most common procedure is coronary angioplasty. Arteries in the abdomen (renal artery, mesenteric artery, aorta, iliac arteries) and in the legs (femoral, popliteal, tibial arteries) have been addressed using this procedure. Angioplasty is also used to treat carotid artery stenosis and some cases of venous stenosis.

Whilst not wishing to be bound by theory, it is believed that arginine methylation inhibitors can be used to treat any one or more of the diseases listed herein by virtue of their ability to disrupt platelet aggregation and/or other platelet function.

Examples

Experiments have been designed to determine the importance of the post-translational modification, arginine methylation, in the function of blood platelets, to identify the mechanism that arginine methylation (and inhibition thereof) has in the regulation of platelet function and to establish the use of arginine methylation inhibitors as antiplatelet drugs.

Experiments make use of human platelets isolated from voluntary donors. Unless otherwise specified, experiments make use of washed platelets. All methodology required for the study of platelet function and biochemistry is disclosed in the following references, the entire contents of which are incorporated herein by reference:

Platelets and Megakaryocytes: Volume 1: Functional Assays. Methods in Molecular Biology Vol. 272 Edited by J. M. Gibbins and M. P. Mahaut-Smith. Humana Press. 2004;

Platelets and Megakaryocytes: Volume 2: Perspectives. Methods in Molecular Biology Vol. 273. Edited by J. M. Gibbins and M. P. Mahaut-Smith. Humana Press. 2004; and

Platelets and Megakaryocytes: Volume 3: Additional Methods and Perspectives. Methods in Molecular Biology Vol. 788. Edited by J. M. Gibbins and M. P. Mahaut-Smith. Humana Press. 2012.

The scope of protein arginine methylation in human platelets was determined by searching available proteomic data consisting of the proteome of human platelets from healthy adult individuals [Kim etal., Nature 2014]. Using MaxQuant and recently published workflow [Onwuli et a/., Proteomics Clin App 2017], it was found that there was 268 monoArgMe sites and 160 diArgMe sites in a total of 211 proteins. A search was then carried out for enrichment in Gene Ontology (GO) terms in the subset of methylated proteins to determine the extent that platelet protein ArgMe was relevant to platelet physiology. GO terms related to platelet aggregation were specifically (P<10 ¹⁰ ) enriched in methylated proteins suggesting a fundamental role of ArgMe in platelet function (Table 1)

Table 1. Enrichment in GO terms in proteins undergoing mono-ArgMe in platelets (a total of 173 proteins), compared to the total platelet proteome (3534 proteins). GO term enrichment analysis was done using GOrilla. Process GO terms with a P value lower than 10E-5 are shown.

To demonstrate the relevance of ArgMe in platelets, the effect of AMI-1, a well known PRMT inhibitor (Cheng et a/., J. Biol. Chem. 2004, 279, 23892-23899), on platelet function was investigated. Human platelets were treated with AMI-1 ex vivo for 4 hours and a reduction in the levels of ArgMe was observed (Fig. 1 B). To explore the participation of proteins modified by ArgMe in platelet aggregation, platelets were stimulated with thrombin. A decrease in the signal for ArgMe was observed (Figure 1C), suggesting that proteins undergoing ArgMe participate in the response of platelets to thrombin. This decreased ArgMe signal was only observed in proteins recognised by the #8015 a-monoArgMe antibody (Figure 1D), which supports the specificity of these findings. Significantly, platelet aggregation was delayed in response to thrombin and collagen in a dose dependent manner (Fig. 3). This behaviour resembles the effects of treating platelets with substances that cause an increase of cyclic nucleotide levels and, in fact, a significant dose-dependent increase in phospho-VASP-S ¹⁵⁷ in platelets treated with AMI-1 (Fig. 2E) was observed, reflecting activation of the protein kinase A pathway.

Together, these data indicate that ArgMe occurs in platelets and plays an important role in platelet homeostasis. Understanding the mechanism associated with the balance between inhibitory and activatory signals in platelets represents an important aspect of the pathogenesis of atherothrombosis. Conclusion

The data described above demonstrates that ArgMe occurs in platelets: analysis of available proteomic data suggests that hundreds of platelet proteins undergo arginine methylation in platelets, PRMTs are expressed (Fig. 1A) and ArgMe can be demonstrated immunologically and can be specifically inhibited (Fig. 1 B). Furthermore it has been shown that ArgMe inhibition by AMI-1 dramatically affects the dynamics of platelet activation by thrombin and collagen, to the extent that platelet aggregation is significantly delayed.

Example 2

VASP plays a central role in platelet signalling pathways (Massberg et al. Blood. 2004; 103(1): 136-42). VASP phosphorylation correlates with platelet inhibition and is paralleled by the inhibition of fibrinogen receptor GPIIb-llla (Aszodi et al. EMBO J. 1999;18(1):37-48). It is demonstrated here, for the first time, that VASP is modified by ArgMe in platelets. VASP was immunoprecipitated using an anti-VASP antibody (from Abeam) from washed platelet lysates (isolated from healthy human donors, and subjected to no treatment). VASP was recognised by an anti- ArgMe antibody (commercially available from Cell Signalling Technologies, product reference #8015, Figure 2A). VASP is predicted to be modified by ArgMe at sites including R10 (Figure 2B). Immunoprecipitated VASP was isolated by SDS- PAGE and the coomassie stained band was digested with Lys-C protease post reduction with DTE and alkylation with iodoacetamide. The resulting peptides were analysed by LC-MS/MS using our Orbirap Fusion mass spectrometer with elution from an easy spray 50 cm PepMap column over a 35 min gradient. Thermo .raw files were converted to .mgf using MSconvert before submitting to Mascot database searching against the human subset of the SwissProt database. The Mascot .DAT result file was imported into Scaffold and a second search of the same database was performed using X!Tandem. Peptide and protein identifications were filtered in Scaffold to require a global false discovery rate of <1 % at both the protein and peptide level. Protein matches also required a minimum of two unique peptide identifications. These experiments indicated that the VASP N-terminal peptide, including R10, is methylated in human platelets (Figure 2C). This is important because R10 contributes to stabilise the VASP EVH1 domain (Figure 2D), while R10 methylation can block a hydrogen bond and thus impair the close packing of the EVH1 C-terminal a-helix against the β-barrel. The VASP EVH1 domain is in fact a critical regulator of actin dynamics and platelet aggregation. Of note, platelet treatment with ArgMe inhibitors increased VASP phosphorylation at S157, a hallmark for platelet quiescence (Figure 2E).