Protein Arginine Methyltransferases (PRMTs) catalyze the addition of a methyl group from S- adenosylmethionine to guanidino nitrogen atoms on arginine residues. Arginine methylation of NIP45 (NFAT- interacting protein, 45kD) by PRMT1 augments its interaction with NFAT and results in elevated cytokine production in T helper cells. Covalent modification of NIP45 by arginine methylation is a novel mechanism of regulating the expression of NFAT-dependent cytokine genes. We have identified a novel PRMT inhibitor (Cpd 4) that disrupts the interaction between PRMT1 and NIP45, resulting in abrogated NFAT-driven transcription. Indeed, Cpd4 treatment reduces expression of T helper cytokines, such as IFN? and IL-17A, leading to diminished inflammation in the delayed type hypersensitivity model, suggesting that PRMT inhibitors may be useful for treating immune-mediated disease. Despite its promising inhibitory profile, Cpd 4 is only biologically active at high concentrations (100?M), making it unfavorable for therapeutic development. In response to PAR-09-129, in conjunction with the nearby Scripps MLPCN center, we propose to identify PRMT 1 inhibitors in a high throughput screen of the 300,000+ NIH small molecule library, measuring changes in the kinetics of active-site labeling with fluorescently labeled maleimide probe in the presence of inhibitors by monitoring the fluorescence polarization signal. We will rule out false-positive and non-selective primary hits by incorporating our fluopol assay into a secondary gel-based screen. Selective inhibitors will then be tested for their ability to inhibit PRMT1 activity using in vitro methylation assays and using antibodies that specifically recognize cellular targets of PRMT1. Identification of specific PRMT inhibitors would provide us with important tools to probe the importance of PRMT activity in T helper cell function. Since aberrant PRMT1 activity has been associated with cardiovascular, malignant, infectious, and autoimmune disease, it may be a viable therapeutic target for several indications.

Public Health Relevance

The Protein Arginine Methyltransferase 1 (PRMT1) enzyme has been implicated in inflammatory processes and in cardiovascular disease, suggesting that specific PRMT1 inhibitors may be therapeutically valuable. The goal of this proposal is to generate small molecule PRMT1 inhibitors and that will provide the research community with valuable tools and may, potentially, generate a potent and cost effective therapies for cardiovascular and inflammatory diseases.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Small Research Grants (R03)
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Special Emphasis Panel (ZRG1-BST-F (50))
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Singh, Hari
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Scripps Research Institute
La Jolla
United States
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Dillon, Myles B C; Bachovchin, Daniel A; Brown, Steven J et al. (2012) Novel inhibitors for PRMT1 discovered by high-throughput screening using activity-based fluorescence polarization. ACS Chem Biol 7:1198-204
Arandjelovic, Sanja; McKenney, Katherine R; Leming, Sunamita S et al. (2012) ATP induces protein arginine deiminase 2-dependent citrullination in mast cells through the P2X7 purinergic receptor. J Immunol 189:4112-22