An Activity-Based Assay to Screen for PRMT1 Inhibitors

基于活动的 PRMT1 抑制剂筛选试验

• 批准号: 8460828
• 负责人: KERRI A MOWEN
• 金额: $ 4.6万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460828
• 关键词: 4 hydroxynonenal; Accounting; Active Sites; Antibodies; Arginine; Autoimmune Diseases; Binding; Biological Assay; Biological Process; CD4 Positive T Lymphocytes; Cardiovascular Diseases; Cardiovascular system; Cell physiology; Cells; Charge; Communities; Cysteine; Cytokine Gene; DNA Repair; Delayed Hypersensitivity; Disease; Effectiveness; Elements; Enzymes; Exhibits; Family member; Fluorescence Polarization; Future; Gel; Genetic Transcription; Goals; Helper-Inducer T-Lymphocyte; Immune; Immunoblotting; In Vitro; Inflammation; Inflammatory; Interferons; Interleukin-17; Kinetics; Label; Laboratories; Lead; Lysine; Maleimides; Malignant - descriptor; Measures; Mediating; Methylation; Methyltransferase; Modeling; Modification; Molecular Bank; Monitor; Mutation; Nitrogen; Parents; Permeability; Positioning Attribute; Process; Production; Protein-Arginine N-Methyltransferase; Proteins; RNA Processing; Recombinant Proteins; Reporter; Research; S-Adenosylmethionine; Signal Pathway; Signal Transduction; Specificity; Structure; Sulfhydryl Compounds; Testing; Time; United States National Institutes of Health; arginine methyltransferase; base; cellular targeting; cofactor; cost effective; cytokine; drug candidate; fluorophore; high throughput screening; in vivo; inhibitor/antagonist; methyl group; novel; novel therapeutics; prevent; response; small molecule; small molecule libraries; therapeutic development; therapeutic target; tool

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：蛋白质精氨酸甲基转移酶（PRMT）催化S-腺苷甲硫氨酸的甲基加成至精氨酸残基上的胍基氮原子。 PRMT1 对 NIP45（NFAT 相互作用蛋白，45kD）的精氨酸甲基化增强了其与 NFAT 的相互作用，并导致 T 辅助细胞中细胞因子的产生增加。通过精氨酸甲基化对 NIP45 进行共价修饰是调节 NFAT 依赖性细胞因子基因表达的新机制。我们发现了一种新型 PRMT 抑制剂 (Cpd 4)，它可以破坏 PRMT1 和 NIP45 之间的相互作用，从而消除 NFAT 驱动的转录。事实上，Cpd4治疗降低了T辅助细胞因子的表达，例如IFNτ和IL-17A，导致迟发型超敏反应模型中的炎症减轻，这表明PRMT抑制剂可能可用于治疗免疫介导的疾病。 尽管其抑制特性很有前景，但 Cpd 4 仅在高浓度 (100 µM) 下才具有生物活性，这使其不利于治疗开发。为了响应 PAR-09-129，与附近的 Scripps MLPCN 中心合作，我们建议在 300,000 多个 NIH 小分子库的高通量筛选中鉴定 PRMT 1 抑制剂，通过监测荧光偏振信号，测量抑制剂存在下荧光标记马来酰亚胺探针标记活性位点的动力学变化。我们将通过将我们的氟醇测定纳入基于凝胶的二次筛选来排除假阳性和非选择性初级命中。然后，将使用体外甲基化测定和使用特异性识别 PRMT1 细胞靶标的抗体来测试选择性抑制剂抑制 PRMT1 活性的能力。 特定 PRMT 抑制剂的鉴定将为我们提供重要工具来探讨 PRMT 活性在 T 辅助细胞功能中的重要性。由于 PRMT1 活性异常与心血管、恶性、感染和自身免疫性疾病有关，因此它可能是多种适应症的可行治疗靶点。