An Activity-Based Assay to Screen for PRMT1 Inhibitors

基于活动的 PRMT1 抑制剂筛选试验

基本信息

批准号：
8324861
负责人：
KERRI A MOWEN
金额：
$ 4.74万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
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/8324861
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

项目摘要

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）通过 PRMT1 催化 NIP45（NFAT 相互作用蛋白，45kD）的精氨酸甲基化从 S-腺苷甲硫氨酸向精氨酸残基上的胍基氮原子添加甲基，从而增强其与NFAT 并导致 T 辅助细胞中细胞因子的产生增加。 NIP45 的精氨酸甲基化是调节 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)，它会破坏 PRMT1 和 NIP45 之间的相互作用，从而导致 NFAT 驱动的转录被废除。治疗后 T 辅助细胞因子的表达减少，例如 IFN¿和 IL-17A，导致迟发型超敏反应模型中的炎症减轻，表明 PRMT 抑制剂可能可用于治疗免疫介导的疾病，尽管 Cpd 4 具有良好的抑制作用，但仅在高浓度 (100 µM) 下才具有生物活性。，使其不利于治疗开发。针对 PAR-09-129，我们建议与附近的 Scripps MLPCN 中心一起鉴定高浓度的 PRMT 1 抑制剂。 300,000+ NIH 小分子库的通量筛选，通过监测荧光偏振信号来测量荧光标记马来酰亚胺探针在抑制剂存在下的活性位点标记动力学的变化，我们将排除假阳性和非选择性初级。通过将我们的 Fluopol 测定纳入基于凝胶的二级筛选中，然后使用体外抗体甲基化测定并使用特异性识别细胞来测试其抑制 PRMT1 活性的能力。特定 PRMT 抑制剂的鉴定将为我们探讨 PRMT 活性在 T 辅助细胞功能中的重要性提供重要工具，因为异常的 PRMT1 活性与心血管、恶性、感染性和自身免疫性疾病有关。多种适应症的可行治疗靶点。