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：
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
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/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

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（由应用提供）：蛋白精氨酸甲基转移酶（PRMTS）催化从s-腺苷硫氨氨酸添加甲基在精氨酸保留时向鸟尼迪诺诺氮原子添加。通过PRMT1，NIP45（NFAT相互作用蛋白，45KD）的精氨酸甲基化增加了与NFAT的相互作用，并导致T辅助细胞中细胞因子产生升高。精氨酸甲基化对NIP45的共价修饰是调节NFAT依赖性细胞因子基因表达的新机制。我们已经确定了一种新型的PRMT抑制剂（CPD 4），该抑制剂破坏了PRMT1和NIP45之间的相互作用，从而消除了NFAT驱动的转录。实际上，CPD4治疗降低了T辅助细胞因子（例如IFN¿和IL-17A）的表达，从而导致延迟类型的超敏反应模型的注射减少，这表明PRMT抑制剂可能有助于治疗免疫介导的疾病。尽管CPD 4仅在高浓度（100€）的生物学上具有抑制作用，但使其不利于治疗性发育。 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.我们将通过将我们的Fluolopol分析编码为二级凝胶屏幕来排除假阳性和非选择性的主要命中率。然后，将测试选择性抑制剂的能力，其能力使用体外甲基化测定和使用专门识别PRMT1细胞靶标的抗体抑制PRMT1活性。特定PRMT抑制剂的识别将为我们提供重要的工具，以探测PRMT活性在T辅助细胞功能中的重要性。由于异常PRMT1活性与心血管，恶性，传染性和自身免疫性疾病有关，因此它可能是多种迹象的可行治疗靶标。