An Activity-Based Assay to Screen for PRMT1 Inhibitors

描述(申请人提供)：蛋白质精氨酸甲基转移酶(PRMTs)催化从S-腺苷蛋氨酸到精氨酸残基上的胍基氮原子上的甲基加成。PRMT1使NIP45(NFAT相互作用蛋白，45kD)的精氨酸甲基化增强其与NFAT的相互作用，导致辅助性T细胞产生细胞因子的增加。精氨酸甲基化修饰NIP45是调控NFAT依赖的细胞因子基因表达的一种新机制。我们已经发现了一种新的PRMT抑制剂(CPD 4)，它可以破坏PRMT1和NIP45之间的相互作用，导致NFAT驱动的转录被取消。事实上，Cpd4治疗减少了T辅助细胞因子的表达，如干扰素和IL-17A，导致迟发性超敏反应模型中的炎症减轻，这表明PRMT抑制剂可能对治疗免疫介导的疾病有用。尽管CPD 4具有良好的抑制活性，但它只有在高浓度(100M)时才具有生物活性，因此不利于治疗开发。作为对PAR-09-129的响应，我们与附近的斯克里普斯MLPCN中心合作，建议在300,000+NIH小分子文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来测量在抑制剂存在的情况下用荧光标记的马来酰亚胺探针标记活性部位的动力学变化。我们将通过将我们的Fluopol分析整合到基于凝胶的二次筛查中来排除假阳性和非选择性的一次点击。然后，将使用体外甲基化试验和使用特异性识别PRMT1细胞靶点的抗体来测试选择性抑制剂抑制PRMT1活性的能力。识别特定的PRMT抑制剂将为我们探索PRMT活性在T辅助细胞功能中的重要性提供重要工具。由于PRMT1活性异常与心血管疾病、恶性疾病、感染性疾病和自身免疫性疾病有关，因此它可能是几种适应症的可行治疗靶点。

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.