An Activity-Based Assay to Screen for PRMT1 Inhibitors

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与精氨酸残基上胍基氮原子的加成。通过PRMT 1对NIP 45（NFAT相互作用蛋白，45 kD）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产生升高。通过精氨酸甲基化共价修饰NIP 45是调节NFAT依赖性细胞因子基因表达的新机制。我们已经鉴定了一种新的PRMT抑制剂（Cpd 4），其破坏PRMT 1和NIP 45之间的相互作用，导致废除NFAT驱动的转录。事实上，Cpd 4治疗降低了T辅助细胞因子如IFN γ和IL-17 A的表达，导致迟发型超敏反应模型中炎症减少，表明PRMT抑制剂可用于治疗免疫介导的疾病。 尽管其具有良好的抑制作用，但化合物4仅在高浓度（100 μ M）下具有生物活性，因此不利于治疗开发。为了响应PAR-09-129，与附近的Scripps MLPCN中心合作，我们建议在300，000 + 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下用荧光标记的马来酰亚胺探针标记活性位点的动力学变化。我们将排除假阳性和非选择性的初步命中，将我们的荧光检测纳入二级凝胶为基础的屏幕。然后使用体外甲基化测定和使用特异性识别PRMT 1的细胞靶标的抗体来测试选择性抑制剂抑制PRMT 1活性的能力。 特异性PRMT抑制剂的鉴定将为我们提供重要的工具来探测PRMT活性在T辅助细胞功能中的重要性。由于异常的PRMT 1活性与心血管、恶性肿瘤、感染性和自身免疫性疾病相关，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.