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（PRMTs）催化从S-腺苷蛋氨酸到精氨酸残基上胍基氮原子的甲基添加。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升高。精氨酸甲基化介导NIP45共价修饰是调控nfat依赖性细胞因子基因表达的一种新机制。我们已经确定了一种新的PRMT抑制剂（Cpd 4），它破坏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治疗降低了辅助性T细胞因子的表达，如IFN¿和IL-17A，导致延迟型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性疾病有用。尽管cpd4具有良好的抑制作用，但它只有在高浓度（100¿M）时才具有生物活性，这对治疗发展不利。为了响应PAR-09-129，我们联合附近的Scripps MLPCN中心，提出在30万+ 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，测量抑制剂存在时荧光标记的马来酰亚胺探针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原发命中，将我们的氟波检测纳入二次凝胶筛选。然后使用体外甲基化测定和特异性识别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。特异性PRMT抑制剂的鉴定将为我们探索PRMT活性在T辅助细胞功能中的重要性提供重要的工具。由于异常的PRMT1活性与心血管、恶性、感染性和自身免疫性疾病有关，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.