Enzymatic Screen for RGS Protein Modulators

描述（由申请人提供）：“G蛋白信号传导调节因子”（RGS）蛋白超家族共有一个确定的RGS结构域，该结构域加速异源三聚体G蛋白α亚基的固有GTP水解速率-一种终止激活的G蛋白偶联受体（GPCR）信号传导的酶活性。由于GPCR构成了现有药物的最大单一类别的蛋白质靶点，RGS蛋白作用的小分子调节剂应该为开发新的治疗方法带来巨大希望，但尚未开发出真正的原理验证小分子。我们最近创建了一种新的，强大的，简便的方法，测量RGS结构域GTP酶加速功能，使用荧光偏振。代替传统的、繁琐的、放射性的“单周转”测定，我们开发了基于检测具有改变的核苷酸结合和水解速率的G亚基的RGS加速的GDP产生的高通量筛选（HTS）方法。该酶促测定与先前已通过MLPCN筛选的RGS/G蛋白-蛋白相互作用测定显著不同。我们要求提供资金，将这种HTS检测试剂（包括所需的蛋白质试剂）交付给MLPCN，并促进我们实验室使用各种中等通量二级和反筛选检测试剂对命中进行筛选后分析。 公共卫生相关性：一种特殊的细胞表面蛋白，G蛋白偶联受体超家族，几十年来为各种临床需求和疾病的药物发现提供了有价值的靶标。这些受体的负调节剂（“RGS蛋白”）的大家族在十多年前被发现，但是由于目前缺乏其作用的化学调节剂，它们作为额外药物发现靶标的潜力尚未被测试。在这里，我们描述了一种新的，酶促反应的小分子库的高通量筛选调节RGS蛋白的行动，我们的目的是筛选NIH分子库小分子库（MLSMR）的帮助下，一个节点内的NIH分子库探针生产中心网络（MLPCN）。我们的长期目标是开发新的化学探针和（最终）药物，在RGS蛋白水平上发挥作用，以改善异常GPCR信号转导引起的多种病理条件的治疗。

DESCRIPTION (provided by applicant): The superfamily of "regulator of G-protein signaling" (RGS) proteins share a defining RGS domain that accelerates the intrinsic GTP hydrolysis rate of heterotrimeric G-protein alpha subunits - an enzymatic activity that terminates signaling by activated G-protein coupled receptors (GPCRs). As GPCRs constitute the largest single class of protein target for existing drugs, small molecule modulators of RGS protein action should hold great promise for the development of novel therapeutics, yet no bona fide proof-of-principle small molecule has yet been developed. We recently created a novel, robust, and facile method of measuring RGS domain GTPase-accelerating function using fluorescence polarization. In lieu of the traditional, cumbersome, radioactive "single-turnover" assay, we have developed a high- throughput screening (HTS) method based on detecting RGS-accelerated GDP production by a G subunit with altered nucleotide binding and hydrolysis rates. This enzymatic assay differs considerably from the RGS/G protein-protein interaction assays that have previously been screened by the MLPCN. We request funding to deliver this HTS assay (including required protein reagents) to an MLPCN, as well as to facilitate our lab's post-screening analysis of hits using various medium-throughput secondary and counterscreen assays. PUBLIC HEALTH RELEVANCE: A particular class of cell-surface proteins, the G protein-coupled receptor superfamily, has for many decades provided valuable targets for drug discovery across a variety of clinical needs and diseases. A large family of negative regulators of these receptors (the "RGS proteins") was discovered over ten years ago, but their potential as additional drug discovery targets has yet to be tested owing to the present dearth of chemical modulators of their action. Here we describe a novel, enzymatic reaction for the high-throughput screening of small molecule libraries for modulators of RGS protein action; our intent is to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) with the assistance of one of the nodes within the NIH Molecular Libraries Probe Production Centers Network (MLPCN). Our long-term goal is to develop novel chemical probes and (ultimately) drugs that act at the level of the RGS protein to improve therapy of multiple pathological conditions caused by aberrant GPCR signal transduction.