Enzymatic Screen for RGS Protein Modulators

描述(申请人提供)：“G-蛋白信号转导调节因子”(RGS)蛋白的超家族共享一个明确的RGS结构域，该结构域可加速异源三聚体G-蛋白α亚基的固有GTP水解率--一种通过激活的G-蛋白偶联受体(GPCRs)终止信号的酶活性。由于GPCRs是现有药物中最大的单一蛋白质靶点，RGS蛋白作用的小分子调节剂应该会为新的治疗药物的开发带来巨大的希望，但还没有真正的原理证明小分子被开发出来。我们最近创造了一种新的、稳健的、简便的方法来测量RGS结构域GTP酶加速功能的荧光偏振。我们开发了一种高通量筛选(HTS)方法，取代了传统的、繁琐的、放射性的“单周转”分析方法，该方法基于检测核苷酸结合和水解率改变的G亚基加速的RGS GDP产生。这种酶活性测定与MLPCN以前筛选的RGS/G蛋白-蛋白质相互作用测定有很大的不同。我们申请资金将HTS分析(包括所需的蛋白质试剂)交付给MLPCN，并使用各种中通量二次和反筛选分析来促进我们实验室的HITS筛查后分析。 与公共健康相关：一类特殊的细胞表面蛋白，G蛋白偶联受体超家族，几十年来一直为各种临床需求和疾病的药物开发提供有价值的靶点。这些受体(RGS蛋白)的一大类负调控因子在十多年前就被发现了，但由于目前缺乏它们作用的化学调节剂，它们作为额外药物发现靶点的潜力尚未得到测试。在这里，我们描述了一种新颖的酶反应，用于高通量筛选RGS蛋白质作用调节器的小分子文库；我们的目的是在NIH分子文库探针生产中心网络(MLPCN)中的一个节点的帮助下筛选NIH分子文库小分子文库(MLSMR)。我们的长期目标是开发新的化学探针和(最终)作用于RGS蛋白水平的药物，以改善由异常的GPCR信号转导引起的多种病理疾病的治疗。

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.