Enzymatic Screen for RGS Protein Modulators

描述（由申请人提供）：“g蛋白信号调节因子”（RGS）蛋白超家族共享一个定义的RGS结构域，该结构域加速异三聚体g蛋白α亚基的内在GTP水解速率-一种通过活化的g蛋白偶联受体（gpcr）终止信号传导的酶活性。由于gpcr是现有药物中最大的一类蛋白质靶点，因此RGS蛋白作用的小分子调节剂应该对新疗法的开发有很大的希望，但目前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颖、稳健、简便的方法，利用荧光偏振测量RGS结构域gtpase加速功能。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“单周转率”测定，该方法基于检测rgs加速GDP产生的G亚基，其核苷酸结合和水解速率发生了改变。这种酶测定法与MLPCN先前筛选的RGS/G蛋白-蛋白相互作用测定法有很大不同。我们请求资助将HTS测定（包括所需的蛋白质试剂）提供给MLPCN，并促进我们实验室使用各种中等通量二次和反筛测定对hit进行筛选后分析。

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.