Enzymatic Screen for RGS Protein Modulators

RGS 蛋白调节剂的酶法筛选

基本信息

批准号：
8066323
负责人：
David P. Siderovski
金额：
$ 3.59万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8066323
关键词：
Agonist Anxiety Attenuated Binding Biochemical Reaction Biological Assay Brain Cell Surface Proteins Cells Central Nervous System Diseases Chemicals Clinical Collaborations Development Disease Family Fluorescence Polarization Funding G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors G-substrate GTP-Binding Protein Regulators GTP-Binding Protein alpha Subunits GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Goals Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Heterotrimeric GTP-Binding Proteins Human Hydrolysis In Vitro Knowledge Lead Measures Methods Molecular Bank Nucleotides Parkinson Disease Pharmaceutical Preparations Pharmacotherapy Production Proteins RGS Domain RGS Proteins Radioactive Reagent Schizophrenia Screening procedure Signal Pathway Signal Transduction Solubility Specificity Synthesis Chemistry Testing Therapeutic United States National Institutes of Health analog aqueous base clinically relevant counterscreen drug development drug discovery high throughput screening improved inhibitor/antagonist member novel novel therapeutics protein function protein protein interaction public health relevance receptor repository response small molecule small molecule libraries tool

项目摘要

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.

描述（由申请人提供）：“ G蛋白信号传导调节剂”（RGS）蛋白的超家族共享一个定义的RGS结构域，可加速固有的GTP GTP水解速率GTP g蛋白Alpha亚基的固有GTP水解速率 - 一种通过激活的Gotote copplote consiners cypletors（gpplect）终止的综合作用（一种示例性的信号传导）。由于GPCR构成了现有药物最大的单类蛋白质靶标，因此RGS蛋白质作用的小分子调节剂应对新型疗法的发展具有巨大的希望，但是尚未开发出真正的原理证明。最近，我们创建了一种新颖，健壮，便捷的方法，用于使用荧光极化测量RGS域GTPase加速功能。代替传统的，繁琐的放射性“单转”测定法，我们开发了一种高吞吐量筛选（HTS）方法，基于检测RGS加速GDP的GDP产生，G亚基具有改变的核苷酸结合和水解速率。该酶试验与以前由MLPCN筛选的RGS/G蛋白 - 蛋白质相互作用分析有很大不同。我们要求资金将此HTS分析（包括所需的蛋白质试剂）传递给MLPCN，并使用各种中型次要二级和尺寸测定法进行了实验室的屏幕后分析。公共卫生相关性：G蛋白偶联受体超家族的一类特定的细胞表面蛋白已经在数十年中为跨多种临床需求和疾病的药物发现提供了宝贵的靶标。十年前发现了这些受体（“ RGS蛋白”）的大量负调节剂（“ RGS蛋白”），但是由于目前的作用化学调节剂的缺乏，它们作为其他药物发现靶标的潜力尚未进行测试。在这里，我们描述了用于对RGS蛋白质调节剂的小分子库进行高通量筛选的新型酶促反应。我们的目的是筛选NIH分子库小分子存储库（MLSMR），并在NIH分子库中的一个节点探测生产中心网络（MLPCN）中。我们的长期目标是开发新的化学探针和（最终）在RGS蛋白水平上起作用的药物，以改善因异常GPCR信号转导引起的多种病理状况的治疗。