Enzymatic Screen for RGS Protein Modulators

RGS 蛋白调节剂的酶法筛选

• 批准号: 7928424
• 负责人: David P. Siderovski
• 金额: $ 3.7万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 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/7928424
• 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; Signal Pathway; Signal Transduction; Solubility; Specificity; Synthesis Chemistry; Testing; Therapeutic; United States National Institutes of Health; analog; aqueous; base; clinically relevant; counterscreen; 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

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) 蛋白超家族共享一个定义的 RGS 结构域，该结构域可加速异源三聚体 G 蛋白 α 亚基的内在 GTP 水解速率，这是一种通过激活的酶活性终止信号传导的酶活性。 G 蛋白偶联受体 (GPCR)。由于 GPCR 构成了现有药物最大的单一类别蛋白质靶标，RGS 蛋白质作用的小分子调节剂应该为新型疗法的开发带来巨大希望，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颖、稳健且简便的方法，使用荧光偏振测量 RGS 结构域 GTPase 加速功能。代替传统的、繁琐的放射性“单周转”测定，我们开发了一种高通量筛选（HTS）方法，该方法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。这种酶促检测与 MLPCN 之前筛选的 RGS/G 蛋白-蛋白相互作用检测有很大不同。我们请求资金将该 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级和反筛选测定对命中进行筛选后分析。 公共卫生相关性：一类特殊的细胞表面蛋白，即 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点。这些受体的负调节因子（“RGS 蛋白”）的一大家族在十多年前就被发现了，但由于目前缺乏其作用的化学调节剂，它们作为其他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促反应，用于高通量筛选 RGS 蛋白作用调节剂的小分子库；我们的目的是在 NIH 分子库探针生产中心网络 (MLPCN) 内的节点之一的协助下筛选 NIH 分子库小分子存储库 (MLSMR)。我们的长期目标是开发新型化学探针和（最终）在 RGS 蛋白水平发挥作用的药物，以改善由异常 GPCR 信号转导引起的多种病理状况的治疗。