Multiplexed flow cytometry screens for RGS inhibitors

信号转导过程是药物发现的主要靶点，G蛋白偶联受体是一种 目前许多治疗药物的主要作用部位。然而，最近的研究表明，信号 通路不仅是线性信息链，而且是相互作用的调节分子的网络，在其中 蛋白质支架、细胞内接近性和抑制控制是信号有效性的主要决定因素 和专一性。抑制G蛋白信号转导的二十个蛋白家族成员 蛋白质信号转导代表了药物干预的新领域，但是：1)它们的生理功能 2)RGS功能的小分子抑制剂尚未见报道。这个 鉴定选择性RGS抑制剂将提供两个方面：1)研究细胞中RGS功能的工具 和体内，以及2)治疗药物开发的起点。一种新的流式细胞术定量检测方法 RGS/GA相互作用将适用于RGS抑制剂的多路高通量筛选。这个 荧光标记的GA蛋白亚基与纯化的RGS蛋白在珠子上的结合将优化为 允许同时检测与RGS 4、6、7、8和9结合的多个GA。这种多路复用方法将 两者都加快了筛查的速度，并将提供有关化学品专一性的即时信息 在主屏幕中标识。细胞培养中功能活性和效应的二次分析 对模型进行了描述。该项目的最终目的是鉴定选择性小分子。 RGS作用的抑制剂。这将提供重要的化学工具，加速新奇的发展 治疗学。

Signal transduction processes are major targets of drug discovery with G protein-coupled receptors being a primary site of action of many current therapeutic agents. Recent work, however, has shown that signaling pathways are not just linear chains of information but are webs of interacting regulatory molecules in which protein scaffolding, intracellular proximity, and inhibitory control are major determinants of signaling efficacy and specificity. The twenty Regulator of G protein Signaling (RGS) protein family members which inhibit G protein signaling represent a novel site of pharmacologic intervention but: 1) their physiological functions remain incompletely understood and 2) there are no reported small molecule inhibitors of RGS function. The identification of selective RGS inhibitors would provide both: 1) tools for the study of RGS function in cells and in vivo and 2) a starting point for therapeutic drug development. A flow cytometry method for quantitating RGS/Ga interactions will be adapted for multiplexed high-throughput screening for RGS inhibitors. The binding of fluorescently labeled Ga protein subunits to purified RGS proteins on beads will be optimized to permit multiple simultaneous assays of Ga binding to RGS 4, 6, 7, 8, and 9. This multiplexed method will both speed the throughput of screens and will provide immediate information on specificity of the chemicals identified in primary screens. Secondary analyses to demonstrate functional activity and effects in cell culture models are described. The ultimate aim of this project is the identification of selective small molecule inhibitors of RGS action. This will provide important chemical tools and accelerate the development of novel therapeutics.