Multiplexed flow cytometry screens for RGS inhibitors

描述（由申请人提供）：信号转导过程是药物发现的主要靶点，G蛋白偶联受体是许多当前治疗剂的主要作用位点。然而，最近的研究表明，信号通路不仅仅是线性的信息链，而是相互作用的调节分子的网络，其中蛋白质支架、细胞内接近性和抑制控制是信号有效性和特异性的主要决定因素。抑制G蛋白信号的20个G蛋白信号调节因子（RGS）蛋白家族成员代表了一个新的药物干预位点，但是：1)它们的生理功能仍然不完全清楚；2)没有报道的RGS功能的小分子抑制剂。选择性RGS抑制剂的鉴定将提供：1)研究RGS在细胞和体内功能的工具；2)治疗药物开发的起点。定量RGS/Ga相互作用的流式细胞术方法将适用于RGS抑制剂的多路高通量筛选。荧光标记的Ga蛋白亚基与纯化的RGS蛋白在微球上的结合将被优化，以允许同时检测Ga与RGS 4、6、7、8和9的结合。这种多路复用方法既可以加快筛选的吞吐量，又可以提供关于初级筛选中确定的化学物质特异性的即时信息。描述了在细胞培养模型中演示功能活性和效果的二次分析。本项目的最终目的是鉴定RGS作用的选择性小分子抑制剂。这将提供重要的化学工具并加速新疗法的发展。

DESCRIPTION (provided by applicant): 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.