Multiplexed flow cytometry screens for RGS inhibitors

描述(申请人提供)：信号转导过程是药物发现的主要目标，G蛋白偶联受体是许多当前治疗药物的主要作用部位。然而，最近的工作表明，信号通路不仅是线性信息链，而且是相互作用的调控分子的网络，其中蛋白质支架、细胞内接近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蛋白信号转导的20个G蛋白信号调节蛋白(RGS)家族成员是药物干预的新靶点，但它们的生理功能仍不完全清楚，RGS功能的小分子抑制剂尚未见报道。选择性RGS抑制剂的鉴定将提供：1)研究RGS在细胞和体内的功能的工具；2)治疗药物开发的起点。一种定量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蛋白亚基与珠体上纯化的RGS蛋白的结合将得到优化，以允许同时检测与RGS 4、6、7、8和9结合的GA。这种多重方法既加快了筛查的速度，又将提供关于初级筛查中鉴定的化学物质的特异性的即时信息。二次分析，以证明功能活性和影响的细胞培养模型被描述。该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.