Biochemical and Cell-Based HTS Assays for RGS17 Inhibitors

描述(由申请人提供)：这项建议是对PA-10-213题为“高通量筛查分析的发展”的回应，用于探针和治疗前的发现。我们的总体目标是开发新的生化和基于细胞的分析方法，用于识别G蛋白信号转导17调节因子(RGS17)的抑制物。RGS17已被发现在肺癌和前列腺癌中过表达，并促进肿瘤细胞生长。报告表明，在肺癌和前列腺癌细胞系中RGS17基因的敲除会降低它们的生长速度，并导致裸鼠异种移植瘤的缩小。不幸的是，没有关于小分子RGS17抑制剂的报道，只有少数几个小分子靶向任何RGS蛋白的报道。因此，RGS17为治疗前的开发提供了一个未知的靶点，并代表了一条开发抗癌先导分子的新途径。因此，RGS17抑制剂的发现将为理解RGS17在肿瘤进展中的作用提供新的途径，并为抗癌药物的开发提供新的小分子先导。为了促进RGS17抑制剂的发现和优化，我们建议使用新的生化和基于细胞的分析方法来开发HTS活动的分析方法。具体地说，我们的目标是建立以下高密度(384-1536井)RGS17：G蛋白α亚单位蛋白：蛋白质相互作用的生化分析方法，以及2)两种新的基于细胞的分析方法，用于实时检测RGS对cAMP水平和GIRK通道动力学的影响。这些目标的实现将为在HTS活动中查询RGS抑制剂的小分子文库提供新的方法，并为RGS17蛋白在癌细胞模型中的功能提供新的化学探针。在开发和验证之后，我们的目标是进行适度的内部HTS筛查，以确定和优化作为RGS17抑制剂的铅分子。该项目的结果将是RGS蛋白配体的新筛选方法和新的探针分子，这些分子将作为研究RGS17蛋白功能的工具，并为治疗前的优化提供初始分子。

DESCRIPTION (provided by applicant): This proposal is in response to PA-10-213 titled "Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic discovery." Our overall goal is develop novel biochemical and cell-based assays that will be used to identify inhibitors of Regulator of G protein signaling 17 (RGS17). RGS17 has been identified as being over-expressed in lung and prostate tumors, and facilitates tumor cell growth. Reports indicate that knockdown of RGS17 in lung and prostate cancer cell lines reduces their rate of growth and results in shrinkage of xenograft tumors in nude mice. Unfortunately, there are no reports of small molecule RGS17 inhibitors, and only a handful of reports of small molecules that target any RGS protein. Therefore, RGS17 presents an unexplored target for pre-therapeutic development and represents a novel path to develop lead molecules in the fight against cancer. Thus, the discovery of RGS17 inhibitors would provide a new avenue for both understanding the role of RGS17 in cancer progression as well as provide new small molecule leads for anticancer drug development. In order to facilitate the discovery and optimization of RGS17 inhibitors, we propose to develop assays for an HTS campaign using novel biochemical and cell-based assays. Specifically, we aim to develop the following 1) a high-density (384-1536 well) biochemical assay for the RGS17:G protein alpha subunit protein: protein interaction, and 2) two novel cell based assays for detection of RGS effects on cAMP levels and GIRK channel kinetics in real-time. Achievement of these aims will provide both novel methodology for use in interrogating small molecule libraries for RGS inhibitors in HTS campaigns as well as provide new chemical probes for RGS17 protein function in cancer cell models. After development and validation, our goal is to perform a modest in-house HTS screen to identify and optimize lead molecules as RGS17 inhibitors. The outcome of this project will be both novel screening methodology for RGS protein ligands and new probe molecules that will serve as tools for studying RGS17 protein function and provide initial molecules for pre-therapeutic optimization.