Biochemical and Cell-Based HTS Assays for RGS17 Inhibitors

描述（由申请人提供）：本提案是对PA-10-213的回应，标题为“开发用于探针和治疗前发现的高通量筛选试验。“我们的总体目标是开发新的生物化学和基于细胞的检测方法，用于鉴定G蛋白信号调节因子17（RGS 17）的抑制剂。RGS 17已被鉴定为在肺和前列腺肿瘤中过表达，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RGS 17的敲低降低了它们的生长速率，并导致裸鼠中异种移植肿瘤的缩小。不幸的是，没有小分子RGS 17抑制剂的报道，并且只有少数小分子靶向任何RGS蛋白的报道。因此，RGS 17为治疗前开发提供了一个未探索的靶点，并代表了开发抗癌先导分子的新途径。因此，RGS 17抑制剂的发现将为理解RGS 17在癌症进展中的作用以及为抗癌药物开发提供新的小分子先导物提供新的途径。为了促进RGS 17抑制剂的发现和优化，我们建议使用新的生物化学和基于细胞的测定来开发用于HTS活动的测定。具体而言，我们的目标是开发以下1）RGS 17：G蛋白α亚基蛋白：蛋白相互作用的高密度（384-1536孔）生物化学测定，和2）用于实时检测RGS对cAMP水平和GIRK通道动力学的影响的两种新型基于细胞的测定。这些目标的实现将提供用于在HTS活动中询问RGS抑制剂的小分子文库的新方法，以及提供用于癌细胞模型中RGS 17蛋白功能的新化学探针。在开发和验证之后，我们的目标是进行适度的内部HTS筛选，以识别和优化作为RGS 17抑制剂的先导分子。该项目的成果将是RGS蛋白配体的新型筛选方法和新的探针分子，这些探针分子将作为研究RGS 17蛋白功能的工具，并为治疗前优化提供初始分子。

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.