Biochemical and Cell-Based HTS Assays for RGS17 Inhibitors

RGS17 抑制剂的生化和基于细胞的 HTS 测定

• 批准号: 8462940
• 负责人: David L. Roman
• 金额: $ 29.45万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-24 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462940
• 关键词: Achievement; Adenylate Cyclase; Advanced Development; Antineoplastic Agents; Biochemical; Biological Assay; Biopsy Specimen; Biosensor; CREB1 gene; Cancer Biology; Cancer cell line; Cell Proliferation; Cell model; Cells; Chemicals; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Detection; Development; Fluorescence; G Protein-Coupled Receptor Signaling; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Housing; Kinetics; Laboratories; Lead; Ligands; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Measures; Mediating; Methodology; Mission; Mus; Nude Mice; Outcome; PC3 cell line; Pathway interactions; Pharmaceutical Chemistry; Phenotype; Play; Prostatic Neoplasms; Proteins; Public Health; RGS Proteins; Reporting; Research; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Therapeutic; Time; Tumor Cell Line; United States National Institutes of Health; Validation; anti-cancer therapeutic; assay development; base; cancer cell; cell growth; density; drug development; fight against; high throughput screening; human disease; inhibitor/antagonist; neoplastic cell; novel; overexpression; protein function; protein protein interaction; receptor coupling; response; screening; second messenger; small hairpin RNA; small molecule; small molecule libraries; therapeutic development; tool; tumor; tumor progression; tumor xenograft

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.

描述（由申请人提供）：本提案是对题为“用于探针和治疗前发现的高通量筛选的测定的开发”的 PA-10-213 的回应。我们的总体目标是开发新型生化和细胞检测方法，用于鉴定 G 蛋白信号传导调节因子 17 (RGS17) 的抑制剂。 RGS17 已被确定在肺和前列腺肿瘤中过度表达，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敲除 RGS17 会降低其生长速度，并导致裸鼠中异种移植肿瘤的缩小。不幸的是，目前还没有关于小分子 RGS17 抑制剂的报道，并且只有少数关于针对任何 RGS 蛋白的小分子的报道。因此，RGS17 为治疗前开发提供了一个尚未探索的靶标，并代表了开发抗癌先导分子的新途径。因此，RGS17抑制剂的发现将为了解RGS17在癌症进展中的作用提供新途径，并为抗癌药物开发提供新的小分子先导物。为了促进 RGS17 抑制剂的发现和优化，我们建议使用新型生化和细胞检测方法来开发 HTS 活动的检测方法。具体来说，我们的目标是开发以下方法：1) 针对 RGS17:G 蛋白 α 亚基蛋白：蛋白质相互作用的高密度（384-1536 孔）生化测定，以及 2) 两种新颖的基于细胞的测定，用于检测 RGS 对 cAMP 的影响实时水平和 GIRK 通道动力学。这些目标的实现将为在 HTS 活动中研究 RGS 抑制剂小分子库提供新的方法，并为癌细胞模型中 RGS17 蛋白功能提供新的化学探针。经过开发和验证后，我们的目标是进行适度的内部 HTS 筛选，以识别和优化作为 RGS17 抑制剂的先导分子。该项目的成果将是 RGS 蛋白配体的新颖筛选方法和新的探针分子，它们将作为研究 RGS17 蛋白功能的工具，并为治疗前优化提供初始分子。