Real-Time Fluorescence Assay:RGS Domain GAP Activit(RMI)

描述（由申请人提供）：“G蛋白信号传导调节剂”（RGS）蛋白超家族的成员已成为特定G蛋白偶联受体（GPCR）信号转导途径的关键调节剂。通过其“GTP 酶加速蛋白”(GAP) 活性，RGS 蛋白使异三聚体 G 蛋白 α 亚基失活，从而减少 GPCR 信号转导。将现有的 GPCR 激动剂与特定的 RGS 结构域抑制剂相结合应该会增强细胞对这些药物的反应。 RGS 蛋白在人脑中具有高度局部化和动态调节分布的多样性，使其成为帕金森病等中枢神经系统疾病药物治疗的有吸引力的靶点。 不幸的是，没有 RGS 蛋白 GAP 活性的小分子抑制剂（或激活剂）可供公开研究。因此，为了识别小分子工具，进一步加深对特定 GPCR 信号通路中 RGS 蛋白功能的了解，并促进识别用于开发 RGS 蛋白定向疗法的先导化合物，我们将修改和验证新颖的、实时的、基于荧光的 RGS 蛋白功能测定法，用于自动高通量分子筛选：基于荧光共振能量转移 (FRET) 的结合测定法，采用青色荧光蛋白标记的 G-α 亚基和黄色荧光蛋白标记的 RGS 蛋白，这是一种单周转 GTP使用荧光传感器进行无机磷酸盐生产的水解测定，以及使用氟修饰核苷酸 BODIPY(r) FL 2'-(或-3')-O-(N-(2-氨基乙基)氨基甲酸酯)鸟苷 5'-三磷酸进行 G-α 核苷酸结合和水解测定。许多有用的药物通过结合细胞表面特定类型的蛋白质受体发挥作用：G 蛋白偶联受体。 我们的小组发现了一个新的蛋白质家族——RGS 蛋白质——可以干扰这些受体。 我们希望创造方法来筛选新的药物化合物，以阻止 RGS 蛋白的干扰，从而使现有药物发挥更有效的作用。

DESCRIPTION (provided by applicant): Members of the "regulator of G-protein signaling" (RGS)-protein superfamily have emerged as critical modulators of specific G-protein-coupled receptor (GPCR) signal transduction pathways. Via their "GTPase accelerating protein" (GAP) activity, RGS proteins deactivate heterotrimeric G-protein alpha subunits and thereby reduce GPCR signal transduction. Combining existing GPCR agonists with specific RGS domain inhibitors should potentiate cellular responses to these drugs. The diversity of RGS proteins with highly localized and dynamically regulated distributions in the human brain, makes them attractive targets for pharmacotherapy of central nervous system disorders such as Parkinson's disease. Unfortunately, no small molecule inhibitor (or activator) of RGS protein GAP activity is publicly available for study. Therefore, to identify small molecule tools for further advancing knowledge of RGS protein function in specific GPCR signaling pathways, and also to facilitate identification of lead compounds for developing RGS protein directed therapeutics, we will modify and validate novel, real-time, fluorescence-based assays of RGS protein function for automated high throughput molecular screening: a fluorescence resonance energy transfer (FRET)-based binding assay that employs cyan fluorescent protein-labeled G-alpha subunits and yellow fluorescent protein-labeled RGS proteins, a single-turnover GTP hydrolysis assay using a fluorescent sensor for inorganic phosphate production, and an assay of G-alpha nucleotide binding and hydrolysis that employs the fluor-modified nucleotide BODIPY(r) FL 2'-(or-3')-O-(N-(2-aminoethyl)urethane)guanosine 5'-triphosphate. Many useful drugs act by binding a particular type of protein receptor on the cell's surface: a G-protein coupled receptor. Our group has discovered a new family of proteins-the RGS proteins-that interfere with these receptors. We wish to create ways to screen for new drug compounds that can stop RGS proteins from interfering and thereby allow existing drugs to act more potently.