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

描述(申请人提供)：“G蛋白信号调节因子”(RGS)-蛋白质超家族的成员已成为特定G蛋白偶联受体(GPCRR)信号转导通路的关键调节器。RGS蛋白通过其“GTP酶加速蛋白”(GAP)活性，使异源三聚体G蛋白α亚单位失活，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蛋白在人脑中的高度定位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靶点。不幸的是，目前还没有RGS蛋白GAP活性的小分子抑制物(或激活剂)可供研究。因此，为了进一步了解RGS蛋白在特定的GPCR信号通路中的功能，也为了便于鉴定开发RGS蛋白导向疗法的先导化合物，我们将修改和验证用于自动化高通量分子筛选的新颖的、实时的、基于荧光的RGS蛋白功能分析方法：基于荧光共振能量转移(FRET)的结合分析方法，其使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，使用用于生产无机磷的荧光传感器的一次周转GTP水解法，以及使用氟修饰的核苷酸BODIPY(R)FL 2‘-(or-3’)-O-(N-(2-aminoethyl)urethane)guanosine 5‘-三磷酸的G-α核苷酸结合和水解的分析。许多有用的药物通过结合细胞表面的一种特定类型的蛋白质受体来发挥作用：G蛋白偶联受体。我们的团队发现了一个新的蛋白质家族--RGS蛋白质--它可以干扰这些受体。我们希望创造方法来筛选可以阻止RGS蛋白干扰的新药化合物，从而允许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.