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

描述（由申请人提供）：“g蛋白信号调节因子”(RGS)-蛋白超家族成员已成为特定g蛋白偶联受体（GPCR）信号转导途径的关键调节剂。通过其“GTPase加速蛋白”（GAP）活性，RGS蛋白使异源三聚体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结构域抑制剂联合使用，可以增强细胞对这些药物的反应。RGS蛋白在人脑中具有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有吸引力的靶点。不幸的是，没有公开的RGS蛋白GAP活性的小分子抑制剂（或激活剂）可供研究。因此，为了确定小分子工具，以进一步推进对特定GPCR信号通路中RGS蛋白功能的了解，并促进鉴定用于开发RGS蛋白定向治疗的先导化合物，我们将修改和验证新的、实时的、基于荧光的RGS蛋白功能检测方法，用于自动化高通量分子筛选。一种基于荧光共振能量转移（FRET）的结合试验，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，一种使用荧光传感器进行无机磷酸盐生产的单次转化GTP水解试验，以及一种使用荧光修饰核苷酸BODIPY(r) FL 2'-(or-3')- o -（N-（2-氨基乙基）氨基甲酸乙酯）鸟苷5'-三磷酸的g - α核苷酸结合和水解试验。许多有用的药物是通过结合细胞表面的一种特殊类型的蛋白质受体起作用的：一种g蛋白偶联受体。我们的研究小组发现了一个新的蛋白质家族——RGS蛋白——它会干扰这些受体。我们希望找到一种方法来筛选能够阻止RGS蛋白干扰的新药物化合物，从而使现有药物更有效地发挥作用。

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.