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

描述（由申请人提供）：“G蛋白信号转导调节因子”（RGS）蛋白超家族成员已成为特异性G蛋白偶联受体（GPCR）信号转导途径的关键调节剂。通过其“GPCR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激动剂与特异性RGS结构域抑制剂组合应增强对这些药物的细胞应答。RGS蛋白在人脑中具有高度局部化和动态调节的分布的多样性，使其成为中枢神经系统疾病如帕金森病的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）可公开用于研究。因此，为了鉴定小分子工具以进一步推进RGS蛋白在特定GPCR信号传导途径中的功能的知识，并且还为了促进鉴定用于开发RGS蛋白定向治疗剂的先导化合物，我们将修改和验证用于自动化高通量分子筛选的RGS蛋白功能的新型实时基于荧光的测定：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蛋白的基于荧光共振能量转移（FRET）的结合测定，使用荧光传感器的单转换GTP水解测定用于无机磷酸盐产生，以及使用荧光修饰的核苷酸BODIPY（r）FL 2 ′-（或-3 ′）-O-（N-（2-氨乙基）氨基甲酸酯）鸟苷5 ′-三磷酸的G-α核苷酸结合和水解的测定。许多有用的药物通过结合细胞表面上的特定类型的蛋白质受体而起作用：G蛋白偶联受体。 我们的研究小组已经发现了一个新的蛋白质家族-RGS蛋白质-干扰这些受体。 我们希望创造方法来筛选新的药物化合物，可以阻止RGS蛋白的干扰，从而使现有的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.