HTS Assays for Modulators of GPCR Signaling

说明书(申请人提供)：市场上超过50%的药物针对G蛋白偶联受体(GPCRs)。其中最重要的是用于治疗神经疾病的药物，如止痛药、抗抑郁药和抗精神病药物，以及用于神经退行性疾病的药物，如帕金森氏症和阿尔茨海默病。最近发现的一个被称为G蛋白信号调节因子(RGS)的蛋白质家族，通过增加相关G蛋白的GTPase活性来减弱GPCR信号，为调节内源性和给药的GPCR配体的活性开辟了一条新的途径。然而，由于缺乏可靠的高通量筛选(HTS)方法，RGS蛋白抑制剂的开发一直受到阻碍。在第一阶段，我们开发了基于荧光的GDP检测方法来测量修饰G蛋白的稳态GTPase活性的RGS依赖的增加。对G蛋白进行突变，以提高相对于GTP的GDP解离速率，从而可以直接测定RGS蛋白的GTPase加速活性。这些开发包括第一个与HTS兼容的用于测量RGS催化活性的生化分析系统。在第二阶段，我们将把这种方法扩展到更多的RGS和G蛋白，并使用新的分子工具与化学信息学相结合来开发RGS选择性小分子抑制剂。这些抑制剂将在生物化学和细胞环境中进行表征。这些新的HTS检测方法和抑制剂的问世将加速以RGS蛋白为重点的药物发现，并阐明它们在GPCR信号转导中的作用。我们将在第二阶段继续与北卡罗来纳大学医学院药理学系的David Siderovski博士成功合作。公共卫生相关性：目前超过一半的药物通过一种名为G蛋白偶联受体的蛋白质家族发挥作用，这些受体的靶向对用于治疗精神分裂症、抑郁症和帕金森病等神经疾病的药物的开发特别有用。为了加速发现针对这些疾病和其他疾病的更具选择性的治疗方法，我们正在开发和验证一系列蛋白质的新筛选分析方法，该家族被称为“G蛋白信号调节因子”(RGS)，它以组织特异性的方式调节GPCRs配体的作用。

DESCRIPTION (provided by applicant): More than 50% of drugs on the market target G protein-coupled receptors (GPCRs). Among the most important of these are drugs used to treat neurological disorders, such as pain relievers, antidepressants and anti-psychotics, as well drugs used for neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. The relatively recent discovery of a family of proteins called "regulator of G protein signaling" (RGS), that attenuate GPCR signals by increasing the GTPase activity of associated G proteins, has opened up a new avenue for modulating the activity of endogenous and administered GPCR ligands. However, development of inhibitors to RGS proteins has been hampered by the lack of robust assay methods for high throughput screening (HTS). In Phase I we developed fluorescence based GDP detection assays to measure RGS-dependent increases in the steady state GTPase activity of modified G proteins. The G proteins were mutated to increase the rate of GDP dissociation relative to GTP hydrolysis so that the GTPase accelerating activity of RGS proteins could be directly measured. These developments comprise the first HTS-compatible biochemical assay system for measuring RGS catalytic activity. In Phase II we will extend the approach to additional RGS and G proteins, and use the novel molecular tools in combination with cheminformatics to develop RGS-selective small molecule inhibitors. The inhibitors will be characterized biochemically and there in a cellular context. The availability of these novel HTS assays and inhibitors will accelerate drug discovery focused on RGS proteins and delineation of their roles in GPCR signal transduction. We will continue our successful collaboration with Dr. David Siderovski at the Department of Pharmacology, University of North Carolina Medical School in Phase II. PUBLIC HEALTH RELEVANCE: Over half of current drugs exert their effects through a family of proteins called G protein- coupled receptors, and the targeting of these receptors has been especially useful for the development of drugs used to treat neurological disorders such as schizophrenia, depression, and Parkinson's disease. To accelerate discovery of more selective therapies for these and other diseases, we are developing and validating novel screening assays for a family of proteins, called 'regulator of G-protein signaling' (RGS) that modulate the effects of GPCR ligands in a tissue-specific manner.