HTS Assays for Modulators of GPCR Signaling

描述（由申报人提供）：市场上超过50%的药物靶向G蛋白偶联受体（gpcr）。其中最重要的是用于治疗神经系统疾病的药物，如止痛药、抗抑郁药和抗精神病药，以及用于治疗神经退行性疾病的药物，如帕金森病和阿尔茨海默病。最近发现的一个名为“G蛋白信号调节因子”（RGS）的蛋白质家族，通过增加相关Ga蛋白的GTPase活性来减弱GPCR信号，为调节内源性和给药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）检测方法，RGS蛋白抑制剂的开发一直受到阻碍。为了克服这一技术障碍，我们建议使用基于GDP的荧光免疫检测来测量修饰Ga蛋白的rgs依赖性稳态GTPase活性的增加。基于BellBrook实验室先前用于开发ADP检测分析的方法，将开发用于检测GDP的高度特异性单克隆抗体和荧光示踪剂。Ga蛋白将发生突变，以增加相对于GTP水解的GDP解离速率，使前者不再是速率限制。这些新的HTS检测方法的可用性将加速关注RGS蛋白的药物发现，并描述它们在GPCR信号转导中的作用。目前超过一半的药物是通过G蛋白偶联受体家族发挥作用的，而这些受体的靶向性对于治疗神经系统疾病（如精神分裂症、抑郁症和帕金森病）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多选择性治疗方法，我们正在开发一种新的蛋白质家族筛选方法，称为“g蛋白信号调节因子”（RGS），它以组织特异性的方式调节GPCR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 Ga 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). To overcome this technical hurdle, we propose to use fluorescence based immunodetection of GDP to measure RGS-dependent increases in the steady state GTPase activity of modified Ga proteins. Based on the approach used previously at BellBrook Labs for development of an ADP detection assay, a highly specific monoclonal antibody and fluorescent tracer will be developed for detection of GDP. The Ga proteins will be mutated to increase the rate of GDP dissociation relative to GTP hydrolysis so that the former is no longer rate limiting. The availability of these novel HTS assays will accelerate drug discovery focused on RGS proteins and delineation of their roles in GPCR signal transduction. 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 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.