Development of cAMP Biosensor, High Content Imaging, and Functional GLP-1R aSSAYS

描述(申请人提供)：这项工作的目标是开发一系列新的高通量的胰高血糖素样肽1受体(GLP-1R)分析，能够推动针对这一临床验证的II型糖尿病(T2 DM)靶标的药物发现计划。除了GLP-1R检测外，我们还将开发另外两种密切相关的受体，即胰高血糖素样肽2受体(GLP-2R)和胰高血糖素受体(GCG-R)的新检测方法，这将作为选择性与GLP-1R的辅助筛查。美国糖尿病协会报告称，有2080万美国人，即7%的人口患有糖尿病(www.diabetes.org)。FDA最近批准了艾塞那肽，一种合成肽GLP-1类似物，用于治疗T2 DM。虽然埃塞那肽非常有效，但它受到药代动力学和剂量的限制，这使得发现小分子口服GLP-1R激动剂或增效剂是一项有价值的临床进步。GLP-1R是一种被广泛研究的G蛋白偶联受体，也是制药行业许多药物发现计划的靶点。尽管在GLP-1R激动剂的发现上投入了大量的资源和努力，但还没有发现GLP-1R的小分子激动剂的报道，也没有小分子激动剂进入临床开发，所有对初级筛查数据的访问仍然是专有信息。在本申请中，我们提出了三种可能有助于克服发现GLP-1R小分子激动剂的障碍的新方法。这三种方法将包括：1)初级cAMP生物传感器筛选，以检测GLP-1R的选择性激动剂、增强剂和拮抗剂；2)功能性高含量分析，其目的是监测受体内化，并区分小分子的药理学；以及3)具有生理意义的高含量分析，用于测量原代β细胞凋亡。这些分析将产生一个工作流程，为HITS鉴定、HITS确认以排除伪影、计数器筛查以确定选择性以及HITS的受体和生理功能分析提供主要筛查以用于铅的开发。

DESCRIPTION (provided by applicant): The goal of this work is to develop a series of novel high throughput assays for the glucagon-like peptide 1 receptor (GLP-1R) which are capable of driving a drug discovery program for this clinically validated target of type II diabetes mellitus (T2DM). In addition to the GLP-1 R assays, we will also develop novel assays for two other closely related receptors, glucagon-like peptide 2 receptor (GLP-2R), and glucagon receptor (GCG-R) which will serve as counter screens for selectivity versus GLP-1 R. The American Diabetes Association reports that there are 20.8 million Americans, or 7% of the population, who have diabetes (www.diabetes.org). The FDA has recently approved exenatide, a synthetic peptide GLP-1 analog for the treatment of T2DM. While highly efficacious, exenatide suffers pharmacokinetic and dosing limitations that would make the discovery of small molecule orally available GLP-1 R agonists or potentiators a valuable clinical advance. GLP-1 R has been a widely studied G-protein coupled receptor (GPCR) and the target of many drug discovery programs within the pharmaceutical industry. Despite the significant resources and effort devoted to discovery of GLP-1 R agonists, there have been no reports for the discovery of small molecule agonists for GLP-1 R, no small molecule agonists have entered clinical development, and all access to primary screening data remains proprietary information. In this application we are proposing three novel assays which may help overcome the hurdles for discovering small molecule agonists of GLP-1 R. The three assays will encompass: 1) a primary cAMP biosensor screen to detect selective agonists, potentiators, and antagonists of GLP-1 R, 2) a functional high content assay that is G-protein independent and is designed to monitor receptor internalization, and distinguishes pharmacology of small molecules, and 3) a high content assay of physiological significance that measures primary beta-cell apoptosis. These assays will generate a work flow that provides primary screens for hit identification, confirmation of hits to rule out artifacts, counter screening to determine selectivity, and receptor and physiological functional analysis of hits for lead development.