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

描述（由申请人提供）：本工作的目标是开发一系列针对胰高血糖素样肽1受体（GLP-1 R）的新型高通量检测方法，这些方法能够推动针对II型糖尿病（T2 DM）这一临床验证靶点的药物发现计划。除GLP-1 R试验外，我们还将开发其他两种密切相关的受体（胰高血糖素样肽2受体（GLP-2 R）和胰高血糖素受体（GCG-R））的新型试验，其将作为对GLP-1 R选择性的反筛选。美国糖尿病协会报告说，有2080万美国人，或7%的人口，谁患有糖尿病（www.diabetes.org）。FDA最近批准了艾塞那肽，一种用于治疗T2 DM的合成肽GLP-1类似物。虽然高度有效，但艾塞那肽具有药代动力学和剂量限制，这将使小分子口服可用的GLP-1 R激动剂或增效剂的发现成为有价值的临床进展。GLP-1 R是一种广泛研究的G蛋白偶联受体（GPCR），也是制药行业许多药物发现计划的目标。尽管投入了大量资源和努力来发现GLP-1 R激动剂，但尚未有发现GLP-1 R小分子激动剂的报告，没有小分子激动剂进入临床开发，并且所有对初步筛选数据的访问仍然是专有信息。在本申请中，我们提出了三种新的检测方法，这些方法可能有助于克服发现GLP-1 R小分子激动剂的障碍。这三项试验将包括：1）检测GLP-1 R的选择性激动剂、增效剂和拮抗剂的主要cAMP生物传感器筛选，2）功能性高含量试验，其不依赖于G蛋白，旨在监测受体内化，并区分小分子的药理学，以及3）具有生理学意义的高含量试验，其测量主要β细胞凋亡。这些测定将产生一个工作流程，该工作流程提供用于命中识别的初步筛选、用于排除伪影的命中确认、用于确定选择性的计数器筛选以及用于先导开发的命中的受体和生理功能分析。

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.