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. ? ? ?