Type 2 diabetes mellitus is one of the most challenging health problems of the 21st century. Global health care expenditures to treat diabetes and its complications are estimated to have been $376 billion in 2010, of which $198 billion was spent in the United States. Efficacious and convenient pharmaceutical interventions are urgently needed to slow this global pandemic. Glucagon-like peptide 1 (GLP-1) and its receptor have emerged as one of the most promising avenues for intervention. GLP-1 is a 30 amino acid peptide hormone that binds to the GLP-1 receptor (GLP-1R), a G-protein coupled receptor (GPCR) that stimulates insulin secretion and controls blood glucose. Modified GLP-1 and GLP-1 analogs are approved for the treatment of type 2 diabetes in all major markets. However, these drugs must be injected daily, limiting their acceptance by patients. An orally available small molecule GLP-1 receptor agonist would be a significant advance in diabetes treatment. The long-term objective of this proposal is to develop orally available drugs that activate GLP-1R. Carmot has identified multiple GLP-1R agonists with potential for oral bioavailability. As Carmot advances these agonists toward the clinic, implementation of biological assays to identify the best drug candidates will be crucial, especially in light of growing evidence that GPCRs may engage subsets of available signaling pathways, a concept known as biased agonism. Indeed, the GLP-1R has been shown to signal through both G-protein and b-arrestin mediated pathways. However, the relative importance of these distinct pathways in promoting insulin secretion is not understood, primarily due to a lack of pharmacological agents that differentiate between these two pathways. Carmot is in the unique position to address this question, having identified chemically diverse agonists that, in preliminary studies, show varying degrees of biased agonism. The goal of this Phase I proposal is to clarify the role of biased agonism in GLP-1R signaling and insulin secretion, and then to use these insights to not only identify compounds that closely mimic the agonism bias of GLP-1 but also to discover molecules with different profiles. Compounds with distinct profiles will be tested in rodent diabetes models as part of a Phase II proposal to characterize their pharmacological properties. Importantly, compounds with different profiles than GLP-1 could have superior efficacy for treating diabetes and other metabolic disorders. The specific aims of this Phase I proposal are 1) to analyze Carmot's more than 24,000 GLP-1R directed molecules for biased agonism, especially current lead molecules; 2) to determine the importance of biased agonism in stimulating insulin secretion from pancreatic islets; 3) to determine internalization kinetics and endocytic trafficking of select compounds, including GLP-1. To ensure the success of this endeavor, Carmot is proposing to bring in an experienced PI, who currently works in academia and has more than a decade of experience working with GPCR signals and endocytic sorting in cell based and animal models.