Discovery of peptides with balanced glucagon antagonism and GLP-1 agonism, supported by an investigation of their molecular basis for such action

Glucagon and glucagon-like peptide 1 (GLP-1) have opposing effects in blood glucose regulation. Glucagon increases plasma glucose by stimulating gluconeogenesis and glycogenolysis in the liver while GLP-1 lowers glucose levels by promoting glucose dependent insulin secretion in the pancreas. Diabetes is characterized by impaired insulin function and excessive hepatic glucose production. Consequently, there has been tremendous interest in the independent identification of glucagon receptor antagonists and GLP-1 receptor agonists. A bi-functional peptide that simultaneously served as a glucagon antagonist and a GLP-1 agonist possesses additional therapeutic potential. However, the identification of such a peptide constitutes a sizable challenge as glucagon and GLP-1 share fifty percent sequence identity and their receptors possess high homology.;This thesis presents the discovery of high potency, balanced peptides of mixed glucagon antagonism and GLP-1 agonism. These peptides were identified and refined by structure-activity relationship (SAR) studies that characterized in vitro biological activities, and eventually demonstrated glucose lowering in diabetic animals. We also explored site-specific chemical conjugation of the bi-functional peptides to improve their pharmacokinetic properties. The in vitro potency was observed to be a function of conjugation site, spacer length, chemical nature of the linker, and the specific starting peptide sequence. These novel bi-functional peptide analogs with extended half-lives constitute a novel class of diabetes drug treatment that simultaneously addresses both impaired insulin function and excessive glucagon production.;A final aspect of this thesis pertains to receptor mutagenesis studies that were conducted to explore the inverse mechanism of action at these two homologous receptors. A series of chimeric receptors were constructed with distinct segments of the glucagon receptor substituted with the corresponding segments of the GLP-1 receptor, and vice versa. Three distinct receptor elements were identified to serve different roles in receptor binding and activation. The receptor N-terminal extracellular domain was observed to determine specificity and binding affinity. The seven-helix transmembrane domain stabilizes the receptor structure, and the three extracellular loops are the key segments that serve to determine receptor activation versus receptor inhibition.;In total, the work reported in this thesis provides novel insights in 7TM-receptor function and a set of unique drug candidates.