GLP‑1 Val8: A Biased GLP‑1R Agonist with Altered Binding Kinetics and Impaired Release of Pancreatic Hormones in Rats

Biased ligands that selectively confer activity in one pathway over another are pharmacologically important because biased signaling may reduce on-target side effects and improve drug efficacy. Here, we describe an N-terminal modification in the incretin hormone glucagon-like peptide (GLP-1) that alters the signaling capabilities of the GLP-1 receptor (GLP-1R) by making it G protein biased over internalization but was originally designed to confer DPP-4 resistance and thereby prolong the half-life of GLP-1. Despite similar binding affinity, cAMP production, and calcium mobilization, substitution of a single amino acid (Ala8 to Val8) in the N-terminus of GLP-1(7–36)­NH2 (GLP-1 Val8) severely impaired its ability to internalize GLP-1R compared to endogenous GLP-1. In-depth binding kinetics analyses revealed shorter residence time for GLP-1 Val8 as well as a slower observed association rate. Molecular dynamics (MD) displayed weaker and less interactions of GLP-1 Val8 with GLP-1R, as well as distinct conformational changes in the receptor compared to GLP-1. In vitro validation of the MD, by receptor alanine substitutions, confirmed stronger impairments of GLP-1 Val8-mediated signaling compared to GLP-1. In a perfused rat pancreas, acute stimulation with GLP-1 Val8 resulted in a lower insulin and somatostatin secretion compared to GLP-1. Our study illustrates that profound differences in molecular pharmacological properties, which are essential for the therapeutic targeting of the GLP-1 system, can be induced by subtle changes in the N-terminus of GLP-1. This information could facilitate the development of optimized GLP-1R agonists.