N-terminally modified Ac-GLP1 and Ac-semaglutide achieve G protein bias by displacing GLP1R's ECL3

The glucagon-like peptide-1 receptor (GLP1R), a class B G protein-coupled receptor (GPCR), is a crucial therapeutic target for type 2 diabetes mellitus and obesity. Current GLP1R agonists signal through both G protein and β-arrestin pathways, which are associated with distinct receptor conformations and trafficking states. Understanding and engineering biased agonism—preferentially activating one pathway over another—offers a strategy to optimize therapeutic effects by enhancing desired outcomes (e.g., prolonged cAMP signaling) while potentially mitigating undesirable ones (e.g., receptor desensitization or side effects linked to β-arrestin recruitment). This study addresses the structural basis for achieving such G protein bias.

Researchers developed novel G protein-biased GLP1R agonists through N-terminal modifications, including acetylation or amino acid substitutions, using a reference ligand for comparison. They employed cryo-electron microscopy (cryo-EM) to determine the structure of the GLP1-Y11-GLP1R-Gs complex at 2.64 Å resolution. In vitro experiments assessed G protein signaling, β-arrestin recruitment, and cAMP signaling. For in vivo evaluation, Ac-semaglutide was administered to diet-induced obese mice, with glucose-lowering activity monitored for three days post-administration.

N-terminally modified GLP1R agonists, including Ac-GLP1 and Ac-semaglutide, demonstrated preferential G protein signaling compared to the reference ligand.

Cryo-EM structural analysis of the GLP1-Y11-GLP1R-Gs complex at 2.64 Å resolution revealed that outward displacement of extracellular loop 3 (ECL3) is the critical structural feature associated with this signaling bias. This distinct conformation differentiates the biased complex from β-arrestin-biased agonist-bound states. Furthermore, Ac-GLP1 and Ac-semaglutide exhibited altered receptor trafficking, significantly attenuated β-arrestin recruitment, and prolonged cAMP signaling. In vivo, Ac-semaglutide successfully maintained its glucose-lowering activity in diet-induced obese mice for three days post-administration.