Disulfide-Directed\nMulticyclic Peptides with N‑Terminally\nExtendable α‑Helices for Recognition and Activation of\nG Protein-Coupled Receptors

Many peptide hormones adopt long α-helical structures\nupon\ninteracting with their cognate receptors but often exhibit flexible\nconformations when unbound. Strategies that can stabilize long α-helices\nwithout disrupting their binding to receptors are still lacking, which\nhinders progress in their biological applications and drug development.\nHere, we present an approach that combines rational design with library\nscreening to create and identify a unique disulfide-directed multicyclic\npeptide (DDMP) scaffold, which could effectively stabilize N-terminally\nextendable α-helices while displaying exceptional efficiency\nin disulfide pairing and oxidative folding. This DDMP scaffold was\nthen utilized for stabilizing the α-helical structure of glucagon-like\npeptide-1 (GLP-1), resulting in a potent GLP-1 receptor (GLP-1R) agonist\nwith a significantly improved α-helicity and proteolytic stability.\nBy incorporating external α-helices into the DDMP scaffold,\nwe can effectively preserve the native N-terminal α-helical\nstructures while allowing for extensive evolution of the C-terminal\ndisulfide-rich domain for enhancing target binding, as demonstrated\nby the generation of the DDMP-stabilized GLP-1 (g1:Ox). The cryo-electron\nmicroscopy structure of the g1:Ox–GLP-1R in complex with heterotrimeric\nG<sub>s</sub> reveals the molecular basis for the potent binding between\ng1:Ox and GLP-1R. Specifically, the DDMP moiety establishes additional\ninteractions with the extracellular domain of GLP-1R, which are absent\nin the case of GLP-1. Thus, this work offers a novel and effective\napproach for engineering therapeutic peptides and other peptide α-helices,\nensuring that both the N- and C-terminal regions remain essential\nfor target recognition and activation.