Novel peptidic glucagon receptor (GCGR) and glucagon-like peptide 1 receptor (GLP-1R) dual agonists are reported to have increased efficacy over GLP-1R monoagonists for the treatment of diabetes and obesity. We identified a novelGLP-1-based dual GLP-1R/GCGR agonist (xGLP/GCG-13) designed with a proper activity ratio favoring the GLP-1R versus the GCGR. However, the clinical utility of xGLP/GCG-13 is limited by its shorthalf-life. Starting from xGLP/GCG-13, dual Cys mutation was performed, followed by covalent side-chain stapling and serum albumin binder incorporation, resulting in a stabilized secondary structure, enhanced agonist potency at GLP-1R and GCGR, and improved stability. The lead peptide(stapled xGLP/GCG-13 analogue with a palmitic acid albumin binder) exhibits balanced GLP-1R and GCGR activations and potent, long-lasting effects onglucose control.was further explored pharmacologically in diet-induced obesity androdent models. Chronic administration ofpotently induced body weight loss and hypoglycemic effects, improved glucose tolerance, increased energy expenditure, and normalized lipid metabolism and adiposity in relevant animal models. These results indicated thathas potential for development as a novel antidiabetic and/or antiobesity drug. Furthermore, we propose that the incorporation of a proper serum protein-binding motif into a di-Cys staple is an effective method for improving the stabilities and bioactivities of peptides. This approach is likely applicable to other therapeutic peptides, such as glucose-dependent insulin-tropic peptide receptor (GIPR) and GLP-1R dual agonists or GLP-1R/GCGR/GIPR triagonists. Xenopus in vivo in vivo db/db2c2c2c2c
