Peptide-based therapeutics such as tirzepatide and semaglutide target the neuroendocrine system to remediate both diabetes and obesity. In addition to integrating the complementary actions of two endogenous metabolically related hormones targeting glucagon-like peptide-1 and glucagon receptors (GLP-1R and GCGR), the synergistic targeting of a third G protein-coupled receptor (GPCR), namely the gastric inhibitory polypeptide receptor (GIPR), may achieve optimal control of blood glucose levels and weight loss in patients with diabetes and obesity. In this work, we harness the predictive power of sequence-derived in silico mutagenesis to rationally design effective single-molecule peptides with triple-receptor-agonist activities. We model the binding profiles of our designed peptides to the GIP, GLP-1 and GCG receptor triad through multiple, long, repeat molecular simulations and effective binding enthalpy calculations to assess their predicted multi-receptor engagement profiles. Our data predict a balanced, highly favourable effective binding enthalpy profile of our newly designed peptides for the three receptors compared to endogenous peptides and the experimental reference peptides, with high targeted specificity for the receptors. The peptides are further engineered to structurally support metabolic stability by substituting sites prone to proteolytic cleavage. The predicted triple-hormone-receptor agonist peptide mechanism opens new avenues for the synthesis and activity testing of multi-targeting peptides with improved stability, offering a computational design strategy for testing the potential reduction of adverse gastrointestinal effects of peptide therapeutics and support future development of lower-dose oral formulations.
