A Multi-Angle Approach to Predict Peptide-GPCR Complexes: The N/OFQ-NOP System as a Successful AlphaFold Application Case Study

With nearly 700 structures solved and a growing number of customized structure prediction algorithms being developed at a fast pace, G protein-coupled receptors (GPCRs) are an optimal test case for validating new approaches for the prediction of receptor active state and ligand bioactive conformation complexes. In this study, we leveraged the availability of hundreds of peptide GPCRs in the active state and both classical homology and artificial intelligence (AI) based protein modeling combined with docking and AI-based peptide structure prediction approaches to predict the nociceptin/orphanin FQ-NOP receptor active state complex (N/OFQ-NOPa). The In Silico generated hypotheses were validated via the design, synthesis, and pharmacological characterization of novel linear N/OFQ(1–13)-NH₂ analogues, leading to the discovery of a novel antagonist (3B; pK_B = 6.63) bearing a single ring-constrained residue in place of the Gly²–Gly³ motif of the N/OFQ message sequence (FGGF). While the experimental validation was ongoing, the availability of the Cryo-EM structure of the predicted complex enabled us to unambiguously validate the generated hypotheses. To the best of our knowledge, this is the first example of a peptide–GPCR complex predicted with atomistic accuracy (full complex Cα RMSD < 1.0 Å) and of the N/OFQ message moiety being successfully modified with a rigid scaffold.