Not Quite Folded: Challenges in Predicting the hNPS‐hNPSR‐Ile107 Complex With AlphaFold2 Multimer

The human neuropeptide S (NPS) receptor (NPSR) is a Class A peptide G protein-coupled receptor expressed in the centralnervous system and endogenously activated by NPS, a 20-mer peptide. NPSR activation promotes cellular excitability via Gqand Gs signalling. Studies suggest that receptor antagonists may reduce drug-seeking behaviours, whilst agonists representinnovative non-sedating anxiolytics with memory-enhancing effects. Despite its therapeutic potential, NPSR remains poorlycharacterised, with neither experimental receptor structures nor drug-like clinical candidates available. To fill this gap, we applieda previously validated AlphaFold2 Multimer-based protocol to model the hNPS–hNPSR complex. The model showing higherstability in molecular dynamics simulations and consistency with known structure–activity relationships served as templateto design novel hNPS analogues. However, experimental validation through synthesis and in vitro pharmacological evaluationof 20 novel truncated cyclic peptides revealed the model’s inability to capture hNPS bioactive conformation, as most analogueswere inactive as agonists. By exploiting the stereochemical switch in hNPS hinge region, we identified four novel cyclic antag-onists (17–20, pA2 in the 6.10–6.20 range). Our findings highlight strengths and limitations of current peptide-GPCR modellingstrategies and underscore the need for integrating AI predictions with experimental refinement to advance ligand discovery forchallenging targets like NPSR.