Design and Synthesis of a Structurally Stabilized B‑Chain Antagonist Targeting Relaxin Family Peptide Receptor 3 (RXFP3).

Relaxin-3, a neuropeptide in the insulin/relaxin superfamily, signals via the G protein-coupled receptor RXFP3 and regulates motivated behaviors such as feeding, arousal, exploration, and reward-seeking. Despite its therapeutic promise for neuropsychiatric and metabolic disorders, the complex two-chain (A and B), three-disulfide structure of relaxin-3 hampers drug development. To overcome this, simplified B-chain-only analogues have been explored and developed, including the stapled RXFP3 agonist H3B10-27-(13/17αF) and the linear antagonist H3B1-22R. Here, we report a novel stapled antagonist, H3B10-22R-(13/17αF), a 14-residue single-chain peptide with high RXFP3 selectivity and binding affinity, enhanced helicity and antagonist potency, and 12-fold improved serum stability compared to linear unstructured H3B1-22R, the current best RXFP3 antagonist. Importantly, H3B10-22R-(13/17αF) significantly inhibited RXFP3 agonist-induced food intake in rats, confirming its in vivo efficacy. These results establish H3B10-22R-(13/17αF) as a potent, stable, and drug-like RXFP3 antagonist, supporting its further development as a therapeutic candidate for feeding disorders (e.g., obesity).