Relaxin-3 is a highly conserved neuropeptide hormone that is the ancestor of the relaxin/insulin superfamily. Through interactions with its cognate receptor, relaxin family peptide receptor-3 (RXFP3), it is involved in regulation of a number of behaviours including food intake, stress, arousal and sleep. These findings suggest that RXFP3 may be a novel target for obesity and anxiety disorders.

Relaxin-3 is a complex two-chain peptide and it shows poor selectivity for RXFP3, also being a high affinity ligand for RXFP1 and RXFP4, the receptors of the related peptides relaxin-2 and INSL5, respectively. This has significantly complicated the analysis of the relaxin-3 signalling system, and better agonists and antagonists clearly need to be developed. Structure-activity relationships (SAR) have revealed that the critical features for receptor binding are located in the relaxin-3 B-chain. We recently showed that a selective high affinity single chain antagonist can be achieved by truncating the B-chain C-terminus, while incorporating an extra C-terminal Arg residue. Interestingly this peptide is unstructured in solution, raising questions about how it binds RXFP3.

We have through extensive mutational efforts generated detailed SAR for the single chain RXFP3 antagonist R3 B1-22R. The non-native Arg is the key driver of the affinity, with additional contributions from a number of hydrophobic residues. Introduction of Pro residues or helix-stabilising ‘staples’ are detrimental for function suggesting that although it likely does bind in a helical conformation, it engages the receptor in a manner that is strikingly different from its parent peptide. These data are highly relevant for the future development of this antagonist towards the clinic.