Developing Insulin-Like Peptide 5-Based Antagonists for the G Protein-Coupled Receptor, RXFP4

Abstract

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (~35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. Our findings detail the creation of a series of A13-nR-modified analogues, among which compounds 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (~3 nM) with reduced partial agonist activity, enhanced antagonist potency (~10 nM) and maximum agonist inhibition (~80%) when compared with the recently reported lead antagonist, A13-nR. These highlight their potential as compelling candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

Keywords: RXFP4, INSL5, peptides, Insulin, RXFP3, Relaxin