Insulin-like peptide 5 (INSL5) is a hormone produced exclusively by colonic L-cells of the colon and acts as the native ligand for the relaxin family peptide receptor 4 (RXFP4). This receptor-ligand system has been implicated in the regulation of gut motility, particularly in the control of colorectal propulsion,1 making it a promising target for treating colon motility disorders such as constipation. However, the clinical application of INSL5 is limited by synthesis challenges,2 resulting in a low yield (0.8%) and thus highlighting the need for more accessible yet potent mimetics of INSL5.
To address this, our laboratory recently engineered an INSL5 analogue, A13:B7-24-GG, featuring a simplified two-chain, two-disulfide scaffold with 32 amino acids, as opposed to the 45 amino acids found in native INSL5 (two-chain, three-disulfide), improving the synthesis yield by 19.5-fold.3 A13:B7-24-GG also demonstrates approximately four-fold higher potency than native INSL5, establishing it as the lead two-chain, two-disulfide analogue of INSL5 to date.
Based on prior structure-activity relationship studies and the recently available INSL5/RXFP4 Cryo-Electron Microscopy structure,4 we identified the B-chain of INSL5 as the sole determinant for RXFP4 binding and activation. Therefore, we hypothesised that bioactive INSL5 analogues could be designed as B-chain-specific compounds. Here, using the B-chain of A13:B7-24-GG as a template, we present new designs and chemical approaches to generate a series of B-chain-specific INSL5 analogues. Using our recently optimised NanoBiT complementation binding assay,5 we found that one of these analogues demonstrated high affinity for RXFP4 (~1 nM). Additionally, the forskolin-induced cAMP assay confirmed its potency at ~9 nM, establishing this new compound as a novel lead for further in vivo studies and a promising therapeutic candidate for constipation.