Engineering of\nNovel Analogues That Are More Receptor-Selective\nand Potent than the Native Hormone, Insulin-like Peptide 5 (INSL5)

Insulin-like\npeptide 5 (INSL5) targets the G protein-coupled receptor,\nrelaxin family peptide receptor 4 (RXFP4), predominantly coexpressed\nin the colorectum. While INSL5 also binds to the related receptor\nRXFP3, it does not activate it. The INSL5/RXFP4 axis is a promising\ntarget for treating gastrointestinal disorders such as constipation.\nDespite its therapeutic potential, the clinical application of INSL5\nhas been hindered by synthesis complexities, necessitating the need\nfor more accessible yet potent mimetics. In this study, we engineered\nan INSL5 analogue A13:B7-24-GG, featuring a simplified two-chain,\ntwo-disulfide scaffold with 32 amino acids, as opposed to the 45 amino\nacids found in native INSL5 (two-chain, three-disulfide), improving\nthe synthesis yield by 19.5-fold. Additionally, A13:B7-24-GG demonstrates\n∼4-fold higher potency (EC<sub>50</sub> = 1.17 nM vs 4.57 nM)\nand ∼11 times greater selectivity than native INSL5, with significantly\nreduced RXFP3 binding affinity, positioning it as a promising new\ntherapeutic candidate for the treatment of constipation.