Design and development of analogues of dimers of insulin‐like peptide 3 B‐chain as high‐affinity antagonists of the RXFP2 receptor

Insulin-like peptide 3 (INSL3) is one of 10 members of the human relaxin–insulin superfamily of peptides. It is a peptide hormone that is expressed by fetal and postnatal testicular Leydig cells and postnatal ovarian thecal cells. It mediates testicular descent during fetal life and suppresses sperm apoptosis in adult males, whereas, in females, it causes oocyte maturation. INSL3 has also been shown to promote thyroid tumor growth and angiogenesis in human. These actions of INSL3 are mediated through its G protein-coupled receptor, RXFP2. INSL3, a two-chained peptide, binds to its receptor primarily via its B-chain, whereas elements of the A-chain are essential for receptor activation. In an attempt to design a high-affinity antagonist with potential clinical application as an anticancer agent as well as a contraceptive, we have previously prepared a synthetic parallel dimer of INSL3 B-chain and demonstrated that it binds to RXFP2 with high affinity. In this work, we undertook full pharmacological characterization of this peptide and show that it can antaogonize INSL3-mediated cAMP signaling through RXFP2. Further refinement by truncation of 18 residues yielded a minimized analogue that retained full binding affinity and INSL3 antagonism. It is an attractive lead peptide for in vivo evaluation as an inhibitor of male and female fertility and of INSL3-mediated carcinogenesis. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 81–87, 2011.