Design, synthesis and structural studies of relaxin receptor modulators

Abstract

The relaxin family of peptides consists of seven structurally related peptides, with a wide variety of biological functions, ranging from involvement in reproductive processes to functions as neuroendocrine modulators. Because of the complex, two-chained structure of the relaxins, and their lack of specificity for the various relaxin family receptors (RXFPs), design of simpler, more selective analogues is important for further investigation of their biological functions and as potential drug leads. The aim of this project was to introduce a helical structure around the receptor-binding region of single-chain relaxin analogues. This was approached by utilising helix capping sequences on truncated relaxin B-chains and by grafting of residues important for binding onto a stable peptide scaffold. Helix capping enhanced the helical properties compared to previous single- chain analogues, but was unable to introduce a sufficiently stable helix. Despite the increased helical tendencies, no high-affinity analogues were found. As a result, the importance of Arg8 was investigated, and we have demonstrated that this residue might be more involved in binding than previously thought. We successfully synthesised a relaxin-3/chlorotoxin analogue, which appeared to fold correctly. However, the yield following oxidation was poor, and no bioactivity data or structural data confirming the correct fold was obtained within the time limits of the project. A secondary aim was to probe for favourable mutations around the receptor- binding region by synthesising a combinational library. We were able to successfully synthesise a library of peptides with mutations in one position by inserting a mixture of amino acids at this coupling step in the solid phase peptide synthesis (SPPS) procedure. Although no significant improvement in binding was seen for the analogues generated, important methodological advances were made that will be used to scan different positions for new contact points with the receptor.