Developing peptidomimetic ligands for relaxin family peptide receptors

Abstract

Relaxin family peptides that target G protein-coupled receptors (GPCRs), have important therapeutic applications (e.g., heart failure, fibrosis, cancer, and disorders related to psychiatry, drug addiction, eating, or colon motility). However, these family peptides have an insulin-like complex heterodimeric structure that is difficult to produce and modify to improve their pharmacokinetic properties (e.g., half-life). I have carried out structure-activity relationship (SAR) studies on these peptides leading to the generation of simplified agonists and/antagonists for the GPCRs, RXFP1, RXFP3, and RXFP4. These novel peptides are easier to prepare in larger yields and retain high affinity for their receptors. They are important research tools, and potential drug leads for the treatment of human diseases as mentioned above. Chapter 1 summarises the literature on the SAR studies of H2 relaxin, H3 relaxin and human INSL5 leading to the design and development of simplified agonist and antagonist. Chapter 2 describes all the materials and methods used in this study. Chapter 3 summarises the SAR studies on H2 relaxin. H2 relaxin is known to have strong anti-fibrotic, vasoprotective, angiogenic, and vasodilatory effects. The receptor for H2 relaxin, RXFP1, is a potential target for the treatment of heart failure, fibrosis and related disorders, including liver cirrhosis, and preeclampsia. Our laboratory has designed a B-chain-only analogue, B7-33, which was shown to be a potent RXFP1 agonist in cells endogenously expressing RXFP1 and in several animal models of fibrosis (mice vs rats). B7-33 is 27 residues long and has short circulation time in serum (t1/2= ~6 min). I have shown that B7-33 can be further truncated up to 4 residues, and it’s in vitro half-life in human serum can be increased from 6 minutes to 60 minutes. I also have developed a B7-33-based single-chain RXFP1 antagonist for the first time. These analogues are important research tools, and drug leads for the treatment of human diseases (e.g. fibrosis-related diseases, prostate, and other cancers). Chapter 4 summarises the SAR studies on the neuropeptide H3 relaxin. The receptor for the H3 relaxin hormone, RXFP3, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviours. This chapter has two parts. In the first part, I have developed four novel analogues based on two chains, and two disulfides bonded H3 relaxin analogue, also known as A2 analogue. I have introduced amide, alcohol, carbamate, and ester functionality at the C-terminus of the B-chain of A2 analogue. In terms of binding to RXFP3, the carboxylic acid remains to be the most suitable functionality. However, the cAMP response seems to be better with the C-terminal ester function. Such results highlight the possibility of generating drug candidates, as applied to H3 relaxin, to slow down the release or generating a long-acting analogue of H3 relaxin. In the second part, I have developed an alpha-helix stabilising chemical method. Using this method, I have produced a high yielding single-chain RXFP3 agonist and antagonist. The agonist peptide, H3B10-27-13/17alphaF, is 20-fold more stable in vitro in human serum compared with the control peptides (H3B10-27-13/17EA and H3B10-27-13/17FF). The novel RXFP3 antagonist, H3B10-22R-13/17alphaF, is only 14 amino acid long compared with the previously reported 23 residues antagonist. These analogues will facilitate the identification of physiological roles of RXFP3 and drug leads for the treatment of neurological disorders such as stress and anxiety. Chapter 5 summarises the SAR of insulin-like peptide 5 (INSL5), a gut hormone. Its receptor RXFP4 is a potential target for the treatment of anorexia, obesity, and colon motility disorders (e.g. constipation and diarrhoea). Our laboratory was the first to design and develop an RXFP4-specific agonist peptide known as analogue 13. However, there was no RXFP4-specific antagonist reported in the literature. The focus of this study was to utilise the non-specific RXFP3/RXFP4 antagonist, deltaR3/I5, as a template to design an RXFP4 specific antagonist rationally. Based on analogue deltaR3/I5, I have developed an analogue 17, which is RXFP4-selective. Analogue 17 is an ideal template for further development into a specific high-affinity RXFP4 antagonist as tool and drug leads. Chapter 6 summarises the findings in this study and provides future directions.