School of Chemistry - Theses
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ItemStructure-function relationship studies on human relaxin-2 leading to the development of novel RXFP1 receptor-selective analogues(2013)The insulin/relaxin superfamily comprises of ten members, insulin, IGFs 1 and 2, relaxin-1, -2, -3 and INSL peptides 3, 4, 5 and 6. All members possess similar tertiary structures but each assumes unique biological functions. The focus of this thesis is to undertake a comprehensive structure-function relationship study to develop novel receptor-selective analogues of a particular member of this family, namely relaxin-2 (relaxin) and to further elucidate the mechanisms behind relaxin ligand-receptor interactions. Relaxin is a 53 amino acid peptide containing six conserved cysteine residues which form the three distinct disulfide bonds that are unique to all the insulin/relaxin superfamily members, one within the A-chain and two others holding the A- and B-chains together. It is the major stored and circulating form within the human body. Apart from its well-known reproductive roles, it also possesses other non-reproductive functions such as a cardioprotective agent and potent anti-fibrotic candidate. Relaxin is able to bind to two of the relaxin family peptide receptors, RXFP1 and RXFP2. Both receptors are leucine-rich repeat containing G-protein coupled receptors (GPCRs). There are two modes of interaction between relaxin and its cognate receptor, RXFP1. These interactions regulate the binding of the ligand to the receptor and also the activation of the cAMP signaling pathway involved. The primary mode of interaction involves the binding cassette present within the B-chain of relaxin which is often referred to as the RXXXRXXI motif that binds to the leucine-rich repeats of the receptor. A secondary interaction requires the A-chain of relaxin to promote activation of the receptor leading to downstream signaling. Detailed studies reported in Chapter 3 including extensive point mutations spanning from the N- to C-termini of the A-chain have led to key residues being identified that are important for native relaxin structure and function. The results, together with the data obtained from circular dichroism and solution NMR spectroscopy have provided new biochemical insights into the mechanism of secondary interaction between relaxin and RXFP1 or RXFP2. In particular, two key residues, Tyr3 and Phe23, which are present at opposite ends of the A-chain, are essential for the overall folding of relaxin. The mutation and/or removal of either or both of these residues cause a significant reduction in RXFP2 responses due to the destabilization of the hydrophobic core structure of relaxin. In combination with truncation studies performed previously, the series of relaxin analogues generated ultimately led to the development of a potent and RXFP1-selective analogue, H2:A(4-24)(F23A). In the next study (Chapter 4), the RXFP1-selective analogue, H2:A(4-24)(F23A) was examined in both in vitro and in vivo experimental models of fibrosis to evaluate its anti-fibrotic potential when compared to native relaxin. H2:A(4-24)(F23A) was found to inhibit TGF-β-stimulated collagen production in human dermal fibroblasts, demonstrating its anti-fibrotic capabilities in vitro. This work was further extended using an in vivo model where isoproterenol was used to induce cardiac fibrosis in mice leading to the accumulation of collagen in the left ventricles which corresponded to high levels of picrosirius red staining for collagen during histological analyses. The administration of H2:A(4-24)(F23A) helped to diminish isoproterenol-induced collagen accumulation in the left ventricles. A reduction in red staining correlated with a decrease in collagen production in the presence of H2:A(4-24)(F23A). Similar to native relaxin, H2:A(4-24)(F23A) was able to inhibit collagen accumulation both in vitro and in vivo experimental models of fibrosis. In the final study described in Chapter 5, the RXFP1-selective analogue, H2:A(4-24)(F23A), was further modified for receptor distribution studies within the brain by employing click chemistry methods to attach a Cy5.5 fluorophore. With the Cy5.5-labelled relaxin and INSL3 analogues, the localization of receptors can be discriminated within the cortex. For the very first time, the accessibility of the analogues can be seen in brain regions known to express either RXFP1 or RXFP2. In addition, the behavioural responses (dipsogenic effects) could also be monitored simultaneously. The use of sophisticated drug design and solid phase peptide synthesis protocols has enabled the successful production of relaxin analogues and, furthermore, helped to answer some of the many questions regarding the mechanism of relaxin-receptor action and biological activity. These novel relaxin analogues serve as exciting initial templates for the further development of novel therapeutics in the near future.