School of Chemistry - Theses

Permanent URI for this collection

Search Results

Now showing 1 - 1 of 1
  • Item
    Thumbnail Image
    Human Relaxin-2: design, synthesis and development of novel RXFP1 agonists and antagonists
    BALAKRISHNAN NAIR, VINOJINI ( 2014)
    The ten members of the human insulin-relaxin superfamily comprise insulin, IGFs 1 and 2, relaxin-1, 2 and 3, and INSL3, 4, 5 and 6. Members of this superfamily share a common tertiary structure, but each has unique physiological functions. This thesis focuses on human relaxin-2 (relaxin) and efforts towards the design, synthesis and development of novel agonists and antagonists of its cognate receptor. The six cysteines found within the A- and B-chain of human relaxin forms characteristic disulfide bonds, which are a structural feature shared by this superfamily. One disulfide bond is held within the A-chain and another two between the A- and B-chains. These cysteines are conserved amongst these peptide hormones. Relaxin, a 53 amino acid peptide, is the major stored and circulatory form found within the human body. Although able to bind to two relaxin family peptide receptors, class 1 (RXFP1) and 2 (RXFP2), its cognate receptor is RXFP1. The relaxin-RXFP1 interaction is regulated in two modes. Both binding of the ligand to the receptor and its downstream biochemical activation is activated by this interaction. The primary interaction mode involves interaction of the binding cassette within the relaxin B-chain (the RXXXRXXI binding motif) with the leucine rich repeats of RXFP1. A secondary interaction involves the relaxin A-chain with the RXFP1 exoloops, hence, activating the receptor and consequently the downstream signalling cascade. The focus of this thesis was the primary binding interaction between relaxin and RXFP1. Relaxin passed Phase III clinical trials for the treatment of acute heart failure in late 2012 and, if it enters the clinic, will be the first novel therapy in more than 20 years. Despite its high specificity and excellent safety profile with no major adverse effects reported so far, like many other native peptides, it has poor pharmacokinetic properties. It is not orally available and survives in the blood only for a very short period (half-life ca. 10 minutes). Chapter 3 focuses on a short-term goal of this thesis which is to improve the pharmacokinetic properties of this peptide hormone using established approaches. Chemical modifications, specifically by oligomerisation and PEGylation, are employed to slow down enzymatic degradation. Dimerisations were carried out as preliminary studies to ensure the RXFP1 binding and activation properties of the analogues were retained. Relaxin dimers were formed with the via a disulfide bond and by click chemistry techniques. Both dimers were found to bind and activate RXFP1 similar to native relaxin. The PEGylated relaxin dimer was shown to be significantly more stable in serum than native relaxin and the disulfide bond-dimerised relaxin. Numerous studies have shown that relaxin is expressed by tumors in mammary, endometrial, thyroid and prostate cells and can act in both autocrine and paracrine manner on RXFP1 receptors in these cells. In human males, relaxin is exclusively produced in prostate secretory epithelial cells and, correspondingly, from prostate cancer cells. There is clear evidence that progression of aggressive prostate cancer can be stimulated by relaxin and/or RXFP1 overexpression. A significant decrease in tumor size and fewer metastases are reported for siRNA targeting of RXFP1 expression in the PC-3 xenograft model. These observations highlight the significant potential for the inhibition of relaxin physiological actions via RXFP1 antagonists to block prostate cancer growth and metastasis. Detailed studies in Chapter 4 focused on the design and development of RXFP1 antagonists specific to the primary relaxin-RXFP1 binding site via the RXXXRXXI binding motif. These two residues, Arg13 and Arg17 together with IleB20 within relaxin B-chain, were found to be essential for binding to and activating RXFP1 receptor. However, when these Arg were mutated to Lys, the analogue exhibited antagonistic properties both in vitro and in vivo by interfering with relaxin-induced signalling and, hence, impairment of prostate tumour growth by reduced affinity to RXFP1. The results from this chapter highlight efforts to increase the affinity of chemical analogues with RXFP1 whilst retaining antagonistic propensity. In particular, the more isosterically similar homo-Arg (HR) to Arg analogue, H2: BR13.17HR, showed improved RXFP1 affinity compared to the current antagonist, H2: BR13.17K whilst being a full antagonist in primary LNCaP cells. Relaxin also has other non-reproductive functions including cardioprotective and anti-fibrotic roles. To develop a highly specific next generation peptide drugs based on relaxin, three main concerns need to be addressed: large size (53 amino acids), complex structure (three disulfide bonds) and short half-life in blood. Simpler relaxin analogues need to be developed that are easier to prepare and modify, highly selective for RXFP1 and able to retain their activity for an extended therapeutic time-frame in patients. Chapter 5 addressed the effects of elimination of the A-chain intradisulfide bond on relaxin interaction with RXFP1. Various truncations were carried out within the A- and B-chains to elucidate the highest affinity RXFP1 analogue. H2: A(C10.15S) at nanomolar concentrations was found to activate RXFP1, which demonstrated that the intramolecular disulfide bond is not necessary for RXFP1 interaction and activation but instead maintained the overall 3D structure of relaxin. Development of these new analogues highlighted that the chemical syntheses of the complex, two chain and two- or three disulfide bond, relaxin peptides as an extremely laborious process. Assembly of the two chains is notoriously challenging due to the poor solubility of the B-chain and post-synthesis handling and purification of the peptide often results in low yields. From knowledge of the binding motifs of relaxin with RXFP1, efforts to develop RXFP1-specific single chains were explored in Chapter 6. The relaxin B-chain has most, if not all, of the amino acids responsible for RXFP1 binding and activation. The single chain peptide, B7-33, was shown to activate primary cells in vitro like relaxin, albeit with lower affinity. Importantly, B7-33 was shown also to mimic the anti-fibrotic actions of relaxin in a mouse model of acute heart failure disease. Together, the results of this thesis have identified novel relaxin agonists and antagonist that possess high affinity RXFP1 binding and potency. These analogues promise to be important lead analogues for further identifying the structural mechanism of relaxins actions in vivo and for development as potential therapeutic agents.