Molecular Pharmacology of C3aR Modulators MRS RANEE SINGH D.V.M. (Doctor of Veterinary Medicine) M.Sc. (Pharmacology) M.Vet.Studies (Laboratory) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2012 Institute for Molecular Bioscience i ABSTRACT The complement system plays key roles in both the innate (non-specific) and acquired (specific) immune response. Complement activation is a tightly regulated process, which takes place in a sequential manner by at least three pathways. All of these pathways generate a small anaphylatoxin protein called C3a. C3a signals immune cells which infiltrate to sites of infection or injury and mount inflammatory responses leading to elimination of pathogens or damaged cells and induction of adaptive immunity. Genetic deficiency of C3 results in susceptibility to bacterial infection. However, in excess C3a can be harmful and catalyses the pathogenesis of many disease conditions. To date, there have been no truly selective and potent C3a receptor agonists/antagonists. Therefore, the purpose of this thesis was to explore the potential to develop a small molecule C3aR agonist/antagonist that can be a useful as a pharmacological probe or biomarker for studying the physiological and pharmacological roles of C3a. Chapter 1 briefly describes basic pharmacology terminology and reviews current knowledge of GPCRs, GPCR intracellular signalling pathways, C3a and C3a-C3aR related intracellular signalling pathways. Understanding the pro-inflammatory roles of C3a and its receptor in vitro and in vivo may provide a new therapeutic target for autoimmune diseases or inflammatory conditions. Chapter 2 reinvestigated reported C3a receptor ligands in a competitive binding assay and a functional assay. Compound affinity was measured by competitive binding against [125I]-C3a (80 pM). Compound functional potency (agonist or antagonist) was measured by fluorescent detection of intracellular calcium release in differentiated U937 cells. Receptor selectivity was measured by receptor desensitization using C3a or C5a, and by competitive binding with [125I]-C5a. C3aR superagonist peptide (WWGKKYRASKLGLAR) and newly reported pentapeptides WPLPR (15) and YPLPR (14) (oryzatensin derivatives) were found to have no selectivity or agonist potency for C3aR. Four decapeptides (8-11) reported as C5aR agonists were shown to be more selective for C3aR than C5aR in a competitive binding assay on human isolated monocytes. Structure-activity relationships (SAR) were studied for new hexapeptides by calcium release, FWTLAR (20) (EC50 350 nM) and FLTLAR (17) (EC50 320 nM). They bound selectively to C3aR (IC50 82 nM, IC50 42 nM respectively) on human PBMCs with no binding to C5aR. Selectivity was confirmed by receptor desensitisation experiments. A new C3aR peptide antagonist (F1LTChaAR6) was obtained by modifying the leucine at position 4 with the bulkier group Cha, showing no agonist activity and binding tightly to C3aR (IC50 238 nM). Chapter 3 introduces an oxazole heterocycle into short peptides to restrict conformation freedom in an attempt to mimic the C3aR-binding conformation of the C-terminal activating domain of human ii C3a. This approach produced multiple potent C3aR agonists, including some with equal affinity to C3a itself and selectivity for C3aR over C5aR on isolated monocyte derived macrophage cells (HMDM). There was a linear correlation between the binding affinity of the compounds and their agonist potency. Chapter 4 describes two approaches for obtaining antagonists of C3aR and shows some structure-activity relationships. Firstly, a potent new C3aR agonist (56) from Chapter 3 was modified by incorporation of a phenyl group at the 5-position of oxazole ring, which improved C3aR binding affinity and led to a novel and potent nonpeptidic C3aR antagonist. Secondly, new non-peptidic agonists of C3aR were developed from the first reported C3aR antagonist by modification of either the linker or hydrophobic region of SB290157 with retention of the terminal arginine. Several potent C3aR antagonists were developed from an oxazole-containing compound by incorporating a phenyl group at the 5-position of oxazole ring. Two other compounds were derived by modifying SB290157 at the linker region through incorporation of a thiazole or oxazole ring. Chapter 5 explores the antagonist mechanism of non-peptidic C3aR antagonist, SB290157 and newly developed antagonist 146, as well as the peptidic antagonist FLTChaAR (25). It reports competition with three different classes of C3aR agonists: hC3a, two potent new hexapeptide analogues (17 and 20) and two potent new non-peptide agonists (63 and 80). SB290157 and 146 show competitive and surmountable antagonism against the hexapeptide agonists (17 and 20) and non-peptide agonist (63 and 80), supporting binding at the same site(s) on C3aR. Compound 25 showed insurmountable antagonism of all agonists (hC3a, 17, 20, 63 and 80), suggesting that 25 binds different sites from these compounds or it may be caused by C3aR internalisation or (pseudo) irreversible binding to C3aR. However, 25 is unlikely to be an allosteric antagonist due to competition with C3a radioligand in a competitive binding assay. In summary, this study has investigated and reported new C3aR agonists and antagonists that can be used to probe influences of C3a/C3aR in physiology and pharmacology. iii DECLARATION BY AUTHOR This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature Conor C. G. Scully; Jade S. Blackeney; Ranee Singh; Huy N. Hoang; Giovanni Abbenante; Robert C. Reid and David P. Fairlie. Selective Hexapeptide Agonists and Antagonists for Human Complement C3a Receptor. J. Med. Chem. 2010, 53, 4938-4948. Conference Abstracts: Singh, R., Scully, C., Chu, P.,Blackeney, J.,Hoang,H., Abbenante, J., Reid, R. and Fairlie, D.P. (2010). Agonists and Antagonists of the human C3a Receptor. Proceedings of Chemistry and Structural Biology Division Symposium (IMB), UQ. Singh, R., Reid, R., Chu P., Reed T. and Fairlie D.P. (2011) Towards human C3a Receptor Antagonists. Proceedings of Chemistry and Structural Biology Division Symposium (IMB), UQ. Publications included in this thesis Conor C. G. Scully; Jade S. Blackeney; Ranee Singh; Huy N. Hoang; Giovanni Abbenante; Robert C. Reid and David P. Fairlie. Selective Hexapeptide Agonists and Antagonists for Human Complement C3a Receptor. J. Med. Chem. 2010, 53, 4938-4948. – Partially incorporated as paragraphs in Chapter 2. Contributor Statement of contribution Singh, R. Designed and performed cell pharmacology experiments (90%) Analysis of the data (90 %) Fairlie, D.P. Intellectual input, project conception and planning, scientific guidance and review and analysis of results (70%) Reid, R.C. Intellectual input, project conception and planning, scientific guidance and review and analysis of results (30%) Scully, C.G.C. Structural design, synthesis and characterisation of compounds (80%) NMR studies (50%) Hoang, H.N. NMR studies (50%) Blakney, J.S. Structural design, synthesis and characterisation v of compounds (20%) Performed experiments some compounds in calcium release assays (10%) Analysis of the data (10%) Contributions by others to the thesis My supervisors, Prof. David Fairlie, Dr. Robert Reid and Dr. Conor Scully all contributed intellectually to this thesis through data analysis, discussion, scientific guidance and critical review of the project; Dr. Scully and Dr. Reid have contributed by manufacturing the majority of the compounds tested in this thesis. Statement of parts of the thesis submitted to qualify for the award of another degree “None.” vi ACKNOWLEDGEMENTS I would like to acknowledge the assistance of a number of people who have helped me throughout my PhD. First, my supervisor who gave me opportunities to do the interesting projects. I gratefully acknowledge his wisdom, advice and support. I also would like to thank my co- supervisors Dr. Scully and Dr. Reid for their kind advice and assistance, without which I would never have made it to the end. I also would like to thank all the members of the Fairlie