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Discovery and Mode of Action of Cyclotides Anjaneya Swamy Ravipati Msc, M.Phil

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Discovery and Mode of Action of Cyclotides Anjaneya Swamy Ravipati Msc, M.Phil Discovery and mode of action of cyclotides Anjaneya Swamy Ravipati MSc, M.Phil A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 Institute for Molecular Bioscience Abstract Cyclotides are plant derived macrocyclic peptides featuring a cyclic cystine knot (CCK) formed by six cysteine residues on a head-to-tail cyclic backbone. Their unique structural features make them exceptionally stable against thermal, enzymatic or chemical degradation. Naturally isolated cyclotides have many pharmaceutically important activities, including uterotonic, anti-HIV, antimicrobial and anticancer properties. The combination of structural features and biological activities make cyclotides an attractive framework in drug design applications. To date over 300 cyclotides have been discovered in five plant families: Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae and Solanaceae. However, their origin and distribution in the plant kingdom remains unclear. In this thesis a total of 206 plants belonging to 46 different plant families have been screened for expression of cyclotides. In this screening program, 50 novel cyclotides were discovered from 31 plant species belonging to the Rubiaceae and Violaceae families. Interestingly, all the Violaceae species possess cyclotides supporting the hypothesis that the Violaceae is a rich source of cyclotides. As a matter of fact, cyclotides have been found in every plant species belonging to the Violaceae family screened so far. It is noteworthy that a novel suite of Lys-rich cyclotides were discovered from Melicytus chathamicus and M. latifolius belonging to the Violaceae family which are endemic to remote islands of Australia and New Zealand. Unlike generic cyclotides, Lys-rich cyclotides possess higher positive charge, which correlates with their earlier retention times in RP-HPLC indicative of lower hydrophobic properties. i The broad spectrum of biological activities of cyclotides suggests the involvement of a common mechanism of action. Earlier studies have demonstrated that cyclotides exert biological activities through interaction with biological membranes. In order to understand the mode of action of novel cyclotides, membrane binding, and membrane permeabilization properties were evaluated on model membranes using a range of biophysical techniques including surface plasmon resonance (SPR), fluorescence spectroscopy and cryo- transmission electron microscopy (cryo-TEM). Regardless of their sequence diversity all of the cyclotides showed affinity towards phospholipids containing a phosphatidylethanolamine (PE) headgroup, which is in agreement with previous studies. In comparison to typical cyclotides, Lys-rich cyclotides display relatively lower haemolytic activities and correspondingly these peptides also showed lower affinities with model membranes. These results support the theory that membrane binding affinities influence the biological activities. The spectroscopic studies clearly show that cyclotides do target and disrupt membranes. However, the complete membrane disruption mechanism has not been evaluated yet. In this thesis, cyclotide-membrane interactions were investigated using cryo-TEM imaging. The results suggest that the membrane disruption mechanism is sequence dependent. Overall, the thesis has provided an increased understanding of the natural diversity of cyclotides and has added to our understanding of their mode of action. In particular, the discovery of a novel family of Lys-rich cyclotides suggests that there might be other new types of cyclotides yet to be discovered. Hence, discovery and characterization of cyclotides in this field of research has much to offer. ii 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 policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. 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. iii Publications during candidature Peer-reviewed papers 1. Cemazar, M., Kwon, S., Mahatmanto, T., Ravipati, A. S, and J Craik, D. (2012). Discovery and applications of disulfide-rich cyclic peptides, Current Topics in Medicinal Chemistry 12, 1534-1545. 2. Fensterseifer, I. C., Silva, O. N., Malik, U., Ravipati, A. S., Novaes, N. R., Miranda, P. R., Rodrigues, E. A., Moreno, S. E., Craik, D. J. and Franco, O. L. (2014). Effects of cyclotides against cutaneous infections caused by Staphylococcus aureus. Peptides. 63, 38-42. 3. Ravipati, A. S., Henriques, S. T., Poth, A. G., Kaas, Q., Wang, C. K., Colgrave, M. L., and Craik, D. J. (2015). Lysine-rich cyclotides: A new subclass of circular knotted proteins from Violaceae. ACS Chemical Biology 10, 2491-2500. 4. Ravipati, A. S., Henriques, S. T., Poth, A. G., Kaas, Q., Wang, C. K., Colgrave, M. L., and Craik, D. J. (2015). Discovery and mechanistic studies of cyclotides. Proceedings of 24th APS compilation of materials. iv Conference abstracts 1. Poster presentation: “Discovery and mechanistic studies of cyclotides” at 24th American Peptide Symposium, Orlando, Florida, USA. (20 – 25 Jun 2015) 2. Poster presentation: “Discovery, mechanistic studies and applications of cyclotides”, at Nanotechnology Entrepreneurship Workshop for Early Career Researchers, Griffth University, Gold Coast. (10 -11 Jun 2015) 3. Poster presentation: “Discovery and characterization of lysine rich cyclotides from critically endangered species from Australian continent” at ASMR QLD Postgraduate Student Conference, Brisbane, QLD, Australia. (29 May 2013) 4. Poster presentation: “Novel cyclotides from Palicourea guianensis Aubl. (Showy cappel)” at International Conference on Circular Proteins 2012, Heron Island, Australia. (14 - 17 October 2012) 5. Oral and poster presentation: “Evaluation of Antioxidant and Anti-inflammatory Activities of Chinese medicinal plants Extracts” at 27th International Symposium on the Chemistry of Natural Products, Brisbane, QLD, Australia. (10-15 July 2011) 6. Oral presentation: “Isolation and identification of biomarkers and cyclic peptides from medicinal herbs” at Student Conference and Careers day, University of Western Sydney. (Feb 2011) v Publications included in this thesis 1. Ravipati, A. S., Henriques, S. T., Poth, A. G., Kaas, Q., Wang, C. K., Colgrave, M. L., and Craik, D. J. (2015). Lysine-rich cyclotides: A new subclass of circular knotted proteins from Violaceae. ACS Chemical Biology 10, 2491-2500. (incorporated as Chapter 3). Contributor Statement of contribution Ravipati, A. S. (Candidate) Designed experiments (80%) Plant species collection (90%) Peptide isolation and purification (100%) Peptide sequencing (90%) Statistical analysis of data (60%) Membrane binding studies (90%) Structure-activity relationship (80%) Wrote and revision of paper (70%) Preparation of figures (80%) Henriques, S. T. Designed experiments (20%) Membrane binding studies (10%) Revised the paper (10%) Revision of figures (10%) Poth, A. G. Peptide sequencing (5%) Kaas, Q. Statistical analysis of data (40%) Wang, C. K. Structure-activity relationship (20%) Colgrave, M. L. Peptide sequencing (5%) Revision of figures (5%) Craik, D. J. Plant species collection (10%) Revision of figures (5%) Revised the paper (20%) vi Contributions by others to the thesis Amino acid analysis of peptides was carried out by Linda Chan at Florey Neuroscience Institutes, The University of Melbourne. Murari Bhandari and Akello helped with purification of cyclotides described in Chapter 4. Bacterial assays described in Chapter 3 were conducted by Dr. Sonia Henriques. Cytotoxicity assays against cultured mammalian cells described in Chapters 3 and 5 were conducted by Stephonie Chaousis and Dr. Sonia Henriques. Statistics on cyclotides described in Chapter 3 were conducted by Dr. Quentin Kaas. Dr. Aaron Poth and Michelle Colgrave confirmed the peptide sequences Garry Morgan conducted TEM imaging described in Chapter 6. Dr. Conan Wang helped with the modelling of Lys-rich cyclotides (Chapter 3) Statement of parts of the thesis submitted to qualify for the award of another degree None vii Acknowledgements During my PhD, many people offered me valuable guidance and support directly or indirectly. I would like to wholeheartedly
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