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Discovery and Characterization of Nav Modulatory Venom Peptides

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Discovery and Characterization of Nav Modulatory Venom Peptides Discovery and characterization of NaV modulatory venom peptides Joshua Seth Wingerd B.Sc. of Molecular Biology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2013 Institute for Molecular Biosciences Abstract Voltage-gated sodium channels (NaV) are integral membrane proteins that are responsible for the increase in sodium permeability that initiates and propagates the rising phase of action potentials, carrying electrical signals along nerve fibers and through excitable cells. NaV channels play a diverse role in neurophysiology and neurotransmission, as well as serving as molecular targets for several groups of neurotoxins that bind to different receptor sites and alter voltage-dependent activation, inactivation and conductance. There are nine NaV channel isoforms so far discovered, each of which display distinct functional profiles and tissue-specific expression patterns. The modulation of specific isoforms for therapeutic purposes has become an important research objective for the treatment of conductance diseases exhibiting phenotypes of chronic pain, epilepsy, myotonia, seizure, and cardiac arrhythmia. However, because of the high sequence similarity and structural homology between NaV channel isoforms, many current therapeutics that target NaV channels – the vast majority of which are small molecules – lack specificity between isoforms, or even other voltage-gated ion channels. The current push for greater selectivity while maintaining a relevant degree of potency has led the focus away from small molecules and towards the discovery and development of peptidic ligands for therapeutic use. Venom derived peptides have proven to be naturally potent and selective bioactive molecules, exhibiting inherent secondary structures that add stability through the formation of disulfide bonds. Organisms such as cone snails and spiders have evolved venom for the purpose of prey capture and defense, therefore many of the components exhibit paralytic qualities and specifically target NaV channels. Much of the discovery process has focused on screening crude venoms for a particular function and then isolating the molecule responsible for that function using assay guided purification. The first section of this thesis describes the development of a cell-based, high-throughput functional assay for the detection of NaV modulating venom peptides in crude venom. This assay was successfully implemented and resulted in the isolation and sequencing of MVIA from Conus magus. Initial results and sequence similarity placed MVIA into the δ-conotoxin family, a poorly described class of peptides that inhibits fast inactivation. However, multiple solid-phase synthesis and bacterial recombinant expression methods were unsuccessful in producing enough of the extremely hydrophobic δ-MVIA for further characterization. As no more C. magus crude venom was available, this project was left until optimized methods of production for highly hydrophobic peptides could be developed. i An optimized method of bacterial recombinant expression was successful in producing large yields of another venom peptide, β/δ-TRTX-Pre1a, isolated from the spider Psalmopoeus reduncus. The same recombinant expression method was also used to produce uniformly labeled 13C/15N-peptide for determination of a heteronuclear NMR solution structure. Pre1a was revealed to be a sub-micromolar inhibitor of both NaV1.2 and NaV1.7 peak currents, yet demonstrated potent inhibition of fast inactivation of NaV1.3. This dual mode of action on different NaV isoforms had not yet been reported for any known venom peptide. Further, Pre1a exhibited structural heterogeneity as determined by analysis of rpHPLC and NMR 15N-HSQC, which was traced to the contribution of residues making up the all aromatic, hydrophobic face of the peptide. The high sequence similarity of Pre1a to previously discovered spider venom peptides allowed comparative functional analysis and the identification of key residues contributing to NaV isoform selectivity. Mutagenesis focusing on both structural and functional aspects of Pre1a was performed incorporating both alanine and non-alanine substitutions. Through a single mutation a residue critical for the observed conformational heterogeneity was identified. This mutation served as a model for the obtainment of a high-resolution solution structure for comparison with the active Pre1a. Lastly, we identified a single residue on the C-terminus critical for NaV channel isoform selectivity. Substitution with amino acids exhibiting different properties of charge, polarity, and size could modify selectivity and in the process create a highly selective inhibitor of NaV1.2. This study not only revealed a unique mode of action for a venom peptide, but also demonstrated novelty as a proof-of-concept for the rational design and engineering of venom peptides using available information and non-standard methods of selective mutagenesis. 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 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. iii Publications during candidature Peer-reviewed publications Vetter, I., Mozar, C.A., Durek, T., Wingerd, J.S., Alewood, P.F., Christie, M.J., Lewis, R.J. Characterisation of NaV types endogenously expressed in human SH-SY5Y neuroblastoma cells (2012) Biochemical Pharmacology 83 (11) , pp. 1562-1571 Muttenthaler, M., Dutertre, S., Wingerd, J.S., Aini, J.W., Walton, H., Alewood, P., Lewis, R. Abundance and diversity of Conus species (Gastropoda: Conidae) at the northern tip of New Ireland province of Papua New Guinea (2012) The Nautilus 126 (2), pp.47-56 Book Chapters Wingerd, J.S., Vetter, I. and Lewis, R.J. (2012) Voltage-Gated Sodium Channels as Therapeutic Targets, in Therapeutic Targets: Modulation, Inhibition, and Activation (eds L. M. Botana and M. Loza), John Wiley & Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/9781118185537.ch3 Posters/Abstracts Wingerd, J.S., Vetter, I. Rash, L. and Lewis, R. (2011, May 15-20) Exploring the Nav Activity of ICK Venom Peptides Using TRTX-Pre1a and δ-MVIA as Models. Poster at the Fourth Conference on Venoms to Drugs, Heron Island, QLD, Australia. Wingerd, J.S. (2011, Mar 23) TRTX Pre1a: A Tale of Two Pharmacologies. Poster #P2J20-1 at The 11th Southeast Asian Western Pacific Regional Meeting of Pharmacologists, Yokohama, Japan. Publications included in this thesis No publications included. iv Contributions by others to the thesis Significant input regarding design, development, and implementation for fluorescent based assays in Chapter II was given by Dr. Irina Vetter, especially regarding the development of FLIPR methods in section 2.8 and 2.9 and the primer design for the cell line characterization for section 2.1. The initial isolation, sequencing, and some activity on NaV1.3 and NaV1.7 for TRTX-Pre1a were completed by Dr. Lachlan Rash. Dr. Rash also contributed significantly to the experimental design of Chapters III and IV, training in the use of TEVC oocyte electrophysiology, and interpretation of the e- phys results. Dr. Rash was also instrumental in the critical review and revision of Chapters III, IV and V of this thesis. NMR instrumentation was handled and experiments were run by Dr. Mehdi Mobli. Significant input regarding interpretation of the NMR results, as well as critical revision of relevant sections of Chapters III and IV of the thesis, was also done by Dr. Mobli. Patient training in the use of Sparky NMR analysis software was provided by Dr. Yanni Chin. Dr. Chin also gave her support regarding data interpretation, running TALOS statistics, and the development of the 15N-HSQC overlay figures, Figure 41, Figure 42, and Figure 64. The pLIC shuttle vector was originally designed by Dr. Natalie Saez. Statement of parts of the thesis submitted to qualify for the award of another degree None v Acknowledgements This thesis is a compilation of years of work that could not have been possible without the personal and professional support of family, friends, and colleagues. First and foremost I would like to thank my parents, Mala and Bruce, for their constant support in all realms and undying positive reinforcement. Although there have been no shortage of hard times, their love and encouragement has helped me to succeed through it all. I would also like to thank my supervisor, Prof. Richard Lewis for his continual support during the entirety of my PhD studies at the Institute for Molecular Bioscience. Not only has Richard allowed me to study in an amazing lab, but he has opened up many opportunities outside the lab and in the field. I would also like to thank my co-supervisor Dr. Irina Vetter for her wisdom, advice and friendship from the onset of this degree. Many thanks go to Dr. Lachlan Rash, who gave his support as a co-supervisor and a mentor for the final half of this degree.
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