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Inhibiting Voltage Gated Sodium Channel 1.7 with Spider-Venom Peptides

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Inhibiting Voltage Gated Sodium Channel 1.7 with Spider-Venom Peptides Developing novel analgesics to treat chronic pain: Inhibiting voltage gated sodium channel 1.7 with spider-venom peptides Darshani Buddhika Rupasinghe Arachchilage BSc in Biochemistry and Microbiology (Honors) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in February 2014 Institute for Molecular Bioscience ABSTRACT Chronic pain is responsible for great physical, mental and economic loss. While there are diverse methods available for managing pain, such as NSAID, opioids and local anesthetics, common issues associated such as addiction and tolerance highlights the unmet demand for a novel analgesic aimed at a novel target. Numerous reports on individuals suffering from loss of function mutations in SCN9A marker, which leads to complete inability to feel pain, yet otherwise normal physiology, has led to the recognition of voltage gated sodium channel 1.7 (NaV1.7) as a prime pain target. Other reports of gain of function mutations that lead to conditions such as paroxysmal extreme pain disorder and primary erythromelalgia confirms the validity of this target. However the presence of nine closely related subtypes of voltage gated sodium channels, NaV1.1-NaV1.9 introduces complications as any cross inhibition may lead to detrimental results. For example inhibition of NaV1.5 subtype could result in cardiac arrhythmia to complete cardiac arrest and death. Therefor it is of utmost importance that any pharmaceutical agent used to inhibit NaV1.7 is highly specific for NaV1.7 and display no cross reactivity on other voltage gated channels. Spiders are one of the most successful terrestrial predators; their venoms have evolved over many millions of years to selectively and potently inhibit nervous system targets in order to rapidly paralyze their prey. Spider-venom peptidomes have been identified as one of the largest known libraries of compounds with very high potency and specificity towards nervous system targets. Spider toxins listed in the Arachnoserver database have been classified into 12 families of sodium channel toxins (NaSpTx), denoted NaSpTx Families 1–12, based on the level of sequence conservation and intercystine spacing. NaSpTx family 2 is the largest toxin family; it comprises 34 peptides of theraphosid origin. This thesis primarily aims at discovering novel inhibitors of NaV1.7. However the intriguing fact that people suffering form congenital indifference to pain also suffers form anosmia, has lead to efforts of understanding the role of NaV1.7 in olfaction. This study has been ii completed with the finding that NaV1.7 is located along axons of olfactory neurones. These findings have been published and research article is attached as chapter 4. During the initial discovery phase of this project, an assay guided fractionation method was used to identify NaV1.7 inhibitors form six spider venoms. This resulted in nine fully sequenced peptides where four of these belonged to NaSpTx family 2. This included the well-known β/ω-TRTX-Tp1a (Protoxin1). Three novel peptides discovered were named β- TRTX-Pe1a, µ-TRTX-Pe1b, and U-TRTX-Pa1a. A complete alanine scan of β/ω-TRTX-Tp1a on NaV1.7 was conducted using a Xenopus laevis oocyte based tethered toxin method. Residues that were important for NaV1.7 binding were mapped onto the three-dimensional structure of β/ω-TRTX-Tp1a. Three novel toxin peptides β-TRTX-Pe1a, β/µ-TRTX-Pe1b, and U-TRTX-Pa1a were recombinantly expressed using an Escherichia coli (E. coli) based system. Channel modulating activities of these toxins were assessed using a Xenopus laevis oocyte based two-electrode voltage clamp system (TEVC). µ-TRTX-Pe1b demonstrated a high degree of selectivity for NaV1.7. It is about 20 times more selective over NaV1.2, which is abundant in the central nervous system, and appears to mildly potentate NaV1.5, which is the cardiac subtype. This is despite the complete set of active residues uncovered in β/ω- TRTX-Tp1a alanine scan being fully conserved in β/µ-TRTX-Pe1b. β-TRTX-Pe1a and U1- TRTX-Pa1a have shown very weak NaV1.7 inhibition, hence not investigated in great detail. The introduction of the mutation S35 to the sequence of β/µ-TRTX-Pe1b has resulted in the β/µ-TRTX-Pe1b-S35, which has shown increased NaV1.7 activity that is almost comparable to β/ω-TRTX-Tp1a. Structure activity relations (SAR) of β/ω-TRTX-Tp1a and β/µ-TRTX-Pe1b-S35 are currently under investigation. It is highly promising that better understanding of residues responsible for the high potency of β/ω-TRTX-Tp1a and higher NaV selectivity of β/µ-TRTX-Pe1b-S35 would lead to engineering a superior NaV1.7 inhibiting analgesic agent. This thesis has widened our understanding of NaSpTx family 2 peptides, and residues, which may be important in achieving NaV1.7 selectivity by inhibitor peptides. 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 Peer reviewed papers Rupasinghe, D.B., Knapp, O., Blomster, L.V., Schmid, A., Adams, D.J., King, G.F., and Ruitenberg, M.J. (2012) Localization of NaV1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction, pheromone sensing and immune function. Channels. Klint, J.K., Senff, S., Rupasinghe, D.B., Er, S.Y., Herzig, V., Nicholson, G.M., and King, G.F. (2012) Spider-venom peptides that target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads. Toxicon Conference Abstracts Rupasinghe, D.B., Knapp, O., Blomster, L.V., Schmid, A., Adams, D.J., King, G.F., and Ruitenberg, M.J. (2011) Localization of NaV1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction, pheromone sensing and immune function. 4th Venoms to Drugs Meeting, Heron Island, Australia, May 15-20 (poster). Darshani b. Rupasinghe, Norelle Daly, Mehdi Mobli, Junhong Gui, Michael N. Nitabach and Glenn F. King (2013) A tethered toxin approach to understand the NaV1.7 binding pharmacophore of Protoxin-1. Chemistry and Structural biology symposium, Institute for molecular Bioscience, University of Queensland, Brisbane, Australia, November 7th (poster). v Publications included in this thesis Introduction: Selected sections of the review indicated below that I coauthored are incorporates into the introduction of this thesis, especially the section on NaSpTx family 1, which was written exclusively by me. Klint, J.K., Senff, S., Rupasinghe, D.B., Er, S.Y., Herzig, V., Nicholson, G.M., and King, G.F. (2012) Spider-venom peptides that target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads. Toxicon Contributor Statement of contribution Rupasinghe, D.B., (Candidate) Designed experiments (50%) Wrote and edited paper (35%) Klint, J.K Designed experiments (25%) Wrote and edited paper (30%) Senff, S Wrote and edited paper (10%) Er, S.Y Wrote and edited paper (10%) Herzig, V Edited the manuscript Nicholson, G.M. Edited the manuscript King, G.F. Wrote and Edited the paper (15%) Designed experiments (25%) Chapter 3: The complete published paper is attached as the Chapter 3 Rupasinghe, D.B., Knapp, O., Blomster, L.V., Schmid, A., Adams, D.J., King, G.F., and Ruitenberg, M.J. (2012) Localization of NaV1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction, pheromone sensing and immune function. Channels. Contributor Statement of contribution Rupasinghe, D.B (Candidate) Designed experiments (70%) Wrote the paper (80%) Knapp, O Designed experiments (10%) Wrote the paper (5%) Blomster, L.V., Designed experiments (5%) Schmid, A., Designed experiments (5%) Adams, D.J Edited the manuscript King, G.F Edited the manuscript Wrote the paper (5%) Ruitenberg, M.J Designed experiments (10%) Wrote the paper (10%) Edited the manuscript vi Contributions by others to this thesis Chapter two: ProTx1 was chemically synthesized by Mr Zoltan Dekan. The structure was determined using 2D homonuclear NMR spectroscopy in collaboration with Dr Mehdi Mobli (Centre for Advanced Imaging, UQ) and Prof. Norelle Daly (James Cook University). A/Prof. Frank Bosmans and John Gilchrist performed the kinetics experiments for the NaV1.7/KV2.1 chimeric constructs. Chapter three: The venom based discovery process is a collaboration with the laboratory of Prof Richard Lewis at the Institute for Molecular Bioscience, The University of Queensland. Dr Irina Vetter conducted all FLIPR assays. Chapter four: (Incorporated paper) Dr Linda V. Blomster, and Dr Anina Schmid were involved in the preliminary findings that olfactory tissue contain NaV1.7 and it is detectable by immunohistochemical methods.
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