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Characterization of Novel Synthetic Analgesics Selectively Targeting Α3 Glycine Receptors

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Characterization of Novel Synthetic Analgesics Selectively Targeting Α3 Glycine Receptors Characterization of novel synthetic analgesics selectively targeting α3 Glycine receptors Maleeha Waqar B.S. Bioinformatics A thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2019 Queensland Brain Institute i Abstract Glycine receptors (GlyRs) are ligand-gated ion channel proteins that mediate fast inhibitory neurotransmission by selectively permitting passage of Cl- ions through its pore, which is activated by the binding of the neurotransmitter glycine. The major inhibitory receptors in the spinal cord are the GlyRs, and are therefore physiologically very important. Mutations in the genes that code for the GlyRs result in several disorders such as hyperekplexia or startle disease. However, disruption of the glycinergic currents that are mediated by GlyRs, particularly in the dorsal horn of the spinal cord that receives sensory input from the superficial laminae, has implications in other disorders too, for example chronic inflammatory pain. Potentiating these GlyRs and their activity can reduce the chronic pain sensitization, which is why potent and selective modulators for the GlyRs are strong candidates for analgesic development. Considering the importance of α3 GlyRs as targets for potentiators to alleviate pain, the aim of this project was to model and predict the binding site of a previously developed GlyR potentiator designated as HO1, and the screening of novel cannabinoids for potentiation in GlyRs, with the use of patch-clamp electrophysiological techniques. HO1 selectively potentiates α3 over α1 GlyRs, expressing in both HEK293 cells and artificial inhibitory synapses. The binding site of HO1 on the α3 GlyR was modelled via molecular docking and was verified by performing site directed mutagenesis of the modelled binding residues in the α3 GlyR and then subjecting these mutants to electrophysiological studies. The mutations retained the functionality of the α3 GlyRs but shifted the receptor’s glycine EC50 and showed no potentiation on binding to HO1. The site for HO1 binding modelled and studied here may have properties of a site involved in allosteric regulation, considering the drastic effects of its mutation on both glycine sensitivity and HO1 potentiation. Recent studies have also shown that certain cannabinoids and their derivatives have a strong effect on glycine receptors, producing analgesic effect in animal models. Two novel cannabinoid derivatives, 5-Desoxy-CBD (1803) and 1,2-dimethylheptyl-desoxy CBD (1816) were subjected to electrophysiological studies in artificial inhibitory synapses expressing α1, α3, α1β and α3β GlyRs to measure potentiating effects. The cannabinoids significantly potentiated α3 GlyR, with 1803 also potentiating α1 and α1β, and 1816 showing more selectivity for GlyRs containing α3 subunits over α1. Both drugs showed a stronger affinity for homomeric channels in comparison to heteromeric, suggesting that their possible site of action is present on the α subunits. Based on the experiments in this study, we ii conclude that HO1 and the cannabinoids are potential leads in development of strong analgesic drugs with GlyR isoform selectivity for the treatment of inflammatory pain. 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, financial support 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 higher degree by research 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 and have sought permission from co- authors for any jointly authored works included in the thesis. iv Publications during candidature No publications included. Submitted manuscripts included in this thesis No manuscripts submitted for publication. Other publications during candidature No other publications. Contributions by others to the thesis Significant contribution was made by Lynch JW in conception of the architecture of the study and in designing the experimental methods, in particular the electrophysiological assays. Talwar S had done the preliminary work of screening and identifying the lead compound CMB-C28-HO1 that is further characterized in this study. Other than that no significant contributions were made to the design, research work and writing of this thesis by any other individuals or parties. Statement of parts of the thesis submitted to qualify for the award of another degree None v Acknowledgements First and foremost, I would like to thank my supervisor Joe, for his unlimited support, guidance, critique and humour. These past 2 years have been an incredible privilege, to have had the opportunity to learn from him. Most admirably, Joe has never taken a day off from being a diligent supervisor, not even when he is rock climbing or recovering from a serious health problem. I also want to thank my co-supervisor Angelo, for always answering any and all questions I had, even when I would be afraid to ask them. More than that, I have truly found the greatest inspiration in Angelo’s commitment to this lab and in being a dedicated teacher. I cannot thank enough Justine Haddrill, the greatest lab manager that any lab could have. It is safe to say that Lynch lab could not run as smoothly as it does without her, and most of my experiments would not have worked if she wasn’t there to guide me properly on where everything is and how it all works. Chen, Nela, Atif and Parnayan, I owe you greatly for always offering great advice, feedback and support wherever and whenever I needed it. A special thanks to Dr. Helen Cooper and Dr. Bruno Van Swinderen for being very supportive in this program and always having their doors and emails open for any problems. I am greatly thankful to my amazing friends Drew, Chai Chee and Consuelo, who have been my family here in Brisbane, have constantly made sure that I not only survive but enjoy my time here, and have also been the smartest and funniest people to be around. I owe my life and all the privileges to my parents, who raised me to be a strong and independent Muslim woman, understand and acknowledge my roots and always raise my head high in the face of any challenge. To my siblings, I am thankful for the constant fights and laughs, for them always reminding me where my true strength comes from, and for always looking up to me. I have relied greatly on the support of all my friends back home, and am truly thankful to all of them. Lastly, but mostly, I am thankful to my wonderful husband, Qasim. Thank you for always being the voice of reason and support, for loving me unconditionally from seven thousand miles away, for always meeting me halfway in every adventure, and for being my best friend. vi Financial support This research was supported by an Australian Government Research Training Program Scholarship Keywords Glycine receptor, Chronic Inflammatory pain, Electrophysiology, Analgesics, Drug Discovery Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 060110, Receptors and Membrane Biology, 30% ANZSRC code: 111504, Pharmaceutical Sciences, 40% ANZSRC code: 110903, Central Nervous System, 20% ANZSRC code: 030402, Biomolecular Modelling and Design, 10% Fields of Research (FoR) Classification FoR code: 0601, Biochemistry and Cell Biology, 30% FoR code: 1115, Pharmacology and Pharmaceutical Science, 40% FoR code: 1109, Neuroscience, 30% vii Table of Contents Title page .................................................................................................................................... i Abstract ................................................................................................................................ ii, iii Declaration by author ................................................................................................................ iv Publications during candidature ................................................................................................. v Submitted manuscripts included in this thesis ........................................................................... v Other publications during candidature ....................................................................................... v Contributions by others to the thesis .........................................................................................
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