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Identifying Druggable Sites in the Pain-Sensing TRPA1 Ion Channel

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Identifying Druggable Sites in the Pain-Sensing TRPA1 Ion Channel Identifying Druggable Sites in the Pain-Sensing TRPA1 Ion Channel The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Skinner III, Kenneth Arthur. 2017. Identifying Druggable Sites in the Pain-Sensing TRPA1 Ion Channel. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41140248 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA © 2017 Kenneth Skinner All rights reserved. Dissertation Advisor: Professor Rachelle Gaudet By: Kenneth Arthur Skinner III Identifying druggable sites in the pain-sensing TRPA1 ion channel Abstract Transient receptor potential (TRP) channels, found in all eukaryotes, are a family of cation channels vital for critical physiological processes, such as mediating the sensation of unanticipated pain. Most TRP channel activities are modulated by a diverse set of exogenous and endogenous ligands, several of which are important for human life. For example, general anesthetics, which are widely used in surgical procedures, have undesired effects by activating TRPA1, which functions as a pain-sensing ion channel. Upon administration, several noxious general anesthetics stimulate peripheral sensory neurons, which transduce pain and inflammation elicited by these widely used drugs. Although evidence points to TRPA1 as the major player in this pain pathway, where propofol binds in the channel is unclear. Here I present the first photocrosslinking strategy to identify the propofol binding sites in TRPA1. I designed synthetically expedient routes to three crosslinkable analogues of propofol, two of which are also functionalized with an alkyne affinity handle for bioorthogonal chemistry. Using mouse TRPA1 heterologously expressed in insect cells and fluorescence-based functional assays to compare propofol and the synthesized analogues, I determined that ortho-substitution, relative to the hydroxyl group, still allows for TRPA1 activation. Using the same activity assay, I conducted small molecule structure-function studies, introducing substituents replacing the phenolic hydroxyl group of propofol as well as adding or replacing substituents at the ortho and para positions. My results demonstrate the importance of hydrogen bonding in propofol activation of TRPA1 and provide evidence that halogen substitution can affect channel function. Photoaffinity labeling with a well-characterized propofol analogue, AziPm, which demonstrates iii the same dose-dependent and bimodal TRPA1 activation profile as propofol, identified two crosslinked residues in TRPA1. Both residues lie on the S6 helix, which, together with S5, forms the channel pore and gate of TRPA1. V954 faces a pocket previously identified as the A- 9607079 antagonist binding site, while E969 lies near the intracellular mouth of the pore at the junction of S6 and the “TRP-domain” -helix, which is important for channel gating. These results provide the first direct identification of general anesthetic binding sites leading to TRPA1 activation. iv Table of Contents Title Page i Copyright Page ii Abstract iii Table of Contents v Acknowledgments vii List of Figures ix List of Tables xiii Chapter 1 – TRPA1 mediates sensation to reactive and nonelectrophilic compounds 1 References 17 Chapter 2 – Design, synthesis, and characterization of propofol photoaffinity probes Abstract 26 Introduction 26 Materials and Methods 28 Results 41 Discussion 51 References 53 v Chapter 3 – Biological evaluation of propofol analogues as TRPA1 modulators Abstract 57 Introduction 57 Materials and Methods 59 Results 62 Discussion 76 References 79 Chapter 4 – Towards a model of propofol-induced activation of TRPA1 Abstract 85 Introduction 85 Materials and Methods 87 Results 90 Discussion 95 References 100 Chapter 5 – Discussion 103 References 108 Appendix – Supplementary 1H and 13C NMR spectra 112 vi Acknowledgments Over the past 6 years of graduate school, I have been fortunate to meet great scientists and colleagues. First and foremost, I thank my advisor Rachelle Gaudet for supporting me as a scientist and young professional. Rachelle has challenged me to keep improving and I am forever grateful for her mentorship. I would also like to thank the members of the Gaudet lab. Jinn Inada, a former postdoctoral fellow in the lab, first taught me how to conduct functional assays. Also, Jeff McArthur, also a former postdoctoral fellow in the Gaudet lab, was a great bench mate and he is still a helpful friend; I hope to visit him someday in Australia. My fellow grad students were also very supportive. I thank Lukas Bane for scientific discussions and friendship while we were in the lab together. Sri Srikant is a great colleague to discuss scientific ideas and experiments. Jack Nicoludis is a great scientist as well and keeps me informed of all local and national news. My DAC committee has also been very helpful during my time here. Professors Dan Kahne, Ralph Mazitschek, and Steve Blacklow provided valuable feedback and were willing to discuss scientific ideas and proposals outside of meetings. I also thank members of the Harvard scientific community. Joe Wzorek was an insightful mentor who always made himself available to answer chemical inquiries. Jason Millberg, a director of the Chemical Biology graduate program is always on top of things administratively and is readily available to talk basketball anytime. I also thank Phil Snyder, who allowed me to come to Harvard for a little over a month and make small molecules that probe TRP channels. Outside of the lab, I would not be here if it were not for my family. My parents, Agnes Oates and Kenneth Skinner, have continually supported me instilled in me the importance of education since I was young. I also would like to thank my brothers and sisters and for the good vii times when I am in New Jersey for the holidays. I also thank my girlfriend, Christina Saak, for being a supportive partner that I can bounce ideas off and share experiences outside of graduate school. This graduate school journey has taught me that everything seems impossible until it’s done. viii List of Figures Chapter 1 Figure 1.1 Intertwined subunit organization of most six-transmembrane (6-TM) channels 3 Figure 1.2 Primary structure organization of a TRPA1 subunit with major domains 6 Figure 1.3 CryoEM structure of TRPA1 7 Figure 1.4 Chemical structures of photoreactive analogues of propofol 12 Chapter 2 Figure 2.1 Synthetic scheme of XPRO and o and p-XPRO-Click 43 Figure 2.2 1H NMR of XPRO 44 Figure 2.3 1H NMR of o-XPRO-Click 46 Figure 2.4 1H NMR of p-XPRO-Click 47 Figure 2.5 Characterization of XPRO crosslinking 49 Figure 2.6 Characterization of o and p-XPRO-Click clickability 50 Chapter 3 Figure 3.1 Robust TRPA1 expression in baculovirus-infected insect cells 64 Figure 3.2 Propofol is a stronger TRPA1 agonist than o-XPRO-Click 66 Figure 3.3 XPRO and propofol share similar TRPA1 potency 68 Figure 3.4 p-XPRO-Click and propofol share similar TRPA1 potency 69 Figure 3.5 AziPm activates TRPA1 similarly to propofol 71 Figure 3.6 2,6-diisopropylaniline is a stronger TRPA1 agonist than propofol 72 ix Figure 3.7 Fropofol does not activate TRPA1 73 Figure 3.8 2,4-diisopropylphenol activates TRPA1 in a bimodal fashion 75 Figure 3.9 4-bromopropofol does not activate TRPA1 77 Chapter 4 Figure 4.1 TRPA1 peptides detected via MS projected onto TRPA1 topology 91 Figure 4.2 CryoEM structure of TRPA1 with residues modified by AziPm 94 Figure 4.3 Close-up view of V954 and E969 96 Appendix Figure A.1 1H NMR of 4-(4-Hydroxy-3,5-diisopropylphenyl)butan-2-one 113 Figure A.2 13C NMR of 4-(4-Hydroxy-3,5-diisopropylphenyl)butan-2-one 114 Figure A.3 1H NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3,5-diisopropylphenyl)butan-2-one 115 Figure A.4 13C NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3,5-diisopropylphenyl)butan-2-one 116 Figure A.5 1H NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3,5-diisopropylphenyl)-3-methyl-3H- diazirine 117 Figure A.6 13C NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3,5-diisopropylphenyl)-3-methyl- 3H-diazirine 118 Figure A.7 13C NMR of 2,6-diisopropyl-4-(2-(3-methyl-3H-diazirin-3-yl)ethyl)phenol (XPRO) 119 Figure A.8 1H NMR of 4-(4-hydroxy-3-isopropylphenyl)butan-2-one 120 Figure A.9 13C NMR of 4-(4-hydroxy-3-isopropylphenyl)butan-2-one 121 x Figure A.10 1H NMR of 4-(3-bromo-4-hydroxy-5-isopropylphenyl)butan-2-one 122 Figure A.11 13C NMR of 4-(3-bromo-4-hydroxy-5-isopropylphenyl)butan-2-one 123 Figure A.12 1H NMR of 4-(4-hydroxy-3-isopropyl-5-((trimethylsilyl)ethynyl)phenyl)butan-2- one 124 Figure A.13 13C NMR of 4-(4-hydroxy-3-isopropyl-5-((trimethylsilyl)ethynyl)phenyl)butan-2- one 125 Figure A.14 1H NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3-isopropyl-5- ((trimethylsilyl)ethynyl)butan-2-one 126 Figure A.15 13C NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3-isopropyl-5- ((trimethylsilyl)ethynyl)butan-2-one 127 Figure A.16 1H NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3-isopropyl-5- ((trimethylsilyl)ethynyl)phenethyl)-3-methyl-3H-diazirine 128 Figure A.17 13C NMR of 4-(4-((tert-butyldimethylsilyl)oxy)-3-isopropyl-5 -((trimethylsilyl)ethynyl)phenethyl)-3-methyl-3H-diazirine 129 Figure A.18 13C NMR of 2-ethynyl-6-isopropyl-4-(2-(3-methyl-3H-diazirin-3-yl)ethyl)phenol (o-XPRO-Click) 130 Figure A.19 1H NMR
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