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A New Target for Pain: Development of Tools to Study Human Histidine Triad Nucleotide Binding Proteins a DISSERTATION SUBMITTED

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A New Target for Pain: Development of Tools to Study Human Histidine Triad Nucleotide Binding Proteins a DISSERTATION SUBMITTED A New Target for Pain: Development of Tools to Study Human Histidine Triad Nucleotide Binding proteins A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Rachit Shah IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Dr. Carston Rick Wagner, Adviser September 2017 © RACHIT SHAH (2017) Acknowledgements I would first like to extend my sincerest thanks to my thesis advisor Dr. Carston R. Wagner. I am very greatful for his patience, encouragement and guidance. His enthusiasm for science, patience as a mentor has and will always keep inspiring me. I am very grateful to my committee members, Dr. Courtney Aldrich, Dr. Carrie-Haskell Leuvano and Dr. Mark Distefano for their valuable suggestion and feedback throughout my graduate studies. I would like to thank my colloborators Dr. Javier Garzon, Dr. Barry Finzel and Dr. George Wilcox. I want to thank Dr. Brock Matter and Dr. Peter Villata for help with LC-MS studies. I want to specially thank former members of Dr. Wagner’s laboratory, Dr. Adrian Fegan, Dr. Sidharth Kumaraperuma and Dr. Amit Ganger for their support and help. I would like to thank Jing Jing Shen, Anieken Okon, Trent West, Alex Strom, Ozgun Kullic, Clifford Czimar and other members in the Wagner laboratory for their generous help and friendship. I owe my special thanks to my most important friends Nayan Patel, Kalpit Patel, Vimal Patel and Manish Gandhi for their strong support and encouragement that had carried me all through difficult times during my graduate study. Lastly I owe my deepest gratitude to my parents and my loving younger brother Rahul Shah. This dissertation is dedicated to my family and my friends. i Table of Contents List of Tables……………………………………………………………x List of Figures…………………………………………………………..xii List of Abbreviations…………………………………………………...xviii Chapter 1: Literature Review…………………………………………...1 Histidine Triad Nucleotide Binding Proteins…………………………...2 i. History of Nucleoside Phosphoramidases………………………….....2 ii. Physiological Roles of Human Histidine Nucleotide Binding Proteins a. Neuronal Cell Biology…………………………………………...9 b. Cancer Cell Biology…………………………………………….14 c. Mast Cell Biology………………………………………………17 d. Mitochondrial Functions………………………………………..22 iii. Structural Characterization of Human Hint Proteins………………...25 iv. Intracellular regulators/substrates of Human Hint proteins………….26 v. Trends………………………………………………………………..30 vi. Outstanding questions………………………………………………..31 vii. Current Research……………………………………………………..31 Chapter 2: A New Target For Pain: Inhibition of Hint1 as a novel strategy to modulate the cross talk between μ-Opioid and NMDA receptor in the central nervous system…………………………………37 i. Introduction………………………………………………………...38 ii. Results……………………………………………………………...41 ii a. Synthesis and in vitro characterization of TrpGc……………….41 b. TrpGc increases the analgesic effect of opioids in mice…….......42 c. TrpGc prevents/rescues the development of morphine tolerance in mice……………………………………………………………...47 d. Inhibition of Hint1 reduces neuropathic pain in CCI model of mice…………………………………………………………..53 e. Ex-vivo western blot analysis demonstrated that TrpGc reduces activation of NMDA receptors………………………………….53 f. TrpGc prevents endomorphin-2 tolerance……………………....57 g. Inhibition of Hint1 antagonizes the effect of morphine on NMDA evoked behavior in mice………………………………………...57 iii. Discussion…………………………………………………………..59 iv. Experimental procedures…………………………………………...61 a. Protein Expression and Purification…………………………….63 b. Isothermal Titration Calorimetry……………………………….64 c. Evaluation of the antinociception and production of acute tolerance of morphine……………………………………65 d. Evaluation of the prevention of the acute tolerance to endomorphin-2 by TrpGc………………………………………65 e. Chronic Constriction Nerve Injury Pain Model………………..66 f. Western blot analysis…………………………………………...67 g. Studies on NMDA evoked behavior in mice…………………...68 h. Synthetic procedure for the preparation of TrpGc……………...68 v. References………………………………………………………….71 iii Chapter 3: A Design, Synthesis and in vivo evaluation of nucleotidomimetic inhibitors of hHint1………………………………....74 i. Introduction…………………………………………………………75 ii. Results………………………………………………………………78 a. Synthesis and in vitro characterization of nucleoside carbamate inhibitors of Hint1………………………………………………78 b. Synthesis and in vitro characterization of nucleoside acyl- sulfamate inhibitors of Hint1……................................................81 c. Impact of a hydrophobic nucleoside in the molecular recognition of ligands by hHint1…….............................................................89 d. HPLC studies to determine the stability of Hint1 Inhibitors in aqueous solutions……………………………………………….94 e. X-ray crystallographic analysis of Hint1 inhibitors ……………94 f. Evaluation of nucleoside acyl sulfamate inhibitors in vivo…….98 g. Evaluation of cellular cytotoxicity of Hint1 inhibitors………..100 iii. Discussion………………………………………………………...102 iv. Experimental procedures…………………………………………105 a. Protein crystallography………………………………………..106 b. Isothermal Titration Calorimetry……………………………...106 c. HPLC and stability studies on Hint1 inhibitors ………………106 d. MTS Cellular Cytotoxicity Studies……………………………107 e. Studies on NMDA evoked behavior in mice………………….107 f. Synthetic procedure for the preparation nucleoside carbamates and acyl-sulfamate inhibitors of Hint1 ……………………………108 v. References………………………………………………………...126 iv Chapter 4: Caught Before Release: An Alternate Slow Substrate for Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)……………………………………………..129 i. Introduction………………………………………………………..130 ii. Results……………………………………………………………..134 a. Design of the slow substrate and synthesis…………………….134 b. Capture of Nucleotidylated-Histidine complex……..................141 c. Hint1-product complex (EP)…………………………………...147 d. Solvent kinetic isotope studies…………………………………148 e. Proton Inventory studies ………………………………………151 f. Role of structural water molecules…………………………….156 iii. Discussion…………………………………………………………158 iv. Experimental procedures………………………………………….160 a. Synthetic procedure for the preparation of 3…………………..160 b. General kinetic methods……………………………………….162 c. Protein purification ……………………………………………162 d. Structural Biology……………………………………………...163 e. Solvent kinetic isotope effect…………………………………..165 f. Proton inventory studies………………………………………..166 v. References…………………………………………………………168 Chapter 5: Extended Conformer Driven Small Molecule Switch-on Fluorescent Probes to Study Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)………………………………………………………..172 i. Introduction………………………………………………………...173 ii. Results……………………………………………………………..175 v a. Design and synthesis of intramolecularly quenched fluorescent nucleotidomimetic probes for hHint1..………………………...175 b. Both probes exhibit intramoleular static and dynamic quenching of fluorescence in an aqueous solution……...................................178 c. Both probes exhibit switch-on fluorescence properties upon incubation with hHint1 in an aqueous solution ……………….183 d. X-ray crystal structure analysis of compound 7 bound with hHint1…………………………………….................................184 e. Utility of switch-on probes in hHint1-ligand displacement studies………………………………………………………….187 f. A fluorescent switch-on FRET probe …………………………192 g. Selectivity of switch-on fluorescence probes………………….193 h. Utility of 8 in monitoring hHint1-substrate complex under steady- state conditions………………………………………………...196 i. Label free detection of hHint1-substrate complex via monitoring transit turnover time……………………………………………201 iii. Discussion…………………………………………………………203 iv. Experimental procedures…………………………………………. 208 a. Ligand binding and displacement studies ……………………..208 b. Quantum yield, fluorescent lifetime and quenching studies …..209 c. Mean residence transit turnover assay….………………………211 d. Synthesis and characterization of compound 8 ………………..212 v. References…………………………………………………………218 vi Chapter 6: Structure and Functional Characterization of Human Histidine Triad Nucleotide Binding Protein 1 mutations associated with Inherited Axonal Neuropathy with Neuromyotonia…………………..221 i. Introduction………………………………………………………..222 ii. Results……………………………………………………………..225 a. Expression and purification of hHint1 mutations associated with peripheral neuropathy.…………………………………………225 b. Size exclusion analysis of hHint1 mutants…….........................226 c. Secondary structure analysis…………………………………..235 d. Characterization of a catalytically inactive hHint1 H112N mutant…………………………………….................................235 e. Steady-state kinetic characterization of hHint1 mutations…….237 f. Ability of hHint1 mutations to switch-on fluorescent probes…240 g. X-ray crystal structure analysis of the hHint1 mutations……...244 h. Mapping structural features that are critical for the molecular recognition of ligands by hHint1……………………………...245 iii. Discussion………………………………………………………...245 iv. Experimental procedures………………………………………….251 a. Cloning and construction of the MBP-hHint1 plasmid ………251 b. Protein expression and purification …………………………..252 c. X-ray crystallography ………………………………………...254 d. Steady-state kinetic measurement using fluorescent assay …..254 e. Circular Dichroism……………………………………………255 f. Size Exclusion Chromatography……………………………...255 g. hHint1-mutants ligand binding studies……………………….255 v. References………………………………………………………..257 vii List of Tables Chapter 1 Table 1: Substrate specificity of nucleoside phosphoramidates by Hint1…28 Chapter
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