Non-Natural Nucleotides As Modulators of Atpases
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NON-NATURAL NUCLEOTIDES AS MODULATORS OF ATPASES BY KEVIN ENG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Anthony J. Berdis Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. This work is dedicated to my previous mentors, Silvana Gaetani, Danielle Piomelli, and Steve Barnett. You are the ones responsible for bringing me this far. Table of Contents Signature Sheet i Dedication ii Table of Contents 1 List of Figures 4 List of Tables 7 List of Abbreviations 8 Acknowledgements 9 Abstract 11 Chapter 1: Introduction 1.1 Overview of ATP and ATPases 13 The chemical properties of ATP and its role in biological systems Structural analysis of ATP binding pocket Existing drugs that target the ATP binding site 1.2 Non-natural Nucleotides as Therapeutic Agents 19 Non-natural nucleotides and translesion DNA synthesis Clinical importance of nucleoside analogs 1.3 Model Systems for Evaluating the Influence of 1 Non-Natural Nucleotides on ATP-dependent Processes 22 Sliding clamp loader ATPase P-glycoprotein 1.4 Statement of Purpose 31 Figures 32 Tables 68 Chapter 2: Selective inhibition of DNA replicase assembly by a non-natural nucleotide: Exploiting the structural diversity of ATP-binding sites 2.1 Abstract 69 2.2 Introduction 71 2.3 Materials and Methods 72 2.4 Results and Discussion 76 Figures 89 Tables 112 Chapter 3: A Novel Non-Natural Nucleoside that Influences P- glycoprotein Activity and Mediates Drug Resistance 3.1 Abstract 115 2 3.2 Introduction 116 3.3 Materials and Methods 118 3.4 Results 121 3.5 Discussion 128 Figures 134 Tables 150 Chapter 4: Conclusions and future directions 4.1 Overview 154 4.2 Chapter 2 Conclusions 155 4.3 Chapter 2 Future directions 156 4.4 Chapter 3 Conclusions 163 4.5 Chapter 3 Future directions 164 Figures 170 References 179 3 List of Figures Figure 1.1 Structures of ATP and dATP 33 Figure 1.2 Schematic of a molecular motor and actin assembly 35 Figure 1.3 Representation of a typical ATPase P-loop 37 Figure 1.4 Chemical mechanism of ATP hydrolysis. 39 Figure 1.5 Structures of ATP competitive inhibitors 41 Figure 1.6 Imatinib and cAbl kinase 43 Figure 1.7 Structures and electron density maps of non-natural nucleobases. 45 Figure 1.8 Schematic of translesion DNA synthesis 47 Figure 1.9 Structures of nucleoside analogs used in cancer chemotherapy 49 Figure 1.10 Structures of nucleoside analogs used in anti-viral chemotherapy 51 Figure 1.11 Schematic of replicase formation 53 Figure 1.12 Subunit compositions of clamp loaders from different species 55 Figure 1.13 Sample of the structural diversity of P-gp substrates 57 Figure 1.14 Model of P-glycoprotein transport 59 Figure 1.15 Structure of P-glycoprotein 61 Figure 1.16 ATP switch model 63 Figure 1.17 The occluded nucleotide model 65 Figure 1.18 Structures of the most recent P-glycoprotein inhibitors 67 Figure 2.1 Natural and non-natural nucleobases used in this study 90 Figure 2.2 Hydrolysis of nucleotide substrates by gp44/62 and the effect of r5-NITP and d5-NITP on gp44/62 ATPase activity 92 Figure 2.3 Effects of d5-NITP on T4 replicase formation and gp45 opening 94 4 Figure 2.4 Plot of K i values for various non-natural nucleotides as a function of their respective nucleobase size 96 Figure 2.5 Molecular modeling of the active sites of gp44/62 and γ-complex. 98 Figure 2.6 Alignment of the ATP-binding region of the bacteriophage T4 gp44/62, Saccharomyces cerevisiae RFC, and Escherichia coli γ-complex 100 Figure 2.7 Inhibition of T4 plaque formation by d5-NI 102 Figure 2.8 d5-NI is not bactericidal or bacteriostatic against E. coli . 104 Figure 2.9 Kinetic model for the effects of a competitive inhibitor on the concurrent activity of two independent enzymes that possess different K m values for a common substrate and different K i values for a common competitive inhibitor 106 Figure 2.10 Comparison of the DNA replication mechanisms of E.coli and T4 bacteriophage 111 Figure 3.1 Structures and electrostatic potential models of non-natural nucleosides used in this study 135 Figure 3.2 Stimulation of P-gp ATPase activity by non-natural nucleosides 137 Figure 3.3 Stimulation of P-gp ATPase activity by increasing concentrations of calcein-AM in the presence and absence of d5-CHI or CsA 139 Figure 3.4 Modulation of drug resistance by d5-CHI in KB-V1 cells 141 Figure 3.5 Effects of d5-CHI, d5-CEI, and d5-PhI on the cytotoxicity of paclitaxel and vinblastine in KB-V1 cells 143 Figure 3.6 Effects of d5-CHI on cell viability. 145 Figure 3.7 Comparison of the structures of P-gp interacting compounds 147 Figure 3.8 Trend in ATPase V max /K m ratios and MDR modulation 149 5 Figure 4.1 Schematic of chapter 2 conclusions 171 Figure 4.2 Kinase profiling of non-natural nucleo(s)tides 173 Figure 4.3 Chemical structures of ligands for the 5HT2, histamine H2, adenosine, and melatonin receptors. 175 Figure 4.4 Schematic of model correlating changes in p-glycoprotein ATPase catalytic efficiency with changes in p-glycoprotein mediated drug resistance 177 6 List of Tables Table 1.1 Subunit compositions and functions of clamp loaders from various species 68 Table 2.1 Summary of inhibition constants for natural and non-natural nucleotides against the ATP-dependent clamp loaders from bacteriophage T4 (gp44/62) and Escherichia coli (γ-complex) 112 Table 2.2 Summary of inhibition constants for various non-natural nucleotides against wild-type, R175K, and R175L mutants of gp44/62 114 Table 3.1 Kinetic parameters for the stimulation in ATPase activity by drug substrates 150 Table 3.2 Permeability coefficients of substrates across MDCK and MDCK-MDR monolayers 151 Table 3.3 Effects of d5-CHI on the catalytic efficiency of drug-stimulated P-gp ATPase activity 152 Table 3.4 Effects of d5-CHI on the cytotoxicity of vinblastine (VBL), doxorubicin (DOX), colchicine (COLC), and paclitaxel (TAX) in KB-V1 cells and parental KB-3-1 cells 153 Table 4.1 ABC transporters involved in multidrug resistance and their known substrates 178 7 List of Abbreviations d4-NITP, 4-nitro-indolyl-2’-deoxyriboside triphosphate; d5-NITP, 5-nitro-indolyl-2’- deoxyriboside triphosphate; r5-NITP, 5-nitro-indolyl-2’-ribose triphosphate; d5-NI, 5-nitro- indolyl-2’-deoxyriboside; d6-NITP, 6-nitro-indolyl-2’-deoxyriboside triphosphate; d5-EtITP, 5-ethyl-indolyl-2’-deoxyriboside triphosphate; d5-EyITP, 5-ethylene-indolyl-2’-deoxyriboside triphosphate ; d5-EyInd, 5-ethylene-indolyl-2’-deoxyriboside; d5-FITP, 5-fluoro-indolyl-2’- deoxyriboside triphosphate; d5-FI, 5-fluoro-indolyl-2’-deoxyriboside; dITP, indolyl-2’- deoxyriboside triphosphate; Ind, indolyl-2’-deoxyriboside; d5-CHITP, 5-cyclohexyl-indolyl- 2’-deoxyriboside triphosphate; d5-AITP, 5-amino-indolyl-2’-deoxyriboside triphosphate; d5- CEITP, 5-cyclohexene-indolyl-2’-deoxyriboside triphosphate; d5-CITP, 5-carboxylate- indolyl-2’-deoxyriboside triphosphate; d5-PhITP, 5-phenyl-indolyl-2’-deoxyriboside triphosphate; AMP-PNP, 5'-adenylyl-beta,gamma-imidodiphosphate; gp44/62, bacteriophage T4 sliding clamp loader; gp45, bacteriophage T4 sliding clamp; gp43 exo -, exonuclease-deficient mutant of bacteriophage T4 DNA polymerase; γ complex, E. coli sliding clamp loader; β clamp, E. coli sliding clamp. d5-CEI, 5-cyclohexene indolyl 2’- deoxyribose; d5-CHI, 5-cyclohexyl indolyl 2’-deoxyribose; d5-PhI, 5-phenyl indolyl 2’- deoxyribose; AP→BL, apical to basolateral; BL→AP, basolateral to apical; CsA, cyclosporine A; MDR, multi drug resistance; MDCK, Madin Darby Canine Kidney; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NIMH, National Institutes of Mental Health; PDSP, Psychoactive Drug Screening Program; Peff , permeability coefficient; P-gp, P-glycoprotein; SAR, structure activity relationship. 8 Acknowledgements This work would not be possible without the contributions of several individuals. First and foremost, my advisor Dr. Anthony Berdis for providing support and guidance throughout my training. I would also like to thank my committee members Dr. Chris Dealwis, Dr. Shigemi Matsuyama, and Dr. Robert Bonomo whose insights were invaluable to my professional development and Dr. John Mieyal for his guidance. Special thanks to Edward Motea, Xuemei Zhang, and Sandra Craig for their time and effort in providing non- natural nucleotides of the highest quality used throughout all my studies. I am also grateful to Dr. Irene Lee for teaching me the principles of protein purification. I would also like to thank Dr. Babho Devadoss for providing me with the invaluable guidance of a senior graduate. I am also indebted to my undergraduate student, Raphael Bendriem, for his fresh insights to the P-glycoprotein project which helped shape the project as it is today. Special thanks to members of the Berdis lab (past and present); Jackelyn Golden, Andrea Ramos, Asim Sherrif, Robert Bowers, Dave McCutcheon, and Kevin Costanzo for making every workday more enjoyable. Several researchers have provided me with their advice and expertise throughout my studies: Philip Kiser, David Lodowski, Jim Fairman, Jay Prendergast, Seunghwan Lim, Vivian Gama, Jose Gomez, Reema Wahdan, Andrea Moomaw, and Monica Montano. During my time here at Case, I have been truly blessed with many great friends: Eric Lam, Vaibhav Pathak, Chohee Yun, Charlotte Chung, Alejandro Colozo, Paul Park, Debarshi Mustafi, Rod Nibbe, Dasha Hajkova-Leary, Ndiya Ogba, the Marksz family, Stasha Razack, Jared Klaus, and Joe Dea.