CHEMINFORMATIC AND MECHANISTIC STUDY OF DRUG SUBCELLULAR TRANSPORT/DISTRIBUTION by Nan Zheng A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmaceutical Sciences) in The University of Michigan 2011 Doctoral Committee: Associate Professor Gustavo Rosania, Chair Professor Gordon L. Amidon Associate Professor Kerby A. Shedden Associate Research Professor Meihua Rose Feng © Nan Zheng 2011 To Dad and Mom With All My Love ii ACKNOWLEDGEMENT I would like to thank my mentor, Dr. Gustavo Rosania, for his patient and insightful guidance throughout my Ph.D. study. I feel fortunate to have met Dr. Rosania at the beginning of career, for he has set up a role model of great scientist with his diligence, creativity, persistence and dedication to the ultimate cure of human diseases. As an adviser, he is always open for discussion with me and my fellow lab mates, and actively seeks opportunities for us to improve our weak points – I am especially grateful to Dr. Rosania’s consistent support and encouragement for practicing my presentation skills and showcasing my work at domestic and international conferences. I want to express my special gratitude to Dr. Rosania for having walked me down the aisle when my parents were not able to attend my wedding ceremony in the US. I would also like to thank my dissertation committee members, Dr. Gordon L. Amidon, Dr. Meihua Rose Feng and Dr. Kerby A. Shedden, for their valuable effort and input. Discussions with Dr. Gordon Amidon always inspired me to dig further and wider into the significance and many aspects about my projects. Dr. Feng has encouraged me to expand my thinking and skills from the pure cell- culture-based laboratory setting to the more clinically relevant problem solving. Dr. Shedden from the Department of Statistics of the University of Michigan demonstrated to me how statistics could greatly facilitate the studying and characterization of scientific problems. I also want to thank Dr. Duxin Sun at the iii College of Pharmacy, the University of Michigan, for having given me a chance to expand my technical skills in the analytical field. During my Ph.D. study, I received a lot of help from my lab mates, fellow classmates, alumni and colleagues from the College of Pharmacy. Dr. Xinyuan Zhang, Dr. Jingyu Yu, Dr. Vivien Chen Nielsen, Jason Baik have lent me tremendous help to initiate my project and fulfill my research goals. I would like to thank Kyoung-Ah Min, Arjang Talattof, Dr. Ke Ma, Dr. Li Zhang, Dr. Neal Huang, Dr. Yiqun Jiang, Dr. Tao Zhang, Juhee Lee, Chinmay Maheshwari, Cara Hartz Nelson, Lindsay White, and Shu-Pei Wu, for their friendship and support. I could not have had my work going forward so smoothly without the assistance from the staff of the College of Pharmacy, especially those from Lynn Alexander, Gail Benninghoff, Dr. Cherie Dotson, Jeanne Getty, Pat Greeley, Maria Herbel and L.D. Hieber. I would like to thank the financial support from the College of Pharmacy, the Elizabeth Broomfield Foundation, and the Predoctoral Fellowship from the University of Michigan. Finally I would like to thank my parents and my husband, Peng Zou, for their love, encouragement and support. iv TABLE OF CONTENTS DEDICATION ....................................................................................................... ii ACKNOWLEDGEMENT …………………………………………………………….. iii LIST OF TABLES .............................................................................................. vii LIST OF FIGURES ........................................................................................... viii LIST OF APPENDICES ...................................................................................... xi ABSTRACT ………………………………………………………………..………… xii CHAPTER I .......................................................................................................... 1 INTRODUCTION ……………………………………..……..………………….... 1 Abstract ………………………………………………………….….………. 1 Introduction ………………………..………………………………..……….. 3 Pharmacological effects as evidence for specific organelle accumulation ..………………………………………………………….. 5 Chemical analysis as evidence for specific organelle accumulation …………………………………………………………. 7 Whole cell based microscopic imaging studies as evidence for intracellular localization ……………………………………...……… 10 Computational models to frame quantitative hypotheses and analyze subcellular distribution patterns …………………...……………….. 13 Conclusion ………………………………………………………...………. 17 Specific aims …………………………………………………………….... 20 References ………………………………………………………………… 24 CHAPTER II ....................................................................................................... 43 THE SUBCELLULAR DISTRIBUTION OF SMALL MOLECULES: A META ANALYSIS …………………………………………………………… 43 Abstract ………………………………………………………………...….. 43 Introduction ……………………………………………………..……..….. 45 Methods ……………………………………………………..…………….. 46 Results ……………………………………………………..………..…….. 50 Discussion ……………………………………………………………….… 55 v References ………………………………………………………………… 60 CHAPTER III ...................................................................................................... 74 SIMULATION-BASED ANALYSIS OF ORGANELLE-TARGETED MOLECULES: LYSOSOMOTROPIC MONOBASIC AMINES ……….…… 74 Abstract ………………………………………………………………...….. 74 Introduction ……………………………………………………..……..….. 76 Methods …………………………………………………………………….. 79 Results ………………………………………………………………..…….. 86 Discussion ……………………………………………………………….… 93 References ……………………………………………………………..… 102 CHAPTER IV ................................................................................................... 122 THE INTRACELLULAR ACCUMULATION OF CHLOROQUINE: SIMULATION-BASED ANALYSIS OF THE PHOSPHOLIPIDOSIS EFFECT ……………………………………………………………………...… 122 Abstract ………………………………………………………...……….... 122 Introduction ……………………………………………………...…….... 124 Materials and Methods ………………………………………...………... 126 Results ……………………………..…………………………...…..…….. 136 Discussion ……………………………………………………………...… 143 References ……………………………………………………………..… 148 CHAPTER V .................................................................................................... 163 SIMULATION-DRIVEN ANALYSIS FOR ASSESSING THE LATERAL INTER-CELLULAR TRANSPORT OF SMALL MOLECULES ON MICRO- FABRICATED PORE ARRAYS …………………………………………….. 163 Abstract ………………………………………………………………....... 163 Introduction ……………………………………………………..……...... 165 Materials and Methods ………………………………………………... 167 Results ……………………………..……………………..………..…….. 172 Discussion ……………………………………………………………...… 175 Conclusions ……………………………………………………………… 178 References ……………………………………………………………..… 179 CHAPTER VI ................................................................................................... 192 SUMMARY ………………………………………………………..…………… 192 APPENDICES .................................................................................................. 199 vi LIST OF TABLES Table 1.1. Features of major subcellular compartments that affect the intracellular distribution pattern of small chemicals ………………………..…………….... 40 Table 1.2. A summary of experimental methodologies …………..………….….. 41 Table 2.1. Summary of the subcellular localization data set …….…………….... 63 Table 2.2. Summary of experimental methods …………………………………… 64 Table 2.3. Drug-likeness based on Lipinski’s Rule of Five and lead-likeness based on Oprea’s Rules of compounds with reported subcellular localizations ………………………………………………………….…………. 65 Table 2.4. Physicochemical property trends of small molecules stratified into lower (<500 Daltons) and higher (>500 Daltons) molecular weight categories, and associated with various subcellular localizations……………………….. 66 Table 3.1. The reference set of ninety-nine lysosomotropic monobasic amines ………..………………………………………………………………………..… 108 Table 4.1. Cellular parameters obtained during 4-hour incubation with different concentrations of CQ. ……………………………………………………..… 152 Table 4.2. Parameter ranges for Monte Carlo simulations ………………..…… 153 Table 5.1. Properties of MDCK cell monolayers on patterned membranes …. 182 vii LIST OF FIGURES Figure 1.1. MDCK cells treated with 50 μM chloroquine concentration for 4 hours prior to staining with Lysotracker Green (yellow, lysosomes), Mitotracker Red (blue, mitochondria) and Hoechst (red, nuclei) ………………………… 42 Figure 2.1. Descriptor distributions of molecules with reported subcellular localization (filled gray area) and a random PubChem sample (solid line) . 68 Figure 2.2. Descriptor distributions of molecules with reported subcellular localization (filled gray area) and random DrugBank dataset (solid line) … 69 Figure 2.3. Descriptor distributions of lower molecular weight (filled gray area; <500 Daltons) and higher molecular weight (solid line; > 500 Daltons) molecules with reported subcellular localization ……………...………….…. 70 Figure 2.4. Calculated, formal charge distributions of lower molecular weight (filled gray area; <500 Daltons) and higher molecular weight (solid line; >500 Daltons) compounds with reported subcellular localization, at three different pH values ………………………………………………………………………... 71 Figure 2.5. Linear discriminant analysis of low (<500 Daltons) and high (>500 Daltons) molecular weight compounds with reported subcellular localizations ……………………………………………………………...……… 72 Figure 2.6. The major subcellular localization categories are represented by diverse subsets of molecules ………………………………………………….. 73 Figure 3.1. Diagrams showing the cellular pharmacokinetic phenomena captured by the two mathematical models used in this study: the