Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2018 Elucidation of Substrate Binding Interactions for Human Organic Cation Transporters 1 (SLC22A1) and 2 (SLC22A2) Using In Silico Homology Modeling in Conjunction with In Vitro Site-Directed Mutagenesis and Kinetic Analysis Raymond E. Lai Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medicinal and Pharmaceutical Chemistry Commons, Other Pharmacy and Pharmaceutical Sciences Commons, and the Pharmaceutics and Drug Design Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/5593 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. © Raymond Lai, 2018 All Rights Reserved ELUCIDATION OF SUBSTRATE BINDING INTERACTIONS FOR HUMAN ORGANIC CATION TRANSPORTERS 1 (SLC22A1) AND 2 (SLC22A2) USING IN SILICO HOMOLOGY MODELING IN CONJUNCTION WITH IN VITRO SITE-DIRECTED MUTAGENESIS AND KINETIC ANALYSIS A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University by Raymond Eugene Lai Bachelor of Science, College of William and Mary, Williamsburg, Virginia, USA Master of Science, Virginia Commonwealth University, Richmond, Virginia, USA Doctor of Pharmacy; Virginia Commonwealth University, Richmond, Virginia, USA Director: Douglas H. Sweet, Ph.D. Professor, Chair Department of Pharmaceutics, School of Pharmacy Virginia Commonwealth University Richmond, Virginia August, 2018 iii ACKNOWLEDGMENTS My long and arduous academic journey toward the completion of my dissertation project and ultimately the achievement of my PhD would not have come to fruition without the support, guidance, and encouragement from a number of incredible people. First and foremost, I would like to thank my research advisor, Dr. Douglas Sweet, for instilling into me not only the knowledge of conducting cutting-edge pharmaceutical research, but also the confidence in me as I a grew as a scientist during my time working in his lab. His unwavering passion for research, teaching, and service at VCU is nothing short of inspiring. He provided me the opportunity, resources, and unparalleled guidance in steering me toward success as I navigated through numerous hurdles while completing my research. A significant amount of praise also goes out to my dissertation committee members, Dr. Keith Ellis, Dr. Phillip Gerk, Dr. Adam VanWert, and Dr. Jürgen Venitz, for offering their time and effort in helping me complete my project as well as challenging me to think critically. Much thanks should also be given to Dr. Philip Mosier of the Department of Medicinal Chemistry for his major contributions in teaching and guiding me through the computationally intensive molecular modeling work that played an integral role in my research. I would also like to acknowledge Dr. MaryPeace McRae and Dr. Frances White for their generosity in lending their fluorescent and confocal microscopes to help us with our cell imaging studies. Additionally, I would like to recognize all the VCU School of Pharmacy faculty for their efforts teaching and coordinating the challenging courses that I endured and completed as part of the graduate curriculum. Much thanks should also be offered to the administrative staff, especially Keyetta Tate, Laura Georgiadis, and Shakim Craft, for their tireless commitment and attention to detail that have resulted in a smoothly run department. A big thank you goes out to all my fellow lab colleagues, Dr. Aditi Mulgaonkar, Dr. Li Wang, Dr. Christine Farthing, Dr. Xiaolei Pan, Dr. Hebing Liu, and Christopher Jay, all of whom have been extremely supportive and helpful during my time at VCU. I would finally like to express my infinite gratitude to all my friends, family, and loving wife, Caroline, who have offered me continuous support, faith, and love through the course of everything I have been able to accomplish. ii TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………………………………………. ii TABLE OF CONTENTS………………………………………………………………………. iii LIST OF TABLES……………………………………………………………………………... vi LIST OF FIGURES…………………………………………………………………………….viii ABBREVIATIONS……………………………………………………………………………… x ABSTRACT…………………………………………………………………………………… xiv CHAPTERS 1. OVERVIEW OF EXPRESSION AND FUNCTION OF ORGANIC CATION AND ANION TRANSPORTERS…………………………………………………………………….. 1 1.A SOLUTE CARRIER 22 TRANSPORTER FAMILY……………………………. 1 1.B MAJOR ORGANIC CATION AND ANION TRANSPORTERS………………. 8 1.C CURRENT SCOPE OF MOLECULAR MODELING…………………………. 20 2. RESEARCH OBJECTIVES AND SPECIFIC AIMS……………………………………. 30 2.A RESEARCH OBJECTIVES AND HYPOTHESIS…………………………….. 30 2.B SPECIFIC AIMS TO ADDRESS HYPOTHESIS……………………………… 30 3. IDENTIFYING STRUCTURE ELEMENTS OF HUMAN ORGANIC CATION TRANSPORTER 2 (SC22A2) MEDIATING SUBSTRATE TRANSPORTER INTERACTIONS………………………………………………………………………………. 33 3.A INTRODUCTION ………………………………………………………………… 33 3.B MATERIAL AND METHODS…………………………………………………… 36 3.B.1 Chemicals and reagents………………………………………………. 36 3.B.2 Homology modeling and docking studies…………………………… 37 3.B.3 Bacterial transformation……………………………………………….. 40 iii 3.B.4 Point mutation of plasmid DNA……………………………………… 40 3.B.5 Cell line transfection and maintenance……………………………... 44 3.B.6 Cell accumulation assays …………………………………………….. 44 3.B.7 Genomic DNA integration confirmation…..…………………………...46 3.B.8 Cell harvest for immunoblotting………………………………………. 46 3.B.9 SDS-PAGE and Immunoblotting…………………………………….. 47 3.B.10 Immunocytochemistry………………………………………………... 48 3.B.11 Green fluorescent protein (GFP) plasmid construction…………… 48 3.B.12 Microscopic imaging………………………………………………….. 49 3.B.13 Statistics……………………………………………………………….. 49 3.C RESULTS………………………………………………………………………… 50 3.C.1 Identification of a hOCT2 model……………………………………… 50 3.C.2 Identifying amino acid residues important for MPP+ hOCT2 interaction……………………………………………………………………… 56 3.C.3 Substitution of hOCT2 amino acid residues in putative binding pocket…………………………………………………………………………... 62 3.C.4 Critical amino acid confirmation through kinetic assays…………… 69 3.C.5 Genomic integration of non-functional hOCT2 mutant constructs... 73 3.C.6 Immunodetection of non-functional hOCT2 mutants………………. 73 3.C.7 Membrane targeting of hOCT2-GFP fusion construct……...……… 74 3.D DISCUSSION…………………………………………………………………….. 81 4. IDENTIFYING STRUCTURAL ELEMENTS OF HUMAN ORGANIC CATION TRANSPORTER 1 (SLC22A1) MEDIATING SUBSTRATE-TRANSPORTER INTERACTIONS……………………………………………………………………………… 88 4.A INTRODUCTION…………………………………………………………………. 88 4.B MATERIAL AND METHODS…………………………………………………… 92 iv 4.B.1 Chemicals and reagents………………………………………………. 92 4.B.2 Homology modeling and docking studies……………………………. 92 4.B.3 Bacterial transformation……………………………………………….. 95 4.B.4 Point mutation of plasmid DNA……………………………………….. 95 4.B.5 Cell line transfection and maintenance.……………………………… 99 4.B.6 Cell accumulation assays……………………………………………... 99 4.B.7 Genomic DNA integration confirmation…………………………..… 101 4.B. 8 Green Fluorescent Protein (GFP) plasmid construction…………. 101 4.B.9 Microscopic imaging………………………………………………… 102 4.B.10 Statistics……………………………………………………………… 102 4.C RESULTS……………………………………………………………………….. 103 4.C.1 Identification of a hOCT1 model…………………………………….. 103 4.C.2 Identifying amino acid residues important for MPP+ hOCT1 interaction…………………………………………………………………….. 109 4.C.3 Substitution of hOCT1 amino acid residues in putative binding pocket………………………………………………………………………… 116 4.C.4 Critical amino acid confirmation through kinetic assays………….. 123 4.C.5 Genomic integration of non-functional hOCT1 mutant constructs. 127 4.C.6 Membrane targeting of hOCT1-GFP fusion construct….………… 127 4.D. DISCUSSION…………………………………………………………………... 132 5. COMPARISON OF SUBSTRATE BINDING INTERACTIONS BETWEEN HUMAN ORGANIC CATION TRANSPORTERS 1, 2, AND 3……………………………………. 139 LITERATURE CITED……………………………………………………………………….. 148 VITA…………………………………………………………………………………………… 162 v LIST OF TABLES Table 1.1 Example compound interactions associated with SLC22 transporters……… 12 Table 1.2 Clinical concentrations of example compounds……………………………….. 15 Table 1.3 Absolute native tissue protein expression levels for human SLC22 transporters……………………………………………………………………………………. 17 Table 1.4. Summary of the SLC family homology model template recommendations… 24 Table 1.5 Summary of critical residues discovered through initial OCT modeling studies………………………………………………………………………………………….. 27 Table 3.1 Primers for hOCT2 site directed mutagenesis…………………………………. 42 Table 3.2 Summary of hOCT2 model evaluation scores…………………………………. 53 Table 3.3 hOCT2 docking interaction summary…………………………………………… 59 Table 3.4 Summary of hOCT2 residue substitutions……………………………………… 65 Table 3.5 hOCT2-MPP+ interaction based conservative substitutions………………….. 66 Table 3.6 hOCT2-MPP+ interaction based non-conservative substitutions…………….. 67 Table 3.7 Summary of Km estimates for hOCT2 constructs……………………………… 72 Table 4.1 Primers for hOCT1 site directed mutagenesis…………………………………. 97 Table 4.2 Summary of hOCT1 mutant evaluation scores……………………………….. 106 Table 4.3 hOCT1 docking interaction summary………………………………………….. 112 Table 4.4 Summary of hOCT1 residue substitutions…………………………………….. 119 Table 4.5 hOCT1 MPP+ interaction based conservative substitutions………………… 120 Table 4.6 hOCT1-MPP+ interaction based non-conservative