DISCOVERING THE MOLECULAR AND CELLULAR MECHANISMS UNDERLYING FENFLURAMINE-INDUCED CARDIOPULMONARY SIDE EFFECTS Vincent Setola Dissertation Advisor: Bryan L. Roth, MD, PhD Department of Biochemistry Case School of Medicine Cleveland, OH August 12, 2005 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS List of Tables v List of Figures vi Acknowledgements viii List of Abbreviations xii Abstract xvi CHAPTER 1: INTRODUCTION 1.1 G Protein-Coupled Receptors: Overview 1 1.1.1 G Protein-Coupled Receptor Signal Transduction 1 1.1.2 G Protein-Coupled Receptor Topology/Structure 2 1.2 Serotonin (5-HT) Receptors 8 1.2.1 5-HT2 Receptors 11 1.2.1.1 5-HT2B Receptors 14 1.2.1.2 Tissue Distribution of 5-HT2B Receptors 14 1.2.1.3 Signal Transduction of 5-HT2B Receptors 15 1.2.1.4 Biochemical Consequences of 5-HT2B Receptor 19 Activation 1.3 The Rise of Fenfluramine 22 1.4 The Fall of Fenfluramine 29 1.5 Biological Activity of Fenfluramine: Generalities of Amphetamine Action 34 1.6 Evidence Linking the 5-HT2B Receptor to VHD 37 i CHAPTER 2: MATERIALS AND METHODS 2.1 Materials 40 2.1.1 Chemicals 40 2.1.2 Transfection Reagents, Cell Culture, and Transfection 41 2.1.3 cDNA Constructs 43 2.1.3.1 Construction of pUniversal-Signal 43 2.1.3.2 Sub-cloning of Human 5-HT2 Receptors 47 2.1.3.3 Generation of Mutant 5-HT2 Receptors 50 2.2 Methods 53 2.2.1 Radioligand Binding Assay 53 2.2.2 Functional Assays 58 2.2.2.1 Inositol Phosphate Accumulation Assay 58 2.2.2.2 [3H]Thymidine Deoxyribose Incorporation Assay 60 2.2.2.3 Activated Mitogen-Activated Protein Kinase Assay 60 2.2.2.4 Molecular Modeling, Ligand Docking Simulations, and 61 Molecular Dynamics Simulations CHAPTER 3: ESTABLISHING THAT FENFLURAMINE CAUSES MITOSIS IN PRIMARY CULTURES OF VALVULAR INTERSTITIAL CELLS VIA ACTIVATION OF 5-HT2B RECEPTORS 3.1 Introduction and Rationale 65 3.2 Results 69 ii 3.2.1 Screening the Receptorome Reveals the 5-HT2B Receptor as a 69 Molecular Target for MDMA and MDA 3.2.2 Valvulopathic Drugs Induce Prolonged Mitogenic Responses 82 in Human Heart Valve Interstitial Cells 3.2.3 Valvulopathic Drugs Induce a Mitogenic Marker— 87 Phosphorylation of Mitogen-Activated Protein Kinase— in Human Heart Valve Interstitial Cells 3.3 Discussion 87 CHAPTER 4: MOLECULAR DETERMINANTS FOR THE INTERACTION OF THE ANOREXIGEN NORFENFLURAMINE WITH THE 5-HT2B RECEPTOR 4.1 Introduction and Rationale 91 4.2 Results 93 4.2.1 Effect of Point Mutations on Ligand Affinity 93 4.2.2 Modeling, Ligand Docking Simulations, and Molecular 105 Dynamics Simulations of Ligand Binding to 5-HT2B Receptors 4.2.3 Effect of Point Mutations on Agonist Potency and Efficacy 124 4.3 Discussion 130 CHAPTER 5: IMPLICATIONS AND FUTURE DIRECTIONS 5.1 Summary 142 5.2 High-Throughput Screening Efforts to Identify Potential 143 Valvulopathogens Among Current and Future Pharmacotherapies iii 5.3 Design of “Second Generation” Phenylisopropylamine Anorexigens 147 5.4 Conclusion 153 BIBLIOGRAPHY 157 iv LIST OF TABLES 1.1 Clinical studies of the efficacy of appetite suppressants 23 2.1 PCR primer sequences used to amplify and sub-clone human 5-HT2 receptor 48 cDNA 2.2 Sequence of sense primers used for site-directed mutagenesis of 5-HT2 receptors 51 3.1 MDMA, MDA, and other valvulopathic drugs bind to recombinant human 74 5-HT2B receptors 3.2 MDMA and MDA, similar to other valvulopathic drugs, activate human 79 5-HT2B receptors in vitro 4.1 Affinity constants (Ki’s) for SNF at wild type and mutant 5-HT2 receptors 97 4.2 Affinity constants (Ki’s) for other 5-HT2B receptor agonist ligand sat wild type 101 and mutant 5-HT2 receptors 4.3 Affinity constants (Ki’s) for SNF and congeners at wild type and V2.53L 5-HT2B 115 receptors and wild type 5-HT2C and 5-HT2A receptors 4.4 Potency (EC50) and relative efficacy (Emax) values for SNF and RNF at wild type 125 and mutant 5-HT2 receptors v LIST OF FIGURES 1.1 G protein-coupled receptor signal transduction: the phospholipase C pathway 3 1.2 Schematic representation of a plasma membrane GPCR 5 1.3 Chemical structures of the indolamine 5-hydroxytryptamine (5-HT, serotonin), 9 and the phenylisopropylamines 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”), 3,4-methylenedioxyamphetamine (MDA), fenfluramine, norfenfluramine, and phentermine 1.4 Signal transduction pathways modulated by 5-HT2B receptors 17 1.5 Amphetamine actions on biogenic amine reuptake, storage, and release 32 2.1 Schematic of the plasma membrane protein expression vector pUniversal-Signal 44 2.2 Sequences of the 5-HT2 receptors sub-cloned into pUniversal-Signal 54 3.1 Large-scale screening of the receptorome reveals that MDMA preferentially 70 interacts with the human 5-HT2B receptor 3.2 MDMA and MDA potently activate 5-HT2B receptors in vitro 77 3.3 MDMA and MDA induce mitogenesis in human heart valve interstitial cells 83 in vitro 4.1 3-D molecular model of the human 5-HT2B receptor showing putative ligand 94 binding residues that are non-conserved among 5-HT2 family receptors 4.2 Representative competition binding isotherms for SNF at wild type and mutant 99 5-HT2 receptors 4.3 Competition binding isotherms for several 5-HT2B receptor agonist ligands at 103 wild type and V2.53L 5-HT2B receptors vi 4.4A 3-D molecular models showing the results of ligand docking simulations that 107 are consistent with conserved features of biogenic amine ligand binding 4.4B Representative energy-minimized structures from ten rounds of computer- 109 simulated annealing of solution 1 (A,B) and solution 2 (C,D) after insertion of the V2.53L mutation 4.5 Representative energy-minimized structure from ten rounds of computer- 112 simulated annealing of solution 1 after insertion of the V2.53I mutation 4.6 Representative energy-minimized structure from ten rounds of computer- 118 simulated annealing of solution 1 bearing the V2.53L mutation after addition or removal of SNF α-carbon substituents 4.7 Competition binding isotherms for SNF at wild type and V2.53A 5-HT2B 122 receptors 4.8 Concentration-response isotherms for agonist-stimulated inositol phosphate 127 accumulation vii ACKNOWLEDGEMENTS I wish to acknowledge the contributions of some of those who have made my work at Case possible and pleasurable: I am profoundly grateful to my advisor, Dr. Bryan Roth, for his outstanding mentorship. Since joining the Rothlab in 2001, I have learned from Bryan’s guidance and from his example an enormous, invaluable amount about the practice and the art of scientific research and analytical thought. Whatever scientific successes I may achieve in the future will be in no small part the result of Bryan’s involvement in my training. I will remember him with fondness and gratitude. I am thankful to my colleagues and friends in the Rothlab, whose talent and motivation have been inspirational to me. I sincerely hope for chances to work with this gifted group of individuals in the future. I deeply appreciate the intellectual and personal support of my fellow graduate students in the Rothlab. Since my first day in the lab, Douglas Sheffler has been helpful in so many ways. I am grateful to him for sharing his many talents, his kindness, and his friendship during my years at Case. I am also thankful to him for being an “uncle” to Maxime, Madeleine, and Maurice. viii I am thankful to my former bay-mate, fellow former graduate student, and friend Zongqi Xia for countless hours of intriguing conversation. Whatever the topic— science, politics, economics, art, cuisine, culture—he never failed to provide unique, thought-provoking perspectives and ideas. Ryan Strachan and Atheir Abbas joined the Rothlab towards the end of my tenure. I thank them for being supportive colleagues and friends. Je suis profondemment reconnaissant pour l’amitié et le soutien de Duna Massillon, Fredéric Bone, et Nadia Rachdaoui. Ils sont parmi les premiers amis que j’ai rencontré à Cleveland et ils sont devenus une « famille » loin de la mienne. J’apprécie egalement l’amitie de France David, Anne-Laure Bulteau, Laurent Chavatte, Michele Le Moing, et Hossein Izeml : les soirées, les anniversaires, les week-end de ski, les sorties sont de tres beaux souvenirs. Wesley Kroeze’s friendship and guidance have made me a better scientist and a better person. Durinsg the best and worst of times, Wes has celebrated with me, stood by me, and counseled me. Wes’s personal and professional stories have inspired me to work hard, to strive for excellence, to make time to enjoy life, and to be a good friend. In addition, Wes has shared with me one of his life’s loves: the great sport of sailboat racing. During our adventures on Ensigns—in Lake Erie and off the Gulf and Atlantic Coasts of Florida—Wes taught me so much about sailing and about competition. For everything Wes has given me, shared with me, and taught me, I am profoundly grateful. ix I have the distinct honor to call Anushree Bhatnagar my dear friend. We met as graduate students in the Rothlab, where we frequently discussed our science and our lives.