STRUCTURE ACTIVITY RELATIONSHIPS OF NICOTINE ANALOGS AND Erythrina ALKALOIDS ON THE ALPHA 4 BETA 2 NICOTINIC ACETYLCHOLINE RECEPTOR By KRISTIN MARIE WILDEBOER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005 Copyright 2005 by Kristin Marie Wildeboer To those I hold dearest: my parents ACKNOWLEDGMENTS At the completion of my graduate career I will have spent over three-fourths of my life in an academic environment. That amounts to many, many hours that teachers and professors have spent educating, guiding, listening, understanding and encouraging me both in and out of the classroom. I feel it is important to acknowledge and thank them all for their time and dedication to their students, and for helping to mold me into someone who possesses an intellectual curiosity that has driven me to where I am today. During my graduate studies I have turned to numerous people that for assistance, whether for experimental advice or for help with purchase orders. I would like to thank the Pharmacology Department staff: Donna Desmond-Kuhn, Barbara Reichert, Judy Adams, Lynn Rogers, Cookie Mundorff, Lynn Raynor, LaTrelle Davis, and Kari Bastow. I also thank Debbie Otero for lending binding assay supplies and advice over the years. I also need to thank BJ Streetman (in the Neuroscience Department) for help with registration and other such questions, which have been numerous. My family deserves much credit and recognition for being extremely supportive and understanding. I thank my father, Stanley; my mother, Judy; and my brothers, Todd and David. Although they may not understand what I have been studying, have always encouraged me. I also thank my dear friends who always provided laughter and compassion when needed. I especially thank Susan LeFrancois, my partner in crime during my years in the lab. She is one of the kindest, funniest, most caring people I have ever met and I am glad I had the opportunity to get to know her. iv I would like to thank current and past members of my supervisory committee: Dr. Stephen Baker, Dr. Thomas Vickroy, Dr. Laszlo Prokai, and Dr. Neil Rowland. I give special thanks to Dr. Roger Papke, who has gone above and beyond the role of a committee member, set aside many hours over the years to answer my questions (no matter how big or small), and always challenged me to better understand all that is membrane bound. I extend many thanks to Dr. Ferenc Soti for the isolation and syntheses of the many compounds for my study. I could always count on him to correct my chemical structures and names. I also thank Dr. Jon Lindstrom for graciously supplying the tsA201 cells used in functional assays. Finally, I would like to express my deep gratitude to the person who has guided and helped shape my research most significantly: my mentor, Dr. William Kem. I would like to thank him for giving me the opportunity to be a member of his lab. It has been an educational, enjoyable ride that I know I will miss when it ends. v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES.............................................................................................................x ABSTRACT.......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 Superfamily of Ligand-Gated Ion Channels.................................................................1 Characterization of Nicotinic Acetylcholine Receptors (nAChRs)..............................2 Studies of the Muscle-type nAChR.......................................................................2 Muscle-type nAChR Stoichiometry......................................................................3 Using Electron Microscopy to Study the Two-Dimensional Structure.................4 Studies of Neuronal nAChRs................................................................................5 Structure of nAChRs.....................................................................................................7 Site-Directed Mutagenesis of the Receptor and Substituted Cysteine Accessibility Method.........................................................................................7 The ACh-Binding Protein......................................................................................8 Pharmacology and Function of nAChRs....................................................................11 Pharmacology of nAChRs...................................................................................11 Distribution and Physiology of nAChRs in the Mammalian CNS......................15 Function of nAChRs............................................................................................16 Functional states of the nAChR...................................................................19 Neuronal nAChR Involvement in Disease..........................................................21 Structure Activity Relationships of Neuronal nAChRs.......................................24 2 MATERIALS AND METHODS...............................................................................28 Radioligand Binding...................................................................................................28 Compound Syntheses..........................................................................................28 Rat Brain Membranes..........................................................................................29 Human Embryonic Kidney Subclone Cell (tsA201) Membranes.......................29 Binding Assay Protocol.......................................................................................30 Binding Assay Data Analysis..............................................................................31 vi Functional Measurements...........................................................................................32 Cell Culture.........................................................................................................32 Membrane Potential Dye, Cell Loading and Compound Plate Preperation........32 Membrane Potential Measurements....................................................................33 Functional Assay Data Analysis..........................................................................34 High Performance Liquid Chromatography (HPLC) Chiral Separation of Nicotine Analogs.............................................................................................34 3 RESULTS...................................................................................................................36 Nicotine Analogs........................................................................................................36 (S)-Nicotine and (S)-Nornicotine........................................................................40 5-Pyridyl Substituted Analogs.............................................................................44 5’-Pyrrolidine Substituted Analogs.....................................................................46 1’-N-Pyrrolidine Substituted Analogs.................................................................47 3’-Pyrrolidine Substituted Analogs.....................................................................50 4 RESULTS...................................................................................................................54 Separation of Nicotine Analog Enantiomers..............................................................54 5 RESULTS...................................................................................................................61 Erythrina Alkaloids....................................................................................................61 β-Erythroidines....................................................................................................63 Aromatic Alkaloids.............................................................................................70 6 DISCUSSION.............................................................................................................74 Substituents at Four Different Positions on Nicotine Decrease Affinity for the α4β2 nAChR and Confer Partial Agonist and Antagonist Properties....................75 Separated Nicotine Analog Enantiomers Display a Difference in Affinity for α4β2 Unlike Nicotine.............................................................................................83 Substituents on the D-ring of the Erythrina Alkaloids Allow for High Affinity Binding to the α4β2 nAChR and Inhibition of the α4β2 Acetylcholine Response.................................................................................................................84 Conclusions.................................................................................................................89 REFERENCES..................................................................................................................90 BIOGRAPHICAL SKETCH...........................................................................................107 vii LIST OF TABLES Table page 3-1 Inhibition of 125I-α-bungarotoxin (btx) and 3H-cytisine binding to rat brain membranes or inhibition of 3H-cytisine binding to human α4β2 expressing tsA201 cell membranes by (S)-nicotine or (S)-nornicotine.....................................41 3-2 Inhibition of 125I-α-btx and 3H-cytisine binding to rat brain membranes or inhibition of 3H-cytisine binding to human α4β2 expressing tsA201 cell membranes by 5-pyridyl substituted analogs...........................................................45 3-3 Inhibition of human α4β2 receptor acetylcholine (ACh) responses by 5-pyridyl substituted analogs...................................................................................................46 3-4 Inhibition of 125I-α-btx and 3H-cytisine binding to rat brain membranes or inhibition of 3H-cytisine binding to human α4β2 expressing tsA201 cell membranes by 5’-pyrrolidine substituted analogs...................................................47 3-5 Inhibition of human α4β2 receptor ACh responses by 5’-pyrrolidine substituted analogs......................................................................................................................47 3-6 Inhibition of 125I-α-btx and 3H-cytisine binding to rat brain membranes or inhibition of 3H-cytisine binding to human α4β2 expressing tsA201 cell membranes by 1’-N- pyrrolidine substituted analogs..............................................48 3-7 Inhibition of human α4β2 receptor ACh responses by 1’-N-pyrrolidine substituted analogs...................................................................................................50 3-8 Inhibition of 125I-α-btx and 3H-cytisine binding to rat brain membranes or inhibition of 3H-cytisine binding to human α4β2 expressing tsA201 cell membranes by 3’-pyrrolidine substituted analogs...................................................52 3-9 Inhibition of human α4β2 receptor ACh responses by 3’-pyrrolidine substituted analogs......................................................................................................................53 4-1 Inhibition of 3H-cytisine binding to rat brain or to human α4β2 expressing tsA201 cell membranes by nicotine enantiomers.....................................................58 5-1 Structures, nomenclature and binding results for β-erythroidine, dihydro-β-erythroidine and tetrahydro-β-erythroidine............................................64 viii 5-2 Structures, nomenclature and binding results for 3-desmethoxy-β-erythroidine, N-methyl-β-erythroidine and β-erythroidinediol.....................................................65 5-3 Inhibition of human α4β2 receptor ACh responses by natural product and semi- synthetic β-erythroidines..........................................................................................69 5-4 Structures, nomenclature and binding results for the aromatic alkaloids erysovine, erysodine and erythraline........................................................................71 5-5 Structures, nomenclature and binding results for the aromatic alkaloids erysotrine, and glucoerysodine.................................................................................72 5-6 Inhibition of human α4β2 receptor ACh responses by natural product aromatic Erythrina alkaloids...................................................................................................73 ix LIST OF FIGURES Figure page 3-1 Signal acquired for membrane potential fluorescence using tsA201 cells expressing human α4β2 nAChRs............................................................................43 3-2 Concentration response curves of nicotine, ACh and nornicotine for human α4β2 receptors expressed in tsA201 cells as measured by changes in membrane potential....................................................................................................................44 3-3 Average effects of the 1’-N-pyrrolidine substituted analogs on human α4β2 receptors expressed in tsA201 cells as measured with membrane potential dye.....50 3-4 Average effects of the 3’-pyrrolidine substituted analogs on human α4β2 nAChRs expressed in tsA201 cells as measured with membrane potential dye......53 4-1 HPLC chiral separation of 1’-N-ethyl-(S,R)-nornicotine........................................57 4-2 Concentration response curves for the enantiomers of nicotine and nornicotine for human α4β2 receptors expressed in tsA201 cells as measured by changes in membrane potential..................................................................................................60 5-1 The common heterocyclic structure of Erythrina alkaloids.....................................62 5-2 Concentration response curve of dihydro-β-erythroidine (DHβE) for human α4β2 receptors expressed in tsA201 cells as measured by changes in membrane potential....................................................................................................................66 5-3 Concentration response curve of ACh in the presence and absence of 1 µM DHβE for human α4β2 receptors expressed in tsA201 cells as measured by changes in membrane potential................................................................................67 5-4 Signal acquired for α4β2 nAChRs upon simultaneous application of DHβE and ACh..........................................................................................................................68 5-5 Structure of a Phelline alkaloid, O-methylisophellibiline........................................70 x