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Identification, and Characterization of Novel Inhibitors of the Presynaptic, Hemicholinium-3-Sensitive Choline Transporter

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Identification, and Characterization of Novel Inhibitors of the Presynaptic, Hemicholinium-3-Sensitive Choline Transporter IDENTIFICATION, AND CHARACTERIZATION OF NOVEL INHIBITORS OF THE PRESYNAPTIC, HEMICHOLINIUM-3-SENSITIVE CHOLINE TRANSPORTER By Elizabeth Ann Ennis Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmacology May, 2016 Nashville, Tennessee Randy Blakely, Ph.D. Craig Lindsley, Ph.D. Kevin Currie, Ph.D. Eric Delpire, Ph.D. David Robertson, M.D. For those who told me I could do anything I think I can ACKNOWLEDGEMENTS I am greatly humbled by the contributions of the many whom have aided me in my completion of my doctor of philosophy in pharmacology. I was given a great privilege by the Interdisciplinary Graduate Program when I was accepted as a Graduate Student at Vanderbilt University. I am extremely grateful for the support that I received from all of the teachers at this great University, particularly to Dr. Sebastian Joyce whose guidance led me to my mentor, Dr. Randy Blakely. Dr. Randy Blakely is a brilliant scientist and an inspiring mentor. The experience of working in his lab will be unparalleled for the rest of my professional life. He has provided me with immeasurable support through constructive criticism, funding, intellectual and scientific freedoms, and a blatant confidence in me regardless of my quirks. Thank you Randy. Before I relinquish this text to more formal matters, there are a few personal acknowledgements I would be remiss to exclude. I would like to acknowledge Dr. Ken Cohen without whom I wouldn’t have been here and wouldn’t have had the inspiration to pursue such a career. I thank my parents, Chris and Cherie Ennis, who are pillars in my life, fought for me to have a chance at life, and supported me, without contest, on every endeavor I have pursued. Finally, I want to mention my gratitude to Kristen Ennis, Noah Green, Christine Petersen, Cassie Retzlaff, Jane Wright, and the rest of my family, and friends who have shown me love and support, for it is those people who I pursue scientific endeavors such as this one. These scientific endeavors that lead to my dissertation began with a high- throughput screen (HTS) which was dreamt up and executed by a team including Dr. Alicia Ruggiero, Dr. Corey Hopkins, numerous colleagues at the Molecule Library Probe iii Production Center Network (MLPCN), Dr. Randy Blakely, and Dr. Craig Lindsley. I cannot thank these researchers enough for all of their hard work and guidance. Of those that initiated the HTS, I would like to especially acknowledge Dr. Craig Lindsley who has provided me with invaluable support throughout my entire graduate school career. Also, I would like to thank Dr. David Robertson and his team for their generous contributions to the ongoing cardiovascular studies, and Dr. Maureen Hahn who spent precious time editing this document. Finally, thank you to my committee, Dr. Kevin Currie, Dr. Eric Delpire, Dr. David Robertson, Dr. Craig Lindsley, and Dr. Randy Blakely. The time you have dedicated to me has shaped my graduate education and made this experience a positive one. Additionally, none of this would have been possible without the research grants from the National Institutes of Health (MH086530, RDB), the Alzheimer’s Association, the training grant that funded my education NIH T32 Award (GM007628), and the Dystonia Medical Research Foundation (DMRF) that funded this research. iv TABLE OF CONTENTS Page DEDICATION ................................................................................................................... ii ACKNOWLEDGMENTS .................................................................................................. iii TABLE OF CONTENTS .................................................................................................. v LIST OF FIGURES ........................................................................................................ viii LIST OF TABLES ............................................................................................................ xi LIST OF ABBREVIATIONS ............................................................................................ xii Chapter I. Choline on the Move: Perspectives on the Molecular Physiology and Pharmacology of the Presynaptic, Hemicholinium-3-sensitive Choline Transporter (CHT) A. Introduction ............................................................................................ 1 B. Putting our Advances in Cholinergic Pharmacology in Context Before Acetylcholine (ACh) was a Neurotransmitter ....................... 5 Discovery of ACh and the Fundamentals of Chemical Neurotransmission .......................................................................... 7 C. ACh, High Affinity Choline Uptake and the Birth of CHT An Overview of the Mechanism of ACh Synapses .......................... 8 From ACh to HACU ......................................................................... 9 Hemicholinium-3: Key Reagent in the Definition of HACU ............ 14 HC-3 Radioligand Binding: HACU enter the Molecular Era ........... 15 D. CHT Molecular Biology and Regulation Early Efforts to Identify CHT Proteins ............................................ 18 Cloning and Characterization of CHT cDNAs and Genes ............. 19 Molecular Mechanism of CHT Regulation ..................................... 23 E. CHT Contributions to Cholinergic Function and Dysfunction In Vivo CHT Genetic Animal Models ......................................................... 27 CHT Gene Contributions to Human Disorders .............................. 35 F. Advances in CHT Pharmacology The Search for Novel CHT Modulators: MKC-231 ........................ 37 The Search for Novel CHT Modulators: ML352 ............................ 40 H. Outline of the Dissertation ................................................................... 42 v II. The Identification of ML352 through SAR Studies Based on HTS Lead Compounds A. Introduction .......................................................................................... 45 B. Materials and Methods ........................................................................ 52 C. Results and Discussion Original HTS Hits .......................................................................... 55 Chemical Synthesis for Diversification of Lead Scaffold ............... 59 Chemical Diversification of Lead Scaffold ..................................... 59 D. Conclusions ......................................................................................... 66 III. The In Vitro and Ex Vivo Characterization of ML352 A. Introduction .......................................................................................... 67 B. Materials and Methods ........................................................................ 68 C. Results and Discussion Potency of ML352 in Heterologous and Ex Vivo Preparations ..... 75 Specificity of ML352 in Heterologous and Ex Vivo Preparations ................................................................................. 77 Michaleis-Menten Kinetics of ML352 in Heterologous and Ex Vivo Preparations ......................................................................... 77 ML352 Inhibition of CHT is Rapid and Reversible ........................ 82 Binding Interactions of ML352 and HC-3 in Heterologous and Ex Vivo Preparations .............................................................. 84 Evaluation of CHT Surface Expression of following ML352 Treatment using In Vitro Preparations .......................................... 84 D. Conclusions ......................................................................................... 86 IV. The In Vivo Characterization of ML352 in Mouse Models A. Introduction .......................................................................................... 89 B. Materials and Methods ........................................................................ 90 C. Results and Discussion Pharmacokinetics of ML352 in Rat, Mouse, and Human Models .. 97 Lethal Dose of ML352 Scales with the Levels of CHT Protein .... 101 ML352 Brain and Plasma Levels over Time in the Mouse .......... 104 ACh, DA, and Choline Levels in Synaptosomes after Acute ML352 Administration ............................................................................. 105 ML352 does not Change Locomotor Activity in Wild Type Mice at Subtoxic Doses ........................................................................... 107 D. Conclusions ....................................................................................... 109 V. Conclusions and Future Directions .............................................................. 114 APPENDIX vi Appendix I Other Structural Classes of HTS Lead Compounds for the Potential Development of CHT Inhibitors .............................................................. 124 Appendix II HTS Lead Compounds for the Development of CHT Activators ............ 132 Appendix III Investigation into the Utility of CHT inhibitors as Novel Therapeutics in Hypercholinergic Disorders through the use of the DYT1 Mouse Line of Dystonia ................................................................................................. 142 REFERENCES ............................................................................................................ 154 vii LIST OF FIGURES Figure 1. Support of acetylcholine (ACh) synthesis sustained by high affinity choline transporter (CHT)-mediated choline uptake ................................................................
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