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Discovery and Characterization of Selective Negative Allosteric Modulators Of Discovery and Characterization of Selective Negative Allosteric Modulators of Human α4β2 Neuronal Nicotinic Receptors DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Brandon J. Henderson, B.S. Graduate Program in Pharmacy The Ohio State University 2011 Dissertation Committee: Dennis McKay, Ph.D., Advisor R. Thomas Boyd, Ph.D. Chenglong Li, Ph.D. Anthony Young, Ph.D. Lane Wallace, Ph.D. Copyright Brandon J. Henderson 2011 ABSTRACT One of the difficulties in understanding the physiological role and connection to diseases and disorders of neuronal nicotinic acetylcholine receptors (nAChRs) lies in the distinction between the specific subtypes involved. It is of great importance to understand how specific nAChR subtypes are involved with normal physiology, diseases, and disorders. α4β2* nAChRs have been implicated in a growing number of diseases and disorders; the most prominent being nicotine addiction. The studies presented in this dissertation characterize a novel, human α4β2 (H α4β2) selective antagonist as well as an allosteric site in which it binds. The primary contributions of this study are: 1) the discovery and characterization of the negative allosteric modulator (NAM), KAB- 18, using calcium accumulation assays on recombinant H α4β2 and H α3β4 nAChRs, 2) the identification of the allosteric site in which KAB-18 binds using homology modeling, blind docking, and molecular dynamics (MD) simulations, 3) the validation of the allosteric site which was identified using computational modeling through the use of site-directed mutagenesis, 4) the construction of 3D models to predict the important binding interactions between this novel class of NAMs with H α4β2 and H α3β4 nAChRs, and 5) the identification of novel compounds that have similar “pharmacophores” as KAB-18 using ligand-based ii virtual screening. These studies provide much information regarding ligands that can interact selectively and potently with H α4β2 nAChRs. These results also provide a novel mechanism for the negative allosterism of this class of NAMs on Hα4β2 and H α3β4 nAChRs. The work presented herein provides a bridge to the design of new molecules from both a ligand based and structure based approach that preferentially bind and inhibit H α4β2 nAChRs. This work will contribute to the field of nAChRs by providing a means to produce novel molecules; whether as molecular tools or as potential therapeutic drug candidates that target H α4β2 nAChRs. iii Dedicated to my friends and family for their continual support throughout my education especially to Laura for putting up with me through this entire process iv ACKNOWLEDGEMENTS First of all, I thank Jesus for letting me get to where I am today. Without my faith, I would have given up long ago. I want to especially thank Dr. Dennis McKay for the support and guidance over these past few years. He has been an exceptional mentor and I thank God that I had the opportunity to learn from him. I specifically want to thank him for the patience he’s had for me over these few years. I want to thank some of the fellow students that have helped and encourage me over the years: Tatiana González-Cestari, Bitna ‘Amy’ Yi, Alex Jaeger, Jared Allen, Ryan Pavlovicz, Raeann Carrier, Ryan Schnieder, Martin Dalefield, Kiran Mahasanen and Dan Carper. I also want to thank my dissertation committee: Dr. R. Thomas Boyd, Dr. Chenglong Li, Dr. Anthony Young, and Dr. Lane Wallace. Thank you Dr. Boyd for teaching me ‘real’ molecular biology. Thank you Dr. Li for always reminding me that chemistry is important, and I shouldn’t forget it as I learn to think as a pharmacologist. Thank you Dr. Young for professional and musical guidance. Thank you Dr. Wallace for all the CNS related talks we’ve had over the years. I want to thank the National Institute of Drug Abuse for supporting my education. I want to send a special thanks to my father, James Henderson, and brother, Justin Henderson, for being so understanding about the fact that I can’t visit home to often. Last but definitely not least, I want to thank my beautiful wife, Laura, for support and understanding. v VITA Undergraduate: The Ohio State University, Columbus, OH, 2002-2006, B.S., with distinction, Chemistry. Graduate: The Ohio State University, Columbus, OH, 2006-Current, Publications Henderson, B.J.; Pavlovicz, R.E.; Allen, J.A.; González-Cestari, T.F.; Orac, C.M.; Bonnell, A.B.; Zhu, M.X.; Boyd, R.T.; Li, C.; Bergmeier, S.C., and McKay, D.B. Negative allosteric modulators that target human α4β2 neuronal nicotinic receptors. J Pharmacol Exp Therap 334, 761-774. 2010 González-Cestari, T.F.; Henderson, B.J.; Pavlovicz, R.E. McKay; S.B., El-Hajj, R.A.; Pulipaka, A,B.; Orac, C.M.; Reed, D.D.; Boyd, R.T.; Zhu, M.X.; Li, C.; Bergmeier, S.C.; and McKay, D.B. Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: Implications for drug discovery. J Pharmacol Exp Therap 328, 504-515. 2009 Fields of Study Major Field: Pharmacy vi TABLE OF CONTENTS ABSTRACT............................................................................................................ii ACKNOWLEDGEMENTS......................................................................................v VITA......................................................................................................................vi List of Figures........................................................................................................xi List of Tables......................................................................................................xvii List of Schemes..................................................................................................xxii STATEMENT OF THE PROBLEM........................................................................1 CHAPTERS 1. Introduction.................................................................................................4 1.1. Nicotinic receptor structure and classification.................................4 1.2. α4β2 Nicotinic Receptors................................................................7 1.3. nAChRs in Nicotine Addiction.........................................................9 1.4. Allosteric interactions of nicotinic receptors..................................11 2. Materials and methods..............................................................................17 2.1. Materials.......................................................................................17 2.2. Fluo-4 calcium accumulation assay..............................................17 2.3. Calcium 5 assay...........................................................................20 2.4. GASP alignment...........................................................................21 2.5. Construction of CoMFA models....................................................21 vii 2.6. Construction of CoMSIA models...................................................22 2.7. Docking and Molecular Dynamics (MD).......................................23 2.8. Transient transfection of HEK tsa cells.........................................24 2.9. Calculations and Statistics............................................................26 3. Characterization and SAR of KAB-18.......................................................28 3.1. Introduction to Study.....................................................................28 3.2. Results..........................................................................................29 3.2.a. Lead Molecule, KAB-18........................................................29 3.2.b. NAM Alignment and Pharmacophore...................................34 3.2.c. Region 1 SAR.......................................................................37 3.2.d. Region 2 SAR.......................................................................38 3.2.e. Region 3 SAR.......................................................................41 3.2.f. Region 4 SAR........................................................................43 3.2.g. Computational Modeling.......................................................45 3.2.e. Novel Design of Antagonists................................................55 3.3. Discussion....................................................................................60 4. Validation of KAB-18 binding site: site-directed mutagenesis study.........67 4.1. Introduction to Study.....................................................................67 4.2. Results..........................................................................................68 4.2.a. Site-directed Mutagenesis with Model Iteration 8.................68 4.2.b. Site-directed Mutagenesis with Model Iteration 9.................72 4.2.c. NAM binding to Phe118……................................................80 viii 4.2.d. NAM binding to Thr58..........................................................86 4.2.e. Future Site-directed Mutagenesis Studies...........................90 4.3. Discussion....................................................................................92 5. 3D-QSAR and 3D-QSSR..........................................................................96 5.1. Introduction...................................................................................97 5.2. Results..........................................................................................97
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