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Using Molecular Tools to Study the Structure and Functional States of the Human Alpha7 Nicotinic Acetylcholine Receptor

Permanent Link: http://ufdc.ufl.edu/UFE0043639/00001

Material Information

Title: Using Molecular Tools to Study the Structure and Functional States of the Human Alpha7 Nicotinic Acetylcholine Receptor
Physical Description: 1 online resource (274 p.)
Language: english
Creator: Wang, Jingyi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: activation -- desensitization -- ligands -- nachr
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels, which mediate cell-to-cell signal transduction chemically. Among the various subtypes of nAChRs, the homomeric alpha7 subtype has been suggested as an important drug target for Alzheimer's Diseases, schizophrenia and inflammatory disorders. The alpha7 receptors feature low open probability (Popen) and rapid desensitization. There are at least two different kinds of desensitized states for alpha7 nAChRs, i.e., the PNU-120596 sensitive desensitized state (Ds) and the PNU-120596 insensitive desensitized state (Di). Moreover, structurally diverse agonists can activate alpha7 nAChRs and impact the rate of the receptors to enter and remain in the different desensitized states. However, details of the interactions between the ligand and receptor that regulate the receptor functions are still in question. As a membrane protein, there is no high-resolution structure of the whole receptor. Therefore, this study utilized different molecular tools, including mutant forms of the receptor, complemented by the structural homology models of alpha7 nAChRs to investigate the structural features of ligands that regulate the nAChR functions. Firstly, to test the impact of the ligands' binding orientations on channel functions, the receptor was tethered with structurally diverse ligands at different sites to mimic different binding orientations. Reactive cysteines were introduced into the alpha7 ligand binding domain (LBD) to allow binding of sulfhydryl-reactive (SH) agonist analogs or control reagents onto specific positions. The tethering of the SH reagents blocked further acetylcholine-evoked activation of the receptor in four alpha7 mutants (S36C, L38C, W55C and L119C). After selective reaction with SH agonist analogs, the type II allosteric modulator PNU-120596 could reactivate the L119C and W55C mutants, and receptors with a reduced or modified C-loop. However, regardless of reactions with agonist analogs or alternative SH reagents, modified S36C and L38C mutants were insensitive to the reactivation by PNU-120596. Molecular modeling showed that in the W55C and L119C mutants, the ammonium pharmacophore of the agonist analog methanethiosulfonate-ethyltrimethylammonium would be in similar positions underneath the C-loop. In the S36C mutant, the ammonium pharmacophore would locate on the edge of the C-loop and possibly prevent the closure of the C-loop. These results suggested that a single ligand can bind within the receptor in different ways and, depending on the specific binding orientation, may variously promote activation, Ds, Di, or alternatively, function as a competitive antagonist. Secondly, to test the influence of H-bonding interaction in channel function, various H-bonding probes were designed and synthesized to systematically investigate the H-bonding interaction in the alpha7 selectivity pocket. Aldo-type condensations between anabaseine dihydrochloride and appropriate carboxaldedhydes yielded arylidene anabaseines with a unique H-bonding property in their alpha7 selective motifs, including two pyrrolyl derivatives as H-bonding donors (2PyroAB and 3PyroAB), two furanyl derivatives as H-bonding acceptors (2FAB and 3FAB), and two thiphenyl derivatives as hydrophobic/steric probes (2TAB and 3TAB). In the receptor, Q57 was mutated to amino acids with unique H-binding property, including lysine (H-bonding donor), glutamate and aspartate (H-bonding acceptors), and leucine (hydrophobic/steric probes), to pair with the synthetic probes. In Q57K and Q57L mutants, the efficacy of 2PyroAB decreased to 14 +/- 2% and 15 +/- 1% relative to acetylcholine, respectively, about half compared to the wild type. However, channel activations by the other arylidene anabaseines were less sensitive to the Q57 mutants. These suggested that channel activation could be facilitated by H-bonding interaction between 2PyroAB and the glutamine 57 residue in the receptor. To study channel desensitization, the type II modulator PNU-120596 was co-applied with the arylidene anabaseines to render the Ds state as conductive. If maintaining the H-bonding partner in the receptor (WT and Q57K) for the two FABs, a larger PNU-120596-stimulated peak response with a smaller net charge to peak response ratio was observed for 2FAB compared to 3FAB. However, such pattern disappeared in Q57D and Q57E mutants. These suggested that H-bonding effect could modulate the receptor desensitization via H-bonding, and depending on the orientation, change the energy barrier and/or energy level of the Ds state. These data suggested that both the orientation and pattern of the H-bond in the alpha7 selectivity pocket would regulate the receptor activation and desensitization, which can be utilized in drug design targeting specific functional states of alpha7 nAChRs. The third project involved investigations of an alternative pharmacophore for ligandi binding to the receptor. A positively charged nitrogen has been considered as the minimum pharmacophore for agonists and some competitive agonists of the nAChRs. A series of novel molecules were synthesized: (E)-3-(6-Benzylidenecyclohex-1-en-1-yl)pyridine (BHP) and its derivatives. These molecules lack the charged nitrogen pharmacophore, but could antagonize the various types of nAChRs. It was observed that BHP competitively antagonized the muscle type nAChR, with an IC50 value of 9 +/- 2 microM. The IC50 of the parent compound, 3-(cyclohex-1-en-1-yl)pyridine (PyHexe), was 261 +/- 52 microM, much smaller compared to that of BHP. These suggested that without the charged pharmacophore, but with the help from the extended aromatic ring, BHP could bind at the agonist binding site and competitively antagonized the receptor. In conclusion, the data reported in this thesis support the hypothesis that nAChR function may be differently regulated by single ligands binding in multiple alternative ways and that specific point-to-point interactions between ligands and receptor can selectively modulate the induction and stabilization of specific conformational states. These new insights may provide new approaches for drug development.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jingyi Wang.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Horenstein, Nicole A.
Local: Co-adviser: Papke, Roger L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043639:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043639/00001

Material Information

Title: Using Molecular Tools to Study the Structure and Functional States of the Human Alpha7 Nicotinic Acetylcholine Receptor
Physical Description: 1 online resource (274 p.)
Language: english
Creator: Wang, Jingyi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: activation -- desensitization -- ligands -- nachr
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels, which mediate cell-to-cell signal transduction chemically. Among the various subtypes of nAChRs, the homomeric alpha7 subtype has been suggested as an important drug target for Alzheimer's Diseases, schizophrenia and inflammatory disorders. The alpha7 receptors feature low open probability (Popen) and rapid desensitization. There are at least two different kinds of desensitized states for alpha7 nAChRs, i.e., the PNU-120596 sensitive desensitized state (Ds) and the PNU-120596 insensitive desensitized state (Di). Moreover, structurally diverse agonists can activate alpha7 nAChRs and impact the rate of the receptors to enter and remain in the different desensitized states. However, details of the interactions between the ligand and receptor that regulate the receptor functions are still in question. As a membrane protein, there is no high-resolution structure of the whole receptor. Therefore, this study utilized different molecular tools, including mutant forms of the receptor, complemented by the structural homology models of alpha7 nAChRs to investigate the structural features of ligands that regulate the nAChR functions. Firstly, to test the impact of the ligands' binding orientations on channel functions, the receptor was tethered with structurally diverse ligands at different sites to mimic different binding orientations. Reactive cysteines were introduced into the alpha7 ligand binding domain (LBD) to allow binding of sulfhydryl-reactive (SH) agonist analogs or control reagents onto specific positions. The tethering of the SH reagents blocked further acetylcholine-evoked activation of the receptor in four alpha7 mutants (S36C, L38C, W55C and L119C). After selective reaction with SH agonist analogs, the type II allosteric modulator PNU-120596 could reactivate the L119C and W55C mutants, and receptors with a reduced or modified C-loop. However, regardless of reactions with agonist analogs or alternative SH reagents, modified S36C and L38C mutants were insensitive to the reactivation by PNU-120596. Molecular modeling showed that in the W55C and L119C mutants, the ammonium pharmacophore of the agonist analog methanethiosulfonate-ethyltrimethylammonium would be in similar positions underneath the C-loop. In the S36C mutant, the ammonium pharmacophore would locate on the edge of the C-loop and possibly prevent the closure of the C-loop. These results suggested that a single ligand can bind within the receptor in different ways and, depending on the specific binding orientation, may variously promote activation, Ds, Di, or alternatively, function as a competitive antagonist. Secondly, to test the influence of H-bonding interaction in channel function, various H-bonding probes were designed and synthesized to systematically investigate the H-bonding interaction in the alpha7 selectivity pocket. Aldo-type condensations between anabaseine dihydrochloride and appropriate carboxaldedhydes yielded arylidene anabaseines with a unique H-bonding property in their alpha7 selective motifs, including two pyrrolyl derivatives as H-bonding donors (2PyroAB and 3PyroAB), two furanyl derivatives as H-bonding acceptors (2FAB and 3FAB), and two thiphenyl derivatives as hydrophobic/steric probes (2TAB and 3TAB). In the receptor, Q57 was mutated to amino acids with unique H-binding property, including lysine (H-bonding donor), glutamate and aspartate (H-bonding acceptors), and leucine (hydrophobic/steric probes), to pair with the synthetic probes. In Q57K and Q57L mutants, the efficacy of 2PyroAB decreased to 14 +/- 2% and 15 +/- 1% relative to acetylcholine, respectively, about half compared to the wild type. However, channel activations by the other arylidene anabaseines were less sensitive to the Q57 mutants. These suggested that channel activation could be facilitated by H-bonding interaction between 2PyroAB and the glutamine 57 residue in the receptor. To study channel desensitization, the type II modulator PNU-120596 was co-applied with the arylidene anabaseines to render the Ds state as conductive. If maintaining the H-bonding partner in the receptor (WT and Q57K) for the two FABs, a larger PNU-120596-stimulated peak response with a smaller net charge to peak response ratio was observed for 2FAB compared to 3FAB. However, such pattern disappeared in Q57D and Q57E mutants. These suggested that H-bonding effect could modulate the receptor desensitization via H-bonding, and depending on the orientation, change the energy barrier and/or energy level of the Ds state. These data suggested that both the orientation and pattern of the H-bond in the alpha7 selectivity pocket would regulate the receptor activation and desensitization, which can be utilized in drug design targeting specific functional states of alpha7 nAChRs. The third project involved investigations of an alternative pharmacophore for ligandi binding to the receptor. A positively charged nitrogen has been considered as the minimum pharmacophore for agonists and some competitive agonists of the nAChRs. A series of novel molecules were synthesized: (E)-3-(6-Benzylidenecyclohex-1-en-1-yl)pyridine (BHP) and its derivatives. These molecules lack the charged nitrogen pharmacophore, but could antagonize the various types of nAChRs. It was observed that BHP competitively antagonized the muscle type nAChR, with an IC50 value of 9 +/- 2 microM. The IC50 of the parent compound, 3-(cyclohex-1-en-1-yl)pyridine (PyHexe), was 261 +/- 52 microM, much smaller compared to that of BHP. These suggested that without the charged pharmacophore, but with the help from the extended aromatic ring, BHP could bind at the agonist binding site and competitively antagonized the receptor. In conclusion, the data reported in this thesis support the hypothesis that nAChR function may be differently regulated by single ligands binding in multiple alternative ways and that specific point-to-point interactions between ligands and receptor can selectively modulate the induction and stabilization of specific conformational states. These new insights may provide new approaches for drug development.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jingyi Wang.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Horenstein, Nicole A.
Local: Co-adviser: Papke, Roger L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043639:00001


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1 USING MOLECULAR TOOLS TO STUDY THE STRUCTURE AND FUNCTIONAL STATES OF THE HUMAN 7 NICOTINIC ACETYLCHOLINE RECEPTOR By JINGYI WANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FU LFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Jingyi Wang

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3 To the most important people in my life, my mother Huazhen Dan and my father Guangren Wang

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4 ACKNOWLEDGMENTS My study would no t have been accomplished without the support and help of many people. To begin with, I would like to specially thank my advisor Dr. Nicole Horenstein scientific guidance and support during the course of my studies. I also want to thank my co a dvisor Dr. teaching and advice on neuroscience and pharmacology. He opened his laboratory to me and provide access to his great team to support these studies. I also express my gratitude to my doctoral committee members, Dr. Ronald Castellano, Dr. Gail Fanucci and Dr. Linda Bloom, for their advice and support. I would like to acknowledge Dr. Ion Ghiviriga help on the characterization of the compounds by 2D NMR spectroscopy. grou p members. I want to thank Dr. Fedra Leonik and Dustin Williams scientific advice and discussion. I would like to especially acknowledge Clare Stokes and Shed Abdullah Abbas Al Rubaiy Clare made all of the mutants in my study and Shed did most of the electrophysiological experiments on OpusXpress. I also thank to Lynda Cortes, Sara Copeland, Matthew Kimbrell and Robin Rogers for their technical support. I would also like to acknowledge Lorriane Clark, Yanbin Wu, Kinga Chojnacka and Sandra Duque for the ir friendship and support. I extend my thanks to my colleagues of the Chemistry Department, in particular, Yan Chen, Mario Moral, Xiangling Xiong, Alison Lecher, Yong mo Ahn, Whitney Kellett for lending equipment, instructive scientific conversations and s haring success and frustration. My special acknowledgements go to the financial support from NIH, Alumni Fellowship and University of Florida Chemistry Department.

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5 I would also like to thank my friends for making my stay in Florida an unforgettable experie nce. M y special gratitude goes to Lijuan Huang, Hui Wang, and Peng Guo. We have been supporting each other from the very first day. I would also want to thank Hao Liu, Zheng Zheng and Li Zhao for their great support at the end of my doctoral study. My grea test thank goes to my family for their supports all the time. I could not possibly have completed my doctoral study without the encouragements and love from my parents, Guangren Wang and Huazhen Dan. Finally, I would like to express my eternal gratitude to my cousins, my grandparents and everyone in my big family for believing me all the time, which encouraged me to continue this difficult journey.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 15 ABSTRACT ................................ ................................ ................................ ................... 19 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 21 1.1 Neurotransmitter and Synaptic Receptors ................................ ........................ 21 1.2 Nicotinic Acetylcholine Receptors ................................ ................................ ..... 23 ...................... 25 1.2.1.1 Nicotinic receptors in signal transduction ................................ ....... 25 1.2.1.2 nAChR functional states ................................ ................................ 28 1.2.2 Mo nitoring the Function of the nAChR Channel ................................ ...... 30 1.3 Different Nicotinic Acetylcholine Receptor Subtypes and Functions ................. 33 1.3.1 Dif ferent Nicotinic Receptor Subtypes ................................ ..................... 33 1.3.2 Role of Different Neuronal nAChR Subtypes in Human Diseases ........... 37 1.3.2.1 Nicotinic rec eptors and cognition ................................ ................... 38 1.3.2.2 Nicotinic receptors and anxiety ................................ ...................... 41 1.3.2.3 Nicotinic receptors and epilepsy ................................ .................... 4 2 1.4 Different nAChR Ligands ................................ ................................ .................. 43 1.4.1 Nicotinic Agonists ................................ ................................ .................... 44 1.4.2 Nicotinic Antago nists ................................ ................................ ............... 48 1.4.3 Positive Allosteric Modulators ................................ ................................ .. 49 1.5 The Structure of Nicotinic Receptor ................................ ................................ .. 51 1.5.1 Overview of the Structure ................................ ................................ ........ 51 1.5.1.1 Extracellular domain ................................ ................................ ....... 52 1.5.1.2 Transmembrane domain ................................ ................................ 55 1.5.2 Three Dimensional Structures of Nicotinic Receptors ............................. 56 1.6 Nicotinic Receptor Drug Development ................................ .............................. 60 1.6.1 Different Drug Development Approaches ................................ ................ 60 1.6.2 Challenges in Nicotinic Drug Developments Study of Different Functional States ................................ ................................ .......................... 61 2 USE OF SULFHYDRYL REAGENTS TO TETHER THE RECEPTOR AND STUDY THE FUNCTIONAL STATES OF THE RECEPTOR ................................ .. 66 2.1 Background ................................ ................................ ................................ ....... 66

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7 2.2 Results and Discussion ................................ ................................ ..................... 67 2.2.1 Experimental Design to Characterize the Effects of 7 Receptor Covalent Modifications by SH Reagents ................................ ....................... 67 2.2.2 Cysteine Mutants Selected for Tethering Studies in the LBD .................. 70 2.2.3 Non selective Sulfhydryl (SH) Reagents and Their Electrophysiologic al Evaluation ................................ ................................ ..... 72 2.2.4 Reaction with SH Reagents and the Subsequent ACh Responses ......... 76 2.2.5 Activation of SH modified 7 Mutants by PNU 120596 ........................... 81 2.2.6 PNU 120596 Potentiation of ACh Responses in SH Reacted Receptors ................................ ................................ ................................ ...... 86 2.2.7 The Modification of the Reduced C loop and PNU Potentiation ................................ ................................ ................................ ... 87 2.2.8 Modeling Modifications of the Agonist and the Binding site ..................... 90 2.2.9 Synthetic Eff orts Towards Sulfhydryl labeling Derivatives of 7 Selective Agonists ................................ ................................ ......................... 91 2.3 Summary ................................ ................................ ................................ .......... 96 2.4 Experimental Section ................................ ................................ ........................ 98 3 DESIGN AND USE OF NOVEL MOLECULES TO STUDY THE H YDROGEN BONDING EFFECT IN THE HUMAN 7 NICOTINIC ACETYLCHOLINE RECEPTOR LIGAND BINDING SITE ................................ ................................ ... 108 3.1 Background ................................ ................................ ................................ ..... 108 3.2 Results and Discussion ................................ ................................ ................... 112 3.2.1 Synthesis of the Hydrogen Bonding Probes (Novel Arylidene Anabasei nes) ................................ ................................ .............................. 112 3.2.2 The Human 7 Receptor Mutants ................................ ......................... 117 3.2.3 Activation Profile of H bonding Probes on the Wild Type Human 7 Receptor and Mutants ................................ ................................ ................. 123 7 Receptors and Mutants ................................ ................................ ............... 130 3.3 Summary ................................ ................................ ................................ ........ 144 3.4 Experimental Section ................................ ................................ ...................... 147 4 DESIGN OF NOVEL MOLECULES TO TEST AN ALTERNATIVE PHARMACOPHORE FOR BINDING AND/OR ACTIVATION OF THE NICOTINIC RECEPTORS ................................ ................................ .................... 162 4.1 Background ................................ ................................ ................................ ..... 162 4.2 Results and Discussion ................................ ................................ ................... 165 4.2.1 Synthesis of ( E ) 3 (6 benzylidenecyclohex 1 en 1 yl)pyridine (BHP) and Its Derivatives ................................ ................................ ....................... 165 4.2.2 Synthesis of 3 (Cyclohex 1 en 1 yl)pyridine (PyHexe) .......................... 171 4.2.3 Synthetic Studies Directed towards 3 (3 Phenyl 1 H pyrrol 2 yl)pyridine (BPP) ................................ ................................ ......................... 173 4.2.4 Electrophysiology Evaluation of the Novel Molecules 255 ........................ 175 4.3 Summary ................................ ................................ ................................ ........ 177

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8 4.4 Experimental Section ................................ ................................ ...................... 178 5 Conclusions an d Future work ................................ ................................ ............... 191 APPENDIX A NMR SPECTRA of SYNTHESIZED COMPOUNDS ................................ ............. 194 B E LECTROPHYSIOLOGY ASSAY AND ANALYSIS ................................ ............. 246 LIST OF REFERENCES ................................ ................................ ............................. 250 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 274

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9 LIST OF TABLES Table page 1 1 Subunits compositions of ionotropic receptors ................................ ................... 23 2 1 Positive allosterica modulator PNU 120596 effects: responses to PNU 120596 applied alone, and su bsequent potent iation of ACh evoked responses. ................................ ................................ ................................ .......... 82 2 2 The effects of SH reagents on PNU 120596 primed ACh evoked responses.. .. 87 2 3 The effects of SH reagents on the relative PNU 12059 6 potentiation of ACh responses ................................ ................................ ................................ ........... 87 2 4 Summary of the synthesis of the sulfyhydryl derivative of the 7 selective agonists. ................................ ................................ ................................ ............. 93 3 1 The predicted iminium cation percentages of the arylidene anabaseines. ....... 116 3 2 The potency (EC 50 ) and recovery of acetylcholine with the wild type and h 7Q57 mutants evalu ated by the net charge response ................................ .. 120 3 3 The efficacy (I max ) and potency (EC 50 ) of the six arylidene anabaseines with the wild type h 7 receptor and h 7Q57 mutants evaluated by net charge ...... 125 3 4 Percent recovery of the h 7 wild type receptor and glutamine 57 mutants after treated with 300 M arylidene anabaseines ................................ ............. 138 4 1 The results of m onitoring the Grignard reaction ................................ ................ 169 4 2 Inhibition potency ( IC 50 ) and maximum inhibition percentage of BHP with different types of nACh Rs ................................ ................................ ................. 176

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10 LIST OF FIGURES Figure page 1 1 Small molecules as neurotransmitters. ................................ ............................... 22 1 2 Acetylcholine, nicotine and muscarine ................................ ................................ 23 1 3 Cys loop receptors structure ................................ ................................ .............. 24 1 5 Nicotinic acetylcholine receptor functional st ate models ................................ ..... 29 1 6 Schematic experimental set up for two electrode voltage clamp (A) and representative response when applying ligand (B). ................................ ............ 32 1 7 Distribution of nAChR subunits in the rodent brain and their related diseases ... 35 1 8 Natural and synthetic agonists for acetylcholine receptors ................................ 45 1 9 Multiple classes of nicotinic agonists. ................................ ................................ 47 1 10 Structures of nAChR antagonists ................................ ................................ ....... 49 1 11 Positive allosteric modulators ................................ ................................ ............. 50 1 12 Ribbon diagrams of the muscle type nAChR resolved at 4 ............................ 53 1 13 The hypothetical energy landscape for 7 nAChR state transitions when bound with the full agonist acetylcholine ................................ ............................ 63 2 1 Experimental design ................................ ................................ ........................... 69 2 2 Cyste ine mutations in the LBD of 7 nAChR ................................ ...................... 71 2 3 The structures of the six SH reagents: Br ACh, MTSET, MTSEA, QN SH, EMTS, and MTSACE ................................ ................................ .......................... 72 2 4 Intrinsic agonist activity of SH reagent agonist analogs for wild type 7 and 7 C116S ................................ ................................ ................................ ........... 73 2 5 Activation profiles of Br ACh and MTSET on wild type (C116C) and mutant human 7 nAChRs ................................ ................................ ............................. 75 2 6 Representative traces from voltage clamp experiments, outline in Figure 2 1C ................................ ................................ ................................ ....................... 76 2 7 Kinetics of subsequent ACh blockade eff ects by MTSET and Br ACh. .............. 77 Figure 2 8. Acetylcholine responses after SH reagent application ................................ 80

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11 2 9 Responses to the type II PA M PNU 120596 after SH reagent treatments ......... 85 2 10 The modification and PNU 120596 potentiation of 7 C116S after DTT treatment ................................ ................................ ................................ ............ 88 2 11. Homology models of 7 covalently modified by the SH reagents ........................ 91 2 12 Synthetic route for piperidine sulfhydryl derivatives. ................................ ........... 92 3 1 The structure of the hydrogen bonding probes in comparison with the structure of 4 OH GTS 21. ................................ ................................ ............... 109 3 2 Synthetic schemes for the arylidene anabaseines ................................ ............ 114 3 3 NOE enhancement used for assigning the olefin geometries of 16a, 16b and 19a c ................................ ................................ ................................ ................ 115 3 4 The electrophysiological characterization of the wild type 7 rec eptor and its glutamine 57 mutants. ................................ ................................ ...................... 119 3 5 Comparison of PNU 120596 simulated responses for the different h 7 receptors when co applied with acetylcholine.. ................................ ................ 121 3 6. Concentration response curves of the arylidene anabaseines (0.3 M to 300 M) ................................ ................................ ................................ ................... 124 3 7 Representative traces of the 300 on and desensitization profile on wild type 7 and glutamine 57 mutants. ................... 126 3 8 Summary of the channel activation via H bonding effect. ................................ 129 3 9 Comparison of the PNU 120596 stimulated currents of the wild type 7 and Q57 mutants when co applied with the six arylidene anabaseines. .................. 131 3 10 Hypothetical mechanism of the H bonding effect in modulating the 7 bonds are shown in dash lines ........................... 134 3 11 Hypothetical H bonding effect of the two PyroABs in modulating the energy barrier of D s in glutamine 57 mutants ................................ ............................... 136 3 12. A summary of the 2FAB residual desensitization profile with the h 7 receptor and glutamine mutants evaluated by PNU 120596 ................................ .......... 141 3 13. The energy landscape of the arylidene anabaseines in comparison with acetylcholine in the wild type receptor. ................................ ............................. 146 4 1 The structures of 4 OH GTS 21 a nd BHP. ................................ ....................... 163

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12 4 2 Design for the retrosynthesis route for BHP ................................ ..................... 164 4 3 Synthetic route to the aldehyde, 23. ................................ ................................ 166 4 4 Synthetic route to the BHP and its two derivatives (4 methoxy and 4 hydroxyl). ................................ ................................ ................................ .......... 167 4 5 The carbon and hydrogen chemical shift (ppm) assignments of compound 25 and NOE results of compound 25 and 27. ................................ ........................ 170 4 6 Synthetic scheme of compound 34 (PyHexe). ................................ .................. 172 4 7 Attempted synth esis of compound 40. ................................ .............................. 1 74 5 1 The structure of cysteine reactive 7 selective agonist analogs. ...................... 192 A 1 4 Bromo 1 methylpiperidine ................................ ................................ ............. 195 A 2 4 Bromo 1,1 dimethylpiperidin 1 ium iodide. ................................ .................... 196 A 3 1 Methylpiperidin 3 yl methanesulfonate. ................................ ......................... 197 A 4 1,1 Dimethyl 3 ((methylsulfonyl)oxy)piperidin 1 ium. ................................ ....... 198 A 5 1 Methylpiperidin 4 yl acetate. ................................ ................................ .......... 199 A 6 4 Acetoxy 1 (bromomethyl) 1 methylpiperidin 1 ium bromide. ......................... 200 A 7 Sodium methanesulfonate. ................................ ................................ ............... 201 A 8 Sodium me thanethiosulfonate. ................................ ................................ ......... 202 A 9 1 ((diethylamino)methyl)piperidin 2 one. ................................ .......................... 203 A 10 Sodium Salt of the Aminal of 3 Nicotinoyl 2 piperi done. ................................ ... 204 A 11 Anabaseine dihydrochloride. ................................ ................................ ............ 205 A 12 3 Pyrrolyl carboxaldehyde. ................................ ................................ ............... 206 A 13 2 Pyrrolylmethylene anabaseine (2PyroAB). ................................ .................... 207 A 14 3 Pyrrolylmethylene anabaseine (3PyroAB). ................................ .................... 208 A 15 2 Furanylmethylene anabaseine (2FAB). ................................ ......................... 209 A 16 3 Furanylmethylene anabaseine (3FAB). ................................ ......................... 210 A 17 2 Thiophenylmethylene anabaseine (2T AB). ................................ ................... 211

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13 A 18 3 Thiophenylmethylene anabaseine (3TAB). ................................ ................... 212 A 19 2 Pyrridinylmethylene anabaseine (2PAB). ................................ ...................... 213 A 20 3 Pyrridinylmethylene anabaseine (3PAB). ................................ ...................... 214 A 21 4 Pyrridinylmethylene anabaseine (4PAB). ................................ ...................... 215 A 22 2 Pyrrolylmethylene anabaseine (2PyroAB), NOESY. ................................ ..... 216 A 23 3 Pyrrolylmethylene anabaseine (3PyroAB), 2D NOESY. ................................ 217 A 24 2 Pyrridinylmethylene anabaseine (2PAB), NOESY. ................................ ........ 218 A 25 3 Pyrridinylmethylene anabaseine (3PAB), NOESY. ................................ ........ 219 A 25 4 Pyrridinylmethylene anabaseine (4PAB), NOESY. ................................ ........ 220 A 27 4 Pyrridinylmethylene anabaseine (4PAB), NOESY. ................................ ........ 221 A 28 5 chloro 2,4 di methoxyphenyl isocyanate. ................................ ....................... 222 A 29 1 (5 chloro 2,4 dimethoxyphenyl) 3 (5 methylisoxazol 3 yl)urea (PNU 120596). ................................ ................................ ................................ ........... 223 A 30 2 Chlor ocyclohex 1 enecarbaldehyde. ................................ ............................. 224 A 31 2 (Pyridin 3 yl)cyclohex 1 enecarbaldehyde. ................................ ................... 225 A 32 Phenyl(2 (pyridin 3 yl)cyclohex 1 en 1 yl)methanol. ................................ ........ 226 A 33 (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP). ................................ ...... 227 A 34 (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridin e (BHP), COSY. .......................... 228 A 35 (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), HMBC. .......................... 229 A 36 (E) 6 Benzylidenecyclohex 1 en 1 yl)p yridine (BHP), HMBC. .......................... 230 A 37 (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), HMBC. .......................... 231 A 38 (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), NOESY spectrum (top) in comparison with proton spectrum (bottom). ................................ .................. 232 A 39 (4 Methoxyphenyl)(2 (pyridin 3 yl)cyclohex 1 en 1 yl)methanol. ...................... 233 A 40 ( E ) 3 (6 (4 Methoxybenzylidene)cyclohex 1 en 1 yl)pyridine (4 MeO BHP). ... 234

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14 A 41 ( E ) 3 (6 (4 Methoxybenzylidene)cyclohex 1 en 1 yl)pyridine (4 MeO BHP), NOESY. ................................ ................................ ................................ ............ 235 A 42 (4 ((Tert butyldimethylsilyl)oxy)phenyl)(2 (pyridin 3 yl)cyclohex 1 en 1 yl)methanol. ................................ ................................ ................................ ...... 236 A 43 ( E ) 3 (6 (4 ((Te rt butyldimethylsilyl)oxy)benzylidene)cyclohex 1 en 1 yl)pyridine. ................................ ................................ ................................ ........ 237 A 44 ( E ) 4 ((2 (Pyridin 3 yl)cyclohex 2 en 1 ylidene)methyl)phenol (4 OH BHP). .... 238 A 45 1 Chlorocyclohex 1 ene. ................................ ................................ .................. 239 A 46 1 (Pyridin 3 yl)cyclohexanol. ................................ ................................ ............ 240 A 47 3 (Cyclohex 1 en 1 yl)pyr idine. ................................ ................................ ........ 241 A 48 N (pyridin 3 ylmethyl)benzamide. ................................ ................................ ..... 242 A 49 N (pyridin 3 ylmethyl) N (tert butoxycarbonyl amino)benzamide. .................... 243 A 50 1 Phenyl 2 (pyridin 3 yl) 2 (tert butoxycarbonyl amino)ethanone. ................... 244 A 51 2 Amino 1 phenyl 2 (pyridin 3 yl)ethanone hydrochlor ic salt. .......................... 245 B 1 Representative traces and expression level test of the h 7 receptor and the glutamine 57 mutants. ................................ ................................ ...................... 247 B 2 The a lternative mechanism for H bonding impact on the h and remain in the Ds state. ................................ ................................ ............... 247 B 3 The relationship of the normalized PNU 120596 evoked peak response after 2FAB and the inhibition on ACh evoked response after 2FAB ......................... 248 B 4 PNU 120596 stimulated pe ak current on ACh applied after ............................. 248 B 5. The energy landscape of the acetylcholine and arylidene anabaseines with different receptor types ................................ ................................ ..................... 249

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15 LIST OF ABBREVIATION S ACh acetylcholine AChBP acetylcholine binding protein APCI atmospheric pressure chemical ionization AD Alzh Ar argon gas BA benzylidene anabasiene BHP ( E ) 3 (6 benzylidenecyclohex 1 en 1 yl)pyridine Boc t butyloxycarbonyl b.p. boiling point Br ACh bromoacetylcholine cDNA copy from RNA via reverse transcription (CHO) n paraformaldehyde CI chemic al ionization COSY correlation spectroscopy concentrated CRC concentration response curve cRNA complementary ribonucleic acid DART direct analysis in real time DIP direct insertion probe D i PNU insensitive desensitized state DMAD dimethylacetylenedic arboxylate DMAP 4 dimethylaminopyridine DMF 1,2 dimethoxyethane DMPU 1,3 Dimethyl 3,4,5,6 tetrahydro 2(1H) pyrimidinone

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16 DMSO dimethyl sulfoxide D s PNU sensitive desensitized state DTT dithiothreitol EI electron ionization EMTS ethyl methanethiosulfonate ESI electrospray ionization Et 2 O diethyl ether EtOAc ethyl acetate EtOH ethanol FAB furanylmethylene anabaseine FLIPR fluorescent imaging plate reader GABA aminoburyric acid GPCR G protein coupled receptors GTS 21 2,4 dimethyoxybenzylidene anabaseine HC l hydrochloric acid HEPES 4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid HMBC heteronuclear multiple bond correlation spectroscopy HNEt 2 diethylamine HOAc acetic acid HPLC high performance liquid chromatography IR infrared spectrometry LBD ligand bindi ng domain LDA lithium diisopropylamide LGIC ligand gated ion channel MeO methoxyl

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17 MeOH methanol MMI multimode ion m.p. melting point mRNA messenger ribonucleic acid MS mass spectrometry Ms mesylate MTSACE 2 (aminocarbonyl)ethyl methanethiosulfonate MTSE A 2 (aminoethyl)methanethiosulfonate MTSET methanethiosulfonate ethyltrimethylammonium nAChR nicotinic acetylcholine receptor NaMTS sodium methanethiosulfonate NaOAc sodium acetate NEt 3 triethylamine NMR nuclear magnetic resonance NOE nuclear overhauser ef fect NOESY nuclear overhauser spectroscopy PAB pyridinylmethylene anabaseine PAM positive allosteric modulator PD PDB protein data bank PNU 120596 N (5 chloro 2,4 dimethyphenyl) (5 methyl 3 isoxazolyl) urea P open ion channel open pro bability PyHexe 3 (cyclohex 1 en 1 yl)pyridine PyroAB pyrrolylmethylene anabaseine QN SH 2 (quinuclidinium)ethyl methanethiosulfonate

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18 RID residual inhibition or desensitization r.t. room temperature saturated SCAM substituted cysteine accessibility mu tagenesis S.E.M. standard error SH sulfhydryl TAB thiophenylmethylene anabaseine TBDMSO (tert butyldimethylsilyl)oxyl THF tetrahydrofuran TLC thin layer chromatography TMA tetramethylammonium TOF time of flight WT wild type

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19 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy USING MOLECULAR TOOLS TO STUDY THE STRUCTRUAL AND FUNCTIONAL STATES OF THE HUMAN 7 NICOTINIC A CETYLCHOLINE RECEPTOR By Jingyi Wang December 2011 Chair: Nicole A. Horenstein Cochair: Roger L. Papke Major: Chemistry Homomeric 7 nicotinic acetylcholine receptors (nAChRs) have been suggested chizophrenia and inflammatory disorders. This dissertation study utilized different molecular tools, including mutant forms of the receptor, complemented by the structural homology models of 7 nAChRs to investigate the structural features of ligands/recep tor interactions that regulate binding to the nAChR and the conformational stability of open and desensitized states. reactive cysteines were introduced into the 7 rec eptor to allow binding of sulfhydryl reactive (SH) agonist analogs or control reagents onto specific positions. The tethering of the SH reagents blocked further acetylcholine evoked activation of the receptor in four 7 mutants (S36C, L38C, W55C and L119C) The type II modulator PNU 120596 could reactivate the tethered L119C and W55C mutants but not the tethered S36C and L38C mutants. These results suggested that a single ligand can bind within the receptor in different ways and, depending on the specific b inding orientation, may variously promote activation, desensitization or alternatively, function as a competitive antagonist.

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20 Secondly, to test the influence of H bonding interaction in the 7 selectivity pocket on channel function, various probes with unique H bonding properties were synthesized and paired with the 7 receptor or its Q57 mutants. We hypothesized that when there was a mismatch for H bonding, a change in channel activation wou ld be observed, such as mutant Q57K or Q57L with 2PyroAB. Via utilization of PNU 120596 to render one of the desensitized states as conductive, H bonds were suggested to lead to the different PNU 120596 stimulated responses between 2FAB and 3FAB. These dat a implied that both the orientation and pattern of the H bond in the 7 selectivity pocket could regulate the receptor activation and desensitization. The third project involved investigations of an alternative pharmacophore for ligand binding to the receptor. A series of novel molecules without the core charged nitrogen ph armacophore were synthesized: ( E ) 3 (6 benzylidenecyclohex 1 en 1 yl)pyridine (BHP) and its derivatives. With the help from the extended aromatic ring, BHP was suggested to bind at the agonist binding site and competitively antagonized nAChRs without selec tivity for the subtype.

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21 CHAPTER 1 1 INTRODUCTION 1.1 Neurotransmitter and Synaptic R eceptors In the nervous system, a synapse enables neuron s to pass signal s to another cell electrically or chemically. In a chemical synapse, the communication between these cells is mediated by neurotransmitter s when the gap bet ween neurons is too large for direct electrical transmission. Neurotransmitters are synthesized from plentiful and simple precursors, which are readily available from diet or easily obtained through a small num ber of biosynthetic steps. 1 These neurotransmitters include amino acids, peptides, monoamines and other small molecules, as shown in Figure 1 1. They diffuse across the synapse to the correspo nding receptor, where they bind and trigger an event at the postsynaptic cell with a wide variety of outcomes. If the neuron is proximate to a muscle cell, the signal transmission may induce several intracellular signal cascades resulting in muscle contrac tion. If the postsynaptic cell is part of glandular tissue, the action potential may initiate signal transduction for hormone secretion. 2 Synaptic receptors are proteins to which neurotransmitters bind to initiate ion flow through the cell membrane directly or indirectly. Ion channels are synaptic rece ptors introducing a hydrophilic pore to allow direct ion diffusion through the membrane lipid bilayer. 3 Unlike ionotropic carriers which bind the ion on one side of the cell membrane and release it on t he other side, ion channels span across the cell membrane and create a conduction pathway for ions in a faster way down their electrochemical gradients. Ion channels open and close for a very short period of time in response to different cellular signals. This gating mode of action can be regulated either by changes in the electrical potential across the plasma membrane, or by a variety of ligands

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22 located inside or outside the neuron. Ion channels that respond to membrane potential changes are called voltag e gated ion channels. Ion channels that open for ion flow upon binding a chemical messenger are known as ligand gated ion channels (LGICs), which are also called ionotropic receptors. Figure 1 1. Small molecules as neurotransmitter s LGICs include, bu t are not limited to, a variety of neurotransmitter receptors, which are multimers made up of at least 4 or 5 subunits ( Table 1 1 ) 4 Different combination s of the subunits differentiate the receptors into subtypes, which vary in ligand binding, permeability t o ions, expression loci and other properties For example, among native N methyl D aspartate ( NMDA ) receptors, the NR1/NR2A subtype predominates at many mature synapses while the NR1/NR2B subtype is mostly found at immature neu rons and in extrasynaptic loci 5 These two NMDA receptor subtypes also differ in the

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23 sensitivity to Mg 2+ block 6 M olecules other than neurotransmitte rs can also modulate the activation of LGICs, such as in those calcium activated potassium channels 7 and cyclic nucleotide gated channels 8 Table 1 1. Subunits compositions of ionotropic receptors Receptor AMPA NMDA Kainate GABA Glycine nA Ch Serotonin Purines Subunit types GluR1 GluR2 GluR3 GluR4 NR1 NR2A NR2B NR2C NR2D GluR5 GluR6 GluR7 KA1 KA2 1 7 1 4 1 4 1 2 3 4 2 9 1 4 5 HT 3 P 2X1 P 2X2 P 2X3 P 2X4 P 2X5 Metabotropic receptors compose the second family of synaptic rec eptors, some of which are G protein coupled receptors (GPCR). The GPCRs do not mediate transport of ions across the membrane, but instead activate intracellular G proteins to initiate signal transduction after an extracellular binding event. Metabotropic r eceptors are also structurally distinct from LGICs. They are monomeric proteins with an extracellular domain for neurotransmitter binding, an intracellular domain that binds to G proteins, and a seven helix segment. Metabotropic receptors are mainly presen t as dimmers, 4 but have also been found in monomeric form. 9 1.2 Nicotini c Acetylcholine Receptors Figure 1 2 : Acet ylcholine, n ico tine and m uscarine The neurotransmitter ACh binds to cholinergic receptors in the synapse and on the neuron, including the ionotropic type nicotinic acetylcholine receptor (nAChR) and the metabotropic type muscarinic acetylcholine receptor ( mAChR). 10 The two kinds of acetylcholine receptors derive their names from their different affinities for nicotine and

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24 muscarine (Figure 1 2). The mAChRs are more sensitive to muscarine and mediate the slow metabolic responses to ACh through the different G proteins attached via coupling to second messenger cascades. The nAChRs are ligand gated ion channels that can be activated by nicotine. Some of them can mediate rapid synaptic transmission in neuromuscular and ganglionic junctions. Figure 1 3. Cys loo p receptors structure adapted from the article written by Sine, S.M. and Engel, A.G.. 11 The nAChR are pentameric proteins with a molecular mass of about 250 270 kDa 12 which belong to the ionotropic Cys loop f amily, together with glycine, serotonin and GABA receptors. 11, 13 Cys loop receptors all harbor a signature sequence of 13 residues flanked by two disulfide bonded cysteines, which form a closed loop situated betwee n the extracellular ligand binding domain and the transmembrane channel domain (Figure 1 3). The five subunits of the Cys loop receptors form an intramolecular vestibule, which selectively allows cations or anions to flow into the cell to depolarize or rep olarize the cell membrane, and exert excitatory or inhibitory effects towards the

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25 action potential in electric signal transduction. Therefore, they are further subdivided into cation and anion selective channels, corresponding to the excitatory receptors (serotonin and acetylcholine receptors) and the inhibitory receptors (GABA and glycine receptors). As one of the first ion channels purified, nAChR is a reference for studying the mechanisms and st ructures of other receptors. It is permeable to alkali and alkali earth cations (K + Na + Ca 2+ ) upon activation. The overall structure of the nAChRs is similar to that of the other Cys loop receptors, as shown in Figure 1 3. The details of the structure of the nAChRs will be considered later. 1.2.1 Nicotinic A cet ylcholine the work of the English physiologist John Newport Langley in the early nineteen century. Langley showed that nicotine first caused a contraction and then produced a inhibition effect when applied to neuromuscular preparation, while curare could inhibit the contraction effect of nicotine. 14 He further pointed out that nicotine needs to combine with a substance at the nerve ending to produce local contraction. 15 In modern terms, nicotine is an agonist that activates the channel for signal transduction like the neurotransmitter acetylcholine. Although nicotine binds to the muscle type nAChRs, the muscle contraction cascade is mainly induced by acetylcholine. 16 Upon acetylcholine binding, contraction results from channel activation, while the inhibition effect is an apparent phenomenon of channel desensitization and/or channel ant agonization. 17 Curare is an antagonist which prevents or dampens the agonist mediated response. 1.2.1.1 Nicotinic receptors in signal t ra nsduction One of the main functions of nAChRs is to chemically mediate the transmissi on of electric signals for cell cell communication, as shown in Figure 1 4. A cetylcholine

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26 molecules are synthesized from choline and acetyl CoA by the enzyme choline acety ltransferase (ChAT) in the neuron When the electric signal arrives at the presynaptic axon terminal, various voltage gated ion channels will open Among them, the voltage gated Ca 2+ channels introduce Ca 2+ into the cell and this trig gers the release of A Ch into the syna ptic cleft Those acetylcholine molecules that travel through the synapt ic cleft can bind with nAChRs on the postsynaptic cell membrane to open the channel gate for cations. This cation flow will depolarize the postsynaptic neuron cell memb rane to the threshold potential, which will activate voltage gated Na + channels fire up the action potential and generate a new electrical signal in the postsynaptic cell, thus effecting cell to cell communication. Figure 1 4. Nicotinic acetylcholine r eceptor signal transduction in a s ynapse. (Adapted from the book, From neuron to brain, 4 th ed., written by Nicholls, J. G., Martin, R. A., Wallace, B. G., and Fuchs, P. A.) 18

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27 The nAChRs are present both at synaptic terminals and at nonsynaptic locations on dendrites, soma and axons. They are primarily expressed in muscle cells and neurons, but may also be found in glia and non neuronal tissues 19 The existence of the non neuronal and nonsynaptic nAChRs suggests that activation may require diffus ion of ACh to the remote site. 20 The spread of the ACh is mediated by diffusion, b ut also limited by the hydrolysis of ACh by acetylcholine esterase (AChE) as shown in Figure 1 4. The enzyme is widely distributed in the CNS, but its reported density and location are sometimes inconsistent with the ACh release sites. 21 At the postsynaptic membrane, acetylcholine is quickly hydrolyzed into acetate and choline by the AChE. For most nAChR s, this hydrolysis process generates two inactive metabolites to prepare the cell for next stimulation. However, choline can selectively activate or desensitize some subtypes of nAChRs, 22, 23 thereby, contributing t o a long lived choline signal from the ACh diffuse transmission which may be limited by high affinity reuptake of choline Besides neuron excitation, nAChR initiates direct and indirect intracellular calcium signaling responsible for neurotransmitter rele ase and intracellular enzymatic processes. Unlike channels which only accommodate Ca 2+ influx at depolarized potentials, nAChR can provide direct Ca 2+ influx even at the resting potential. This Ca 2+ influx can initiate calcium release from intracellular st ores. 24 The alternative, indirect way of promoting Ca 2+ influx is through activation of voltage gated calcium channels by nAChR induced membrane depolarization, which is the primary source of calcium ion for intracellular signal pathway processes. Calcium elevation can act as a secondary messenger to modulate synaptic transmiss ion indirectly. This indirect effect can be achieved either by enzyme

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28 modification on the receptor or by boosting the release of many different neurotransmitters, including ACh, dopamine, norepinephrine, serotonin, glutamate and GABA. 12, 25 For example, protein kinases and phosphatases are two known enzymes that are able to modify glutamatergic synapses mediated by calcium signals from nAChRs. Further, activation of nAChRs, via inducing several intracellular signal c ascades, also enhance glutamate release that lasts up to several minutes. 26 The action of nAChRs on glutamate receptors contributes to the induction of synaptic plastici ty, which is an important neurochemical foundation of learning and memory. 12 1.2.1.2 nAChR functional states Nicotinic acetylcholine receptors are allosteric proteins that bind with neurotransmitters extracellularly and initiate opening of their channel gate for cations to flow through the cell membrane via conformational changes. This process is termed non conductive state, the so called desensitized state. Thus the desensitized state pr events continuous receptor activation, which is suggested to be important for neuroprotection, intracellular signal transduction and trafficking. 27 Early quantitative modeling of ligand gated channel functional states relied on analogy with enzymatic catalysis and resulte was formulated by Koshland, Nemethy and Filmer in 1966, and it is therefore named the KNF model. 17, 28 In this model, ligand bind is supposed to instruct a conformational change in individual subunits, implying that the functional units are plastic. The mor e proposed by Monod, Wyman, Changeux (MWC model), 29 in which proteins exist in several discrete quaternary pentamers assembled with a symmetrical subunit

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29 state, open state and desensitized state). The equilibrium of these functional states will be distur bed by ligands having different affinity to each channel state (Figure 1 5). However, asymmetries do exist in nAChRs subunits when transitioning between different functional states, as suggested by asymmetric affinity labeling results both at the extracell ular ligand binding site and in the transmembrane channel region. 30, 31 Therefore, the modified MWC model relaxes the symmetry requirement for nAChRs, so as to permit all combinations of quaternary pentamer containi ng subunits to exist in one of the conformational states. 32 When the ligand binds to the nACh receptors, the li Considering that nAChRs appear to be desensitized with a long exposure to ligand, the desensitized state should have the highest ligand binding affinity (Figure 1 5) Figure 1 5. Nicotinic acetylcholine receptor functional state models adapted from the book, Nicotinic acetylcholine receptors, 1 st Ed, written by Changeux, J.P. and Edelstein, S.J. 17

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30 1.2.2 Monitoring the Function of the nAChR Channel Permeability to cations is the main property utilized in monitor ing the nAChR channel functions, which is the basis for ion based flux assays and electrop hysiology assays. Non electrophysiological assays include detecting either the change of intracellular concen tration of the permeated ions or the downstream consequence of the concentration change Both radioactivity and fluo rescence have been applied to t rack the intracellular cation concentration change in nAChR expressed cells, example s being the 86 Rb + efflux assay 33 and the intracellular Ca 2+ fluorescence assay 34 The la t ter method is safer and is therefore currently widely used for functional studies of ligand s and receptors in both academic and industrial laboratories 35, 36 Measuring the conse quence of the concentration change is based on the change of the membrane potential which can be utilized for analyzing ion channel functions in vitro and/or in vivo both non electrophysiologically (fluorescent method) and electrophysiologically (patch cl amp). Fluorescence measurements require development of fluorescent dyes which are sensitive to membrane potential. The bisoxonol fluorescent dye bis (1,3 dibutylbarbituric acid)trimethine oxonal (DiBAC 4 ) was used earlier, the fluorescence change of which c omes from its redistribution resulting from depolarization or hyperpolarization 37 The FLIPR membrane potential dye is nowadays more widely used since it has faster kinet ics and is less sensitive to temperature. It also correlates well with the membrane potential determined by patch clamp recording 38 potenti of the ion channel embedded in the cell membr ane can be treated as a circuit, with the membrane potenti al as the battery and the ion channels as

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31 law can be rewritten for the ion channel membrane circuit as : I=(E m E rev ) N P open where E m represents the membrane potential, E rev stands for the reversal potential, N is the number of ion channels, P open is the probability for a channel to be open, and is t he conductance (reciprocal of the resistance) for each single channel. In the process of channel activation via ligand binding, all of the parameters above are constantly changing except E rev N and To simplify the system, either the voltage or current is held constant in voltage clamping to represent the functional change of the channel (N P open ) in the other term (current or voltage). Patch clamping can be applied in either single channel or multi channel recording. 39 A single pipette serving as both the voltage electrode and the current electrode is utilized in patch clamping, and different sucking techniques can be applied in comb ination with the pipette movement to make either single or multi channel recordings. 39 However, good patches of membrane for single chan nel recording are hard to obtain, and it is almost impossible to control voltage with a single electrode over the entire surface of larger cells or elaborately branched neurons. This research only makes use of a two electrode voltage clamp, which is the mo st common approach for voltage clamping larger cells. As shown in the scheme in Figure 1 6 A, two electrodes are utilized in the circuit; one is used to measure membrane voltage and the other is to inject current to keep the voltage at the command voltage. 40 When applying an agonist to the receptor in a two electrode voltage clamped system, channels will open for ion flow and generate an apparent ov erall current change for all the plasma membrane channels. For nAChRs, when a neurotransmitter opens the channel pore, cations will flow and generate a negative current through the membrane from outside to inside.

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32 Therefore, a positive current from the cur rent electrode is required to keep the membrane voltage constant, as shown in Figure 1 6 B. Figure 1 6. Schematic experi mental set up for two electrode voltage clamp (A ) and representative response when applying ligand (B ) Both integrated and automated systems have been developed for two electrode voltage clamping and single electrode patch clamping. Systems like PatchXpress are capable of recording membrane potentials for small mammalian cells, including human embryonic kidney 293 (HEK) cells and Chine se hamster ovary (CHO) cells. 41, 42 However, the automated patch clamp method requires the use of cell lines either stably transfected or naturally expressing the receptor of interest. Multiple cell lines must be de veloped and maintained for different targets, which increases the expense and timeline. 43 In contrast, OpusXpress makes high throughput two electrode voltage clamp recording possible on large am phibian oocyte cells. Such cells are capable of expressing heterologous receptors at the plasma membrane. Thus, any number of cDNA or RNAs may be kept for oocytes expression, and once injected, cells can be used as early as the next day in some cases. 40, 43

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33 1.3 Different Nicotinic Acetylcholine Receptor Subtypes and Functions 1.3.1 Different Nicotinic Receptor Subtypes Nicotinic acetylcholine receptors are found in the central nervous system, peripheral nervous syste m and nonneuronal tissues such as muscle. They are even reported in white blood cells. 19, 44, 45 Different combinations of the various subunit types enrich the variety of the nAChRs subtypes, which serve the differe nt functional purposes of nAChRs throughout the neuronal and non neuronal cells. Most nAChR are heteromeric, usually containing two identical alpha subunits plus three others, which was first revealed by SDS gel electrophoresis of muscle type nAChRs then supported by molecular weight measurements, direct quantification and sequence analysis 17 Four subunits were first identified in the 1970 s and 1980s in muscle type nAChRs based on the ir apparent molecular weights : 1, 40,000; 1, 50,000; 60,000; 66,000 Da A fifth subunit type in muscle was named according to the Greek alphabet. There are two ligand binding sites in muscular nicotin ic receptors, one at the 1 subunit interface, and the other at the 1 or 1 interface. The 1 subunit is structural and is not directly involved in ligand binding. Apart from homomeric neuronal receptors, which are built as pentamers of identical s ubunits, the compositions of heteromeric nicotinic receptors are much more complicated. There are twelve nAChR subunit types in the nervous system, nine subunits ( 2 10) and three subunits ( 2 4). The homologue of neuronal 7, 8 or 9 subunits is th e ancestral subunit and further differentiated into type and type in nervous and muscle system. 46 The 2 10 subunits share a pair of adjacent cysteines on the C loop, which is required for the ligand binding site. 47 The subunits also

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34 contribute to a ligand binding, by providing an interface that is complementary to the subunit. The ancestral 7 8 and 9 subunits are able to form functional homopentamers, which potentially co ntain five ligand binding sites. The more recently evolved 2 3, 5 6, 10, and 2 4 cannot form functional oligomers when expressed individually, but may respond to nicotine when co expressed in pairs or as part of larger assemblies. 46 Although it has been reported that 4 subunit s may form functional receptors when expressed alone in Xenopus oocytes 48 it preferentially co assembles with 2 subunit both in heterologous systems as well as in the brain. Knowledge of the precise stoichiometry within a given heteropentamer and definite rules of subunit assembly is sti ll limited. 17 The simplest stoichiometry would be 2[ ]:3[ ] for heteromeric receptors, with two binding sites at the interfaces, as in the muscle type receptor. 49 However, within a given receptor, the two subunits and the t wo (or three) subunits are not necessarily identical, e.g., 3 2 4 receptor. The 5 and 3 subunits cannot form either the principal or the complementary sites for ligand binding, 1 subunit in muscle receptors. For example, they participate in forming the 3 4 5 and 6 3 4 oligomers, contributing as one subunit per pentamer. 50, 51 The 10 subunit is the latest identified member of the family, and requ ires association with 9 to yield functional receptors. The heteromeric 9 10 is pharmacologically non identical but similar to the homomeric 9 receptor. 52 Besides differences in stoichiometry and binding sites, th ese nicotinic acetylcholine receptor subtypes also exhibit different affinities to ligands Some of these combinations have rather low affinity to acetylcholine (e.g., 7, 3 4 and 3 2),

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35 whereas some others bind acetylcholine more tightly (e.g., 4 2, 4 4 and 3 5 2). 17 Even subtypes with very similar constitutions may vary in their ligand affinity and pharmacological properties. For examp le, 4 2 3 or 3 2 2 3 receptors have high sensitivity to nicotine and low Ca 2+ 3 2 receptors behave in an opposite fashion, being low in sensitivity to nicotine and high in Ca 2+ permeability. 53 Figure 1 7. D istribution of nAChR subunits in the rodent brain and their related diseases, adapted from the article written by Jensen A. A. et al. 54 The abbreviations for the different brain areas displayed are as follow : Am, amygdala; Hc, hippocampus; Ht, hypothalamus; SN: substantia nigra; VTA, ventral tegmental area. The fact that many neuronal cells express multiple subtypes of nAChR initially hampers the identification of the different native nAChR subtypes and their specific

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36 roles. However, subtype selective affinity ligands and the availability of genetically engineered knockout or knockin mice enable the localization of specific nAChR subunits and/or subtypes, and make it possible to analyze the pharmacology and f unctional role of nAChRs in complex neurobiological systems. Figure 1 7 shows a summary of the distribution of nAChR subunits and their pharmalogical functions in the brain. The figure reflects the results of radiolabeled ligand binding experiments, immuno precipitation and immunopurification obtained from WT and/or nAChR subunit knockout mice. 54 As shown in Figure 1 7, nAChRs are widely distributed in the brain, and the subunit composition tends to be cell and region specific. Among them, the 4 2 subtype accounts for 90% of the high nicotinic affinity neuronal nAChR in the mammalian brain, which is followed by the homomeric 7 subtype. 55 The 7 containing receptors are ex pressed at high level in the cortex, hippocampus, media habenula and subcortical limbic regions of the brain, and at low levels in the thalamic regions and basal ganglia. The 7 subunit is also reported to be able to assemble in heteromeric form together w ith 2 or 3 subunits, but rare ly has evidence for such assembly been obtained in neuron s 56 except one report reporting the 7 2 assembly in rodent, basal forebrain cholinergic neurons 57 Although 2 10 and 2 4 subunits are mainly found in neuronal cells, they are also present in non neuronal tissues. 7 is highly expressed in mammalian muscle during development and the perinatal period, and decreases later on in adult life. 58, 59 Both B lymphocyte and T lymphocyte cells contain receptors responsive to nicotine. Circulating and thymic T lymphocytes express the 3, 4, 7, 2 and 4 receptor subunits, 19, 44 while either 4 or 7 subunits are present on B cells. 45 Alpha7 nAChRs are also found in human macrophages, which are required for

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37 acetylcholine inhibition of macrophage tumor necrosis factor release in anti inflammatory processes. 60 Antibody binding, Ca 2+ influx and RT PCR experiments indicate the presence of 3, 7, 9, 2 and 4 subunits in human epidermal keratinoc ytes. 61, 62 Nicotinic receptors are also found in lung cells, containing 3, 5, 7 subunits (in bronchial epithelial cells) and 4 subunits (in alveolar epithelial cells). 63 The nAChR are also found in vascular cells, for example in brain endothelial cells, which nicotine alters the permeability of the blood brain barrier through mediating the expression of 7 and 2 subunits. 64 Neuron neuron interactions are also mediated by nAChRs in astrocyte cells. 7 subunits have been found to be important in hippocampal astrocytes together with 3, 4, 3 and 4 subunits, 19, 65 spinal cord astrocytes mainl y contain 3, 5 and 2 subunits, 66 and 4 2 and 3 4 receptors exist in glial cel ls. 65 1.3.2 Role of Different Neuronal nAChR Subtypes in Human D iseases Acetylcholine appear s very early in evolution, even before nervous cells. 56 In non neuronal tissues, n icoti nic acetylc holine receptors are involved in cell to cell communications and control important cell functions such as proliferation, adhesion, migration, secretion, survival and apoptosis. 56 For example, 7 nicotinic receptors are suggested to be influential in inflammatory disorders and lung cancer. 67, 68 Due to the variety of nAChRs subtypes and the circuits they may control, the biological effects of nAChRs may sometimes be opposite. For example, in brain development and aging, nicotinic receptors are involve d in ne rvous system devel opment and neuronal cell death. In the early development of the nervous system, nicotinic receptors may regulate the proliferation and/or survival of neuroblasts 69 newborn neurons 70 and the spinal cord motoneurons from naturally occurring cell death 71 Conversely, excessive

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38 stimulation of nicotinic receptors enhances neuronal cell death as illustrated by the knockin mice expressing mutant forms of 4 or 7 subunits. 72, 73 Nicotinic receptor s are also important in neuroprotection 74 and in brain functions related to consciousness 75 such as reward process es addiction, the central processing of pain signals, working memory, selective attention, anxiety, sleep and wakefulness. Therefore, a lternation or deficits of the nicotinic receptors have been implicated in a wide range of neuronal dysfunctions and mental illness es including but not limited to 76 Parkinson isease, 77 nicotine addiction, 78 pain, 70 anx iety, 79 epilepsy 80 and schizophrenia 81 (Figure 1 7) 54 The 7 nAChRs are suggested to be influential in cognitio n impairment, anxiety and epilepsy, which are discussed in more detail below. 1.3.2.1 Nicotinic receptor s and cognition Cognitive impairment involves problems with thought processes including loss of higher reasoning, forgetfulness, learning disabilities, concentration difficulties, decreased intelligence, and other reductions in mental functions. Cognitive impairment deficit hyperactivity disorder, etc. Nicotine and its agonists selectively acting on nicotinic receptors can improve working memory and attention in both experimental animals and humans. A wide range of vertebrates, including mice, rats, zebrafish, rabbits, and monkeys, have shown improvement in various memory and learning tasks, as well as in attention experiments, when exposed to nicotine or its agonists. 77 Human studies have been carried out both

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39 on smokers and nonsmokers, both of which benefit from nicotine administration on working memory and intensity of at tention. 82, 83 However, the effects of nicotine and nicotinic agonists on aged impairment effect. This may be due to the complex change in aged rats, including decrease in nicotinic and dopaminergic receptor numbers. 84, 85 As nicotinic agents can improve cognitive impairment, they may als o benefit human patients with a wide range of cognitive disorders. Interestingly, epidemiological studies have suggested that smoking may protect against the evolution of neurodegenerative diseases such as s 86 However, human cognitive disorders are complicated, because they may involve nicotinic receptors directly or through interaction with other neurotransmitter receptor systems. of cognitive function, with onset typically after age 60. A lthough a direct relationship has not been established between nicotinic receptors and AD a selective decrease in the number of telencephalic nicotinic receptors has been observed, and the severit y of associated cognitive deficits has been correlated to the loss of cholinergic function. 17 N europrotection effects of nicotine during a ging are now well established and indicate a relationship between the loss of nicotinic receptor and AD Therefore, the cholinergic hypothesis is one of the proposed disparate mechanisms for AD, together with the amyloid deposition hypothesis, the tau p rotein deposition hypothesis and the ApoE4 abnormality hypothesis 87 89 Various types of nicotinic receptors are suggested to be involved in the cholinergic hypothesis for AD (Figure 1 7). The 7 receptor is co

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40 loca lized with and has high affinity for the amyloid peptide. Therefore, it has been suggested that nicotinic agents that block the binding of amyloid to the receptors may prevent downstream apoptotic events. 76 Knocking out 2 from aged mice (2 years old) will also result i n impairments in spatial memory; for example, impaired performance in the Morris water maze learning test 90 and a fear conditioning task 91 Disease (PD), which is considered primarily as a movement disorder and typically has its onset after ag e 55. Although tobacco smoking provides protection against onset of PD, a direct relationship between nicotinic receptors and PD has not been established, although an accepted role of nicotinic receptors is to stimulate the dopaminergic system and/or other neuroprotective pathways. 77 Immunoprecipitation experiments performed on mouse striata l extracts identified heteromeric nAChR subunits in dopaminergic neuron terminal fields, including 4, 6, 2 and 3 (Figure 1 7). Schizophrenia is another kind of mental disorder associated with the nicotinic receptor. Individuals with schizophrenia have a number of elementary psychophysiological abnormalities in filtering sensory stimuli, which manifest as auditory hallucinations, paranoid delusions or disorganized speech and thinking. The onset of symptoms typically occurs in young adulthood. Sensory gat ing has long been known to be deficient in schizophrenia and nicotinic signaling has been shown to be involved in schizophrenia related inhibitory defects on sensory stimuli. 81 Nicotinic receptor deficits have been found mainly in 7 but also in 4 2 in schizophrenic patients (Figure 1 7). 92, 93 A genetic locus on chromosome 15q14 of the 7 nicotinic receptor subunit gene has been associated with the inheritance of a pathophysiogical defect in some

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41 families of schizophrenic individuals. 81, 92 The 7 selective agonists GTS 21 and PNU 282987 were shown to be capable of re storing the amphetamine induced sensory gating deficit in anesthetized rats. 94 In conclusion, although the roles of nicotinic receptors in these mental disorders and diseases are still not completely elucidated, gi ven their effects on cognitive enhancement, nicotinic receptors are important drug targets for these diseases. Nicotine, and numerous nicotinic agonists and antagonists have been applied in clinical trials. 1.3.2.2 Nicotinic receptors and a nxiety Anxiety is considered to be a normal reaction to psychological stress, which can create feelings of fear, worry, uneasiness and dread. When these feelings become excessive, the condition is classified as an anxiety disorder. Although it is still not clear how the cholinergic pathway affects anxiety, various drugs targeting nicotinic receptors have been applied in anxiety treatment. 79 Nicotine has been shown to act as an anxiolytic in three tests of anxiety: the light dark box (reducing responsiveness to the aversive properties of the white area), 95 the elevated plus maze (increasing the time that mice spend in the open arms and the total number of arm entries), 96 a nd the mirrored chamber (reducing the latency to enter the mirrored chamber) 97 M ice having a hypersensitized mut ant form of the nAChR ( 4L9 S ) and mice lacking the 4 subunit have been reported to be more anxious than wild type mice. 98, 99 However, 2 knockout mice show no difference in anxiety and it is still unclear whethe r 2 is present in these receptors. 100 The effect of the 7 subunit on anxiety also remains ambiguous. Whereas mice lacking the 7 subunit appear to have a decreased anxiety level s in the open field

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42 test, they did not show any difference from the wild type mice in the light dark test of anxiety related behavior. 101 1.3.2.3 Nicotinic receptor s and epilepsy Epilepsies are characterized by recurrent seizures manifested from transient abnormal neuronal activity. These seizures last f rom seconds to minutes occur repetitively or in isolation, and can be either focal or spread across the entire brain causing motor, sensory, or cognitive disturbances. Mutation in the transmembrane domain of the 4 subunit (S248F) is associated with auto somal dominant nocturnal frontal lobe epilepsy (ADNFLE) which was the first discovery of a human disease associated with a neuronal nAChR. 102 Insertion of an extra alanine codon into the C terminal end of the secon d transmembrane helix of the human 4 subunit was later found in an Australian family, and mutation of a valine residue to methionine (V287M) in the 2 subunit was also demonstrated in a Scottish family with ADNFLE. 1 03, 104 Both mutations appear to result in reduced permeability to Ca 2+ and enhanced desensitization. 105 Although each mutant displays slightly different characteristics in the electrophysiology test of the Xenopus oocyte expression system, they all have increased sensitivity to ACh. 106, 107 Besides the 4 and 2 subunits which are most populated in the brain, 5 and 7 also affect seizures in various ways. Mice lacking 5, particularly when doubly knocked out with 2, are more resistant to nicotine induced seizures. 108 Although there is no direct evidence of 7 selective agonist, JN403, shows anticonvulsant poten tial in the audiogenic seizure paradigm in DBA/2 mice. 109 Moreover, the homomeric 7 nicotinic receptor is widely

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43 found in the hippocampus and frontal co rtex, which are identified as major susceptibility loci for ju venile myoclonic epilepsy. 1.4 Different nAChR Ligands Numerous nicotine or acetylcholine analogues, which mimic the structures of nicotine and/or acetylcholine, have been identified or synthesized. 110, 111 However not all of these analogues behave like nicotine and acetylcholine in activating the receptor. Moreover, there are also emerging classes of new molecules with structures that are less similar or completely different compared to nicotine and acetylcholine. Based on groups: (1) agonists, which activate the receptor in the same manner as nicotine and of the receptor. Efficacy and potency are the two parameters for evaluating agonism. Efficacy is the maximum response that a ligand can achieve relative to the reference neurotransmitter, typically ACh in the case of the nAChR. Potency is generally repres ented by EC 50 which is the agonist concentration capable of producing half of the maximum response. In contrast, IC 50 50 and its potentiating magnitude are generally investigated. In recent years, allosteric modulators have opened a new field of drug development for the nicotinic receptor. The concept of receptor allostery was developed from enzymatic allosterism, which ref ers to conformational and activity changes of an enzyme that result from molecular binding of a regulatory substance at a site other than the site for substrates or cofactors. 112 Therefore, non competitive antagonists can also be called allosteric modulators, and they have a negative effect on channel activation.

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44 On the other hand, positive allosteric modulators (PAMs) are molecules that bind at a site other t han the agonist binding site, do not activate the receptor by themselves, but result in an enhancement of channel activation by an agonist. Such enhancement on channel activation is also called potentiation. 1.4.1 Nicotinic Agonists Nicotine is an alkaloi d isolated from the tobacco plant and similarly, many of the acetylcholine receptor agonists are either directly isolated from nature or derived from natural products, as shown in Figure 1 8. There is chiral selectivity for the nicotine antipodes, as the n atural product S enantiomer shows greater binding affinity compared to the synthetic R enantiomer. 113, 114 Cytisine, like nicotine, is a very toxic plant alkaloid from laburnum anagyroides and is useful as a highly selective 4 2 radioligand and agonist. 115 Anatoxin is also a very toxic alkaloid, which is isolated from a fresh water algae. 116 The (+) enantiomer of anatoxin has very high, selective affinity for 4 2 receptors, while the ( ) enantiomer is virtually inactive. 117, 118 However, the discovery of epibatidine from an E cuadoran frog, 119 whose two enantiomers have similar affinities for nicotinic receptors and similar functional activity, 120, 121 remarkably impac ted research on nicotinic receptors and development of new synthetics. The natural resources of anabaseine are varied, including plants, ants, and marine nemertine worms. 122, 123 These natural products and synthetic derivatives branch out from their parent molecules with different structural variations, while maintaining the nicotinic agonist property. Some of them only differ from their parents in the side groups, which is the case for the non selective agonist carbamylcholine and the 4 2 selective agonist 6 chloronicotine. 124 Some mimic the shape of the parent natural product, like the cytisine

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45 analog CP 526555 (verenicline), which was developed by Pfizer and approved by the FDA as an aid for smoking cessation. Some synthetic nicotinic agonists are de rived from more than one natural product; e.g., the 7 selective agonist tropisetron 125 which shares the carbonyl group feature with anatoxin, the aromatic ring motif with epibatidine and the bicyclic ring found in both natural products. Some elaborate the natural product with an additional functional group, like the 7 selective agonist GTS 21 which derives from anabaseine. 126 Figure 1 8. Natural and synthetic agonists for acetylcholine receptors; adapted from th e article written by Daly, J. W. 111 All of the compounds show n in Figure 1 8 have at least one hydrogen bond acceptor and a positively charged nitrogen, which for a long time were thought to be the minimum pharmacophore for activating the nicotinic receptor. 127 Howeve r, in the 1990s, tetramethylammonium (TMA) was found to be a full agonist for some neuronal types of nicotinic receptors, including 4 2, 3 4 and 7. 22 TMA was also able to activate the muscle type nicotinic recept or, though, the TMA induced maximum response is less than 40% of that induced by acetylcholine. Therefore, a quaternary ammonium is the

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46 minimum pharmacophore to activate the nicotinic acetylcholine receptor. Protonated tertiary amines and secondary amines can also activate the nicotinic receptor in combination with other structural features, as is the case for nicotine and cytisine. However, the selectivity rules for heteromeric and homomeric nicotinic receptors have not been strictly defined. Even natural nicotinic agonists may differ from their own synthetic derivatives in subtype selectivity. There are two different approaches to identify the selectivity of a nAChR agonist and the results are not always consistent: binding studies, and functional studies such as that obtained from two electrode voltage clamp experiments. This section will focus mainly on selectivity based on functional assays. Heteromeric selective agonists have been extensively developed and discussed in several papers. 54, 111, 128, 129 Efforts were also made to identify an 7 subtype selectivity pharmacophore. 130, 131 Figure 1 9 shows examples of acetylcholine receptor agents, with the agonists selective fo r 7 highlighted. 131 There are three structur ally distinct main groups of molecules selective for 7. The first group contains a choline motif, defined as a quaternary ammonium group proximate to a hydrogen bond acceptor. Choline is the metabolite of acetylcholine and is present at high concentrations at neuronal junctions. It can both bind to the 7 and 4 2 receptors, 111 while selectively activating 7. 22 This choline motif can also be applied to cyclic amines to create 7 selectivity, e.g., N methyl pyrrolidine choline. 132 The second 7 selective motif is the tropane motif, whose selectivity may be due to a small hydrophobic methyl group affixed to a bridgehead nitrogen in a bicyclic system. 131 For example, quinuclidine, a non selective agonist, can become 7 selective if a methyl group is added to the nitrogen. 133 The third 7 selective mo tif is the benzylidene motif. Two

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47 Figure 1 9 : Multiple classes of nicotinic agonists. Circled compounds are 7 selective. Compounds in filled circles have no significant activity for 7, 4 2 or 3 4 receptors. 131

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48 compounds were initially identified as 7 selective i n the anabaseine family, benzylidene anabaseine and cinnnamylidene anabaseine, both of which have an extended aromatic rings conjugated with the non selective anabaseine parent motif. Moreover, such 7 selectivity raised by the extended aromatic ring can a lso be applied to quinuclidines (Figure 1 9), suggesting that the benzylidene motif involves proper placement of an aromatic ring relative to the minimum charge pharmacophore. 131 Aside from these three motifs, other molecules may also be 7 selective agonists. There are several 7 selective agonists in the quinuclid i ne family, and their selectivity is suggested to be based on various combinations of aromatic rings as well as H bonding acceptors and/or H bonding donors. 130 Some nicotinic like compounds and piperidines are also 7 selective, but the pharmacophore for thei r selectivity has not yet been fully delineated 1.4.2 Nicotinic A ntagonists Nicotinic antagonists can inhibit receptor activation via two different mechanisms. The first one occurs in a competitive manner, in which antagonists bind at the same place in th e receptor as agonists, preventing the agonist from activating the receptor. The second mechanism occurs in a non competitive manner, in which an antagonist and an agonist are bound to the receptor at different locations Increasing the concentration of ag onist can exclude the competitive antagonist out of the receptor while it cannot impact the binding of the non compe ti tive antagonist. Therefore, a potency without changing the efficacy, while non competitive agonists will be able to

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49 Certain peptides, like bungarotoxin and conotoxins, can bind and antagonize different nAChR subtypes with high selectivity. 54 Alpha bungarotoxin from an elapid snake, Bungarus multicinctus selectively tags the 7 homopentamer in the brain 134 A t oxin from the marine snails of th e genus Conus conotoxin MIII selectively tags 6 containing receptors 135 Some small natural or synthetic antagonists also have subtype selectivity (Figure 1 10). Methyllycaconitine is both potent and highly sel ective as an 7 competitive antagonist. 118, 136 The competitive antagonist DH E has selectivity for 4 containing nAChR with IC 50 values lower than 0.4 M, but it also has an IC 50 of approximately 8 M with the 7 s ubtype. 137 The noncompetitive antagonist mecamylamine is suggested to be more potent with 3 4 receptors than with 3 2 receptors. 138 Figure 1 10. Structures of nAChR antagonists 1.4.3 Posit ive Allosteric M odulators Although GABA A receptor positive allosteric modulators (PAMs) have been known and used clinically for decades, nAChR PAMs have only been identified in recent years. 139 Both heterome ric and homomeric nAChR PAMs have large structural variety, since they can be as small as divalent cations, or as large as proteins. 139, 140 Many nAChR PAMs are natural products and synthetic molecules (Figure 1 11) Although

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50 there are some exceptions (e.g. galantamine), nAChR PAMs are mostly electrically neutral at physiological pH. These PAMs generally do not activate nicotinic receptors unless they are in the presence of the agonists. However, a PAM derivative was recently identified which can activate the channel when applied alone and also 141 Figure 1 11. Positive allosteric modulators adapted from the arti cle written by Williams, D. K et al (reprinted with permission from biochemical pharmacology). 139 All PAMs potentiate the receptor via increasing its sensitivity to agonists, current magnitudes, and empirica l Hill coefficients. Gronlien et al. proposed that positive allosteric modulators (PAMs) for 7 can be divided into two classes, type I and type II, based on the functional properties of the modulator. 142 The type I PAMs potentiate the receptor with little or no effect on the basic onset and decay kinetics, i.e. shape of the response, while the type II PAMs markedly slow response decay kinetics and can even

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51 activate receptors that have been desensitized by applicatio n o f high agonist concentrations. For 7 nAChRs, positive allosteric modulators (PAMs) are of great interest because they not only open a new field for drug development, but also provide a new tool to study the receptors, which have a low open probability and become non conductive after long term binding with an agonist. Figure 1 11 shows several small organic molecules belonging to the different types of 7 PAMs. Among them, although 1 (5 chloro 2,4 dimethoxyl phenyl) 3 (5 methyl isoxazol 3 yl) urea (PNU 120596) is very potent and selective for the 7 subtype, 143 other 7 PAMs are either not strictly selective for 7, or not potent, or exhibit inhibition effects at a certain concentration. 139 The pharmacophore and potentiation mechanism for different potentiating effects of PAMs is still unclear. Two asymmetric ureas, NS 1738 and PNU 120596 display different potentiation profile. Studies on the 7 chimeric receptors fu sed with the 5 HT 3 receptor suggested different potentiation mecha nisms and/or different binding sites between NS 1738 and PNU 12596. 144 On the other hand, a type I PAM, 5 hydroxylindole has a competitive effect on PNU suggesting the type I PAM and the type II PAM may bind at the same place in the receptor and/or potentiate the rece pathway. 145 1.5 The Structure of Nicotinic Receptor 1.5.1 Ov erview of the Structure Although various structures have emerged for the nicotinic acetylcholine receptor via Xray crystallography, homology modeling and electron microscope imaging, at present

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52 there is still no high resolution structure for the entire mem brane protein. Early stage structural studies identified residues and subunits involved in ligand binding, channel gating, and ion selectivity through mutagenesis, radioactive labeling, cysteine accessibility mutagenesis and other techniques. 54, 147, 148 Such approaches are critically important tools for interpreting structures and functional relationships for nicotinic receptors to aid the interpretation of 3D structures or computational homology models of nicotinic receptors. Results of chemical and molecular biological studies, together with structural biology investigations, all agree that nicotinic acetylcholine receptors are pentameric proteins, with an N terminal extracellular domain (ECD) for binding ligands, a transmembrane (TM) domain for channel gating, and an intracellular domain (ICD) with undefined roles, as shown in Figure 1 12. Although some structural information has been revealed for the extracellular and transmembrane domains of nAChRs, the structure of the intracellular domain is still unclear, because it holds little if any homology to structurally known proteins and has been suggested to have intrinsically disordered regions. 146 1.5.1.1 Extracellular domain As shown in Figure 1 blades, as described by Karlin. 147 The extracellular domain of each subunit starts at the amino termini with a three turn helix, and thereafter forms a bundle of ten strands ( 1 10) with several connecting loops (Figure 1 12 C). The signature Cys loop is extracellular and the tr ansmembrane domains. Affinity labeling also identified another disulfide bond on the C loop in all 150

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53 Figure 1 12. Ribbon diagrams of the muscle type nAChR resolved at 4 146 A. top view of the receptor from the synaptic cleft. B. receptor viewed parallel with the Only one ligand binding domain is highlighted ( red; green; blue; light blue) C. a single subunit ( ) viewed parallel with the membrane plane. The strands composing the sandwich are in blue for the inner strands and red for the outer stands.

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54 Therefore, this region of the extracellular domain of the nicotinic receptor is also ca lled the ligand binding domain (LBD). The C loop, together with two other loops (loop A and B) and three outer strands ( 7 and 9 10), constitute the primary ligand binding face (also called the plus face). Affinity label incorporation has also been note d in the non subunits, 151, 152 which contribute to the complementary ligand binding face (i.e., minus face) including loops D F and seven other strands ( 1 6 and 8). Both agonists and competitive antagonists b ind underneath the C loop as shown in Figure 1 12 A and B. The common feature between the agonists and the competitive antagonists (Figure 1 8 to Figure 1 10) is the charged nitrogen. In the nicotinic receptor, the positively charged ammonium or immonium g roup of an agonist binds in a cage of five aromatic sides consisting of Tyr93, Trp149, Tyr188 and Tyr198 from the plus face, and Trp55 from the minus face. This cage model is supported by affinity labeling on these aromatic amino acids, 151 153 by changes in agonist binding or gating via mutating these sites, 147 or by activation by tethered agonists (i.e. quaternary ammonium moieties attached) at these positions. 154, 155 The cage model has been identifi ed in the homologous snail acetylcholine binding protein crystal structures in complexes with various agonists. Ab initio quantum mechanics suggests a cation interaction between these aromatic residues and the ligand. 156 Moreover, when substituting these tryptophans or tyrosines with fluorinated phenyl groups to decrease their electron densities, a clear correlation has been identified between agonist potenc y and the extent of aromatic ring fluorination. 157

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55 1.5.1.2 Transmembrane domain The transmembrane domain contains four helix segments (M1 4). M1 connects with the 10 strand at the extracellular domain and spans across the transmembrane domain followed by the other three segments (Figure 1 12 C). M3 connects M2 via a small loop which is close to the ligand binding domain, and M3 joins with M4 through the large structurally undefined M3 M4 cytoplasmic loop (i.e., the intracellular domain). Affinity labeling and substituted cysteine accessibility mutagenesis (SCAM) suggest that the channel pore is lined with M2 segments from the five subunits. 158, 159 In SCAM, residues of int erest are mutated to cysteine and tested for reactivity towards small, charged sulfhydryl specific methanethiosulfonate reagents to examine whether they are exposed to water. Residues reacted with MTSEA over the entire length of M2 and an helix topology of M2 was suggested by the fact that every 3 or 4 residues are exposed to water. It has also been suggested that charge selectivity moieties and channel gates locate in the transmembrane domain. However, they are neither regulated by a single residue nor by a cluster of adjacent residues. 54, 147 A minimum of three mutations in the M1 M2 loop and in M2 is required to change the charge selectivity of nAChRs from cationic to anionic. 160 The channel gate for the nicotinic receptor both sterically and s non conductive state. However, the location of the channel gate, revealed by 3D structure analysis, as well as SCAM and mutagenesis functional studies, is still controversial. 54, 147, 161 These complex results may be due to the existence of multiple gates for the receptor in the resting state and the desensitized state, respectively.

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56 1.5.2 Three Dimensional Structures of Nicotinic Receptors The development of various three dimensional structures for nAChRs has gre atly helped the study of function and structure of the receptor. There are four important groups of 3D structures for nAChRs. The first one is a group of acetylcholine binding protein (AChBP) structures in apo form or in complex with various ligands. AChBP is the homologue of the extracellular part of the nicotinic receptors, to which acetylcholine also binds. 162 AChBP can be crystallized with nAChR agonists, antagonists, or even a positive allosteric modulator (gala ntamine). 163 168 These AChBPs were isolated from two molluscan, aplysia californica (ac) and lymnaea stagnalis (ls) which contain 210 amino acids and have overall homology of 20% to 24% with the extracellular domai n of nAChRs. In the AChBP structures, each subunit contains ten strands assembled as a barrel and several loop structures constituting the ligand binding site (Figure 1 12). Ligands bind at the interface of two subunits in AChBP crystals and the residue s identified in the ligand binding sites are consistent with the photoaffinity labeling and mutagenesis results obtained from nAChRs. 163 These structures also suggest that the distortion of the C loop structure is r elated to the different modes of action of an agonist and antagonist on the receptor. 164 However, the structures of AChBP lack the transmembrane domain of nicotinic receptors, and therefore give little, if any, information about the receptor function. However, with the advantage of predicting ligand binding and the large library of structures with different ligands bound revealed in the last ten years, AChBP structures still remain as the main templates for model development used in studying ligand and receptor interaction.

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57 The only structure of the w hole, pentameric nicotinic receptor was obtained via electron microscopy (Figure 1 12) 146 Postsynaptic membranes from the elec tric organ of the Torpedo ray have been the source of material for the structural studies. The isolated membranes convert readily into long tubular crystals, where nAChR can be imaged in its native lipid surroundings and ionic environment, thus avoiding pa rtial denaturation or refolding, as is likely to occur in the presence of detergent and/or exposure to unnatural salts. 169 One advantage of electron microscopy is that it can also examine a physiologically transient process with the rapid freezing technique. This revealed that activating the receptor would result in a widening of the channel pore by about 3 170 With more repli cates of the electromicroscopic scanning and help of the homology modeling, the resolution of the nAChR structure has been improved to 4 146 169 In this 4 structure the to tal length of the protein is about 160 normal to the membrane plane. The ligand binding domain su rrounds a long, ~ 20 diameter central vestibule and has two binding sites for ACh at the interface of the two and non binding domain resulting in the stru c ture for t he extracellular domain, as shown in Figure 1 12. Com paring the structure of the nAChR subunit (without ligand) with the corresponding region of AChBP (bound with carbamylcholine), Unwin also suggested that the C loop tends to close towards the channel p ore after being bound with agonist. A 15 16 twist of the 1 2 loop after binding with an agonist from the LBD may initiate the channel gating in the transmembrane domain. 171, 172 In the torpedo structure, e ach subunit has four helixes spanning across the membrane while the second helix (M2, 40 long) is oriented towards the channel pore to regulate the

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58 channel gate and ion selectivity. 172 Only a curved helix was identified in each receptor subunit f or the small intracellular domain (the stretch sequence between M3 and M4) in M4 appeared to be disordered and remained recepto r structure, the side chain orientation is still quite ambiguous in this low resolution structure. In 2007, a partial nicotinic receptor structure was resolved at 1.94 by Chen and his co workers 173 With three mu tations made in the receptor, they were able to crystallize the mouse bungarotoxin in a monomeric form. This crystal has an overall structure similar to those of AChBP and Torpedo nAChR. In their study, an oligosaccharide was also identified connected to the receptor through asparagine 141 and serine 143, which is suggested to facilitate the folding and trafficking of the receptor. 173, 174 Two highly conserved res idues, one from 2 and one from 6, were identified forming a hydration pocket inside the sandwich core to regulate ligand activation on the receptor. This hydrophilic center is not present in AChBP, suggesting that this 1 LBD crystal structure may be a better template for studying the intrasubunit interactions of nAChR. However, this partial 1 structure is not a good template for the ligand binding site because as a monomer, it lacks the information on the complementary binding face and carries a mutat ion at 149, which is known to be critical for agonist binding and activation of the receptor. 175, 176 Recently, two prokaryotic pentameric receptors, which are homologues of the nicotinic acetylcholine receptor, ha ve been identified and crystallized. 177 180 One of them, which is gated by protons, was cloned from Gloeobacter violaceus bacteria and is

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59 abbreviated as GLIC. GLIC shares 20% amino acid identity with the human 7 ni cotinic acetylcholine and does not desensitize even at the pH eliciting maximum electrophysiological response. 177 The other prokaryotic ion channels were cloned from Erwinia chrysanthemi bacteria (abbreviated as ELI C) and they share 16% sequence identity with the nAChR 1 subunit. 179 The pore diameter of ELIC was determined to be 7 which was suggested to mimic a closed f orm of the channel. 179 Like nAChRs, GLIC and ELIC are both selective for cations, and contain the all sheet hydrophilic domain, four transmembrane segments and the signature Cys loop. The lack of the intracellular domain in these receptors indicates that the extracellular domain and transmembrane domain are adequate for channel function. Structural comparison of GLIC and ELIC suggests a gating mechanism resultin g from a quaternary twist from the sandwich and tertiary deformations of 1 2 loop, 6 7 loop (cys loop), M2 M3 loop, M2 and M3 helices. 178, 180 The recent crystal structures of GLIC complexed with two anestheti cs (targeting Cys loop receptors) are also informative concerning the allosteric potentiating site for GABA and nAChR in the transmembrane domain. 181 Various nAChR PAMs have been suggested to bind inside the four helix cavity of one subunit or at the GLIC anethetics crystal structure described above. 182 184 However, these prokaryotic ion ch annels showed weak conservation with the amino acids in nAChRs that contribute to neurotransmitter binding. They lack the C loop disulfide bond which is critical for channel function and conserved in the nAChR subunits. 150 No ligands have been identified in these prokaryotic ion channel structures. Therefore, GLIC and ELIC are

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60 less informative about agonist bindin g, and their use in interpreting agonist induced gating mechanisms will need further development. 1.6 Nicotinic Receptor Drug Development 1.6.1 Different Drug Development Approaches Nicotine was isolated in 1828 and its structure was reported in 1893, even before the concept of nAChRs was established. 111 The nAChR n on competitive antagonist, mecamylamine (Inversine ) was marketed in the 1950s for treatment of hypertension. Varenicillin (Chantix and Champix ) is another example of a nicotinic drug on the market targeting the nicotinic receptor for aiding smoking cess ation. At present, additional nicotinic receptor ligands are being tested in different phases of various clinical trials. 185 Two main approaches to nAChR drug discovery will be discussed. 186, 187 The first one, generally referred to as a structure activity relationship (SAR) study, is based on the structure of the known active ligands and their functional information to predict new ligands as potential drugs. 188 Those structural motifs identified as responsible for function are normally called pharmacophores. The advantage of this method is that there is no need for the protein structure. SAR studies on acetylcholine and nicotine have been done to exp lore the molecular recognition of the nicotinic receptor long before anything was known about the atomic level structure of nAChR. 127 Recently, the quantitative SAR (QSAR) model was developed for some struct urally similar nicotinic receptor ligands, and the results have helped to identify new drug candidates. 189, 190 Another drug development approach is based on the structure of the protein target. 191 This approach emerged along with in silico drug design applications. The

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61 advanta ge of this method is its ability to take any ligand and predict the ligand receptor Although in principle the first requirement for predicting whether a ligand wi ll bind to a protein is complementarity of the sized and the shape of ligand and binding pocket. However, other factors must be taken into account. Several scoring programs have been developed for evaluating the docking results, all of which mainly conside r van der Waals contacts and electrostatic effects. Some of these scoring programs also take into account H bonding, desolvation energy, hydrophobicity, etc. 192 However, this protein based approach requires the structure of the target protein, and its success is greatly limited by the accuracy of the protein structural model. Various homology models of 1 LBD m onomeric structure as templates. 182, 193, 194 However, the templates have low homology, and without any high resolution structure of the whole nicotinic receptor, homology modeling is less successful for predicting channel function. For example, although computational modeling predicted different electron density profiles for Trp149 in the 4 2 receptor and the 7 receptors, 195 a mutagenesis study indicated this residue is c 176 However, this approach has been applied successfully in explaining ligand binding and is generally used as a gui de for designing experiments. 185 196, 197 1.6.2 Challenges in Nicotinic Drug Developments Study o f Different Functional States Although the ligand and protein approaches have greatly enriched the potential drug library for nicotinic therapeutics, most of the drug candidates identified have failed during, or even before, clinical trials due to variou s side effects. The diversity of the

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62 receptor subtypes, lack of knowledge of the receptor structure and its relationship with channel functions, and limitations of computational modeling are some of the challenges for nicotinic receptor drug discovery at p resent. As mentioned above, various efforts have been made to develop subtype selective therapeutics and acquire high resolution nAChR structures. But the knowledge is very incomplete on how agonists bind to nAChRs and initiate the channel gating or, alter natively, channel desensitization. One question at the core of therapeutic drug development is how a drug will affect the endogenous signaling mediated by the natural activator. The nave assumption is that an agonist will replace or augment the stimulatio n provided by the natural activator. However, for most nAChR subtypes, a primary effect of the prolonged presence of an agonist is to produce desensitization, decreasing the effect of the normal fluctuations in ACh signaling. In this regard, the effect of an agonist may be similar to that of an antagonist, as is the case with two drugs commonly used to paralyze the neuromuscular junction, succinylcholine (a potent neuromuscular receptor agonist), and tubocurarine (a competitive antagonist). Therefore, in th e drug development, studying receptor desensitization is as important as studying receptor activation. 198 The properties of nAChR activation and desensitization depend on the subunit composition, the ligand occupancy and the structure of the bo und ligand. 139, 145, 199 The heteromeric nicotinic receptors, such as the 4 2 receptors, have two distinct open states (O states): one has short lived openings (O*) that occur in isolation, and the other has longer 139 On the other hand, the homomeric 7 receptor has two distinct desensitized states (D states). One is a unique concentration dependent fast desensitization (D s ), which is reversible to the open state

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63 in the presence of the type II positive allosteric modulator, PNU 120596. The other is an intrinsic desensitization state (D i ) which is insensitive to the presence of PNU 120596 and is preferred in high agonist and /or PNU 120596 occupancy. 139, 145 Figure 1 13. The hypothetical energy landscape for 7 nAChR state transitions when bound with the full agonist acetylcholine; adapted from the article written by Williams, D. K e t al (reprinted with permission from biochemical pharmacology). 139 The basic MWC model of the 7 nAChR (Figure 1 5) can be expanded and presented in a schematic energy landscape as shown in Figure 1 13. The revised model assumes that the agonist binding steps proceed from a rapid change in agonist concentration and all receptors are initially in the resting closed state (C state). The key features of 7 activation and desensitization are consistent with the m odel. The P open of 7 receptors is very low under all conditions, and open times are very brief, usually less than 100 s. These features are associated with the steep barrier for entry into the open state, which is located in a very shallow trough. In thi s model, the D s state (rapid desensitization) is more stable at high levels of agonist occupancy. The true intrinsic desensitized state (D i ) of 7 receptors is less stable than that of heteromeric nAChR, and so the absolute free energy difference between t he D i and C states at high levels of

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64 agonist occupancy is less for 7 than for the heteromeric nAChR model, as discussed in our previous study. 139 Moreover, 7 receptors can be activated by structurally dive rse agonists (Figure 1 9) and are highly likely to enter and remain in desensitized states at rates determine by the structures of the agonists. 131, 199 Using different benzylidene anabaseine (BA) analogues, a gener al trend for channel activation indicated that polar substitutents on the phenyl ring of the benzylidene moiety will make an agonist more potent and efficacious. 199 Another feature of 7 receptors is that they do no t convert to a state with high affinity for ACh, as appears to be the case for heteromeric receptors. Once the ACh has been removed or metabolized, a heteromeric receptor rapidly resensitizes and can be activated again by ACh. However, 7 receptors do not readily return to a functional state after the application of nicotine or the 7 selective agonist 2,4 dimethoxybenzylidene anabaseine (GTS 21). We refer to such failure of 7 receptors to fully recover after an agonist application as the manifestation of inhibition effects via blocking the receptor are transient and their RID effects are mainly due to the residual desensitization, which is rever sible in the presence of PNU 120596. Interestingly, diOH GTS 21, a GTS 21 metabolite, is RID negative. This clearly provides a link between how subtle modifications of the structure of the agonist can modulate RID. Understanding channel desensitization, a s well as the channel activation, is important for drug design. Although desensitization appears to be non conductive, it is still functionally important. It has been shown that some forms of 7 mediated

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65 cytoprotection require long periods of treatment wit h concentrations of agonist lower than the threshold concentration required for transient activation of the ion channel. 200 In the same cells, strong transient activation of the 7 receptor is cytotoxic within seconds. 200 Targeting on drugs differently desensitizing the receptor is as importan t as developing drugs to activate the receptor. We traditionally associate the efficacy of specific agents with their relative effectiveness for inducing transitions of the receptors among mutiple conformational states subsequent to binding in a single pr eferred conformation. However, it is unclear how a single ligand binds inside the receptor to initiate channel activation and different desensitized states. Does a ligand adopt different binding modes to activate and desensitize the receptor? How do struct entry into the open state, the D s state and the residual inhibition desensitization (RID)? What are the pharmacophores for these functional modulations? To answer these questions, in this research, several m olecules were designed and synthesized as probes to study the functional states of 7 nicotinic acetylcholine receptors. The data in the following chapters suggest new insights which can be implemented for drug development targeting the 7 nAChRs, and prov ide new methods to study the ligand receptor interaction.

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66 C HAPTER 2 2 USE OF SULFHYDRYL REAGENTS TO TETHER THE RECEPT OR AND STUDY THE FUNCTIONAL STATE S OF THE RECEPTOR 1 2.1 Background As mentioned in chapter 1, 7 nAChRs enter and remain in desensitized s tates in ways that can depend on the structure of the agonist 199 An important goal is to decipher the complex structural interplay between the character and disposition of ligands bound to the receptor and the impa ct of that binding on receptor activation and desensitization. One challenge is to unravel the molecular interactions between bound ligand and protein without a sufficiently high homology protein template for structural modeling. Tethering the agonist to t he receptor is a beneficial strategy because it can limit the number of possible binding modes and the overall freedom of the ligand, hence simplifying the analysis of functional studies. This method derives from the SCAM method, where u nique cysteines are introduced by point mutations at a series of positions, and each mutant is then reacted with a selective sulfhydryl reactive reagent (SH reagents) Functional analyses establish whether the reaction produced a labeled receptor. For the 7 receptor which h as a small P open and a high propensity for desensitization, study of the activation of the receptor by tethered agonist is problematic. Of course this is not the case for heteromeric receptors or the 7 mutants that have higher P open or show less desensiti zation. 201, 202 On the other hand, we have gained new insights into the unique desensitization properties of 7 r eceptors through the use of the Type II PAM PNU 120596 (Figure 1 11), which not only enhances apparen t peak current during agonist application, but can 1 R eprinted with permission of the American Society for Pharmacology and Experimental Therapeutics

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67 transform desensitized states into detectable conductive states. 142, 199 Therefore, this Type II PAM provides a new tool to activate otherwise silent desensitized f orms of the 7 receptor bearing tethering agonists. This chapter describes a systematic study of the geometric and spatial requirements for how bound ligands may activate the 7 receptor, and in some cases promote conversion of the receptor to PNU 120596 sensitive desensitized states (D s ) The experimental data provide a useful approach for integrating nAChR homology modeling, including docking and molecular dynamics simulations, with functional analysis of ligand dependent conformational changes. 2.2 Res ults and Discussion 2.2.1 Experimental Design to Characterize the Effects of 7 Receptor Covalent Modifications by SH Reagents Figure 2 1A illustrates the docking of TMA (tetramethyl ammonium cation) in the ligand binding domain (LBD) of 7 created by Dr. Horenstein. TMA is a full agonist of 7, so this pose represents the hypothetical minimal structure able to induce the array of 7 functional states, including the active ion channel state and ligand bound, non can be converted into a conducting state with the application of the PAM PNU 120596. All mutants used in these studies were conducted in a background of C116S so the newly added cysteine was the sole free cysteine in the ligand binding domain Figure 2 1B shows an example of the predicted placement of the affinity ligand methanethiosulfonate ethyltrimethylammonium (MTSET) bromide covalently bound to the unique cysteine at the L119C mutant. The model suggests that t he agonist analog will be most likely positioned in the same vicinity of the LBD as the docked TMA, but

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68 with important differences. The experimental goal in this chapter is to determine whether MTSET or an alternative agonist analog tethered in such orient ations impact ion channel function, such as the induction and stabilization of functionally relevant nonconducting states. To facilitate interpretation of the experimental data, Figure 2 1 C outlines the basic sequence of experiments with proposed interpre tations of the outcomes. The initial characterization of the cysteine mutants was determined by the action of ACh (experiment 1), which provided a measure of the intrinsic functionality, the expression level of the cysteine point mutant, and a baseline for gauging subsequent treatments of the receptor. Next, the mutants were treated with SH reagents, in experiment 2, which served to determine whether the mutants retained the same pharmacological profile as the control receptors (wild type and C116S 7) with regard to transient activation of the ion channel by the SH reagents. Additional ACh applications were conducted (experiment 3) to make an initial determination of whether a covalent modification of the receptors occurred that resulted in perturbation of function. We anticipated two most likely outcomes: subsequent ACh evoked responses would remain unchanged (case I), or they would be eliminated (or reduced), indicative of functionally significant modification of the receptor (case II). Case I represents t wo possibilities: failure of the SH reagent to react with the receptor (case Ia), or failure of the modification to result in a detectable change in function (case Ib). A third outcome that may also have been anticipated is that receptors would be modified in such a way to have increased sensitivity to ACh (case III). This aspect is still under further investigation and not illustrated in the figure.

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69 Figure 2 1. Experimental design adapted from the article written by Wang, J. et al (reprinted with permis sion of the American Society for Pharmacology and Experimental Therapeutics). 203 A) The docked pose of TMA in the LBD of 7. B) Predicted placement of the affinity MTSET reagent covalently bound to a cysteine (L119C ). C) Schematic of the design and hypotheses related to the possible events in the labeling study with the SH reagents after the initial characterization of functional cysteine mutants

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70 An important caveat for results that appeared intermediate between t hose anticipated for case I and II (partial reduction of receptor response to a post labeling application of ACh) is that there may be significant difference in the reaction rates for various covalent modifications. Therefore, in such cases, kinetic studie s were also conducted. Once receptors were determined to be in a covalently modified state that was unresponsive to further activation by ACh, it was hypothesized that (case IIb) the tethered ligand might be situated as a simple antagonist, either occludin g access of the LBD to agonist, or holding the receptors in a nonactivitable state, alternatively the tethered agonist may function like a receptor/agonist complex resting in a desensitized conformation. If this was the D s state, it could be detected via t he reactivation by the type II PAM PNU 120596. Experiment 4, the application of PNU 120596 was conducted to make this determination. Finally, experiment 5, a follow up application of ACh, probes the PNU 120596 potentiation of this ACh response as affected by the sulfhydryl reacted receptors. 2.2.2 Cysteine M utants Selected for Tethering Studies in the LBD In the human 7 nAChR, a single, nondisulfide bonded free cysteine is found at position 116, which if left intact, could complicate the interpretation of thiol specific labeling studies when cysteine is introduced elsewhere in the receptor. Therefore the 7 C116S mutant was prepared and used as background for all additional cysteine mutants. 204 The mutants used for these studies were selected from a total of 44 7 LBD cysteine mutants reported pre viously. 204 Cysteine mutations on the com plementary face are more functionally competent, while cysteine mutations on the primary face of the LBD are less well tolerated since such mutants are e ither not well expressed or are

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71 poorly responsive to ACh 204 Due to the instrumental detection limit, mutants were considered for further tethe ring agonist studies only if when control applications of ACh gave a response measuring at least 30% of the net charge determined for the C116S receptor at the same time point after mRNA injection into the oocyte (experiment 1 in Figure 2 1C). Figure 2 2 Cysteine mutations in the LBD of 7 nAChR ; adapted from the article written by Wang, J. et al (reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics). 203 A) S ide view of the receptor model as a dimer. The key elements of 7 were modeled using the 2BG9 template for the transmembrane domain fused to the ligand binding domain made from 2PGZ template. The view was generated from the outside of the channel pore, and the four tr ansmem brane helic es (M1 to M4) were lined clockwise as shown, putting the M2 helix toward the channel pore. The intracel lular domain is not shown. B) A close view of the predicted agonist binding site indicating the plus face and minus face of the receptor colored in blue and light sea green, respectively. To select the cysteine mutants used in the labeling study, Br ACh and QN SH were docked to the wild type human 7 nAChR dimer by Yeung Chiu. After docking, different poses within 5 kcal/mol difference fr om the best score were used to predict the

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7 2 potential interacting side chains that could be mu tated to cysteine. This set included five on the minus face (S36, L38, W5 5, L119, and I165. See Figure 2 2 .). All of these mutants were conducted in a background o f C116S. The ACh EC 50 values for wild type (C116C), C116S, S36C, L38C, W55C, L119C, and I165C we re 27 3 131 29.4 0.7 204 20 9, 24 5, 180 20, 30 4 and 60 26, M respectively. 2.2.3 Non selective Sulfhydryl (SH) Reagents and Their Electrophysiological Evaluation Figure 2 3. The structures of the six SH reagents: Br ACh, MTSET, MTSEA, QN SH, EMTS, and MTSACE (reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics). 203 The structure of the six SH reagents used in these ex periments are shown in Figure 2 3. Two of these agents, MTSET and Br ACh, are agonist analogs that have been used previously to characterize muscle and/or T.califonica type nAChR. 205, 206 Because quinuclidine is an agonist for neuronal nAChR, it was hypothesized that if posed in a favorable orientation in the 7 LBD, QN SH might also function as a tethered agonist once covalently bound to the receptor. MTSEA was used as a fourth cationic SH reagent to test for the po tential importance of the positive charge placement in the LBD, independent of the other elements of the presumed pharmacophore. 131 Specifically MTSEA is smaller than MTSET, and lacks the N tetraalkyl functionality. The

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73 noncationic agents MTSACE and EMTS were used as con trols for nonspecific effects of the SH reactions, and could also be considered as non charged steric probes. Figure 2 4. Intrinsic agonist activity of SH reagent agonist analogs for wild type 7 and 7 C116S (reprinted with permission of the American So ciety for Pharmacology and Experimental Therapeutics). 203 Top, representative traces of wild type 7 and 7C116S to Br ACh and MTSET displayed with ACh control responses obtained from the same oocytes. Bottom, conce ntration response curves for Br ACh and MTSET, normalized to the maximal ACh evoked responses. Each point represents the average response of at least four oocytes ( S.E.M.).

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74 As expected, neither MTSEA nor the noncationic SH reagents evoked currents when a pplied to cells expressing either wild type 7 or the cysteine null 7 C116S. The affinity alkylating agent Br ACh was a potent and efficacious full agonist for wild type and C116S 7 with EC 50 values between 12 and 15 M (Figure 2 4). MTSET was a weak par tial agonist for both the 7 control receptors (I max 26%). The EC 50 values for MTSET were 292 62 and 219 104 M for wild type (C116C) and C116S, respectively. Although considered as a possible agonist analog, QN SH had no apparent agonist activity ab ove our limits of detection for either wild type or C116S 7. Likewise, the three other SH reagents (Figure 2 3) showed no detectable agonist activity on wild type or C116S 7 nAChR. The presentation of a SH reagent to a cysteine mutant is predicted to res ult in a covalent modification of the receptor, dependent on the accessibility of the specific mutated residue. However, because several of our reagents are agonist analogs, covalent reaction with the receptor may be preceded by, or coincident to, activati on of the receptor. Therefore, we measured current stimulated by the presentation of the SH reagents (experiment 2 in Figure 2 1C). Not surprisingly, significant currents were detected only for Br ACh and MTSET, the two analogs that had the greatest agonis t activity with the wild type and C116S 7 receptors. A summary of evoked net charge responses stimulated by 1 mM Br ACh and MTSET, after normalization to the ACh maximal response determined previously, 204 is displayed in Figure 2 5. Analysis of variance indicated that for all of the receptors te sted, Br ACh was equally as efficacious as ACh and that MTSET was significantly less efficacious than either ACh or Br ACh ( p <0.001). There were no significant differences in the relative agonist efficacies among

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75 the various mutants. It is noteworthy that QN SH, which showed little if any partial agonism with wild type 7, did show significant responses during the application to 7 L119C nAChR at 1 mM (21% of the net charge response relative to the ACh controls). Figure 2 5. Activation profiles of Br ACh and MTSET on wild type (C116C) and mutant human 7 nAChRs (reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics). 203 Each point represents the average response of at least four oocytes ( S.E.M.).

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76 2.2.4 Reaction with SH Reagents and the Subsequent ACh Responses After characterizing the intrinsic agonist activity of the SH reagents, their inhibition effect on the following ACh evoked responses was studied, outlined in the e xperiment 3 in Figure 2 1C. Representative traces are shown in Figure 2 6 to illustrate the effects of Figure 2 6. Representative traces from voltage clamp experiments, outline in Figure 2 1C, illustrating the effects of 60 s applications of SH reagents on C116S and cysteine mutant 7 receptors and the subsequent effects of PNU 120596 potentiatio n (reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics). 203 Because of the magnitude of PNU 120596 effects, some of the responses are shown at a less sensitive current scale as noted. 203

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77 60 s applications of SH reagents on C116S and cysteine mutant 7 receptors. In initial experiments, the oocytes were treated with the SH reagents at 1mM for 60 s and traces presented in Figure 2 6 indicates that certain combinations of mutant receptor and SH reagent resul ted in complete suppression of response to the subsequent probe ACh application. MTSEA and the L119C and W55C mutants, as well as MTSET and the L119C mutant showed little if any response to a subsequent ACh application. The remaining SH reagent/receptor co mbinations showed a range of responses to ACh in the subsequent ACh application. One possible interpretation of these results is that in certain cases, the labeling reaction kinetics may have been too slow to result in complete labeling at the free thiol. Figure 2 7. Kinetics of subsequent ACh blockade effects by MTSET and Br ACh. To interpret the labeling kinetics for different cysteine mutants, it is important to know the environment of the free cysteine. Environmental factors include accessibility of t he thiol to SH reagents and the charge of the thiol. In general, ionic thiolate (solvent

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78 accessible) is a much better nucleophile than nonionic thiolate (solvent exclusive). 207 The envir onment of the five cysteine mutation sites at positions Ser36, Leu38, Trp55, Leu119, and Ile165 were predicted by evaluating both the solvent accessible surface and the solvent exclusive surface in Chimera 1.4 ( http://www.cgl.ucsf.edu /chimera/download.html ) for the 7 homology model built on the 2PGZ template. Both methods ranked the five amino acids consistently, in an order of Ile165 > Trp55 > Leu119 > Ser36 > Leu38, in regard to relative sol vent accessibility. However, the calculated cysteine accessibility is only a guide for one factor that might determine the experimental outcome of the labeling rates of the different cysteine mutants. In the case of I165C, no block of ACh responsiveness w ere detected with any of the SH reagents tested. In this case, it is possible that labeling was rapid and complete, with the caveat that a label at I165C may be silent with respect to its ability to block the binding of ACh and activation of the channel. I nspection of the homology model suggests that a label at I165 may be too far away to impact access of ACh, especially in a dynamic setting. State dependence of accessibility may also be a factor that may impact the tethering reaction kinetics of the agoni sts. Although we do not know which functional state the homology model represents, the results of I165C were not state dependent. There were no blockade on the subsequent ACh response observed for MTSET or Br ACh treated I165C up to 5 min (data not shown). On the other hand, w hile MTSEA and QN SH produced maximal effects with just 60 s treatments, the agonist like reagents, Br ACh and MTSET required treatments of up to 5 minutes for maximal effect s on subsequent ACh responses (Figure 2 7 ), and even after 5 minutes treatment with

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79 MTSET L38C remained responsive to subsequent applications (Figure 2 7) The L119C and W55C 7 mutants were similar in their sensitivity to Br ACh block of ACh evoked respo nses. A full 3 minute incubation was re quired to achieve thi s effect. However, there were no significant effects of Br ACh applications on the ACh evoked responses of S36C or L38C 7 mutants, even with Br ACh applications of up to 7 minutes. The slowness or failure to react may have been indicative of a dependence of the labeling reaction on the receptor state or a favored binding pose that was not optimal for the covalent reaction. One cannot rule out that labeling did occur, and if it did occur, the labeled receptor was still able to bind and respond to the ACh T hese data c tional states of the receptor might have slowed the reaction rates of the sulf hydryl reagents with some cysteine mutants. To compensate for the relatively slow reaction rates of Br ACh and MTSET (on receptors other than L119C), the applications of these SH reagents were extended to 5 min, which was sufficient to produce optimal effects. Figure 2 8 summarizes the inhibition effects of the SH reagents treated cysteine mutants on the ACh evoked response. Because some mutants showed reduced sensitivity to ACh, the concentration of ACh was 60 M for C116S and L119C 7 mutants, and 300 M for 7S36C, W55C, and I165C. The ACh evoked responses of C116S, and I165C 7 receptors were unchanged relative to their initial A Ch control responses by the application of any of the SH reagents (Figures 2 6 and 2 8 ). Given the lack of a free cysteines for C116S, it is unlikely that there was any covalent modification of this receptor. If there were any reactions with either the wi ld type (C116C, data not shown ) or the I165C 7 receptors

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80 (Figure 2 8 ), they appeared to be functionally neutral, at least in re gard to ACh activation (Figure 2 1 C Case I). Figure 2 8. Acetylcholine responses after SH reagent application (reprinted w ith permission of the American Society for Pharmacology and Experimental Therapeutics). 203 SH reagents were applied at 1 mM for 60 s except MTSET and Br ACh, which were applied for 5 min to achieve maximum effect. A response of 1.0 represents no detectable change made by SH reagents (reflecting case I in Figure 2 1C), whereas a response of 0 .0 represents complete blocking of subsequent ACh activation by covalently attached SH reagents (reflecting case II in Figure 2 1C) A response more than 1.0 may represent potentiation of subsequent ACh activation by covalently attached SH reagent, which is still under further investigation. In contrast, there were functional after effects identified in the other four receptors (W5 5C, L119C, S36C and L38C) that contained mutations in the complementary face of the LBD. Representative traces are shown in Figure 2 6 and a summary of the results is shown in Figure 2 8. As shown in Figure 2 8, not all of the reagents were equally effect ive at inhibiting the ACh evoked responses of the cysteine mutants. Indeed, each mutant had a distinct profile for the six reagents. Reaction of the free cysteine at the

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81 S36 position produced decreases in the ACh response with all of the five methanethiosu lfonate SH reagents but not the alternative r eactive agonist analog Br ACh. However, treatments of 7 S36C with MTSACE, EMTS, QN SH, and MTSEA produced only partial inhibition of 7 S36C ACh evoked responses, while following MTSET treatment no significant AC h responses could be detected. The 7 L38C evoked responses after QN SH. Only the positively charged SH reagents and Br ACh were able to inhibit the 7 W55C mutant. As previously repor ted 204 the ACh evoked responses of 7 L11 9C were very efficiently blocked by all the cationic SH reagent treatments. Following 60 s treatments with QN SH, MTSET or MTSEA, ACh evoked responses decreased essentially to zero. A 60s MTSACE application also blocked 75% of the 7 L119C ACh evoked re sponse. The 7 L119C mutant was also sensitive to Br ACh, however a three minute application was required for the complete blocking effect (Figure 2 8 ) Interestingl y, the ACh response of the EMTS treated 7 L119C receptor appeared enhanced relative to th e response of that mutant to ACh prior to EMTS. This indicates that EMTS itself may be a covalently allosteric modulator acting in a manner distinct from that of PNU 120596. 2.2.5 Activation of SH modified 7 Mutants by PNU 120596 After confirming through kinetic studies that the labeling of the various mutant receptors with the various SH reagents was maximal, PNU 120596 was then used to probe for the presence of D s states (experiment 4 in Figure 2 1C). The t ype II PAM PNU 120596 was used for this purpose because it can convert a previously desensitized receptor into a conducting state.

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82 Firstly, the reactions of the wild type and the mutated receptor to PNU 120596 without the treatment of SH reagents were compared as negative controls. To conduct these exp eriments, cells were typically used with 24 hours of injection, when the control responses to 60 M ACh were very low; otherwise the potentiated responses became too large to maintain a voltage clamp. Under these conditions, PNU 120596 applications produce d small stimulus artifact baseline deflections which, when normalized to small initial controls, gave values for peak deflections of < 10%, which was taken as the limit for detection. The results for different 7 receptors are listed in Table 2 1, in which values are compared by peak currents relative to the peak currents evoked by 60 M ACh before the application of 300 M PNU 120596. Table 2 1. Positive alloteric modulator PNU 120596 effects: responses to PNU 120596 applied alone, and subsequent potentia tion of ACh evoked responses. (N.S. No significant response. a These are the data for 7C116S receptors following a 60 s treatment with 1 mM DTT intended to reduce the vicinal disulfide in the C loop.) 203 Recept or Response to PNU 120596 applied alone (%) ACh response after application of PNU 120596 (%) 7 N.S. 3000 40 C116S N.S. 2000 500 I165C N.S. 1250 240 S36C N.S. 1200 450 L38C 35 9 910 63 W55C N.S. 1200 40 L119C 54 4 3100 460 C1 16S DTT a 26 16 1700 500 PNU 120596 does not produce significant activation of wild type or C116S 7 rece ptors when applied alone (Table 2 1, Figure 2 6 ). However, when ACh or other agonists are applied to receptors previously primed with PNU 120596 199 agonist evoked responses are s ubstantially increased (Figure 2 6). Likewise, the 7 W55C, I165C and S36C mutants showed no significant response to applications of 300 M

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83 PNU 120596 alone in the absence of agonis t or prior to treatments with SH reagents (data not shown). Following the application of SH reagent s to wild type, C116S, or I165C 7 receptors there was no response to subsequent applications of PNU 120596 (r epresentative traces in Figure 2 6). To study the potential effect s of SH reagent modifications to the naturally occurring cysteines in the C loop, dithiothreitol (DTT) was applied to the h 7 C116S receptor to render the vicinal disulfide as free cysteines at positions 190 and 191 for tethering of the sulfhydryl reagents. Interestingly, the 7 L38C and L119C mutants, as well as C116S receptors previously treated with DTT, did show small but significant responses to subsequent applicatio ns of PNU 120596 alone (Table 2 1). Applications of 60 M ACh following priming with 300 M PNU 120596 produced responses that were 9 to 31 fold larger than the responses measured during the prior a pplication of ACh alone (Table 2 1 ). After SH reagent and ACh treatments, 300 M PNU 120596 was applied for 12s. The PNU 120596 peak current response profiles of the different mutants after the SH reagent treatments are shown in Figure 2 9, and some representative tr aces are shown in Figure 2 6. It was hypothesized that if treatment of the cysteine mutant 7 receptors with our SH reagents inhibited subsequent ACh evoked responses because the receptors were being locked into a PNU sensitive form of desensitization, the n they should be directly activated by PNU 120596 after SH treatments. It was further hypothesized that this activity could depend on both the agonist character of the reagent and the specific orientation of the covalently bound reagent in the LBD. The com plete inhibition of the ACh probe pulse as seen with W55C and L119C (Figures 2 6 and 2 8)

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84 is most likely due to exclusion of ACh from the covalently modified agonist binding sites, though one cannot rule out the possibility that the labeled receptor is acc essible to ACh, but is in an alternative functionally blocked state. The most straightforward interpretation is that the ACh binding site is occupied by the bound SH reagent, and that the receptor exists in one or more stable analogous to the D s and D i sta tes that are entered after binding of free agonists. The S36C and L38C mutants had no apparent PNU 120596 evoked responses after application of any of the cationic SH reagents. L38C yielded a small PNU 120596 evoked response after treatment with EMTS; howe ver, this PNU response was intrinsic to L38C (Table 2 1) and did not depend on reaction with a SH reagent. In contrast, W55C showed greatly enhanced responses to applications of PNU 120596 after treatment with Br ACh, QN SH, and MTSET, but no PNU 120596 in duced responses were observed after treatment with MTSACE or EMTS. These results suggest that once reacted with the agonist analogs, the 7 W55C receptors were at least partially converted into a PNU 120596 sensitive desensitized state. Consistent with the previous observation that reactions with either MTSACE or EMTS failed to block the subsequent ACh evoked responses of 7 W55C, these agents clearly did not induce PNU 120596 sensitive desensitization either. It is noteworthy that PNU 120596 did reactivate the MTSEA treated 7 W55C but to a lesser extent than after the thio labeling agonists (Figure 2 3). This observation may suggest that the core pharmacophore for PNU 120596 reactivation may be simpler than the core agonist structure for activation of the unmodified wild type receptors.

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85 Figure 2 9. Responses to the type II PAM PNU 120596 after SH reagent treatments (reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics). 203 Inspection of Figure 2 9 reveals that of all the mutants tested, covalently modified L119C produced the largest PNU induced currents. Based simply on a lack of ACh blockade, 7 L119C seemed insensitive to labeling with EMTS. This agent, which was partial ly effective on 7 S36C, showed no apparent effect on any of the rest of the mutants. Although EMTS treatment did not seem to inhibit the ACh evoked responses of 7 L119C, it did seem to increase responses to PNU 120596 alone from 54% of the ACh peak value (Table 2 1) to approximately 500% ACh peak. This suggests that EMTS did at least partially react and that it increased the reactivity of the mutant receptor to both ACh and PNU 120596. However, the PNU 120596 evoked response

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86 following EMTS treatment was r elatively small, suggesting that this reagent was ineffective relative to the other SH reagents tested in converting receptors to the PNU 120596 sensitive desensitized state. 2.2.6 PNU 120596 Potentiation of ACh Responses in SH Reacted Receptors As shown in Figure 2 6 (C116S traces) and Table 2 1, PAM PNU 120596 can bind with the 7 receptors long enough to potentiate the acetylcholine applied 3.5 minutes later, if the ligand binding site is not blocked. Therefore, further applications of ACh to PNU 120596 primed SH reacted receptors could provide a sensitive probe for the degree of labeling by the SH reagent. Responses were compared to baseline ACh evoked responses, and the res ults are summarized in Table 2 2. Some representat ive traces are shown in Figure 2 6. To represent the inhibition effect of SH reacted receptors, the data in Table 2 2 are compared to the ACh responses after application of PNU 120596 observed i n the absence of SH reactions (Table 2 1), the results of which are listed in Table 2 3. The 7 S36C receptors showed largely diminished post PNU 120596 ACh peak current values, except after Br ACh. The post PNU 120596 ACh peak responses of 7 L38C were most strongly affected by QN SH and MTSEA, and totally unaffected by EMTS treatment. The PNU 1 20596 potentiated ACh responses of 7 W55C were most strongly affected by QN SH and MTSEA, while MTSET, Br ACh, and MT SACE had intermediate effects. EMTS, which seemed to potentiate 7 W55C and L119C ACh evoked responses ( Figure 2 8 ), also increased the PN U 120596 potentiated currents of L119C, suggesting that EMTS, once react ed with L119C, may itself be a covalent allosteric potentiator, possibly working in an additive or synergistic manner with PNU 120596.

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87 The PNU 120596 potentiation of 7 L119C ACh res ponses was strongly reduced by all of the reagents except EMTS and Br ACh, which produced 31% and 75% reductions, respectively (Table 2 3). In general, all of the SH reagent effects on the PNU 120596 potentiated ACh responses were consistent with the inhib ition of the control ACh evoked r esponses, as shown in Figure 2 8. One striking disparity was seen in the effects of MTSACE on 7 W55C. While MTSACE did not block the control ACh evoked responses, it did decreas e the PNU 120596 potentiation (Table 2 3). I n addition for the W55C 7 receptor both MTSET and MTSEA re duced the ACh response (Figure 2 8 ), but after MTSET PNU 120596 still potentiated the ACh response and after MTSEA the potentiation was greatly inhibited (Tables 2 2 and 2 3 ). Table 2 2 The e ffects of SH reagents on PNU 120596 primed ACh evoked responses. Data are expressed relative to initial ACh control responses. 203 The durations of the SH reagent reactions are consistent with the data presented in F igure 2 8. Receptor MTSACE EMTS Br ACh QN SH MTSET MTSEA % S36C 40 20 210 40 810 210 130 40 23 5 8 1 L38C 450 180 990 200 690 110 58 14 630 21 30 16 W55C 460 70 1760 40 220 40 56 12 440 140 7 1 L119C 160 100 2 140 260 760 340 21 10 81 10 71 52 Table 2 3. The effects of SH reagents on the relative PNU 120596 potentiation of ACh responses 203 Values are the ratios of PNU 120596 potentiation in Table 2 1 (before SH treatments) and 2 2 (after SH treatments) Receptor MTSACE EMTS Br ACh QN SH MTSET MTSEA S36C 0.033 0.175 0.675 0.108 0.019 0.007 L38C 0.495 1.09 0.758 0.063 0.692 0.033 W55C 0.383 1.467 0.183 0.046 0.367 0.006 L119C 0.052 0.690 0.246 0.007 0 .026 0.023 2.2.7 The Modification of the Reduced C loop and PNU To st udy the potential effects of SH reagent modification in the C loop, a key structure in the LBD (Figure 2 2 ), reduction of the vicinal disulfide at positions 190 an d 191 in the 7 C116S receptor was attempted by treatment of 1mM dithiothreitol (DTT).

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88 Figure 2 10. The modification and PNU 120596 potentiation of 7 C116S after DTT treatment (reprinted with permission of the American Society for Pharmacology and Exper imental Therapeutics). A) Representative traces illustrating the strong agonist activity of MTSET, QN SH, and Br ACh for 5 min DTT treated 7 C116S, and the induction of a PNU 120596 sensitive desensitization. B) The SH reagent and PNU 120596 activation of DTT treated 7 C116S relative to the average of two ACh controls before DTT treatment. 203

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89 This would then unmask two free thiols that could be reached with an SH reagent. W hen exposed to DTT for increasing periods of time, the receptors became progressively less responsive to activation by ACh, barely showing any response after five minutes of DTT treatment. Interestingly, even when the receptors were no longer able to be activated by ACh, the reduced receptors coul d be activa ted by Br ACh or MTSET (Figure 2 10). A series of ACh and PNU 120596 applications were performed to determine if the agonist analogs showed evidence of covalently modifying the receptors and inducing PNU 120596 sensitive desensitization. After t reatment of the reduced receptors with MTSET or QN SH, there was a small recovery of sensitivity to ACh, while after Br ACh the receptors remaine d unresponsive to ACh (Figure 2 10 A ). However, treatment of the reduced receptors with each of the agonist an alogs appeared to very effectively induce the PNU 120596 sensitive desensitized state, since large currents were stimulated by the applicatio n of PNU 120596 alone (Figure 2 10 B ). In summary, after reduction by DTT, the C loop vicinal cysteines became pote ntial targets for SH reagents, as demonstrated by the effects of MTSET and Br ACh. Before DTT treatment, these agents did not have evoked responses larger than those of the initial ACh controls. But after reduction of the vicinal cysteines, the channels ac tivated strongly during the process of covalent modification. Successful modification was further confirmed by the large subsequent PNU 120596 evoked responses. The results suggest that part of the binding site for ACh is destroyed when the C loop vicinal disulfide is reduced, but that the loss of binding affinity for the LBD is offset by covalent modification with the SH reagents.

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90 2.2.8 Modeling Modifications of the Agonist and the Binding site Dr. Horenstein modeled bound SH reagents by introducing non na tural amino acids into the 7 homology model. These would be equivalent to the covalent modification of the amino acid residues of the receptor (Figure 2 11, A and B). For example, after MTSET reacted with the free cysteine in the LBD, a trimethylamino eth ylthio group was attached to the sulfur of the cysteine mutant. After molecular mechanics minimization, the structures of the MTSET modified mutants (S36C, W55C, and L119C) were superimposed. The side view of the receptor (Figure 2 11 A) shows that MTSET l abeled 36C is farther away from the C loop, whereas MTSET 55C and MTSET 119C place the ammonium pharmacophore further underneath the C loop. MTSET labeled 36C can also place the ammonium group of the label proximate to Glu189, possibly excluding the ammoni um group from the LBD by electrostatic attraction. The covalent reactions of MTSET with either W55C or L119C are predicted to put the agonist like portion of MTSET close to, but still at some distance from the predicted preferred position of the minimal a gonist TMA in the LBD (Figure 2 11, C and D). Notice that the predictions for the two complexes are not identical, although both are associated with similar functional results. Whereas the L119C complex puts the MTSET ammonium against the lip of the C loop the W55C MTSET ammonium sits almost perfectly where the TMA ammonium sits. These may both represent intermediate states that relax to an equivalent PNU 120596 sensitive state Alternatively, both of these intermediate states may, themselves, be stable in the absence of PNU 120596, but, as

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91 suggested by the data in Figure 2 9, the L119C MTSET intermediate state is more readily converted into a stable conducting state. Figure 2 11. Homology models of 7 covalently modified by the SH reagents (reprinted wit h permission of the American Society for Pharmacology and Experimental Therapeutics). 203 A) and B) Superposition of three labeled receptor models: S36C (yellow), W55C (green), and L119C (red) after covalent reaction with MTSET. The structures were optimized with Amber 10. A) Side view from outside the channel pore. B) Top view facing the ion channel pore from the extracellular domain. The C loop is shown in blue, whereas the ammonium of MTSET is shown as a dark blue ball in both. C) and D) Comparisons of modified receptor to a putative lig and bound complex. C) O verlay of the 7 TMA complex (white) with the L119C MTSET (magenta) label. D) The overlay of the 7 TMA complex (white) with the W55C MTSET (magenta) label. 2. 2.9 Synthetic Efforts Towards Sulfhydryl labeling Derivatives of 7 Selective Agonists As introduced in Chapter 1 (Figure 1 9), there are several 7 selective pharmacophores. 131 It was of interest to use 7 selective agonist like SH reagents for further investigation of these pharmacophoric elements. Although QN SH contains the 7 selective tropane motif, QN SH cannot activate the wild type 7 nicotinic receptors,

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92 thus limiting QN 7 selective activation. However, other SH reagents containi ng the tropane motif were not commercially available nor were Figure 2 12. Synthetic route for piperidine sulfhydryl derivatives. their syntheses reported in the literature. Therefore, syntheses of piperidine methane thiosulfonate derivatives (compound s, 3 4 and 6 ) were attempted as shown in Figure

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93 2 12, top, because these compounds were expected to be 7 selective agonist like SH reagents. Generally, methane thiosulfonate derivatives can be obtained via S N 2 substitution between sodium methanethiosulf onate (NaMTS) and the appropriate bromides. 208 Initially, two methods to make the NaMTS, 8 were attempted as shown in Figure 2 12, bottom. 209 Although numerous workers have reported use of method A to synthesize NaMTS, 209 212 addition of methanesulfonyl chloride to the s odium sulfide aqueous solution led to the production of sodium methanesulfonate, 7 instead. This corresponds to the hydrolysis of the sulfonyl chloride. Ethanol was also used as the solvent in method A in another paper. 213 to react, like water, with methanesulfonyl chloride, method B was used to synthesize the sodium methane thiosulfonate, even though it is more costly than method A. 209 Reacting sodium methanesulfinate w ith sulfur produced the desired compound, 8 in 71% yield after purification. Method B also had several other advantages: reaction progress appeared coincident with dissolution of sulfur; the reaction proceeded quickly (within 10 minutes); and the purifica tion was straightforward. Therefore, method B is recommended for the synthesis of NaMTS. Table 2 4. Summary of the synthesis of the sulfyhydryl derivative of the 7 selective agonists. N.R., no reaction occurred. Entry Reaction conditions Result 1 (Route A) 0.06M 2a 0.06M NaMTS, DMF, 20 hours, r.t. then 5hr, 50 C N.R. 2 (Route A) 0.06M 2a 0.06M NaMTS, CH 3 CN, 20 hours, r.t. then 5hr, 50 C N.R. 3 (Route A) 0.06M 2a 0.06M NaMTS, DMSO, 3 days, r.t. then 4hr, 65 C N.R. 4 (Route A) 0.06M 2b 0.06M NaMTS, D MSO, 3 days, r.t. then 4hr, 65 C N.R. 5 (Route B) 0.15M 1a 0.15M NaMTS, DMF, 6 hours, 50 C N.R. 6 (Route B) 0.15M 1a 0.15M NaMTS, CH 3 CN, 6 hours, 50 C N.R. 7 (Route C) 0.05M 5 0.06M NaMTS, DMSO, 3 days, r.t. then 4hr, 65 C N.R.

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94 Three different rout es were attempted for synthesis of compound 3 4 and 6 as shown in Figure 2 12, top. In routes A and B, modifications were introduced on the 3 or 4 position of the piperidine ring to make the precursor 1a and 1b via reaction of 3 or 4 hydroxyl N methy l piperidine with hydrobromic acid and methanesulfonyl chloride, respectively. Then 1a and 1b were reacted with methyl iodide, which yielded their N methyl forms, 2a and 2b The 4 bromo substituents 1a and 2a were obtained in yields of 28% and 27% respecti vely, and characterized by NMR and mass spectrometry. Since the mesylate (Ms) is a better leaving group than bromide, compounds, 1b and 2b were also synthesized. Compound 1b was synthesized via reacting 3 hydroxyl 1 methylpiperidine with methanesulfonyl c hloride in the presence of pyridine. Since pyridine is problematic for the following steps, after the reaction completed, pyridine was removed via flash chromatography using a silica column. Although proton NMR confirmed the presence 1b it was not pure du e to decomposition during chromatography (noted in Appendix A 3). The N methyl form of 1b (compound 2b ) could not be isolated in pure form, possibly due to the instability of the compound. Compound 2b was obtained in 45% yield with an unidentified impurity The presence of 2b was confirmed by mass spectrometry. Then compounds, 1 or 2 were reacted with sodium methanethiosulfonate to synthesize the piperidine sulfhydryl derivatives under various conditions as listed in Table 2 4. Unfortunately, there was no sign of desired in Table 2 4 indicated the presence of the unreacted starting materials. Alternatively, synthesis of a thiosulfonate substituent, compound 6 was attempte d as shown in route C in Figure 2 12, thereby avoiding S N 2 substitution on the hindered piperidine ring as in

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95 routes A and B. Compound 5 4 acetoxy 1 (bromomethyl) 1 methylpiperidin 1 ium bromide was synthesized from acetylation 214 of 4 hydroxyl 1 methyl piperidine followed by alkylation 215 using methylene bromide, with yields of 78% and 61 % for the acetylation step and the alkylation step, respectively (Figure 2 12). However, reaction of 5 with NaMTS did not produce any apparent desired product t (entry 7, Table 2 4). 208 In conclusion, the synthesis o f the piperidine methanethiosulfonate derivatives was unsuccessful under the conditions used. The failure of routes A and B may be due to the difficulty of carrying out S N 2 displacements on the secondary cation of a six membered ring, despite the presumabl y enhanced nucleophilicity of the methanethiosulfinate anion. Therefore, both starting materials, 2a and NaMTS, were recovered. The instability of mesylates ( 1b and 2b ) and/or the resulting impurities may be the alternative reasons for the failure to produ ce the methanthiosulfonate sustituents, which would have shown as white spots against a yellow background on the TLC plate 208 In route C, compound 5 has an extreme case of beta branch ing, which served to lower its reactivity. To make 7 selective agonist like sulfhydryl reagents, syntheses of other derivatives of compound 1 and/or 2 can be attempted using stronger leaving groups, such as a triflate, or by use of somewhat elevated temp eratures (higher than 50 65 C used in the studies reported here). We can also use some catalysis to facilitate the reaction, such as a crown ether or silver nitrate. We can also extend the carbon bridge between the charged nitrogen and the leaving group i n compound 5 producing molecules that have a tether length as in QN SH (Figure 2 3). Due to time constraints,

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96 these syntheses were not pursued further, and commercially available SH reactive reagents were used for the functional study of the nAChR and its mutants. 2.3 Summary In these studies, SH reactive agonist analogs were used with the PAM PNU 120596 to probe the 7 LBD for the functional consequences of tethering agonist like molecules in specific locations. One limitation to th e use of ACh responsive ness as the sole reporter of covalent modification is that it provides little or no insight into the underlying basis for the loss of function; e.g., whether the binding site is simply occluded or whether the receptor is being held in specific nonfunctiona l states, such as those as sociated with desensitization. This distinction could be of particular importance for examining the effects of SH reagents that may b e agonist analogs. Therefore, t ype II PAM PNU 120596 was used as an additional probe for the rec eptor state following SH modification. The results show that a single agent, such as MTSET, covalently bound at different sites, can either convert the receptors to the PNU 120596 sensitive desensitized state, as in 7 W55C, 7 L119C, or the reduced vicin al cysteines, or may simply block ACh activation, as in 7S36C and 7 L38C. The block of ACh evoked responses for 7S36C and 7 L38C was consistent with simple occlusion of the binding site or the induction of PNU 120596 insensitive desensitization. 142, 145 These binding modes suggest how the agonist may bind in the two desensitized states, D s and D i which are identified in the native 7 nAChRs. 145 For 7 W55C, and especially 7 L119C, there is good evidence for the induction of the PNU 120596 sensitive desensitized state but it is unclear how complete that

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97 conv ersion was for the two mutants. Are the differences in the PNU 120596 responses, which were l arger for 7 L119C than for 7 W55C, indicative of more incomplete conversion for 7 W55C, or just a higher P open for the modified 7 L119C? The results suggest the possibility tha t there may be multiple forms of PNU 120596 sensitive and insensitive desensitization, based on similar activity with multiple ag ents and effective reactions at 7 W55C, 7 L119C, and the reduced vicinal cysteines. An agonist like molecule covalently bou nd in the LBD of the 7 nAChRs is likely to have the effect of stabilizing some of the same ligand bound non conducting (i.e., desensitized) states that predominate the state function of the native receptor in the prolonged presence of agonist. Our most ba sic concept of ligand gated ion channel function is that receptors have evolved to react to an activating signal rapidly and to open the channel pore transiently, and then to desensitize to prevent excessive activation. Although this model fits our percept ion of synaptic ion channels, it is still in question whether it is applicable to receptors such as 7 nAChR. The 7 nAChRs perform important functions in non neuronal cells, and it has been suggested that in some cases ion channel activation may not be re quired. 216, 217 As introduced in Chapter 1, 7 mediated cytoprotection does not require the activation of the ion channel, and strong activation of 7 receptors may be even cytotoxic. 200 These alternative modes of signaling suggest th at stable, ligand bound, nonconducting states, such as D s and/or D i may represent additional functional states for the 7 receptor. In conclusion, our results support the hypothesis that a single ligand may adopt multiple binding poses within the ligand binding pocket leading to multiple outcomes with respect to the functional state of the receptor. The non stationary behavior of a

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98 ligand gated ion channel following a jump in agonist concentration may therefore be due to the relative stability and kineti c evolution of various binding poses and not the relative probability of specific conformational transitions arising from a single binding mode Our data suggest that a new approach for drug development can be implemented, by taking advantage of structural models and ligands with more comprehensive co nsideration of where they bind, how they bind, and most importantly the effects of binding. 2.4 Experimental Section 2.4.1 7 nAChR c lones and s ite directed m utants 176 The human 7 clone was obtained from Dr. Jon Lindstrom (Univ. P ennsylvania, Philadelphia, PA). The human RIC 3 clone, obtained from Dr. Millet Treinin (Hebrew University, Jerusalem, Israel), was co injected with the 7 constructs to improve the levels and speed of receptor expression ( work by Lynda Cortes, Sara Braley and Shehd Abdullah Abbas Al Rubaiy of the Dr. Papke lab). Amino acids are numbered as for human 7 (vicinal C loop cystei nes at positions 190 and 191). Mutations were introduced by Clare Sto kes using the QuikChange site directed mutagenesis kit (Stratagene, La Jolla CA) following th In order to obtain a Cys null background, a naturally occurring cysteine in 7 was mutated to serine ( 7C116S). As previously report ed 204 the pharmacology and macroscopic acti vation properties of this Cys null receptor were indistingu ishable from those of wild type 7 in regards to the potency and relative efficacy of diverse agonists including ACh, tetramethylammonium quinuclidine, and 4 OH GTS 21. The mutant also was indis tinguishable from wild type in regard to the rapid concentrat ion dependent desensitization characteristic of 7. 204 The novel cysteine mutants used in these

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99 expe riments were made in the LBD of 7C116S. All mutations were confirmed with aut omated fluorescent sequencing. After linearization and purifi cation of cloned cDNA, RNA transcripts were prepared in vitro using the appropriate mMessage mMachine kit from Ambion Inc. (Austin, TX). 2.4.2 Expression in x enopus o ocytes 43 Mature (>9cm) femal e Xenopus laevis African frogs (Nasco, Ft. Atkinson, WI) were used as the source of oocytes. Before surgery, frogs were anesthetized by laying the animal in a 1.5g/L solution of MS222 (3 aminobenzoic acid ethyl ester; Sigma, St. Louis, MO) for 30min. Oocy tes were removed from an abdominal incision by Shehd Abdullah Abbas Al Rubaiy To digest the follicular cell layer, harvested oocy tes were treated with 1.25 mg/mL collagenase from Worthington Biochemical Cooperation (Freehold, NJ) for 2 hours at room tempe mM NaHCO 3 0.82 mM MgSO 4 15 mM HEPES (pH 7.6), 12 mg/L tetracycline). After that, stage 5 oocytes were isolated and injected with 50 nl (5 20 ng) each of the appropriate subunit cR NAs. Whole cell voltage clamp r ecordings of the oocytes were mad e 2 to 10 days after injection by Lynda Cortes, Shehd Abdullah Abbas Al Rubaiy or me. The experimental response values were normalized to 300 M ACh control to correct for variation in th e abs olute magnitude of the evoked current response over time. 2.4.3 Chemicals The methanethiosulfonate compounds EMTS, MTSACE, QN SH, MTSEA, and MTSET (See Figure 2) were purchased from Toronto Research Chemicals Inc. (North

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100 York, ON, Canada). PNU 120596 was o btained from Tocris (Ellisville, MO) and all of the other solvents and reagents for electrophysiology and synthesis were from Sigma Chemical Co. (St. Louis, MO) and Fisher Scientific (Pittsburg, PA). 1 H and 13 C NMR spectra were obtain ed using VXR 300, Gem ini 300 or Mercury 300 (300 MHz) spectrometers (Varian, Palo Alto, CA) in appropriate deuterated solvents. Mass spectra were obtained on an Agilent 6210 TOF spectrometer operated in the ESI and ( ) ESI ionization mode s EMTS, PNU 120596, and MTSACE stock s olutions were prepared monthly in DMSO stored in refrigerator monthly Working solutions were prepared fresh Other SH r eagent stock solutions were prepared the usage of the rotary evaporator. 4 B romo 1 methylpiperidine 1a. 218 Hydrobromic acid (80 mL; 48% in water) was added to 5.0 g 4 hydroxyl 1 methylpiperidine with stirring, and the solution turned dark red after the addition of the acid. The mixture was heated to reflux for 16 hours, and then was cooled to room temperature and concentrated in vacuo. The obtained mixture was cooled on an acetone dry ice bath and the pH was adjusted with 20% (w/v) sodium hydroxide to approximately 14. The aqueous solut ion was then saturated with sodium chloride and extracted with chloroform (4 50 mL). The organic layers were combined, dried over magnesium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography (silica, 2 0:1 dichloromethane/methanol). Compound 1a was obtained as a light yellow liquid in 28% yield (1.8 g). 1 H NMR (300

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101 MHz, CDCl 3 ) ppm 1.96 2.25 (m, 6 H) 2.27 (s, 3 H) 2.67 (br. s., 2 H) 4.18 (br. s., 1 H) ESI MS: m/z 178.0223 and 180.0210 [M+H]+ (calculated: 178.0226 and 180.0206). 4 B romo 1,1 dimethylpiperidin 1 ium iodide 2a. 4 bromo 1methylpiperidine (0.59 g) was stirred in 3 mL acetonitrile, and 0.70 g methyl iodide in 3 mL of acetonitrile was added. The mixture was then stirred at room temperature for 14 hours. The white precipitate that formed was collected by filtration, and the filtrate was mixed with ethyl ether to recov er more precipitate. The obtained white solids were combined and dried in vacuo, giving the desired compound, 2a in 27% yield 1 H NMR (300 MHz, CD 3 OD ) ppm 2.32 (m, 2 H) 2.59 (m, 2 H) 3.31 (s, 3 H) 3.25 (s, 3 H) 3.63 (m, 4 H) 4.62 (br. s., 1 H) ESI MS: m/z 192.0397 and 194.0376 [M]+ (calculated: 192.0382 and 194.0362). 1 M ethylpi peridin 3 yl methanesulfonate 1b 3 hydroxyl 1 methylpiperidine (2.0 g, 17.4 mmol) and pyridine (1.65 g, 20.88 mmol) were stirred together in 20 mL of dichloromethane, with cool ing in an ice water bath. A solution of 1.5 mL methanesulfonyl chloride in 20mL dichloromethane was added to the mixture dropwise over a period of 40 minutes. After 30 minutes at 0 C, the reaction mixture was quenched by addition of 18 mL of saturated aqu eous sodium bicarbonate. The organic phase was separated and the aqueous phase was extracted with additional dichloromethane (3 20 mL). The organic layers were collected, dried over magnesium sulfate, filtered and then concentrated under reduced pressure The remaining pyridine was removed by flash chromatography (silica gel, 40:1 dichloromethane/methanol). Compound 1b was obtained as a light yellow oil in 43% yield. 1 H NMR (300 MHz, CDCl 3 ) ppm 1.49 1.75 (m, 2 H) 1.76 1.89 (m, 2 H) 2.29 (s, 3 H) 2.3 4 2.51 (m, 3 H) 2.76 2.82 (m, 1 H) 3.04 (s, 3 H) 4.72 4.85 (m, 1 H)

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102 1,1 D imethyl 3 ((methylsulfonyl)oxy)piperidin 1 ium 2b. Compound 1b (400 mg) was dissolved in 2 mL acetonitrile, to which was added methyl iodide (1.7 eq, 0.219 mL). The mixture was stirred for two days at room temperature. The precipitate formed was collected by filtration, redissolved in acetonitrile and precipitated by addition of ethyl ether. Impure 2b was obtained as a faint yellow solid in 45% crude yield. ESI MS: m/z 208.1022 [M]+ (calculated: 208.1002). 1 M ethylpiperidin 4 yl acetate 1c. 4 Hydroxy N methyl piperidine (5.0 g) was added to acetic anhydride (11 mL) containing two drops of concentrated sulfuric acid. The reaction mixture was heated in an oil bath (100 C) for 20 m inutes. Then the mixture was cooled and poured into 5 mL ice cold water. The pH was adjusted to 10 using 7% sodium carbonate. The resulting solution was extracted with ethyl ether. The organic layers were combined, dried with magnesium sulfate and concentr ated under reduced pressure. The crude product was purified by flash column chromatography (silica, 20:1 dichloromethane/methanol). Compound 1c was obtained as yellow oil in 78% yield. 1 H NMR (300 MHz, CDCl 3 ) ppm 1.70 (m, 1 H) 1.82 1.97 (m, 2 H) 2.21 ( t, J=8.03 Hz, 2 H) 2.28 (s, 3 H) 2.55 2.77 (m, 2 H) 4.64 4.86 (m, 1 H) 13 C NMR ( 75 MHz, CDCl 3 ) ppm 21.57, 31.11, 46.35, 53.22, 70.11, 170.79. 4 Acetoxy 1 (bromomethyl) 1 m ethylpiperidin 1 ium bromide 5. Compound 1c (500 mg) was stirred in acetonitril e (1 mL) along with two equivalents of dibromomethane at room temperature for four days. The resulting precipitate was filtered and recrystallized in absolute ethanol to afford 5 as a white solid in 61% yield. 1 H NMR (300 MHz, CD 3 OD ) ppm 2.06 2.17 (m, 2 H) 2.11 (s, 3 H) 2.28 (m, 2 H) 3.32 (s,

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103 3 H) 3.73 (m, 4 H) 5.03 (m, 1 H) 5.48 (s, 2 H) ESI MS: m/z 250.0443 and 252.0417 [M]+ (calculated: 250.0437 and 252.0417). Sodium methanesulfonate 7. 209 Method A. Sodium sulfide nonahydrate (36 g, 0.15 mol) was dissolved in water (40 mL) with gentle heating to about 45 C to facilitate dissolution. The soluti on was cooled to 0 5 C in an ice bath with stirring. Freshly distilled methanesulfonyl chloride (11.65 mL, 0.15 mol) was added dropwise over 35 minutes. The reaction mixture turned yellow, then milky white. The mixture was heated under reflux for 18 hours Some sulphur was precipitated after heating; the reaction mixture turned yellow and then colorless during heating. The solution was cooled to room temperature and evaporated on a high vacuum rotary evaporator. A mixture of white solid and yellow solid wa s obtained and re dissolved in water. The sulfur was removed by filtration and the filtrate was evaporated to dryness and dried in a desiccator for an additional 24 hours. Sodium methanesulfonate 7 was obtained as a white solid in 96% yield (melting point > 295 C ). 1 H NMR (300 MHz, D 2 O ) ppm 2.78 (s ) 13 C NMR ( 75 MHz, D 2 O ) ppm 38.61. HPLC/( )ESI MS: m/z 95.2 [M Na] (calculated: 95.0). Sodium methane thio sulfonate 8 209 Method B. A mixture of sodium methanesulfinate (500 mg, 4.9 mmol) and sulfur (157 mg, 4.9 mmol) in dry methanol (60 mL) was heated to reflux. After 10 minutes, the sulfur had dissolv ed and the reaction completed. The solvent was removed under reduced pressure. The crude product was ground and then dissolved in absolute ethanol. The resulting cloudy solution was filtered, and the filtrate was concentrated to dryness to produce 8 as whi te crystals in 71% yield (melting point: 270 271 C; literature value 209 : 272 273 C). 1 H

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104 NMR (300 MHz, D 2 O ) ppm 3.30 (s ) 13 C NMR ( 75 MHz, D 2 O ) ppm 54.99. ( )ESI MS: m/z 110.9589 [M Na] (calculated: 110.9580). Genera l method s for reactions with sodium methanethiosulfonate 8 208 A 20 mg sample of subs tituted piperidine ( 1 or 2 ) was stirred with sodium methanethiosulfonate 8 at room temperature and then heated in 1mL solvent as listed in Table 2 4. The progress of the reaction was monitored by TLC (silica; eluent with CH 2 Cl 2 /CH 3 OH in ratios of 10:1 (for 1 ) or 2:1 (for 2 reagents. The Dragendorff solution, which was used to track tertiary amines ( 1 and 4 ) and/or quaternary amines ( 2 and 3 ), was prepared by mixing solution A (1.7 g basic bismuth nitrate and 20g tartaric aci d in 80 mL water) and solution B (16 g potassium mM 5,5 dithiobis 2 nitrobenzoic acid and 0.5 mM dithiothreitol in methanol (pH is adjusted to 8 using trimethy l amine), and will yield a white spot against a yellow background for sulfhydryl reagents ( 3 and 4 ). The resulting reaction mixture of entry 1 was added to 10 mL ethyl ether, the precipitate was filtered, collected and showed no presence of the desired pro duct. 2.4.4 Electrophysiology Experiments were conducted using OpusXpress 6000A (Molecular Devices, Unio n City CA). OpusXpress is an integrated system that provides automated impalement and voltage clamp of up to eight oocytes in parallel. Cells were auto matically bath perfused with Ringer's buffer (115 mM NaCl, 10 mM HEPES, 1.8 mM CaCl 2 2.5 mM KCl, 1 M atropine, pH 7.1 to 7.3) and both the voltage and current elect rodes were filled with 3M KCl. The agonist compounds were delivered from a 96 well plate and applied via

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105 disposable tips to eliminate any possi bility of cross contamination. Drug application alternated between ACh controls and experimental applications. Cells were voltage clamped at a holding potential of 60mV. Data were collected at 50Hz and filtered at 20Hz. F low rates were set at 2mL/min. Unless otherwise indicated, drug applications were 12 s in duration followed by 181 s washout periods. Each oocyte received two initial control applications of ACh, a 60s 1mM SH reagent app lication (initia l flow rate 2 mL /min for 10 s, followed by 0.5 mL /min for 50 s), a follow up control application of ACh, then an application of 300 M PNU 120596, and another follow up ACh control a pplication. Control ACh was 60 M for 7 wild type, 7 C116S, and 7 L119C ; 100 M ACh for 7 L38C, and 300 M for the other mutants. These concentrations were empirically determined to give robust reproducible responses with repeated ACh applications and reflected intrinsic differences in t he ACh potency for the mutants 204 Due to the rapid desensitization of 7 nAChR, the net charge values were generally used to compare the experimental responses. 219 However, PNU 120596 was capable of allowing agonists to induce enormous and sustained responses, which introduced la rge variance into the c alculated net charge response. Therefore peak responses were compared for treatments after using PNU 120596. 219 All of the experimental responses were calculated relative to the average o f the first two ACh control responses to normalize the data and compensate for the varying levels of channel expression among the oocytes. Means and S.E.M. were calculated from the normalized responses of at least four oocytes for each experimental concent ration. Individual oocytes were used for no more

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106 than one test concentration, because SH reagents are potentially able to form co valent bonds with the receptor. Whenever PNU 120596 was used, the cells were discarded afterwards; the bath was cleaned with et hanol and flushed with Ringer's buffer for 20 min. The protocol for study of the reduced C190 C191 C loop disulfide was as above, except that after the initial ACh controls, the 7 C116S nAChRs were treated with 1mM dithiothreitol (DTT) for 60 s (initial f low rate 2 mL / min for 10 s, followed by 0.5 mL /min for 50 s) plus additional minutes, as indicated, via static bath (i.e. stopping the flow of Ringers wash to retain the reagent in the bath). Then a follow up ACh test was applied before a 60 s treatment w ith an SH reagent, followed by ACh, PNU 120596, and another ACh application. All of the responses were normalized to the average of the two initial ACh control applications and compared as described above. The SH reagent labeling kinetics experiments simi larly involved incubating receptors for va rious periods of reaction time prior to washout of reagent. The comparative concentration response data between wild type and C116S receptors for Br ACh, MTSET, MTSEA, and QN SH were collected from 100 M to 3mM, u sing 300 M ACh as control prior to and after each application of the reagent. Data were analyzed as described previously 219 2.4.5 Molecular modeling A homology model for the human 7 nAChR was created by Yeu ng Chiu using the Aplysia californica AChBP structure (PDB ID 2PGZ) to select the cysteine mutant candidates 164 A ClustalW 220 alignment of the AChBP and human 7 sequence was generated and submitted to the Swiss Model structure server. The resulting monomeric

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107 model was superimposed twice on the A and B chains of the AChBP pe ntameric crystal structure in order to gener ate a dimer model (Figure 2 2A). The model was then examined for clashes which subsequently resolved by variation of side chain rotomers, or in combination with constrained minimization using the GROMOS force fie ld resident in the SPDB viewer 4.0. This was followed by Amber 10 (Case et al., 2008, University of California, San Francisco) molecular mechanics refinement with the bound 2PGZ ligand (cocaine hydrochloride) included to prevent collapse of the LBD dur ing structural optimization. Docking was performed with the Dock 6.1 program, (Lang et al., 2006, University of California, San Francisco) with evaluation of dock scores based on a grid of 0.3 spacing. Homology models for MTSET and MTSEA labeled cysteine mut ants and energy minimization of these structures in AMBER required definition of atoms and force field parameters, which were obtained by Dr Horenstein via computati on on model labeled cysteines. The labeled adduct formed between N formyl cysteine carboxam ide and MTSEA or MTSET was built I ts structure was m inimized, and charges were calculated with MOPAC and AM1 parameters in ANTECHAMBER. Force constants were identified for the structures with the generalized amber force field, GAFF, and these values were transferred as required to allow recognition of the labeled residues of the protein model in the xleap routine of AMBER10.

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108 CHAPTER 3 3 DESIGN AND USE OF NOVEL MOLECULES TO S TUDY THE HYDROGEN BONDING EFFECT IN TH E HUMAN 7 NICOTINIC ACETYLCH OLINE RECEPT OR LIGAND BINDING SITE 3.1 Background In chapter 2, different binding conformations were found to lead to different functional states of the 7 nAChRs, depending on the deposition of the core charged pharmacophore. However, as introduced in chapter 1, stru ctural variation within the the receptor desensitizes. 199 This chapter presents experiments designed to investigate the mol ecular interactions between the ligand and receptor in the 7 selectivity pocket and their influences on the different functional states of the receptor. A general trend among benzylidene anabaseines is that polar substitutions on the phenyl ring facilita te the activation of the 7 nAChRs (e.g. 4 OH GTS 21 in Figure 3 1). 199 One hypothesis is that the polar group can form a hydrogen bond with the receptor to increase P open Among non bonding interactions, H bonding is one of the modulation of channel gating. 157, 221, 222 However, prior studies mainly focused on the receptor residues interacti ng with the core pharmacophoric features of non selective ligands, namely the charged nitrogen and the H bond acceptor, as found in molecules such as acetylcholine and nicotine. 157, 221, 222 A systematic hydrogen bo nding pharmacophore analysis is reported here both for the ligand and the receptor in the human 7 selectivity pocket. Although there are three 7 selectivity motifs as described in chapter 1, compounds utilizing the selective arylidene motif were focused in order to strictly control whether the aromatic ring was H

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109 bond accepting, donating, or neither (Figure 3 1). H bond effects have been considered in channel activation, desensitization, and the nature of the desensitized state. Figure 3 1. The structu re of the hydrogen bonding probes in comparison with the structure of 4 OH GTS 21. The goal of this section is to provide a semi quantitative description of the free energy profile for the functional states of 7 nAChRs. As introduced in Chapter 1 (Figure 1 13), 7 nAChRs have different free energy diagrams depending on the ligand occupancies. Initially, all receptors are in the resting closed state (C state). When an agonist is applied the apparent functional changes of the receptor arise due to the shift in the receptor equilibrium from the receptor free of ligand to the receptor with a certain ligand occupancy. ( Note : t hese models assume that the agonist binding steps occur as

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110 a result of a rapid change in ag onist concentration) 139 Since 7 nAChRs can potentially accommodate five ligands per receptor, it is difficult to determine when the 7 receptor is in the low or intermediate ligand occupancy levels, and it is also difficult to compare these energy profiles among receptors bound with different ligands. Therefore, all of the experiments in this section have been set up to compare the receptors in high occupancy levels of ligands and all the discussions refer to the energy profile of the receptor in this occupancy level When an agonist is applied to the receptor, the open state is the only state conductive to ions. Therefore, the receptor activation response can be correlated with the ability of an agonist to lead to the open state of the receptor. Before the discovery of positive allosteric modulators the non conductive desensitized states of nAChRs were experimentally unapproachable. In this section, we used type II PAM, PNU 120596, to probe one of the desen sitized states, which can be reactivated by PNU 120596. A s shown in Figure 1 13, the functional states of the receptor are closely related to each other, and the observed experimental response of the receptor is a composite result of all of the functional states. For example, when an agonist is less effective than acetylcholine at activating the receptor, it may be either due to destabilization of the open state of the receptor bound with this agonist, or to a stabilization of the desensitized states relati ve to the acetylcholine bound form. To untangle the complex system and semi quantitatively correlate the free energy profiles of the new ligands with the experimental outcomes, the assumptions described in the following paragraphs were made.

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111 The relative efficacy value of a ligand ( I max ) is used to assign the energy barrier to enter the open state from the closed state. The influenced both by the relative energy level and energy barrier of entering the open state from t he closed state, and the relative barriers to desensitized states and their energy levels. (Figure 1 13). However, the open state has much higher energy than the other functional states of the open The 7 receptor r arely opens, and desensitizes quickly. Therefore, the ability of the receptor to be in the open state (i.e., the size of the activation response or I max ) will be determined mainly by the relative energy barrier instead of the energy level of the open state Note that change of the other functional states, such as the energy level and barrier of the D s state, will also affect I max but those changes will be evaluated by other experimental outcomes. In this chapter, I max is used only to generate a hypothetica l estimate of the energy barrier of the receptor open state. To the best of our knowledge, there is no direct way to evaluate the energy of D s However, as discussed in Chapters 1 and 2, PNU 120596 can not only increase the ivation on the receptor, it can also reactivate th e desensitized receptor. 142 We introduce the hypothesis that application of PNU 120596 to a D s state will instantaneously, without perturbation, report on the D s sta te by rendering it conductive. When PNU 120596 is co applied with an agonist to the receptor, t he peak current is a dynamic parameter, the height of which is correlated with the barrier for entry of the ligand receptor complex into the D s state: the higher the peak, the lower the barrier for entry into D s On the other hand, co application of an agonist with PAM PNU 120596 can produce a sustained current without decay, which results

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112 from the ability of PNU 120596 to reactivate the D s state of the receptor. 142 Therefore, the more stable the D s state (lower energy level) is, the longer the current will be sustained. A larger value will be obtained from the net charge to peak response ratio of the co application response Note that these energy models assume that PNU 120596 does not perturb the open state of the receptor. Regarding the D i state, detection is difficult because it is non conductive under any circumstance. In a previous study, we identified D i during strong episodes of activation, and this state is stable in the presence of the PAM. As mentioned in chapter 1, another feature of the benzylidene anabaseines is their different residual inhibition and response to an acetylcholine probe application. 199 Although the D s state has been suggested to contribute to the RID effect, 199 we used the RI D effect to assign the energy barrier of the D i state, which is assumed to be the lowest energy state and more likely to be dominant in the RID effect up to one half hour. 139 In this section, the contributio ns of the D i and D s states in generating RID have also been investigated in more detail. The relative energy levels of the D i and D s states were estimated based on analysis of RID effects and PNU responses. By use of the H bonding probes shown in Figure 3 1, these experimental data provide a useful approach to decipher the molecular interaction between ligand and protein, and reveal new insights into the pharmacological functions of PAMs. 3.2 Results and Discussion 3.2.1 Synthesis of the Hydrogen Bonding Pr obes (Novel Arylidene Anabaseines) Since benzylidene substituents of anabaseine can activate n AChR selectively 131 compounds with pyridinyl pyrrolyl furanyl, or thiophenyl aryl motif s were

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113 proposed to probe for the H bonding effects in the selectivity pocket (Fi gure 3 1) The synthetic schemes for the arylidene anabaseine s are shown in Figure 3 2. The aryl carboxa ldehydes were purchased from Sigma Aldrich, except for 3 pyrrole carboxaldehyde, which was synthesized from the triflic acid catalyzed isomerization of 2 pyrrole carboxaldehyde in 32% yield 223 The tedious continuous extraction step was omitted in favor of two consecutive chromatographic steps (both using CH 2 Cl 2 /EtOAc at 12:1 ratio as the mobile phase), a dry silica column followed by a normal silica gel column. 223 As shown in Figure 3 2, the gram scale s ynthesis of anabaseine utilized a mixed Claisen type condensation between N protected piperidinone 9 and ethyl nicotinate. 224 The synthesis of 9 proceeded in low yields, so I sought to optimize the Mannich reaction of valerolactam leading to N protected 9 Substitution of paraformaldehyde for aqueous formaldehyde, use of a Dean Stark trap, and use of two equivalents of paraformaldehyde and diethylamine led to protected 9 in 85% yield after vacuum distillation. Mixed Claisen condensation of 1 (diethylaminomethyl) 2 piperidone 9 and ethyl nicotinate afforded the sodium salt, 10 in 71% yield. Compound 10 were identifie d as a mixed salt containing multiple isomers (Appendix A 10), which may include two enolate forms and/or a keto form of compound 10 as illustrated in Figure 3 2. Considering that the isomeric mixture will not impact the hydrolysis and cyclization in the n ext step, the synthesis was continued without further characterization or purification of this compound. Conversion of 10 to anabaseine dihydrochloride was achieved by refluxing in a 5:1 concentrated HCl/acetone mixture. After recrystallization

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114 from absolu te ethanol, the anabaseine dihydrochloride salt 11 was obtained as a white solid in 55% yield. Figure 3 2. Synthetic schemes for the arylidene anabaseines adapted from the article written by Wang, J. et al 227

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115 The general method for synthesis of benzyl idene anabaseines and aryl analogs involves aldol type condensations between an aromatic aldehyde and anabaseine, which presumably reacts via the enamine form of anabaseine. Although the reaction may be catalyzed by acid, for example, HCl, buffer systems s uch as acetic acid/acetate can be utilized for aromatic carboxaldehydes with strong electron withdrawing groups. 225 Thus, condensation of anabaseine dihydrochloride and aryl carboxaldehydes ( 12 15 ) in met hanolic solution optimally produced 16 19 in the presence of acetic acid and sodium acetate (mole ratio: 3 1) at room temperature (Figure 3 2). It was important to conduct these reactions in an inert atmosphere (N 2 or Ar) to minimize side product formation Although some aldol condensation reactions can require heat to effect dehydration, 226 double bond formation can often be readily achieved at room temperature. In the present system with aryl carboxaldehydes, higher temperature tends to adversely affect the yields for condensation. For example, in the synthesis of 19a running the reaction at 60, 40 and 25 C gave 17%, 34% and 58% yields, respectively. The yields for the nine arylidene anabaseines were 35% to 65% as shown in Figure 3 2. Figure 3 3. NOE enhancement used for assigning the olefin geometries of 16a 16b and 19a c adapted from the article written by Wang, J. et al 227

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116 The E double bond geometries for 16a b and 19a c were confirmed by NOESY (Figure 3 3, Appendix A 22 to A 27). In the case of the pyrrole compounds 16a, b irradiation of H5 produced a positive NOE on H 8 of the pyrrole ring, leading to confirmation of the E olefi n geometry for compounds 16a and 16b For 19a c irradiation of H 6 resulted in enhancement of both H2 and H4, which would only arise from the E isomer of 19 seen in all of the three 19 isomers. MOPAC semi empirical calculations reveal ed that the two aromatic rings found in compounds 16 or 19 can stack face to face in the Z isomer, but this does not compensate for other destabilizing effects; the Z isomers for the five 16 and 19 are a ll higher in energy than the E isomers by more than 4 kcal/mol (MOPAC). Table 3 1. The predicted iminium cation percentages of the arylidene anabaseines. The pKa values are calculated from the chemical shift ( obtained from CDCl 3 ) of the proton on the carb on adjacent to the iminium cation (H )= 0.055pKa+4.29 (correlation coefficiency: 0.999). 225 No error bars were reported for the calculated pKa because of limited inf ormation available from the paper that reported the equation above. The protonation percentage of the iminium cation (HA%) is calculated at pH=7.0 via equation Ka= ([H + ][A ])/[HA]. 2FAB 3FAB 2PyroAB 3PyroAB 2TAB 3TAB 2PAB 3PAB 4PAB (H) 3.84 3.82 3.75 3. 78 3.83 3.84 3.91 3.92 3.92 pKa 8.18 8.55 9.82 9.27 8.36 8.18 6.91 6.73 6.73 HA + % 94 97 100 99 96 94 45 35 35 The basicity of the imine nitrogen has been suggested to affect the benzylidene 228, 229 Therefore, the iminium cation percentages (HA + %) of the nine arylidene anabaseines were predicted using the method reported by Zoltewicz, J. A. et al. (Table 3 1). 225 The three PABs ( Figure 3 1) are predicted to be less than 50% protonated, while the other six arylidene anabaseines are almost completely protonated. Initial electrophysiological characterization showed that the three

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117 PABs had no detectable responses below 100 M. To simp ly the system, only the other six arylidene anabaseines were used as the H bonding probes for the following studies, so that the functional differences identified for the compounds would not be due to any protonation state difference at the imine nitrogen. 3.2.2 The Human 7 Receptor Mutants After preparing the H bonding probes, the next step was to identify their hydrogen bonding partners inside the receptor. T he computational prediction derived from the human 7 homology model was first examined using the AChBP crystal structure complex with 4 OH GTS 21 (2WN9) as the template. Among the residues within 5 of the benzylidene motif of 4 OH GTS 21 glutamine 57 (minus face) orients at an ap propriate angle for H bonding. Glutamine is capable of acting as an H bond donor and/or an H bond acceptor, and can form a hydrogen bond with PyroABs and FABs, respectively. Therefore, four mutants at this position with unique hydrogen bonding properties were prepared to further investigate the relationship between the H bon din g pattern and the receptor function. The h 7Q 57 K receptor can form an H bond with the furan ring of the two FABs while the h 7Q57D receptor and the h 7Q57E receptor can form an H bond with the two PyroABs. The h 7 Q57L mutant and the two TABs were prepa red to serve as non hydrogen bonding controls, but they also present a degree of hydrophobicity which may not be present in FABs or PyroABs. For the evaluation of data in a study such as this, the first consideration should be the impact of each mutation o n receptor function and/or assembly. Because acetylcholine is used as a reference response control for the other measurements in all

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118 determined (Figure 3 4 and Table 3 2). An exp ression test was initiated to determine the effects of the mutations on the receptor open probability (P open ) and assembly efficiency to help determine the best time to collect the data. This test involved using the two electrode voltage clamp apparatus on oocytes having the same length of time to express the receptor. The response levels of the mutants with 300 M ACh were compared with the wild type on the same day using oocytes injected with mRNA four days previously. The results are summarized in Figure 3 4 and the representative traces are shown in Appendix B 1. Among them, the h 7Q57L and h 7Q57E receptors showed statistically significant (P < 0.01) decreases in the ACh evoked responses, the net charge responses of which were 12% and 21%, respectively, relative to the wild type receptor. The h 7Q57K and h 7Q57D receptors showed small changes in their expression levels, but they were not statistically different from the wild type. Since acetylcholine is used as reference for all the other experimental co mpounds, a lower response level to acetylcholine can greatly affect the experimental error of the other compounds, especially the response value for some of the partial agonists (partial agonists are molecules inducing smaller response than acetylcholine), such as the H bonding probes used here. Therefore, cells needs to be expressed more than five days for experiments using the h 7Q57L and h 7Q57E mutants, while for the wild type (WT) receptor, the h 7Q57K and h 7Q57D mutants could be used two days after i njecting mRNA. In the concentration response curves (CRCs) of 7 agonists, 300 M acetylcholine was generally used as a control to normalize the difference of the expression level from

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119 cell to cell in the wild type 7 receptor. 176 Higher acetylcholine control concentration has also been used for some mutants to achieve maximum response. 230 To identify the Figure 3 4 The electrophysiological characterization of the wild type 7 receptor and its glutamine 57 mutants. A) The expression level of the wild type and Q57 mutants. The net charge response of each oocyte is normalized to the average net charge response of the wild type receptor. **, p<0.01 The data represent the average responses of at least four ooctyes ( S.E. M.). B ) Concentration response curves plotted at ACh concentration from 0.3 M to 1 mM. C ) Representative traces of the ACh evoked responses. minimum concentration for acetylcholine to achieve maximum response in these glutamine 57 mutants, concentration response curves were plotted for these mutants and displayed with the result of the 7 wild type receptor in Figure 3 4 B. The calculated potencies and efficacies are displayed in Table 3 2. The potencies of the wild type 7 receptor, h 7 Q57L and h 7 Q57E m utants were very similar, with EC 50 values of 31 3 M, 26 3 M and 36 4 M respectively. The EC 50 of the h 7 Q57K mutant

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120 increased two times, compared to that of the wild type receptor (Figure 3 4 B, Table 3 2). Although the potencies varied among the mutants, 300 M acetylcholine was sufficiently concentrated to achieve a maximum response with all of the h 7 receptors tested (Figure 3 4 B), and therefore acetylcholine was used at this concentration as a control for all of the following experi ments. Table 3 2. The potency (EC 50 ) and recovery of acetylcholine with the wild type and h 7 Q57 mutants evaluated by the net charge response For acetylcholine, I max value was fixed at 1.0 to obtain the potency from the Hill equation. WT Q57L Q57K Q57D Q57E EC 50 / M 31 3 26 3 65 3 18 2 36 4 Recovery/% 88 5 87 6 98 2 92 2 96 6 The wild type 7 receptor features low open probability and quick desensitization at high ligand occupancy, which contributes to sharper and sharper response s with increasing agonist concentrations (Figure 3 4 C). 231 This feature was maintained in the mutated receptor. As shown in Figure 3 4 C, the response change of h 7Q57K mutant (from 100 M to 300 M ACh) was similar to that in the wild type receptor (from 30 M to 100 M ACh). The concentration shift in the h 7Q57K mutant corresponds to the potency shift as shown in Table 3 2 and Figure 3 4 B. The desensitization profiles of the 7 receptor and glutamine 57 mutants were evaluated by the type II allosteric modulator, PNU 120596 (Figure 3 5). The potentiation effects of PNU 120596 on the ACh evoked peak response are c ompared in Figure 3 5 A to evaluate the energy barriers to enter the D s state for different h 7 receptor types. The energy levels of the D s state of acetylcholine bound receptors are displayed in terms of the net charge to peak response ratio wh en PNU 120596 was co applied with acetylcholine (Figure 3 5 B).

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121 Figure 3 5. Comparison of PNU 120596 simulated responses f or the different h 7 receptors when co applied with acetylcholine Each oocyte received two initial 300 M ACh controls followed b y a solution of 300 M acetylcholine and 300 M PNU 120596. The peak and net charge responses were normalized to the average of the two initial controls. The net charge to peak response ratio was calculated for each ooctye. Data represent the averages of a t least four oocytes ( S.E.M.) A) Peak current. B) Net charge to peak response ratio. Based on our hypotheses described earlier, a larger peak current value in panel A represents a lower energy barrier to enter the D s state, while a larger value of the ne t charge to peak response ratio in panel B represents a lower energy level for the D s state, i.e., a more stable D s state. None of the glutamine 57 mutants showed a desensitization profile identical to that of the wild type 7 receptor, as illustrated in F igure 3 5. The h 7Q57L mutant displayed higher potentiated peak current by PNU 120596 than the wild type, while the net charge to peak ratio for h 7Q57L was similar to that of the wild type. Based on our hypotheses described earlier, this would suggest the h 7Q57L receptor has a lower energy barrier to enter the D s state than the wild type receptor while the energy levels of their D s states are abou t the same. Therefore, the h 7Q57L receptor will be more likely to enter the D s state than the wild type receptor. On the other hand, the h 7Q57E receptor may disfavor the D s state by increasing the D s state energy level (Figure 3 5 B) while keeping

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122 the en ergy barrier to enter the D s similar to that of the wild type. When mutating glutamine 57 to aspartate, the energy barrier to enter the D s might have increased while the relative energy level of the D s state might have decreased (Figure 3 5). Based on our hypotheses described earlier, the data of the h 7Q57K receptor suggested a decreased energy barrier and an increased energy level for D s due to the mutation. Therefore, the D s state distribution changes in the latter two receptors may depend on how long th e ligand has been in the receptor. The h 7Q57K receptor will enter the D s state quicker while the h 7Q57D receptor will stay in the D s state longer after bound with acetylcholine. The residual inhibition and desensitization (RID) of acetylcholine on differ ent receptor types were evaluated by the recovery test. 199 In a recovery test, the responses of the ACh controls are compared before and after the application an experimental compound to investigate whether the comp ound remains in the receptor to prevent the receptor from activated by ACh. The recovery tests result of 300 M ACh are compared in Table 3 2. However, the run down effect difference in oocytes from batch to batch will cause variation of the recovery data. To set up a standard for the RID effect, 75% was chosen as a cut off value for recovery; lower than 75% is considered as the presence of the RID effect. As shown in Table 3 2, neither wild type 7 receptor nor glutamine 57 mutants showed a RID effect with acetylcholine. Glutamine 57 is not conserved in different nAChR subtypes, but conserved in 7 receptors from different species. 204 It is not close to the positively charged nitrogen of active ligands when they are docked in the homology model. Therefore, the 7 receptor functional changes of th e Q57 mutants reported here may be due to an indirect

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123 influence on the action of acetylcholine via some other residues in the receptor. For example, glutamine 57 is close to tryptophan 55 on the same strand and leucine 119 on the 1 strand (Figure 2 2), both of which are important for agonist binding and PNU 120596 potentiation as illustrated in Chapter 2. Mutation on 57 may change the allosteric communication inside the receptor, thus resulting in di fferent activation and PNU 120596 potentiation profiles of acetylcholine. These have also been considered in the following sections when discussing the arylidene anabaseines among the mutants. 3.2.3 A ctivation P rofile of H bonding Probes on the Wild T ype H uman 7 R eceptor and Mutants Similar to the benzylidene anabaseines, 214 the six arylidene anabaseines were not able to induce detectable responses on the 4 2 or 3 4 receptor subtypes, but they activated 7 receptors to various degrees. 227, 228 The 7 selective benzylidene motif can be further extended and redefined as an 7 selective arylidene motif. The potencies and efficacies of the six arylidene anabaseines with the 7 wild type receptor were obtained from conc entration response curve (CRC) analysis (Figure 3 6) and are compared in Table 3 3. The representative traces are shown in Figure 3 7. All of the six arylidene anabaseines are partial agonists for the h 7 wild type receptor, and induced a maximum net charg e response equal to or less than half of the acetylcholine response. 3FAB was the weakest agonist among the six arylidene anabaseines, activating less than 14% net charge response relative to acetylcholine at 300 M. In contrast, 2FAB, which only differs f rom 3FAB in the H bond acceptor orientation, induced 50% net charge response relative to acetylcholine. The two PyroABs (H bonding donor) also activated 7 receptors. 3PyroAB is as effective as 2FAB in the wild type 7 receptor

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124 (Figure 3 6 and Table 3 3). Although 2PyroAB is less efficacious than 2FAB and 3PyroAB (I max 27%), it is more potent, with a EC 50 value of 5.8 0.9 M Without any H bonding ability, 2TAB and 3TAB yielded similar CRC results (Figure 3 6), with efficacies around 23% and potencies a round 46 M. Based on our hypotheses that the relative height of the energy barrier to enter the open state is determined by the value of I max entry into the open state, the tendency of which is decided by the pattern and the orientation of the H bond in the arylidene motif binding pocket. Figure 3 6. Concentration response curves of the arylidene anabasein e s (0.3 M to 300 M) shown because most of t hem showed detectable responses only at 300 M To investigate whether glutamine 57 is the H bonding partner for these arylidene anabaseines and how the H activation profiles of the six arylidene anabaseines were studied with the four glutamine 57 mutants. The results are compared with the wild type receptor in Figure 3 6 and

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125 Table 3 3. The representative traces are shown in Figure 3 7. Although the expression level of the wild type and glutamine m utants varied (Figure 3 4), all of the arylidene M ACh control, they were also further normalized to the maximum response of acetylcholine with that particular type of receptor mutant. The resul ting efficacies would be valid for interpretation of the H bonding network on receptor activation regardless of each 7Q57D and h 7Q57K mutants (Table 3 2). This will be considered when discussin g the potency change of the arylidene anabaseines among the mutants. Table 3 3 The efficacy (I max ) and potency (EC 50 ) of the six arylidene anabaseines with the wild type h 7 receptor and h 7 Q57 mutants evaluated by net charge. Some drugs show response s o nly at 300 M; their net charge value is reported as I max and efficacy is displayed as N/A WT Q57L I max EC 50 I max EC 50 2FAB 0.50 0.02 16 2 0.55 0.04 11 2 3FAB 0.13 0.00 83 3 12% N/A 2PyroAB 0.27 0.01 5.8 0.9 0.14 0.02 8.8 3.4 3PyroAB 0.44 0.04 17 5 0.45 0.02 4.7 0.9 2TAB 0.23 0.02 46 9 0.17 0.01 54 9 3TAB 0.25 0.00 46 1 0.26 0.02 15 4 Q57K Q57D Q57E I max EC 50 I max EC 50 I max EC 5 0 2FAB 0.29 0.00 49 2 0.40 0.01 6.8 0.6 0.35 0.02 34 4 3FAB 4% N/A 0.14 0.01 45 9 4% N/A 2PyroAB 0.15 0.01 37 8 0.44 0.02 2.1 0.3 0.20 0.01 11 2 3PyroAB 0.25 0.01 28 1 0.65 0.03 5.4 0.8 0.48 0.03 4.4 1.1 2TAB 0.08 0.00 101 1 0.19 0.02 34 9 0.15 0.02 34 12 3TAB 0.11 0.00 98 5 0.34 0.02 18 3 0.18 0.03 38 19 As shown in Figure 3 5 and Table 3 3, the activation profiles of t hese compounds vary differently among the four mu tants compa red to the wild type receptor. But there are some general trends. When mutating glutamine 57 to leucine or lysine, 2PyroAB

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126 Figure 3 7 Representative traces of the 300 desensitization profile on wild type 7 and glutamine 57 mutants. Each oocyte received a 300 M ACh control, a 300 M arylidene anabaseine treatment followed by another 300 M ACh control. All of th e traces were scaled to the ACh control response s applied before the arylidene anabaseines the absolute peak values of which varied from 1.9 A to 11 A. almost failed to activate the receptor (representative traces in Figure 3 7). The efficacy of 2PyroAB decreased by one half if glutamine 57 was mutated to leucine (Table 3 3), while the other observed for 2PyroAB in the Q57K mutant that lacks an H bonding acceptor for 2PyroAB. However, such a decrease was not observed in Q57D and Q57E mutants that

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127 are still able to accept an H bond from 2PyroAB (Figure 3 6, Figure 3 7 and Table 3 3). These data suggested that in the receptor activation by arylidene anabas e ines, glutamine 57 may be crucial for accepting an H bond from the 2 position for the arylidene motif in the 7 selectivit y pocket. The activation profile changes of 2FAB and 3PyroAB to these glutamine mutations are less dramatic than those of 2PyroAB. The 2FAB induced a maximum net charge response similar to that of 3PyroAB did in the wild type receptor. Such similarity was maintained in the h 7Q57L and h 7Q57K receptors (Figure 3 6). However, i f the H bond max values changed to 0.40 0.01 and 0.35 0.02 for the Q57D and Q57E mutants, respectively, about a 20% and 30% decrease compared to the wild type receptor (Figure 3 6 and Table 3 mutant remained similar to that of the wild type. When glutamine 57 was mutated to eased from 44% to 65%. These data suggest that glutamine 57 may donate an H bond to the 2 position of the arylidene motif to facilitate bond from the same position. sults with 3FAB and 3PyroAB are not very indicative of the H bonding effect of the 3 position of the arylidene motif in channel activation. 3FAB was very weak at activating the receptor regardless of whether the glutamine was mutated or not (Table 3 3, rep resentative traces in Figure 3 9 A). There were changes observed in 3PyroAB with the mutants, but they are not sufficiently supportive for the presence of the H bonding.

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128 Since the two TABs lack H bonding ability, they should show the same activation of the mutants if H bonding is the only interaction modulating the receptor activation in the arylidene motif binding pocket. This was the case for the h 7Q57K and h 7Q57E mutants as shown in Figure 3 6. However, in the h 7Q57L and h 7Q57D mutants, 3TAB was both more efficacious and more potent than 2TAB, as shown in Figure 3 5 and Table 3 3. In the h 7Q57L and h 7Q57D mutants, 3TAB was twice as potent as in the wild type receptor, and 3TAB was also more efficacious with the Q57D mutant. Aspartate is smaller than glutamine, lysine and glutamate. Thus the size change of 57 residue in the receptor may be more sensitive to 3TAB than 2TAB, resulting in higher potency and efficacy of 3TAB. The difference in the 2TAB and 3TAB activation profiles may also be due to a ste ric/hydrophobic effect, in which the leucine mutant may have optimized the hydrophobic interaction of 57 with 3TAB. There was a global decrease in the profiles of the h 7 Q57K mutant compared to the wild type ( Figure 3 6 a nd Table 3 3). The I max value s for FABs PyroABs and TABs with this mutant all decreased to half of those for the wild type The 300 M 3FAB induced only 4% net charge response relative to the ACh control. The EC 50 values of the arylidene anabaseines with the h 7 Q57K receptor increase d one or more times compared to their EC 50 values in the wild type receptor. Such decrease in potency was also observed in the ACh activation profile of the h 7Q57K receptor (Table 3 2 and Figure 3 4 C). Therefore, one of the f actors contributing to the potency shift of the arylidene anabaseines may be the mutation impact on the charged nitrogen core pharmacophore. Specifically, the Q57K may have an unfavorable electrostatic interaction with the positively charged iminium nitrog en of

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129 the anabaseine moiety. Therefore, the decrease of the arylidene anabaseine efficacy may be due to several factors, including H bonding, steric hindrances, and charge effects and etc. Figure 3 8. Summary of the channel activation via H bonding effe ct. In summary, glutamine 57 on the minus face of the receptor is suggested to interact with the 2 position of the arylidene motif through H bonding to modulate the receptor activation (Figure 3 8). Although having different H bonding properties, both 2FAB and 2PyroAB are suggested to form an H bond with the glutamine 57. The two ligands (2PyroAB and 2FAB) and the glutamine 57 may adopt similar orientations to optimize the H bond interaction to facilitate the channel activation Without the H bond with glut amine 57, 2PyroAB can barely activate the receptor while 2FAB will activate the receptor to a lesser degree. On the other hand, glutamine 57 is not likely to regulate channel activation via H bonding with the 3 position of the arylidene motif. The relative ly high activation of the receptor by 3PyroAB and low activation by 3FAB may result from the interaction of these anabaseines with other amino acids in the LBD. Such results are also consistent with the homology modeling result, in which the 2 position of 4 OH GTS 21 is in closer contact with glutamine 57 than its 3 position (data not shown).

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130 the 7 Receptors and Mutants The modulation of the H bonding effect on the D s state of the receptor was inv estigated for the six arylidene anabaseines using the type II modulator, PNU 120596. Each oocyte received two initial ACh controls followed by a co application of 300 M PNU 120596 and 300 M of one arylidene anabaseine, the response of which was used to e valuate the energy barrier and energy level of the D s state (Figure 3 9) based on our hypotheses described earlier. The representative traces of the PNU 120596 potentiated arylidene anabaseine evoked responses are displayed along with the traces of the ary lidene anabaseine evoked responses in Figure 3 9 A. The summaries of the peak currents and the net charge to peak response ratios of the PNU 120596 stimulated currents are displayed for the six H bonding probes and different 7 receptor types in Figure 3 9 B and C. Interestingly, in the presence of PNU 120596, the weakest agonist 3FAB (Table 3 3 and Figure 3 9 A) was able to activate the h 7 wild type receptor to a similar level as 3PyroAB (one of the best partial agonist among the six arylidene anabaseine s) After mutation of glutamine 57 to different amino acids, 3FAB and 3PyroAB showed various changes in the PNU 120596 stimulated currents (Figure 3 9 A). For example, in the Q57L mutant, 3FAB showed a longer sustained current than 3PyroAB in the presence of PNU 120596 stimulated peak current was about twice as large as that for 3FAB (Figure 3 9 A and B). Such variations among PNU 120596 potentiated responses of the wild type and h 7Q57 mutants were also observed in the other H b onding probes (Figure 3 9 B and C).

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131 Figure 3 9. C omparison of the PNU 120596 stimulated currents of the wild type 7 and Q57 mutants when co applied with the six arylidene anabaseines. Each oocyte received two 300 M ACh controls, and was then treated wi th one of the arylidene anabaseines at 300 M, either with or without 300 M PNU 120596. Each single trace represents a single experim ent, but all of these traces were normalized to the level of its corresponding ACh control, the absolute peak value of whi ch varied from 0.6 A to 10.2 A. These controls were scaled to the same level and omitted for clear presentation. A) Representative traces. B) Peak current of PNU 120596 stimulated responses. C) Net charge to peak ratio of the PNU 120596 stimulated respon ses. The peak and net charge responses were normalized to the average of the two initial ACh controls. The net charge to peak response ratio was calculated for each ooctye. Data represent the averages of at least four oocytes ( S.E.M.) Based on our hypoth eses described earlier, a larger peak current value in panel B represents a lower energy barrier to enter the D s state, while a larger value of the net charge to peak response ratio in panel C represents a lower energy level for the D s state, i.e., a more stable D s state.

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132 As shown for the wild type h 7 receptor (Figure 3 9 B and C), the six arylidene anabaseines showed distinct profiles of their effects on the energy barrier and energy s state. The energy barrier order to enter the D s state, from the lowest to the highest, is as follows: 2FAB < 2PyroAB < 3FAB < 3FAB 2TAB < 3PyroAB (Figure 3 9 B). However, 2FAB and 2TAB showed the highest energy level for entering the D s state, followed by 2PyroAB and 3TAB, then by 3FAB and 3PyroAB (Figure 3 9 C). These suggest that H bonding effects in the selectivity pocket may also influence the desensitization profile of the receptor. In the following sections, the two parameters (barrier and energy level) of the D s state will each be discussed s eparately for the six H bond probes among the mutants. Note that glutamine 57 mutation itself will impact the potentiation profile of PNU 120596 on acetylcholine, a non selective agonist (Figure 3 5). Moreover, the experimental error bars are large for PNU 120596 involved experiments (Figure 3 5 and Figure 3 9). It will be problematic to diagnose the H bonding effect change in different receptors via comparing the relative values of a single arylidene anabaseine in Figure 3 9. Therefore, all of the six aryl idene anabaseines were compared as a group, serving as controls for each other. The following discussions are based on the changes of their relative responses among the different receptor types. As shown in Figure 3 9 B, all of the six arylidene anabaseine s showed much lower PNU 120596 stimulated peak responses than the ACh in the five receptor types (Figure 3 5 A). Based on our hypotheses de s cribed earlier, these data would suggest that the arylidene anabaseines have higher barriers to enter the D s state c ompared to ACh. In the wild type receptor, compounds with polar groups on the 2 position of the arylidene

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133 motif (2FAB and 2PyroAB) showed PNU 120596 stimulated peak response about twice as large as those compounds with polar groups on the 3 position of the arylidene motif (3FAB and 3PyroAB). The differences between 2FAB and 3FAB, 2PyroAB and 3PyroAB were both statistically significant, with P value less than 0.01 in the student T test analysis (n 6). The relatively lower D s state energy barrier of 2FAB co mparing to 3FAB remained in the h 7Q57K mutant (still statistically significant, P < 0.01, n 5), but such pattern was lost in the other mutants. As shown in Figure 3 9 B, the differences between 2FAB and 3FAB were within experimental error and not statis tically significant for the Q57L, Q57D and Q57E mutants. On the other hand, based on our hypotheses described earlier, after mutating glutamine 57, the energy barrier to enter the D s state of 2PyroAB was no longer lower than that of 3PyroAB (Figure 3 9 B). These data suggested that a specific orientation might be being recognized between the ligand and the receptor via an H to the D s state. Many factors may contribute to the H bonding recognition of a ligand b y the receptor. Neit her ligand nor receptor is constrained in the same orientation in a single functional state or among the different functional states. The experimental outcomes above represent the average of a series of events. A hypothetical mechanism explaining the experimental outcome above is displayed for the wild type 7 receptor in Figure 3 10. In the homology model of the h 7 receptor bound with 4 OH GTS 21, glutamine 57 was H bonding with glutamine 117 on the minus face (data not shown). Therefo re, the orientation of the glutamine 57 is constrained in Figure 3 10 to simplify the discussion, with a same deposition relative to all the four arylidene anbaseines (FABs and PyroABs). Likewise, the ligands were also oriented the same in the LBD, to

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134 emph a size the different H bonding orientations and patterns in the arylidene binding pocket in the receptor. Figure 3 10. Hypothetical mechanism of the H bonding effect in modulating the 7 nds for amino acids interacting with the glutamine 57 (minus face) in the receptor (e.g. Q117).

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135 Because the glutamine mutation itself impacts the D s state of the receptor, which compound is H bonding with the receptor cannot be assigned from the data prese nted in Figure 3 9. For example, it may be either because that 3FAB forms a n H bond with glutamine to increase the energy barrier to enter the D s state or that 2FAB forms a H bond with glutamine to decrease the energy barrier to enter the D s state. The mec hanism in Figure 3 10 presented an example that the H bonding effect was proposed to increase the energy barrier to enter the D s state for all the arylidene anabaseines. The alternative mechanism, in which the H bonding effect decreases the energy barrier to enter the D s state, is illustrated in the Appendix B 2. As proposed in Figure 3 10, 3FAB is able to form an H bond with the wild type receptor while 2FAB is not. In the h 7Q57K mutant, such H bonding difference is also observed for the two FABs. The re lative energy barrier pattern of the two FABs remains similar to that of the wild type. However, when glutamine 57 is mutated to leucine, aspartate, or glutamate, the two FABs may be no longer able to form an H bond with the receptor, resulting in an indis tinguishable energy barrier pattern for the two FABs among these mutants. Such loss of H bonding difference can also be used to explain the results of the two PyroABs with the h 7Q57L and h 7Q57K mutants. However, the mutation impacts of the h 7Q57D and h 7Q57E mutants were slightly different for the two PyroABs. One difference between glutamine and these two negatively charged amino acids is the number of the H bonds they can accept. The lone pair of the amino group of glutamine is conjugated with the sy stem of the carbonyl group, therefore, only its carbonyl group will be able to accept an H bond. Aspartate and glutamate have carboxylate instead, which is able to accept two H bonds at different orientations

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136 (illustrated in Figure 3 11). Therefore, the h 7Q57D and h 7Q57E receptors could form an H bond with both of the two PyroABs to eliminate their difference in the energy barrier of the D s state (Figure 3 9 B). Figure 3 11. Hypothetical H bonding effect of the two PyroABs in modulating the energy barri er of D s in glutamine 57 mutants. The H bonding effect may not only impact the energy barrier to enter the D s state, it may also regulate the energy level of the D s state when the receptor is bound with different ligands. Although the PAM PNU 120596 stimul ated peak responses for the six arylidene anabaseines were all smaller than those of the acetylcholine, their net charge to peak response ratios were not (Figure 3 5 B and Figure 3 9 C). The net charge to peak response ratios of the 3FAB and 3PyroAB were 1 .5 times as large as that of the

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137 acetylcholine in the wild type. The net charge to peak response ratios of 2PyroAB and 3TAB were about the same as that of the acetylcholine in the wild type, while those of 2FAB and 2TAB were about half that of acetylcholin e. In the wild type 7 receptor, both 3FAB and 3PyroAB displayed lower energy levels of the D s state compared to 2FAB and 2PyroAB, respectively. The net charge to peak ratio of 2FAB was statistically different from that of 3FAB in the wild type and the h 7Q57K mutant, with P value less than 0.01 in the student T test analysis (n 5). But the difference between 3PyroAB and 2PyroAB was not statistically significant. The hypothetical mechanisms of the H bonding effect in the energy barrier of the D s state ca n also be applied in explaining how the H bonding effect modulates the energy level of the D s state based on our hypotheses described earlier. As exemplified in Figure 3 10, 3FAB may form a hydrogen bond with glutamine 57 to lower the energy level of the D s while 2FAB cannot. The h 7Q57K mutant can keep the same H bonding pattern for 2FAB and 3FAB, while the other mutants cannot. Therefore, the relative energy level patterns of the D s state for 2FAB and 3FAB changed in the h 7Q57D and h 7Q57E mutants compa red to the wild type receptor. The energy level of the D s state for 2FAB is similar to 3FAB in the h 7Q57D mutant and lower than 3FAB in the h 7Q57E mutant (0.01 < P < 0.05, n 5). Since the experimental results cannot rule out that glutamine 57 may form a H bond with 2FAB instead of 3FAB to increase the energy level of the D s state, an alternative mechanism is illustrated in Appendix B 2 to represent that case. There was a unique change in the net charge to peak response ratio of 3FAB in the h 7Q57L muta nt (Figure 3 9 C). This may be due to a unique change of the binding mode of 3FAB in the h 7Q57L receptor. Glutamine 57 is in van der Waals contact with

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138 leucine 119, but 3FAB may impact this contact via direct H bonding or indirect H bonding via a water mo lecule, which is widely present in the ligand binding domain of AChBP crystals. 232 Mutating glutamine 57 to a non polar leucine might have di srupted the network between 3FAB and the two amino acids, thus resulting in a dramatic decrease of the D s state energy level of the ligand bound receptor. Another feature of the benzylidene anabaseines is their residual inhibition desensitization (RID) eff ect. 199 The six arylidene anabaseines were also used to investigate whether the H bonding interaction in the benzylidene motif would impact the control applied after the 300 M arylidene anabaseines. This post ACh response normalized to the response of the ACh control applied before the arylidene anabaseines is called the recovery; the smaller the recovery value, the higher the RID effect. The rec overy results 7 receptor and glutamine 57 mutants are listed in Table 3 4. Table 3 4. Percent recovery of the h 7 wild type receptor and glutamine 57 mutants after treated with 300 M arylidene anabaseines. RID effect is present when the recovery value is lower than 75%. 2FAB 3FAB 2PyroAB 3PyroAB 2TAB 3TAB WT 63 13 95 3 98 2 66 13 61 7 58 7 Q57L 28 6 88 3 78 5 77 4 53 4 47 11 Q57K 86 6 100 2 94 5 85 3 86 6 9 9 2 Q57D 21 3 77 9 98 16 84 4 68 1 53 6 Q57E 81 11 91 1 88 8 77 6 82 3 80 6 In the wild type, 2FAB showed a stronger RID effect than 2PyroAB, while 3PyroAB showed a stronger RID effect than 3FAB (Table 3 4). The two hydrophobi c probes, 2TAB and 3TAB, showed RID effects similar to those of 2FAB and 3PyroAB, with recovery values ranging from 58% to 66% (representative traces in Figure 3 7). These

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139 results suggested that the H bonding donor at the 2 position (2PyroAB) or H bonding acceptor at the 3 position (3FAB) of the arylidene motif prevented the generation of RID effect on the wild type h 7 receptor. Lower recoveries were observed with some mutant receptors when direct H bonding is eliminated, such as 3PyroAB and 2FAB with the h 7Q57L mutant, and 3FAB with the h 7Q57D mutant (Table 3 4). However, only the change with 2FAB is significant. The other changes are small and the resulting recoveries are at the cut off of the RID effect (75%) considering the experimental variation due to the run down effect of the oocyt es. When glutamine 57 was mutated to leucine, lysine, aspartate or glutamate, 2FAB with the h 7Q57L and the h 7Q57D mutant showed the largest RID change as shown in Table 3 4. Less than 30% of the receptors in these mutan ts recovered to be responsive to acetylcholine after treatment with 300 M 2FAB. On the other hand, when glutamine 57 was mutated to lysine or glutamate, the RID effect of 2FAB decreased to be negligible compared to the wild type (Table 3 4 and Figure 3 7) The RID effect enhancement in h 7Q57L and h 7Q57D mutants was unique for 2FAB, possibly due to a unique modulation on the receptor desensitization. As the contribution of D i to RID is not known, the functional states of the receptor after treatment with 300 M 2FAB was investigated with PNU 120596 to decipher how the D s and D i states contribute to the apparent RID effect. 2FAB was chosen because it showed the strongest RID effect among the different receptor types. Instead of treating with 300 M ACh afte r the 300 M 2FAB (Figure 3 7), oocytes expressing different types of receptors received a solution of 300 M PNU 120596 to allow studying the contribution of the D s state to the RID effect. The results are shown in Figure 3 12 A and

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140 B. If RID effect was c aused only by D s and the PNU 120596 stimulated peak responses were the same for the five 7 receptor types binding with 2FAB at equal abundance, the inhibition effect of 2FAB on the subsequent ACh control (equal to 100% minus the recovery percent) would be proportional to the PNU 120596 stimulated peak response among the different receptor types, as illustrated by the dashed line in Figure 3 12 B. However, as shown in Figure 3 12 B, the inhibition on the ACh evoked response after the 300 M 2FAB treatment w as neither linear nor directly proportional to the peak value of the PNU 120596 stimulated response. A similar relationship was obtained even after considering the variation of the PNU 120596 stimulated peak responses after mutating the receptor (in Append ix B 3). The major difference after normalization is the location of the Q57D receptor in the plot. Note that the run down of the cells may have resulted in a right shift of the inhibition on the ACh evoked response after the application of 2FAB (the x axi s in Figure 3 12). Interestingly, although the h 7Q57K and h 7Q57E receptors did not show a detectable RID effect (recovery 75% in Table 3 4) after treatment with 2FAB, they were stimulated by PNU 120596 after the application of 2FAB (Figure 3 12 A and B). This may be due to a small number of channels occupied by 2FAB, which were masked by the cell run down effect in the ACh recovery evaluation but revealed by PNU 120596. The peak current of the PNU 120596 stimulated response after 2FAB treatment of the h 7Q57E receptor could reach a similar level as that of the h 7Q57D and the h 7Q57L receptors, which are very good at generating RID effect (Figure 3 7). This suggests that the RID effect in the h 7Q57D and the h 7Q57L receptors mainly

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141 Figure 3 12. A su mmary of the 2FAB residual desensitization profile with the h 7 receptor and glutamine mutants evaluated by PNU 120596. A) R epresentative traces. The results are displayed in the order of the experiment protocol from left to right with total time of 3.5 mi n between each step: ACh, 2FAB, PNU 120696, ACh, ACh. All of the compounds were applied at 300 M concentration over 12 seconds. B ) Relationship between the PNU 120596 evoked response and the inhibition on ACh evoked response after 2FAB. The values of the inhibition on ACh evoked response were obtained from the recovery values displayed in Table 3 4 and are presented on the x axis. The peak currents of the PNU 120596 stimulated response after 300 M 2FAB are displayed on the y axis. The ratio of the two axe s of the wild type receptor was used as the slope to generate the dashed line. C) R epeated recovery tests of 2FAB with different h 7 receptor types. The cells were repeatedly treated with eight doses of acetylcholine after a single 300 M 2FAB treatment. T he 3.5 minutes Ringer wash is applied between each application. The first time point (3.5 min) corresponds to the time when the PAM PNU 120596 was applied in panel A.

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142 comes from a stable desensitized state which cannot be activated by PAM PNU 120596, i.e., the D i state. Additional features that can be identified in Figure 3 12 A are the various levels of ACh control responses applied after PNU 120596. Because the off rate of PAM PNU 120596 is small, the response of the subsequent ACh control can also be pot entiated if the agonist binding site is not blocked (Figure 2 6). 203 With PNU 120596 being removed decrease over time. Howeve r, this is only the case for the h 7Q57K receptor as shown in Figure 3 12 A. In the h 7Q57D and h 7Q57L receptors; the responses of the ACh controls after the application of PNU 120596 even increased over time. A summary of the peak currents of the last tw o ACh controls is attached in Appendix B 4. The result for the h 7Q57K receptor indicates that there was little, if any, 2FAB left in the receptor to prevent ACh from being potentiated by PNU 120596, which is consistent with the lowest RID effect of 2FAB i n this mutant (Table 3 4). For the other four receptors, the PNU 120596 potentiated ACh responses (Figure 3 12 A and Appendix B 4) resulted from various effects, including the different occupancies and off rates of 2FAB, and different PNU 120596 off rates from various receptors. The 7 receptor has five potential ligand binding sites. Therefore, although the inhibition effects of 2FAB on the receptor are known, the occupancy of 2FAB inside the receptor cannot be determined. The off rate of 2FAB from the fou r receptors (wild type, h 7Q57L, h 7Q57D and h 7Q57E) was investigated via repeated recovery tests as shown in Figure 3 12 C. At the second time points in Figure 3 12 C (corresponding to application of the first ACh control after PNU 120596 in Figure 3 12 A), 2FAB still blocked the

PAGE 143

143 h 7Q57L and h 7Q57D receptors very efficiently. However, at the third time points (10.5 min), the recovery of the h 7Q57D receptor increased much more than the h 7Q57L receptor (Figure 3 12 C). The different off rates of 2FAB in the two receptors may account for the difference of the last ACh control responses of the h 7Q57L and h 7Q57D receptor shown in Figure 3 12 A. On the other hand the similar off rate of 2FAB in the wild type and h 7Q57E receptor (Figure 3 12 C) is also cons istent with their similar PNU 120596 stimulated ACh responses shown in Figure 3 12 A. In all, 2FAB showed the longest and strongest RID effect with the h 7Q57L receptor, followed by h 7Q57D, wild type, h 7Q57E and h 7Q57K. The RID effect can be affected b y the energy levels of the D s and D i states and by the energy barriers to leave from these desensitized states to the closed (unliganded) state. As discussed above, D i contributes more to the RID effect than D s Therefore, the recovery data in Table 3 4 ca n represent the relative energy level of the D i state. The relative energy levels of D s and D i among the different receptor types will be consistent with the y axis to x axis value ratios of each point plotted in Figure 3 12 B. For example, the energy leve l ratio of D s to D i of the Q57K mutant will be 1.9 times of that of the wild type receptor. On the other hand, assuming that dissociation only occurs from the closed state and leads the receptor directly to states with no ligand occupancy the off rate of 2FAB from the receptor can represent the energy barrier for leaving the D i state to the closed state when the receptor is fully occupied with ligands. For example, in the 2FAB profile, the energy barrier between the closed state and the D i state is higher for the Q57L mutant than for the Q57D mutant. However, these RID effect data are not very supportive for modulation effects via H bonding interaction between Q57 and 2FAB.

PAGE 144

144 The changes described above among Q57 mutants may be due to indirect influences resu In conclusion, based on our hypothese s described earlier, the H bonding effects desensitized states. Compo unds with a polar group on the 2 position of the arylidene motif may lower the energy barrier to enter the D s state while decreasing the stability of the D s state. The compounds with a polar group on the 3 position show the opposite trend. The RID effect i s and the D i state, with a main contribution from the D i state. The mechanism of how the modulation takes effect via H bonding is still in question. Due to limited experimental data, only 2FAB was suggested to have a unique interaction with glutamine 57 to impact the D i state, but the impact of this interaction on RID involves unidentified communication with other amino acids in the ligand binding sites. 3.3 Summary In this chapter, a series of probes was d esigned and synthesized to investigate how the interaction between an agonist and the receptor in the 7 selectivity pocket regulates the activation and desensitization of the receptor. Although both the six membered ring and the five membered ring heteroc yclic arylidene anabaseines were synthesized, only the five membered ring arylidene anabaseines were used to probe their H bonding interaction in the receptor. Based on our hypotheses described at the beginning of this chapter, using the efficacy of these ligands and their results of the PNU 120596 stimulated response, the relative energy barrier and/or the energy level of the different functional states can be

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145 effect on th e subsequent ACh control (RID effect), the relative energy level of the D i state can be calculated. An example of the energy landscape for the wild type receptor is shown in Figure 3 13 and a full version with all different receptor types is attached in Ap pendix B 5. All these arylidene anabaseines are 7 selective partial agonists. Their lower efficacies can be due to relative higher energy barriers to enter the open state than ACh, as shown in Figure 3 13. If the receptor can form an H bond with the ligand in the ligand binding domain similar to 2FAB/2 PyroAB with the glutamine 57 (Figure 3 8), the energy barrier to enter the open state will be lowered, compared to the two TABs (Figure 3 13, a vertical scale expanded version of Appendix B 5) and the other mutants that cannot form H bonds with these two c ompounds (A ppendix B 5). However, such point to point H bonding interaction cannot compensate the weak agonism raised by the arylidene anabaseine motif. Therefore, 2FAB/2PyroAB are still partial agonists for the receptor like the other benzylidene anabasei nes 199 and their efficacies are not dram a tically higher than the two TABs. If the H bond acceptor is moved one carbon away from the anabaseine pharmacophore (i.e. 3FAB), the receptor will preferentially go to the de sensitized state, because of the increased energy barrier to enter the open state and the decreased energy level of the D s state (Figure 3 13). If the H bond donor is move done carbon away from the anabaseine pharmacophore (i.e. 3PyroAB), based on our hypo these s described earlier, the possibility that the receptor will be in the open state will increase compared to the other arylidene anabaseines, because of the decreased energy barrier to enter the open state and the increased energy barrier to

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146 enter the D s state compared to the other arylidene anabaseines (Figure 3 13). The two hydrophobic probes (2TAB and 3TAB) display very similar energy landscape profiles. Figure 3 13. The energy landscape of the arylidene anabaseines in comparison with acetylcholine in the wild type receptor. These energy landscapes represent experimental data obtained using ligands at 300 M (high occupancy). Curves with experimental support are highlighted. The abbreviations for the s 120596 i 120596 insensitive desensitized state. One limitation in using PNU 120596 to interpret the H bonding effect arises from the mutation impact on the PNU 120596 potentiation profile. Although PN U 120596 is suggested to bind in the transmembrane domain, some mutations have also been identified in the ligand binding domain that affect the potentiation of the PAM PNU 120596. 202 Considering that the six arylid ene anabaseines differ only slightly from each other in the selectivity pocket, they can be utilized as controls for each other when comparing their desensitization profiles determined by the PAM PNU 120596. The evidence that 2FAB and 2PyroAB have lower en ergy barriers and higher energy levels for D s than 3FAB and 3PyroAB (Figure 3 9) suggests that the orientation of the H bond is important in channel functions. Several orientation examples have been proposed for such recognization of the H bonding network within the 7 selectivity pocket of the receptor (Figure 3 10, Figure 3 11 and Appendix B 2). Although these mechanisms are

PAGE 147

147 still very hypothetical, they provide examples of how to interpret the native receptor function from mutagenesis results. Although the H bondin g effect of glutamine 57 with 2FAB leads to one of the lowest energy barrier for the open state among the six arylidene anabaseines (Figure 3 13), it does not enable 2FAB to have the lowest energy of the D s state among the six probes. We know from the crys tallography of AChBP that specific ligands will stabilize these ligand binding domain analogs in distinct low energy conformations. 165, 166 A single compound may also adopt different conformations to activate or des ensitize the receptor. Perhaps, in the conformation which determines the energy barrier of the open state, the 2FAB is H bonding with glutamine 57 while in the conformation affecting the D s state, this H bonding interaction may be lost (Figure 3 8 and Figu re 3 10). Alternatively, the H bonding effect may destabilize the D s state, thus increasing the energy level of the D s state of the 2FAB bound receptor (Appendix B 2). In conclusion, the results in this section support the hypothesis that both the orient ation and the H desensitization. These H bonding effects can be specifically associated with changing the relative energy levels and/or the energy barriers of the functional states. The energy lands capes generalized in this section summarizes how an agonist goes into the open, D s and/or D i states. This information is key for designing drugs with specific preference for some of the receptor functions. 3.4 Experimental Section 3.4.1 Chemicals Solve nts and reagents for electrophysiology and synthesis were from Sigma Chemical Co. (St. Louis, MO) and Fisher Scientific (Pittsburg, PA). PNU 120596 was

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148 synthesized as described below. 1 H and 13 C NMR spectra were obtain ed using VXR 300, Gemini 300, Mercury 300 (300 MHz) or Inova 500 (500 MHz) spectrometers (Varian, Palo Alto, CA) in appropriate deuterated solvents. Mass spectra were obtained on an Agilent 6210 TOF spectrometer operated in ESI, DART, TOF, MMI TOF or DIP CI ionization mode s rotary evaporator. 1 ((diethylamino)methyl)piperidin 2 one 9. Delta valerolactam (10.0 g, 0.10 mol), diethyl amine (20.8 mL, 0 .20 mol) and paraformaldehyde (6.1g, 0.07 mol) were stirred in 100 mL toluene and heated in a Dean Stark apparatus at reflux for 22 hours. The reaction mixture was cooled to room temperature and concentrated to a total volume of about 35 mL. Then 35 mL bri ne was added to the reaction mixture and the mixture was adjusted to pH 11 using 4 M sodium hydroxide. The organic phase was separated and the aqueous phase was extracted with ethyl acetate (40 mL 2). The organic layers were combined, dried over magnes ium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by vacuum distillation and compound 9 was obtained as a yellow oil (b.p. 133 134 C at 0.25 mm Hg) in 85% yield. 1 H NMR (300 MHz, CDCl 3 ) ppm 1.02 (t, J=7.16 Hz, 6 H) 1.78 (dt, J=6.86, 3.29 Hz, 4 H) 2.35 2.44 (m, 2 H) 2.57 (q, J=7.06 Hz, 4 H) 3.35 (m, 2 H) 4.15 (s, 2 H) Although the boiling point for compound 9 was different from the literature (lit. value: 113 C at 3.1 mm Hg), the proton chemical shifts repor ted here were identical with the literature. 224 TOF MS: m/z 207.1476 [M+Na]+ (calculated: 207.1468 ).

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149 2 Piperidin one,1 [(diethylamino)methyl] 3 (hydroxyl 3 pyridinylmethylene) sodium salt 10. To a mixture of 9 (8.98 g, 0.05 mol), ethyl nicotinate (7.44 g, 0.05 mol) and 50 mL dry toluene was added 3.88 g (0.10 mol) sodium hydride (57 to 63% dispersion in mineral oil) portionwise in an ice water bath. Then the reaction mixture was heated at reflux for four hours and cooled. The unreacted sodium hydride was carefully removed by suction filtration and then washed with hot toluene (40 mL). The unreacted sodium hydride was destroyed by 95% alcohol carefully. The orange filtrate was concentrated to a total volumn of 40 mL under reduced pressure and put in the refrigerator for 16 hours. Compound 10 was obtained as a yellow precipitate formed from the filtrate, which was filter ed and collected in 71% yield. 1 H NMR (300 MHz, DMSO d 6 ) ppm 1.06 (t, J=6.94 Hz, 10 H) 0.96 1.02 (m, 4 H) 1.69 (m, 4 H) 2.27 (m, 4 H) 2.60 (br. s., 10 H) 3.27 (br. s., 4 H) 3.96 (s, 2 H) 4.16 (s, 2 H) 7.14 (dd, J=7.0 8, 5.18 Hz, 1 H) 7.34 (br. s., 1 H) 7.34 (dd, J=7.30, 4.97 Hz, 1 H) 7.45 (d, J=7.60 Hz, 1 H) 7.61 (d, J=7.59 Hz, 1 H) 8.24 (d, J=3.51 Hz, 1 H) 8.30 (s, 1 H) 8.39 8.49 (m, 2 H). Although the proton chemical shifts were not identical with the literature, 224 compound 10 did lead to the production of compound 11 the identity of which was confirmed by proton NMR and high resolution mass spectrometry. An abaseine dihydrochloride 11. Compound 10 was heated at reflux for 19 hours in a 250 mL round bottom flask in a solution of 120 mL concentrated hydrochloric acid and acetone (5:1, v/v; the acetone was added to aid the precipitation of sodium chloride). The reaction mixture was cooled down to room temperature and the fine white precipitate (sodium chloride) was removed by filtration. After concentrating in vacuo to a total volume about 50 mL, more sodium chloride solid formed, which was filtered again.

PAGE 150

150 The fi ltrate was concentrated in vacuo again and then refluxed in distilled isopropanol and ethanol (1:1) with a few drops of water to help fully dissolve the solid. After cooling and storage in the refrigerator for about two hours, the product was obtained as a white solid, which was filtered under Ar. The filtrate was concentrated and recrystallized to recover more product, which gave a final yield of 55 % for compound 11 (m.p., 145 150 C, with decomposition). 1 H NMR (300 MHz, DMSO d 6 ) ppm 1.66 (m, 4 H) 2.8 1 (q, J=5.99 Hz, 2 H) 3.18 (t, J=6.72 Hz, 2 H) 7.87 (dd, J=8.03, 4.67 Hz, 1 H) 8.02 (br. s., 2 H) 8.63 (dt J=8.11, 1.79 Hz, 1 H) 8.94 (dd, J=5.19, 1.53 Hz, 1 H) 9.27 (dd, J=2.12, 0.66 Hz, 1 H). Although the melting point obtained was different from the va lue reported in the literature (literature value: 175 179 C, with decomposition), the proton NMR results were identical with the literature. 224 ESI MS: m/z 161.1075 [M+H]+ (calculated: 161.1073 ). 3 Pyrrollyl carboxaldehyde 12b. 2 Pyrrolyl carboxaldehyde (2.1g, 0.02 mol) was stirred in 20 mL 1,2 dichloroethane along with 5 mL of trifluoromethanesulfonic acid. The reaction mixture was heated at 75 C for 21 hours and then quenched with saturated sodium acetate. The black junk was removed by filtrations and the filtrate was concentrated to dryness in vacuo. The crude product was first purified by passing through a dr y silica column using a mixture of dichloromethane and ethyl acetate (v/v, 1:1) as the eluent. The semi pure product was purified by flash column chromatography (silica, 12:1 dichloromethane/ethyl acetate) to separate the desired product from the unreacted starting material. Compound 12 a was obtained as a light red brown solid in 32% yield. 1 H NMR (300 MHz, CDCl 3 ) ppm 6.61 6.72 (m, 1 H) 6.86 (q, J=2.39 Hz, 1 H) 7.50 (dt, J=3.18, 1.55 Hz 1 H) 9.79 (s, 1 H) 10.15 (br. s., 1 H). 13 C NMR (75 MHz,

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151 CDCl 3 ) pp m 107.43, 121.41, 126.70, 128.72, 186.91. The proton and carbon NMR results were identical with the values reported in literuature. 233 TOF MS: m/z 96.0444 [M+H]+ (calcu lated: 96.0444). General procedure for preparing arylidene anabaseines (16 19). Anabaseine dihydro chloride 11 (100 mg, 0.312 mmol) was dissolved in 1.1 mL of a methanolic mixture of 0.6 M acetic acid (2.5 eq.) and 0.2 M sodium acetate along with three equi valents of the appropriate carboxaldehyde at room temperature The r eaction was protected under argon and tracked by TLC (silic a ). When the reaction finished (5 hours to 2 days) the mixture was quenched by adding deionized water and its pH was adjusted to 10 with solid sodium carbonate. After extraction with ethyl acetate, the organic phase was dried with magnesium sulfate and evaporated to dryness. The crude product was purified by a silica gel column, using a mixture of dichloromethane and methanol as th e eluent. Then hydrochloric acid was carefully added to the arylidene anabaseine ( 16 18 ) alcoholic solution with cooling in an ice water bath, followed by evaporation to dryness to yield the salt form of the product. These arylidene anabaseine salts were m ade daily i 100 mM 19 a was prepared in DMF as stock solution (or 50 mM in DMSO) and was then 19 b and 19 c were diluted in R Some of the arylidene anabaseines have been reported in several patents, including compound 16a (2PyroAB), 234 compounds 17a (2FAB) and 17b (3FAB), 235 the dihydrochloric form of compound 18a (2TAB) and 18b (3TAB), 234 the trihydrochloric form of compound 19b (3PAB). 236 However, the characterizations of these compounds

PAGE 152

152 were poorly reported with only the proton chemical shifts of hydrochloric acids forms of compounds 18a 18b and 19b documented using deuterated DMSO as the NMR solvent. 234, 236 Detailed characterizations of compounds 16 19 are described as following. 2 P yrrolylmethyl ene anabaseine (2PyroAB, 16 a). After chromatography ( silica, 10:1 CH 2 Cl 2 /CH 3 OH), the title compound was obtained in 39 % yield as a brown solid. 1 H NMR (300 MHz, CDCl 3 1.94 (m, 2 H), 2.75 (t, J =5.77 Hz, 2 H), 3.68 (t, J =5.40 Hz, 2 H), 6.32 (t, J =2.99 Hz, 1 H), 6.55 (s, 2 H), 6.82 (s, 1 H), 7.18 7.29 (m, 1 H), 7.75 (dt, J =7.78, 1.88 Hz, 1 H), 8.36 (dd, J =4.89, 1.53 Hz, 1 H), 8.52 (d, J =1.46 Hz, 1 H), 11.0 0 (br. s., 1 H); 13 C NMR (75 MHz, CDCl 3 113.17, 121.88, 123.33, 124.95, 127.59, 129.28, 136.09, 136.89, 148.97, 149.15, 167.87. TOF MS: m/z 238.1347 [M+H]+ (calculated: 238.1339). 3 Pyrrolylmethylene anabaseine ( 3PyroAB 16 b ). After chromatography ( silica, 8 :1 CH 2 Cl 2 /CH 3 OH) the title compound was obtained in 35 % yield as a yellow green solid. 1 H NMR (300 MHz, CDCl 3 J =6.13, 5.89 Hz, 2 H), 2.77 (t, J =6.13 Hz, 2 H), 3.78 (t, J =5.26 Hz, 2 H), 6.33 (br. s., 1 H), 6.57 (s, 1 H), 6.73 (br. s., 1 H), 6.85 (br. s., 1 H), 7.33 (dd, J =7.67, 4.89 Hz, 1 H), 7.82 (d, J =7.59 Hz, 1 H), 8.62 (d, J =4.38 Hz, 1 H), 8.73 (d, J =0.58 Hz, 1 H), 9.83 (br. s., 1 H); 13 C NMR (75 MHz, CDCl 3 22.01, 26.67, 49.92, 110.00, 119. 29, 1 20.51, 120.93, 123.20, 126.23, 130.72, 136.65, 136.80, 149.44, 149.87, 168.23. TOF MS: m/z 238.1337 [M+H]+ (calculated: 238.1339). 2 F uran ylmethylene anabaseine (2FAB, 17 a). After chromatography ( silica, 25 :1 CH 2 Cl 2 /CH 3 OH) the title compound was obta ined in 48 % yield as a brownish yellow solid (m.p., 101 102 C) 1 H NMR ( CDCl 3 1.96 (m, 2 H) 2.80 (td, J =6.72,

PAGE 153

153 2.19 Hz, 2 H) 3.75 (t, J =1.00 Hz, 2 H) 6.27 6.44 (m, 2 H) 6.38 6.38 (m, 1 H) 7.24 (ddd, J =7.89, 4.82, 0.88 Hz, 1 H) 7.40 (s, 1 H) 7.71 (dt, J =7.81, 2.01 Hz, 1 H) 8.55 (dd, J =4.89, 1.68 Hz, 1 H) 8.64 (dd, J =2.19, 0.88 Hz, 1 H) 13 C NMR (75 MHz, CDCl 3 21.34, 26.13, 49.97, 111.97, 113.61, 122.33, 122.87, 128.24, 135.67, 136.04, 143.58, 149.51, 149.55, 151.79, 166.64. D ART MS: m/z 239.1179 [M+H]+ (calculated: 239.1179). 3 F uranylmethylene anabasei ne (3FA B, 17 b). After chromatography ( silica, 28 :1 CH 2 Cl 2 /CH 3 OH) the title compound was obtained at 57% yield as an orange yellow oil. 1 H NMR (300 MHz, CDCl 3 J =6.10 Hz, 2 H) 2.72 (td, J =6.79, 2.19 Hz, 2 H) 3.82 (t, J =5.48 Hz, 2 H) 6.39 6.56 (m, 2 H) 7.34 (dd, J =7.81, 4.89 Hz, 1 H) 7.44 (t, J =1.68 Hz, 1 H) 7.55 (s, 1 H) 7.81 (dt, J =7.78, 1.81 Hz, 1 H) 8.64 (d, J =3.21 Hz, 1 H) 8.73 (br. s., 1 H) 13 C NMR (75 MHz, CDCl 3 122.94, 125.64, 130.03, 135.86, 1 36.10, 142.88, 143.18, 149.50, 149.61, 166.83. ESI MS: m/z 239.1190 [M+H]+ (calculated: 239.1179). 2 Thiophen ylmethylene anabaseine (2TAB, 18 a). After chromatography ( silica, 25 :1 CH 2 Cl 2 /CH 3 OH) the title compound was obtained in 50% yield as a yellow soli d (m.p., 150 151 C) 1 H NMR ( CDCl 3 J =6.10 Hz, 2 H) 2.85 (td, J =6.68, 1.97 Hz, 2 H) 3.83 (t, J =5.48 Hz, 2 H) 6.85 (s, 1 H) 7.03 7.17 (m, 2 H) 7.35 (dd, J =7.81, 4.89 Hz, 1 H) 7.47 (m, 1 H) 7.83 (m, 1 H) 8.65 (dt, J =4.86, 1.15 Hz, 1 H) 8.75 (m, 1 H) 13 C NMR (75 MHz, CDCl 3 128.19, 128.31, 128.82, 130.85, 135.84, 136.19, 138.93, 149.63, 149.67, 166.98, D ART MS: m/z 255.0941 [M+H]+ (calculated: 255.0950).

PAGE 154

154 3 Thiophen ylmethylene anabaseine (3TAB, 18 b). After chromatography ( silica, 3 0 :1 CH 2 Cl 2 /CH 3 OH) the title compound was obtained at 56% yield as a yellow solid (m.p., 87 88 C) 1 H NMR (300 MHz, CDCl 3 J =6.06 Hz, 2 H) 2.85 (td, J =6.65, 2.04 Hz, 2 H) 3.84 (t, J =5.33 Hz, 2 H) 6.64 (s, 1 H) 7.12 (dd, J =4.82, 1.46 Hz 1 H) 7.29 7.40 (m, 3 H) 7.82 (dt, J =7.78, 1.81 Hz, 1 H) 8.64 (br. s., 1 H) 8.74 (br. s., 1 H) 13 C NMR (75 MHz, CDCl 3 129.08, 129.34, 130.22, 136.15, 136.32, 137.26, 149.69, 149.83, 167.29. ESI MS: m/ z 255.0941 [M+H]+ (calculated: 255.0950). 2 P yridinyl methylene anabaseine (2PAB 19 a): After chromatography ( silica, 15 :1 CH 2 Cl 2 /CH 3 OH) the title compound was obtained in 58% yield as a yellow white solid (m.p., 122 124 ) 1 H NMR (300 MHz, CDCl 3 p pm 1.85 (quin, J =6.13 Hz, 2 H), 3.14 (td, J =6.65, 1.90 Hz, 2 H), 3.91 (t, J =5.55 Hz, 2 H), 6.63 (s, 1 H), 7.16 (dd, J =7.52, 4.89 Hz, 1 H), 7.22 (d, J =8.03 Hz, 1 H), 7.34 (dd, J =7.81, 4.89 Hz, 1 H), 7.66 (td, J =7.70, 1.83 Hz, 1 H), 7.79 7.89 (m, 1 H), 8.5 9 8.69 (m, 2 H), 8.74 8.81 (m, 1 H); 13 C NMR (75 MHz, CDCl 3 133.01, 134.73, 135.55, 135.60, 135.80, 148.98, 149.23, 149.32, 154.88, 166.59. DIP CI MS: m/z 250.1328 [M+H]+ (calculated: 250.1339). 3 P yridinyl methylene anabaseine (3PAB 19 b): After chromatography ( silica, 15 :1 CH 2 Cl 2 /CH 3 OH) the title compound was obtained in 50% yield as a white solid (m.p., 69 71 ) 1 H NMR (300 MHz, CD 3 ppm 1.88 (quin, J =5.99 Hz, 2 H), 2.87 (t, J =6.50 Hz, 2 H), 3.86 (t, J =5.48 Hz, 2 H), 6.66 (s, 1 H), 7.47 (dd, J =7.74, 5 .26 Hz, 1 H), 7.55 (dd, J =7.89, 4.97 Hz, 1 H), 7.87 (d, J =8.18 Hz, 1 H), 7.94 8.01 (m, J =7.89, 1.83, 1.64, 1.64 Hz, 1 H), 8.47 (d, J =4.97 Hz, 1 H), 8.52 (s, 1 H), 8.60 8.70 (m, 2 H);

PAGE 155

155 13 C NMR (75 MHz, CD 3 OD 33.59, 134.24, 135.89, 137.38, 138.61, 138.74, 149.46, 150.09, 150.66, 150.95, 168.86. MMI TOF MS: m/z 250.1338 [M+H]+ (calculated: 250.1339). 4 P yridinyl methylene anabaseine (4PAB 19 c): After chromatography ( silica, 1 5 :1 CH 2 Cl 2 /CH 3 OH) the title compoun d was o btained as light yellow oil in 65% yield 1H NMR (300 MHz, CDCl 3 J =6.18, 5.99 Hz, 2 H), 2.81 (td, J =6.57, 2.04 Hz, 2 H), 3.92 (t, J =5.62 Hz, 2 H), 6.56 (s, 1 H), 7.16 (d, J =6.13 Hz, 2 H), 7.35 (ddd, J =7.78, 4.86, 0.80 Hz, 1 H), 7.83 (dt, J =7.81, 1.94 Hz, 1 H), 8.53 8.69 (m, 3 H), 8.74 (d, J =1.46 Hz, 1 H); 13C NMR (75 MHz, CDCl 3 ppm 22.10, 25.53, 50.34, 123.01, 123.50, 132.28, 134.98, 135.41, 136.09, 143.08, 149.52, 149.73, 149.76, 165.94. TOF MS: m/z 250.1339 [M+H]+ (calculated: 250.1339) 5 chloro 2,4 dimethoxylphenyl isocyanate (20 ) : 237 5 chloro 2,4 dimethoxy aniline (790 mg, 4.21 mmol) and triethylamine (640 L, 4.21 mmol) were stirred together in 25mL dry toluene on an ice water bath under N 2 Then 1.25 g triphosgene was add ed to the mixture in portions and then the reaction mixture was kept in an ice water bath for another 10min before heating to temperature and the unreacted triphosgene was removed by filtration and the filtrate was evaporated to dryness. The crude product was obtained as a purple solid in 99% yield, which was used directly in the next step without further purification. 1 H NMR (300 MHz, CDCl 3 ) 3.89 (s, 3H), 3.93 (s, 3H), 6.47 (s, 1H), 6.94 (s, 1H); 13 C NMR (75 MHz, CDCl 3 ) ppm 56.23, 56.50, 96.59, 113.33, 116.42, 124.45, 130.01, 152.89, 153.12. IR (N=C=O): 2223 cm 1

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156 1 (5 chloro 2,4 dimethoxyphenyl) 3 (5 methylisoxazol 3 yl)urea (PNU 120596, 21): 238 Compound 20 (533 mg, 2.5 mmol) was stirred in 50 mL dry benzene along with 3 amino 5 methyl isoxazole (245 mg, 2.5 mmol) under Ar. Then the reaction mixture was heated at 65 C for four days and a grey purple solid precipitated gradually. The reaction mixture was then concentrated to dryness. The crude product was recrystallized three times from isopropanol. The obtained pa le grey solid was dissolved in ethyl acetate, decolorized by carbon and filtered through Celite. The filtrate was concentrated to dryness and compound 21 was obtained as a fluffy white solid in 39% yield. The melting point ( 219.5 220.5 C) was identical to that of an authentic sample from Tocris. 1 H NMR (300 MHz, acetone d 6 ) : ppm 2.38 (d, J =0.88 Hz, 3 H) 3.89 (s, 3 H) 3.96 (s, 3 H) 6.41 ( br. s. 1 H) 6.87 (s, 1 H) 8.31 (s, 1 H) 8.85 (br. s., 1 H) 9.13 ( s 1 H). ESI MS calculated for C 13 H 14 ClN 3 O 4 (M+Na + ) : 334.0565 ; found: 334.0551 3.4.2 Molecular modeling A homology model f or the human 7 nAChR was created using the Aplysia californica AChBP structure (PDB ID 2WN9 ) as the template. 165 The 7 sequence was modeled with PRIME (Schrodinger2010, Schrodinger Inc.). 239 The resulting monomeric model comple xed with the 2WN9 ligand (4 OH GTS 21) was superimposed five times on each chain of the AChBP pentameric crystal structure in order to generate a pentameric model. The model was then examined for clashes which were subsequently resolved by variation of si de chain rotomers, or in combination with constrained minimization using the GROMOS force field resident in the SPDB viewer 4.0, followed by Amber 10 (Case et al., 2008, University of California, San Francisco) molecular

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157 mecha nics refinement with the bound 2WN9 ligand included by Dr. Horenstein. The model quality was assessed with the Molprobity server. 240 3.4.3 Electrophysiology The 7 nAChR clones, site directed mutants and their expression in the X. laevis oocytes were prepared by Clare Stokes and Sara Copeland as described in Chapter 2. Shehd Abdullah Abbas Al Rubaiy, Sara Copeland, Mathew Kimbrell and Robin Rogers conducted electrophysiology experiments using OpusXpress 6000A The conditions used were also described in Chapter 2. Data were collected at 50Hz and filtered at 20Hz for 7 Flow rates were set a t 2mL/min for 7 Drug applications alternated between ACh controls and experimental applications, unless otherwise indicated A solution of 300 M PNU 120596 was either applied together with 300 M ACh or arylidene anabaseine, or applied alone after 300 M 2FAB as noted for each figure in the Results and Discussion section of this chapter. D rug applications were 12 s in duration followed by 181 s washout periods. Because PNU 120596 has a slow off rate, 20 minutes washing with Ringer was applied after the e xperiments involving PNU 120596 involved experiments. For experiments in which the ACh control responses remained relatively stable, net charge r esponses to experimental drug applications were calculated relative to the preceding ACh control responses to normalize the data. 219 For experiments in wh ich ACh control responses varied through the course of the experiment, because of either RID or PNU used to normalize the data for all subsequent responses. Due to rapid desensiti zation, net charge responses were generally used to compare activation response in 7

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158 nAChRs. 219 However, because PNU 120596 is capable of allowing agonists to induce enormous and sustained responses, both normalized peak responses and the normalized net charge responses were compared for experiment s involving PNU 120596. 203 The net charge to peak response ratio was calculated for each ooctye to evaluate the response curve shape. These normalization procedures compensated for the v arious levels of channel expr ession among the oocytes Mean values and standard errors were calculated from the normalized responses of at least four oocytes for each experimental concentration. The statistical P values were obtained from student T tests that compare two samples assum ing unequal variances. For both the 7 wild type receptor and the Q57 mutants the control ACh concentration was 300 M, a concentration that is sufficient to evoke maximal net charge response. 219 For concentration response relations, data derived from net charge analyses were plotted using Kaleidagr aph 3.0.2 (Abelbeck Software; Reading, PA), and curves were g enerated from the Hill Equation shown as below Where Imax stands for the maximal response for a particular agonist/ subunit combination, and n represents the Hill coefficient. Imax, n, and the EC 50 values were all unconstrained initially for the fitting procedures However, due to the low expression level of the receptor or low activation response of the arylidene anaba seines, the following CRCs curves were generated while constraining the Hill coefficient value at 2 to obtain a curve to compare with the others: 2PyroAB, 3PyroAB and 3TAB with the h 7Q57L receptor; 2PyroAB with the h 7Q57K receptor; 3FAB and 2PyroAB with the

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159 h 7Q57D receptor; 2FAB, 2PyroAB, 3PyroAB and 2TAB with the h 7Q57E receptor. Because ACh is the reference full agonist, for the ACh concentration response curves the data were normalized to the observed ACh maximum, and the I max of the curve fits were constrained to equal 1. 228 Before curve fitting, the CRC responses of the arylidene anabaseines were further normalized to the maximum ACh response obtained from the CRC result of the corresponding receptor type to eliminate the mutation impact. 3.4. 4 Energy landscape Each of the characterized ligand/receptor combinations has a unique energy landscape determining the rates for conversion among the conformational states of the receptors. We have previously proposed a schematic model for ACh bound wild type 7 in terms of the stability of the resting closed state, the native open state (i.e. in the absence PAMs), and two distinct desensitized states, D s (which can be converted into an open state by PNU 120596) and D i (a non conducting state which persists eve n in PNU 120596 modified receptors). 139 This scheme (Figure 1 13) was used as the starting point to describe the effects of the Q57 mutations on activation and desensitization by ACh. The relative height cha nges of the energy barrier and the energy level were assigned based on the natural logs of the ratio of the experimental mutant versus the wild type. The percent changes were all divided by two to fit the energy change in scale in Figure 3 13 and Appendix B 5. The energy barriers for entering the open state of the acetylcholine from the resting closed state were adjusted to reflect the mutation impact on the ACh response data in Figure 3 4 A. Note that these models do not account for observed differences i n

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160 ACh potency, as was seen with the Q57K mutants, because the energy profiles all assume the same level of agonist binding (i.e. maximum occupancy). For these basic models of ACh, the increased currents observed with PNU 120596 co application were used to assign values for the barriers for entering into the D s state, as well as the relative stability of the D s state (Figure 3 5). Specifically, the relative magnitudes of the PNU 120596 modulated peak currents were used to assign the relative heights of the e nergy barriers into D s In the case where the peak values were larger for the mutants than for wild type, the barriers of the D s state were adjusted downward. In order to estimate the relative stability of the mutant receptors in the D s state, the net char ge to peak current ratio for each of the responses evoked by ACh and PNU 120596 co applications was calculated and compared to that of the wild type. In the cases where the net charge/peak values were larger for the mutants than for wild type, the levels o f the D s state were adjusted downward. Since the effects of the experimental ligands on the mutant receptors have been characterized by making measurements relative to ACh activation, the hypothetical ACh energy landscapes for each mutant can be modified t o describe the energy profiles for each mutant/ligand combination (Figure 3 13 and Appendix B 5). The I max values were used to obtain the relative differences in the activation energy barriers based on the natural log of the inverse of the I max ratios. In these two mutants, The data in Figure 3 9 B were used to estimate the relative heights of the barriers into the D s states. In order to estimate the relative stability of the D s states for the receptor ligand pairs, the relative values for the potentiated n et charge to peak were calculated. The energy levels of the D i states were modified based on the inhibition of the subsequent ACh control

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161 calculated from the recovery data in Table 3 4. Changes were made only when the ACh recovery was lower than 75%. Estim ations of residual inhibition/desensitization and subsequent activation by PNU 120596 alone (Figure 3 12 B) were used to estimate the relative stability of D s and D i After setting up the energy levels of the D s and D i states as above, considering the lar ge experimental error, the only data inconsisten t with Figure 3 12 B was the Q57K mutant. The relative energy levels of D s and D i of the Q57K mutant with 2FAB was changed via comparing the single point slope with the wild type receptor in Figure 3 12 B. Si nce there was less D i in the Q57K mutant (larger y to x ratio) than in the wild type, the relative energy level of the D i state was moved upwards accordingly. Because the recovery curves in Figure 3 12 C were not of sufficient quality to obtain a half time value, the energy barrier for leaving D i to the closed state was adjusted upwards qualitatively for Q57L compared to Q57D in Appendix B 5. Additional qualitative adjustments have also been made in the Q57L and Q57E mutants in Appendix B 5. These two muta nts showed greatest impact on the receptor activation by ACh (Figure 3 4 A), which resulted in very high energy barriers for entering the open state for the arylidene anabseines in the energy landscape, such as 3FAB with the Q57L mutant (data not shown). T o maintain the pathway from the closed state to the open state as the preferred path for channel open, the energy barriers between these two states in Q57L and Q57E mutants were lowered qualitatively while keeping the trend of the open probabilities of the arylidene anabaseines the same as before in each mutant. The resulting energy landscape is shown in Appendix B 5.

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162 CHAPTER 4 4 DESIGN OF NOVEL MO LECULES TO TEST AN A LTERNATIVE PHARMACOPHORE FOR BI NDING AND/OR ACTIVAT ION OF THE NICOTINIC RECEPTORS 4.1 B ackground As introduced in Chapter 1, nAChR agonists and small competitive antagonists share a common pharmacophore, the positively charged nitrogen. With addition of a few alkyl groups to the charged nitrogen, ions such as tetramethylammonium, can activat e nAChRs effectively. 131 In the crystal structures of the acetylcholine binding proteins, 163, 166 a few conserved aromatic residues, including Tyr93, Trp149, Tyr188 (Tyr190 for 4 subunit), Tyr198 and Trp55 (Trp57 for 2 subunit), have been identified and suggested to recognize the charged nitrogen via a cation interaction. The cation interaction has been calculated and well characterized in both gas phase and solution for transition metals, alkali cations, ammonium, etc. 241 243 This interaction has been accepted as an important non bonding interaction between small molecules and proteins. Unnatural amino acid mutagenesis studies supported such cation interactions in the ligand binding site of the nAChRs. 244 Agon ists would decrease their potency when the conserved aromatic residues were mutated to unnatural amino acids with lower electron density in the center of the aromatic ring, and the potency decrease was proportional to the change of the electron density. A s a non bonding interaction, cation interaction is not very strong. The cation interaction ability of the charged nitrogen as the case in nAChRs is much weaker than the transition metals. 242, 243 In a binding st udy of two isomeric compounds with a synthetic receptor (cyclophane type), the charged nitrogen isomer does bind more tightly, but only by 2.5 kcal/mol, than the carbon isomer in the cation trap constituted by

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163 four aromatic rings. 245 In a real receptor, such energy difference can be easy compensated by the allosteric conformational change of the whole protein. In a previous study, it was found that 300 M 4 OH GTS 21 activated two mutants of the 7 type receptor (Y188F and D197A) effectively while 300 M acetylcholine did not. 230 This result raised an interesting question as to why ACh lost its ability to activate the receptor. As part of the C loop, Tyrosine 188 was proposed to move down via a cation interaction with the agonist to disrupt a salt bridge between aspartate 197 and lysine 145 residues in the floor of the binding pocket to initiate chann el opening in the muscle type nAChRs. 246 The activation of the Y188F mutants by 4 OH GTS 21 suggested an alternative activation mechanism might be operative that the binding of the extended hydrophobic group of 4 OH GTS 21 compensates for a loss of function, possibly due to loss of recognition of the positively charged nitrogen pharmacophore in Y188F mutant. Figure 4 1. The structures of 4 OH GTS 21 and BHP. Therefore, an alternative pharmacophore was proposed for ligands binding with the nAChRs, such that compounds retained the core shape of the benzylidene anabaseines without the positive charge. These compounds might bind underneath the C loop of the nAChRs, and possibly lead the receptor to enter into various fu nctional

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164 states, such as the open state and the PNU 120596 sensitive desensitized state. Compound ( E ) 3 (6 benzylidenecyclohex 1 en 1 yl)pyridine (BHP) was designed as shown in Figure 4 1 to test this hypothesis. Figure 4 2. Design for the retrosynthesis route for BHP. A) The synthesis route to benzylidene 2 chlorocycloalkenes; adapted from the article written by Schulze, K. et al.. 247 B) Retrosynthesis route for BHP. The dashed rectangles indicate how the retrosynthesis of BHP deri ves from the synthesis of benzylidene 2 chlorocycloalkenes in panel A. To the best of our knowledge, nothing has been reported regarding the synthesis of ( E ) 3 (6 benzylidenecyclohex 1 en 1 yl)pyridine (BHP). Lacking the imidine motif, BHP cannot be synth esized via the aldol condensation, which has been utilized for synthesizing benzylidene anabaseines, such as 4 OH GTS 21. 225, 227 Synthesis of BHP via Wittig type olefinations was not successful either. 248 However, benzylidene 2 chlorocycloalkenes have been synthesized in three steps, including a Grignard reaction, a halogenation step followed by an elimination step (Figure 4 2 A). 247 We envisioned a str aightforward synthesis of BHP and report the results of these studies. A simplified

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165 retrosynthesis route derived from the synthesis of benzylidene 2 chlorocycloalkenes is shown in Figure 4 2 B. Two BHP para site derivatives, 4 MeO BHP and 4 OH BHP, were al so synthesized to mimic the hydrogen bonding interaction of 4 OH GTS 21 (Figure 4 1). 4.2 Result s and Discussion 4.2.1 Synthesis of ( E ) 3 (6 benzylidenecyclohex 1 en 1 yl)pyridine (BHP) and Its Derivatives 2 (Pyridin 3 yl)cyclohex 1 enecarbaldehyde, 23 is a key intermediate to synthesize BHP and its derivatives (Figure 4 3). Surprisingly little has been reported regarding the synthesis of compound 23 Since Suzuki couplings of vinyl carboxaldehydes and aryl boronic acids are well known, 249 it appeared reasonable to consider synthesizing compoun d 23 from Suzuki coupling of pyridine 3 boronic acid and 2 c hlorocyclohex 1 enecarbaldehyde 22 (Figure 4 3). Compound 22 was obtained via a Vilsmeier Haack reaction (Figure 4 3). Considering toxicity and handling issues, triphosgene was initially used to react with DMF to form the chloro iminium ion precursor to react with cyclohexanone. 250 Both DMF and THF were evaluated as solvents for the reaction, but the yields were only 4% and 20%, respectively. The low yields might have resulted from the low efficiency of triphosgene to generate the iminium ion precursor since plenty of cy clohexanone was recovered after the reaction. Therefore, phosphorus oxychloride was utilized, which is a better electrophile. This increased the yield of compound 22 to 54% and the gram scale synthesis provided enough starting material for the next step. 251 Although some Suzuki reactions can take place without heating, 249 the Suzuki reaction to synthesize compound 23 requires heat to reflux. The reaction between

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166 compound 22 and pyridine 3 boronic acid proceeded with minimal side reactions and compound 23 was obtained in 82% yield after chromatographic purification. Figure 4 3. Synthetic route to the aldehyde, 23 BHP, 25 and 4 MeO BHP, 27 were then synthesized from com pound 23 by reacting it with phenylmagnesium bromide and 4 methoxyphenylmagnesium bromide, respectively, followed by dehydrohalogenation as shown in Figure 4 4. 247 The two Grignard reaction products, 24 and 26 were obtained in 79% a nd 98% yield, respectively. 252 The desired conjugated dienes ( 25 and 27 ) were obtained at room temperature when halogenating the alcohols, 24 and 26 using carbon tetrabromide and triphenyl phosphine 247 Th e yields for compounds 25 and 27 are 29% and 52%, respectively.

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167 Figure 4 4. Synthetic route to the BHP and its two derivatives (4 methoxy and 4 hydroxyl).

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168 The literature method upon which this synthesis was based reported that halogenation of the allyli c alcohol yielded isolable halo compound (Figure 4 2 A). 247 Regardless of whether the chloride or bromides were employed, in my hand, I was unable to isolate any halogenated intermediates, and only observed the final, desired diene p roducts. Although the halogenation products were mostly identified as the main products for reactions in such condition, no halo substituents were identified in synthesizing BHP and its derivatives. Comparing with chlorination, bromination increased the yi eld of compound 25 (BHP) from 36% to 52%. Therefore, dehydrobromination was utilized to synthesize BHP and its two derivatives, and no bromo substituted intermediates were observed in their syntheses. The 4 TBDMSO phenyl magnesium bromide 29 was freshly made before reacting with aldehyde as shown in Figure 4 4. Due to the electron donating effect of the TBDMSO group, generation of the Grignard reagent was difficult. No reaction was observed even after refluxing 0.17 M 4 TBDMSO phenyl bromide with 2 equiva lent of magnesium for 16 hours. However, if the 4 TMSO phenyl bromide was concentrated to 0.87 M, the Grignard reagent, 28 could be obtained. However, unlike the preparation of the other Grignard reagents in which extra heat can be problematic, 253, 254 the reaction between 4 TBDMSO phenyl bromide and magnesium powder ( 50 mesh, 2 eq) would not start until 4 hours after reflux (Table 4 1). The common signs of the start of the Grignard reaction are cloudiness of the r eaction mixture and dissolution of the magnesium powder. However, these signs were not observed during synthesis of Grignard compound 28 The reaction mixture only turned a little cloudy and the magnesium powder only turned black when the reaction was stop ped. Therefore, the

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169 extent of the Grignard reaction was estimated by its ability to produce compound 29 via TLC and proton NMR; the resulting data are displayed in Table 4 1. No Grignard reagent was obtained in the first two hours. Four hours is enough for making the Grignard reagent, the amount of which can react with 0.4 equivalent of compound 23 completely and produce compound 29 in 51% yield (calculated by compound 23 ). The lower percentage of reacted compound 23 at 8 hours time point may reflect experi mental error. Table 4 1. The results of monitoring the Grignard reaction. One equivalent of 4 TBDMSO phenyl bromide was taken out from the reaction at different time points as listed below to reacted with one equivalent aldehyde 23 (10 mg) in 1 mL dry THF The reactions were tracked by TLC and stopped when no more compound 28 were generated. The percentages of the reacted aldehyde occurs. Reaction time / hours 1 2 4 8 23 Percentage of the 23 reacted / % NR NR 40 27 42 The TBDMS group can be removed from compound 30 readily with tetrabutyl ammonium floride. 255 Compound 31 (4 OH BHP) was obtained in 50% yield after chromatography. The presence of the hydroxyl was also confirmed by infrared spectrometry with a new broad band centered at 2950 cm 1 observed for compound 31 The proton and ca rbon chemical shifts of BHP, 25 were assigned as shown in Figure 4 5 A via COSY and HMBC (spectra are shown in Appendix A 34 to A 37). Several of these chemical shifts supported the formation of the conjugated diene in BHP. The two proton peaks, one tripl et at 5.90 ppm and one singlet at 6.16 ppm (Appendix A 33), are signature chemical shifts for allylic protons, the splitting patterns of which are consistent with the double bonds configuration of BHP, as shown in Figure 4 5 A. The chemical shifts of the t wo allylic hydrogen attached carbons, 127.6 and 132.4

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170 ppm, also support the structure of BHP, 25 Additional evidence for the structure of BHP instead of the halogenated substituents comes from the number and the splitting patterns of the aliphatic hydroge ns: two hydrogens as a triplet at 2.78 ppm, two hydrogens as a quartet at 2.39 ppm and two hydrogens as a quintet at 1.82 ppm. Figure 4 5. The carbon and hydrogen chemical shift (ppm) assignments of compound 25 and NOE results of compound 25 and 27 A ) Proton (underlined) and carbon chemical shift assignments. B) The NOE results. Although the newly synthesized compounds lack the charged nitrogen featured in the benzylidene anabaseines (Figure 4 1), their overall three dimensional structures are

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171 very si milar. Due to the steric effect, BHPs also prefer the formation of the E olefin geometry as do the benzylidene anabaseines. 225, 227 The E double bond geometry for 25 and 27 were confirmed by NOESY (Figure 4 5 B, App endix A 38 to A 41). In both cases, irradiation of H 9 resulted in enhancement of both H 10 and H 15 leading to confirmation of the E olefin geometry for compound 25 and 27 4.2.2 Synthesis of 3 (C yclohex 1 en 1 yl)pyridine (PyHexe) To investigate whether t he activities of BHPs arise from the extended hydrophobic group or the pyridinyl hexene core, compound 34 was proposed as a structural control. Compound 34 3 (cyclohex 1 en 1 yl)pyridine (PyHexe) is a non charged analog of anabaseine (Figure 1 8), the non selective parent compound of 4 OH GTS 21. It was synthesized as shown in Figure 4 6. The Suzuki reaction tolerates a wide range of functional groups, typically with low toxicity of the reagents and the byproducts. 24 9, 256 Therefore, route A was initially tried to synthesize compound 34 as shown in Figure 4 6. 1 Chlorocyclohex 1 ene 32 was obtained via reacting cyclohexanone with phosphorus pentachloride in 59% yield after purification by fractional distillation. 257 However, refluxing compound 32 and pyridine 3 boronic acid for 40 hours only converted 33% of the compound 32 with accompanying decomposition. The yield of compound 34 from route A was less than 1%. Several factors may contribute to the low reaction efficiency of the Suzuki coupling. The catalysis con dition may need optimization, such as using different pallidium catalysts and solvents, or increasing the basicity of the reaction medium. 25 6 Moreover, there may be some phosphorus containing compounds left in compound 32 that might have poisoned the Suzuki catalyst, 258 thus resulting in the low conversion percentage of compound 32 to compound 34

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172 The traditi onal method to prepare aryl cyclohexene is via reacting cyclohexanone with aryl lithium as shown in route B in Figure 4 6. 259 The method utilized steam distillation to remove the reactants. The heat in the process might have facilitated the hydroxyl elimination of comp ound 33 to yield compound 34 Avoiding the tedious steam distillation, compound 33 was isolated instead. Compound 33 was reported to form compound 34 after addition of concentrated sulfonic acid. 260 However, no desired product was identified following the literature method. 260 On the other hand, ha logenations of compound 33 using methanesulfonyl chloride readily converted the tertiary benzyl alcohol, 33 to the desired conjugated benzyl alkene 34 at room temperature in 65% yield after chromatography. 261 F igure 4 6. Synthetic scheme of compound 34 ( PyHexe ).

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173 4.2.3 Synthetic Studies Directed towards 3 (3 P henyl 1 H pyrrol 2 yl)pyridine (BPP) The iminium cation of 4 OH GTS 21 (Figure 4 6) not only can act as positive charge center, it can also donate a H bond t o one or more polar residues in the ligand binding site of nAChRs. This H bond donating property may also contribute to the activation of the h 7Y188F mutant 4 OH GTS 21. Therefore, compound 40 was proposed to test the hypothesis stating that the extended phenyl group, together with an H bond donor in the center cyclic ring, is sufficient to activate the receptor. The synthesis of compound 40 appeared in a patent with limited information. 262 In the patent, the diarylpyrroles are generally made from reacting dimethyl acetylenedicarboxylate with appropriate 2 amino 1, 2 diaryl ethanone hydrochloric salts. However, the synthetic method was described in detail for a compound 40 analog, 2,3 diphenylpyrrole and the only information reported for compound 40 is its melting point. A four step synthesis had been accomplished to prepare the 2 ( amino) 1 phenyl 2 (pyridin 3 yl)ethanone hydrochloride salt, 38 which is the precursor i n the patent for making compound 40 (Figure 4 7). Compound 35 was synthesized from reacting the benzoyl chloride with 3 aminomethyl pyridine. 263 Although 1.2 equivalents of triethylamine was added to neutralize the hydrochloric acid generated during th e reaction, ~15% of the 3 aminomethylpyridine remained unreacted, presumably as its aminomethyl hydrochloride salt. The unreacted 3 aminomethylpyridine was separated from compound 35 via controlled pH extraction, which afforded compound 35 in 81% yield. Af ter protecting the amino group of compound 35 with a Boc group, 264 the resulting compound 36 was reacted with LDA to produce compound 37 via [1,3] acetyl

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174 migration in 82% yield. 264 After deprotection in weak acid, 265 the hydrochloride salt, 38 was obtained in 82% yield. Figure 4 7. Attempted synthesis of compound 40 Dimethylacetylenedicarboxylate (DMAD) is a reagent widely used t o prepare heterocyclic dicarboxylates. 266 Pyrroles can be synthesized via reactions between amino ketones and DMAD. 262, 267 However, the desired compound 39 was not identified in the reaction when refluxing compound 38 with 4 equivalent DMAD and 2.4 equivalent

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175 of sodium acetate in methanol (step 1) followed by addition of 1 M hydrochloric acid (step 2). Further in vestigation of the first step showed compound 38 was quite stable in the reaction condition without DMAD, but the primary amine of compound 38 reacted immediately upon addition of the DMAD at room temperature via tracking by TLC using Ninhydrin reagent (0. 3 g ninhydrin in 97 mL n butanol along with 3 mL acetic acid ). However, the proton NMR of the products in step 1 did not showed correct patterns or integrals of protons for the possible reaction intermediates (structures not shown) or the desired product 3 9 Various pyrrole syntheses are known to be plagued by side reactions. The electrophilic acetylene (DMAD) may react with the nucleophilic amine of compound 38 268 The resulting product may react with alcohols such as methanol solvent us ed here. 268 The pyridine ring of compound 38 may be also problematic since the pyridine nitrogen is also basic and can react with DMAD, though more weakly than the amine group. 266 Moreover, the formed pyrrole, 39 is not very stable, may not be very stable. However, the reaction product mixtures obtained for step 1 and step 2 were both too complicated to deduce the side reaction products which then might be useful to guide optimization of the reaction. Because of the limited time length of the study, syntheses of compound 39 and compound 40 were not further investigated. 4.2.4 Electrophysiology Evaluation of the Novel Molecules 272 Xenopus oocytes expressing mRNAs corresponding to 7, 3 4, 4 2 subunits or of nAChRs were used to evaluate the activity of the newly synthesized compounds. Although neither BHP or its derivatives was found to activate any of the receptors, they can highly inhibi t the activation of acetylcholine on these receptors except the 4 2

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176 subtype (Table 4 2). BHP is most potent for the muscle type receptor with IC 50 at 9 2 M. BHP can also fully inhibit the 3 4 and 7 receptors, with IC 50 values of 61 9 M and 73 18 M, respectively. On the other hand, the control molecule, PyHexe, is much less potent on the muscle type receptor than BHP, the IC 50 of which is 301 92 M. This indicates that the high inhibition ability of BHP, especially with the muscle type of the r eceptor, arises from the extended aromatic group. Table 4 2. Inhibition potency ( IC 50 ) and maximum inhibition percentage of BHP with different types of nAChRs. The IC 50 was obtained from Hill equation using negative unconstrained Hill coefficient value and fixed I max at 1. The IC 50 of 4 2 was not shown because the inhibition effect of BHP on this receptor subtype was too small to fit in the equation. The ACh control concentrations for the various nAChRs are as follows: 3 4, 100 M; muscle, 30 M; 7, 60 M; 4 2, 30 M. 3 4 muscle 7 IC 50 / M 61 9 9 2 73 18 As introduced in Chapter 1, there are two inhibition modes, i.e., competitive and non inhibit the channel opening competitively, 10 M BHP was co applied with various concentration of acetylcholine and treated to the muscle type nAChR The application of 10 M BHP shifted the EC 50 of the acetylcholine from 18 1 M to 65 12 M without affecting the efficacy of ace tylcholine dramatically, which represents a typical competitive inhibition manner. These data suggest that BHP binds to the same site in the receptor as ACh does. possible f or BHP to activate the receptor in certain conditions such as when mutating certain residues and/or in the presence of PAMs? There are several mutants on the C loop or the cation binding center that behave specifically with 4 OH GTS 21. Those

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177 7 mutants, which can be activated by 4 OH GTS 21 but not ACh, might have been rescued for activation with the extended aromatic ring from 4 OH GTS 21. Therefore, they were first investigated for activation, which include h 7W55V, h 7Y188F, h 7C116S/C190A, h 7C116S/C 190P, h 7C116S/C191A and h 7C116S/C191P. Although 4 OH GTS 21 cannot activate 4 2 wild type receptor, it can activate some 4 2 mutants, which may also result from the specific interaction of the extended aromatic ring binding pocket. 230 Therefore, these mutant s were also tested with BHP and its derivatives, which include 4Y190F 2, 4 2W57A, 4 2W57Y, 4 2W57F, 4 2W57G and 4 2W57V. However, neither BHP nor 4 MeO BHP can induce detectable responses on any of these 7 or 4 2 mutants. The actions of 4 OH BHP, w hich is more structurally similar to 4 OH GTS 21, are still under investigation. PAM PNU 120596 can induce large response without decay in the presence of an agonist. Initial test of BHP and PNU 120596 showed no detectable response with the 7 nAChRs when applying together. The effects of PNU 120596 on the BHP derivatives with the wild type 7 and the 7 mutants are still under investigation. 4.3 Summary In this chapter, three novel non charged analogs of benzylidene anabaseines, BHPs, were successfully s ynthesized. The control molecule, PyHexe, was also obtained in a modified synthetic route. Although the reaction to make the 4 TBDMSO phenylmagnesium bromide requires special conditions, the overall syntheses are simple (within six steps), straight forward and the resulting products are easy to purify. The new series of compounds have kept the E isomeric configuration of the benzylidene anabaseines, and thus may adopt similar binding poses in the receptor. Although the

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178 current data available do not support BHP and its derivatives as having agonist activity, antagonist with the help of the extended hydrophobic group. This is the first time that a non charged molecular has been found to be a competitive antagonist of the nAChRs. 4.4 Experimental Section 4.4.1 Chemicals Solvents and reagents for electrophysiology and synthesis were from Sigma Chemical Co. (St. Louis, MO) and Fisher Scientific (Pittsburg, PA). PNU 120596 was synt hesized as described in Chapter 3. 1 H and 13 C NMR spectra were obtained using VXR 300, Gemini 300, or Mercury 300 or Inova 500 (300 MHz) spectrometers (Varian, Palo Alto, CA) in appropriate deuterated solvents. Mass spectra were obtained on an Agilent 6210 TOF spectrometer operated in ESI, DART, TOF, MMI TOF or DIP CI ionization mode s rotary evaporator. 2 C hloro cyclohex 1 enecarbaldehyde 22. Freshly distilled phosphorus oxychloride (4.0 mL, 0.044 mol) was dropped into a solution of 4.2 mL dry DMF (0.054 mol) dissolved in 10 mL dry trichloroethylene, with stirring at 10 C under Ar for 10 minutes. The reaction mix ture turned a little milky, but no precipitate formed. Then the reaction mixture was warmed up to room temperature. A solution of 4.3 mL dry cyclohexanone (0.041 mol) in 10 mL dry trichloroethylene was added dropwise over 30 minutes. The color of the react ion mixture became orange at this point. After heating at 65 C for 3 hours, the reaction mixture was cooled and poured into a 50 mL saturated

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179 sodium acetate solution. The aqueous phase was extracted with trichloroethylene (6 30 mL). The organic layers w ere combined, dried with sodium sulfate and concentrated under reduced pressure. The crude product was then purified by flash column (silica, 35:1 petroleum ether/ethyl acetate) and compound 22 was obtained as a light yellow oil (3.5 g ) in 54% yield. 250, 251 1 H NMR (300 MHz, CDCl 3 ) ppm 1.60 1.71 (m, 2 H) 1.71 1.86 (m, 1 H) 2.28 (tt, J =6.08, 2.32 Hz, 1 H) 2.47 2.69 (m, 2 H) 10.20 (s, 1 H). IR: C=O, 1678.6cm 1 (literature value 250 : 1678cm 1 ); CH 2 2940.1 cm 1 and 2862.5 cm 1 2 (P yridin 3 yl)cyclohex 1 enecarbaldehyde 23. Compound 22 (1.30 g, 9 mmol) was stirred in 40 mL toluene along with 310 mg tetrakis (triphenylphosphine) palladium at room temperature for 10 min under Ar. Then a suspension of pyridine 3 boronic acid (1.66 g, 13.5 mmol) in absolute ethanol (minimum amount to help transferring the solid) was added to the reaction mixture, followed by a 2 .2 mL sodium carbonate solution (2M). After heating at reflux for 6 hours, the reaction mixture was cooled down to room temperature and quenched with 80 mL 5% (w/v) sodium carbonate solution. The organic phase was separated and the aqueous phase was extrac ted with ethyl acetate (4 50 mL). The organic layers were combined, dried over magnesium sulfate and concentrated in vacuo. After chromatography (silica, 2:1 petroleum ether/ethyl acetate), compound 23 was obtained as a yellow oil (1.33 g ) in 79 % yield. 1 H NMR (300 MHz, CDCl 3 ) ppm 1.69 1.87 (m, 3 H) 2.33 2.43 (m, 2 H) 2.48 2.58 (m, 2 H) 7.34 (ddd, J =7.78, 4.86, 0.95 Hz, 1 H) 7.58 (dt, J =7.89, 1.90 Hz, 1 H) 8.51 (dd, J =2.26, 0.80 Hz, 1 H) 8.62 (dd, J =4.89, 1.68 Hz, 1 H) 9.48 (s, 1 H). 13 C NMR (75 M Hz, CDCl 3 ) ppm 21.06, 22.05, 22.13, 33.70, 122.95, 134.86, 135.66, 137.13, 148.91, 149.34, 154.97,

PAGE 180

180 192.11 IR: C=O, 1668.4 cm 1 ; aryl H, 3028.6 cm 1 ; CH 2 2931.8 cm 1 and 2859.5 cm 1 DART MS: m/z 188.1074 [M+H]+ (cal: 188.1070) P henyl(2 (pyridin 3 yl)cyc lohex 1 en 1 yl)methanol 24. Compound 23 (350 mg, 1.9 mmol) was dissolved in 25 mL dry diethyl ether at 2 C under Ar. Then 1.7 mL phenyl magnesium bromide (2.8 M in diethyl ether) was added dropwise to the reaction mixture. The reaction was kept at 2 C f or 1.5 hours, and then quenched with 15 mL ice cold water and 15 mL cold saturated ammonium chloride solution. After separation, the aqueous phase was extracted with ethyl acetate (3 40 mL). The organic layers were combined, dried over magnesium sulfate, and concentrated in vacuo. The crude product was purified via a flash column (silica, 2:1 petroleum ether/ethyl acetate) and compound 24 was obtained as a light yellow oil which turn ed into a white foaming solid in vacuo (398 mg in 79% yield ). 1 H NMR (300 MHz, CDCl 3 ) ppm 1.62 (br. s., 3 H) 1.67 1.90 (m, 2 H) 2.05 2.31 (m, 1 H) 2.31 2.69 (m, 1 H) 5.34 (s, 1 H) 5.64 (br. s., 1 H) 7.02 7.41 (m, 6 H) 7.57 (d, J =7.59 Hz, 1 H) 8.2 7 (d, J =3.65 Hz, 1 H) 8.43 (s, 1 H). 13 C NMR (75 MHz, CDCl 3 ) ppm 22.21, 22.56, 22.81, 32.42, 71.45, 123.20, 125.43, 126.30, 127.72, 131.67, 136.00, 137.66, 139.05, 143.22, 146.77, 148.50. DART MS: m/z 266.1547 [M+H]+ (cal: 266.1539) ( E ) 3 (6 B enzylidene cyclohex 1 en 1 yl)pyridine 25. Compound 24 (282 mg, 1.06 mmol) and carbon tetrabromide (710 mg, 2.12 mmol) were dissolved in 15 mL dry dichloromethane under argon on an ice water bath. Triphenyl phosphine (560 mg, 2.12 mmol) was added to the reaction mixt ure in five portions over 4 min. Then the reaction mixture was kept at room temperature for 20 hours and then quenched with 10 mL deionized water. The pH of the reaction was adjusted to about 11 using solid sodium

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181 carbonate and extracted with ethyl acetate (3 15 mL). The organic layers were combined, dried over magnesium sulfate and concentrated to dryness. After chromatography (silica, 20:1 2:1 hexane/ethyl acetate), compound 25 was obtained as a white solid (136 mg ) in 52% yield (m.p. 81 82 C). 1 H NMR (300 MHz, CDCl 3 ) ppm 1.79 (quin, J=6.15 Hz, 2 H) 2.28 2.41 (m, 2 H) 2.75 (td, J=6.26, 1.49 Hz, 2 H) 5.87 (t, J=4.25 Hz, 1 H) 6.13 (s, 1 H) 7.09 7.36 (m, 6 H) 7.61 (dt, J=7.79, 2.00 Hz, 1 H) 8.47 8.60 (m, 2 H) 13 C NMR (75 MHz, CDCl 3 ) ppm 22.6 9, 26.30, 27.09, 122.70, 126.31, 127.24, 127.92, 129.03, 129.04, 129.05, 132.01, 136.71, 137.50, 137.54, 137.65, 138.34, 148.04, 150.15. ESI MS: m/z 248.1436 [M+H]+(cal: 248.1434) (4 M ethoxyphenyl)(2 (pyridin 3 yl)cyclohex 1 en 1 yl)methanol 26. This comp ound was synthesized in the same procedure as described for compound 24 while using 4 methoxyphenylmagnesium to react with compound 23 in dry THF instead. After chromatography (silica, 1:1 petroleum ether/ethyl acetate), compound 26 was obtained as a light yellow oil which turned into a white foaming solid in vacuo (462 mg in 98 % yield). 1 H NMR (300 MHz, CDCl 3 ) ppm 1.56 1.87 (m, 5 H) 2.13 2.46 (m, 3 H) 3.77 (s, 3 H) 4.80 (br. s., 1 H) 5.29 (s, 1 H) 6.77 6.87 (m, 2 H) 7.16 7.25 (m, 3 H) 7.55 (dt, J =7.74, 1.90 Hz, 1 H) 8.33 (dd, J=4.97, 1.61 Hz, 1 H) 8.42 (dd, J=2.19, 0.58 Hz, 1 H) 13 C NMR (75 MHz CDCl 3 ) ppm 22.33 22.61 22.94, 32.57, 55.09, 71.43, 113.28, 123.24, 126.66, 131.80, 135.25, 135.95, 137.67, 139.05, 147.07, 148.72, 158.20. ESI MS: m/ z 296.1658 [M+H]+(cal: 296.1645). ( E ) 3 (6 (4 M ethoxybenzylidene)cyclohex 1 en 1 yl)pyridine 27. Compound 27 was synthesized from compound 26 via the same procedure as described for compound 25 After chromatography (silica, 20:1 4:1 petroleum ether/ethy l acetate),

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182 compound 27 was obtained as a light yellow oil in 29 % yield ( 64 mg). 1 H NMR (300 MHz, CDCl 3 ) ppm 1.79 (quin, J=6.21 Hz, 2 H) 2.34 (td, J=6.13, 4.09 Hz, 2 H) 2.74 (ddd, J=7.78, 4.71, 1.53 Hz, 2 H) 3.79 (s, 3 H) 5.83 (t, J=4.16 Hz, 1 H) 6.06 (s 1 H) 6.78 6.89 (m, 2 H) 7.08 7.19 (m, 2 H) 7.25 (ddd, J=7.78, 4.86, 0.80 Hz, 1 H) 7.60 (dt, J=7.85, 1.92 Hz, 1 H) 8.41 8.62 (m, 2 H) 13 C NMR (75 MHz, CDCl 3 ) ppm 22.73, 26.28 27.14 55.17 113.40 122.69 126.79 130.05 130.28 131.31, 136.28, 136.73, 137.70, 138.43 147.94 150.13, 158.04. ESI MS: m/z 278.1552 [M+H]+(cal: 278.1539). 4 ((T ert butyldimethylsilyl ) oxy) phenyl magnesium bromide 28. 253 Magnesium powder ( 50 mesh, 0.169 g, 6.96 mmol) was stirred in 2 mL dry THF. Compound 23 in 2 mL dry THF was added slowly t o the reaction mixture and a small piece of iodine was added to agitate the reaction. The reaction mixture was heated near reflux for four hours. Then the reaction mixture was cooled on an ice water bath and used directly in the next step. (4 ((T ert butyld imethylsilyl)oxy)phenyl)(2 (pyridin 3 yl)cyclohex 1 en 1 yl)methanol 29. Compound 23 (0.26 g, 1.39 mmol) in 2 mL dry THF was added dropwise to the 4 ((t ert butyldimethylsilyl ) oxy )phenyl magnesium bromide solution prepared above on an ice water bath. The re action mixture was kept at room temperature for 16 hours, and then quenched with 10 mL cold saturated NH 4 Cl and 10 mL icy water. The unreacted magnesium was filtered off and the filtrate was worked up in the same procedure as described for compound 24 Com pound 29 was obtained as light yellow oily after purified by a flash column (silica, 2:1 hexane/ethyl acetate), which turned into a white foaming solid in vacuo (280 mg in 51% yield). 1 H NMR (300 MHz, CDCl 3 ) ppm 0.18 (s, 6 H) 0.97 (s, 9 H) 1.48 1.90 (m 5 H) 2.08 2.45 (m, 3 H) 4.10

PAGE 183

183 (br. s., 1 H) 5.28 (s, 1 H) 6.68 6.81 (m, 2 H) 7.05 7.17 (m, 2 H) 7.23 (ddd, J=7.78, 4.86, 0.66 Hz, 1 H) 7.55 (dt, J=7.81, 1.94 Hz, 1 H) 8.36 (dd, J=4.89, 1.68 Hz, 1 H) 8.45 (dd, J=2.04, 0.58 Hz, 1 H) 13 C NMR (75 MHz, CDCl 3 ) ppm 4.21 18.38, 22.61, 22.87 23.21 25.87 32.89 71.97 119.75 123.49 126.88, 132.34 135.93 136.15 137.79 139.27 147.50 149.09 154.54. ESI MS: m/z 396.2360 [M+H]+(cal: 396.2353). ( E ) 3 (6 (4 ((T ert butyldimethylsilyl)oxy)benzylidene)c yclohex 1 en 1 yl)pyridine 30. Compound 30 was synthesized from compound 29 via the same procedure as described for compound 25 After chromatography (silica, 10:1 hexane/ethyl acetate), the desired product was obtained as a colorless oil (256 mg ) in 96% y ield. 1 H NMR (300 MHz, CDCl 3 ) ppm 0.19 (s, 6 H) 0.97 (s, 9 H) 1.66 1.91 (m, 2 H) 2.34 (td, J=6.24, 4.02 Hz, 2 H) 2.75 (ddd, J=7.85, 4.71, 1.61 Hz, 2 H) 5.82 (t, J=4.16 Hz, 1 H) 6.04 (s, 1 H) 6.77 (m, 2 H) 7.08 (m, 2 H) 7.24 (ddd, J=7.78, 4.86, 0.80 Hz, 1 H) 7.59 (dt, J=7.81, 1.94 Hz, 1 H) 8.52 (m, 2 H) 13 C NMR (75 MHz, CDCl 3 ) ppm 4.18, 18.42 22.98, 25.89, 26.53 27.39 119.82, 122.93 127.21 130.50 130.87 131.55 136.5 1, 137.01 137.97 138.73 148.17 150.37 154.40 APCI MS: m/z 378.2261 [M+H] +(cal: 378.2248). ( E ) 4 ((2 (P yridin 3 yl)cyclohex 2 en 1 ylidene)methyl)phenol 31. Compound 30 (184 mg, 0.49 mmol) was stirred on an ice water bath in 1 mL THF. After adding 0.73 mL tetrabutyl ammonium fluoride (1M in THF), the reaction mixture was warmed up to room temperature. After 1 hour, the reaction was quenched with 10 mL deionized water and extracted with ethyl acetate (2 15 mL). The organic layers were combined, washed with 15 mL brine and dried over sodium sulfate. The crude product was purifie d via a flash column (silica, 2:3 ethyl acetate/petroleum ether) and compound 31 was

PAGE 184

184 obtained as a white solid (89 mg) in 69 % yield (m.p. 200 201 C). IR (Ar OH): broad 2400 3500 cm 1 1 H NMR (300 MHz, CDCl 3 ) ppm 1.80 (quin, J=6.17 Hz, 2 H) 2.27 2 .40 (m, 2 H) 2.74 (ddd, J=7.74, 4.75, 1.53 Hz, 1 H) 5.84 (t, J=4.31 Hz, 1 H) 6.01 (s, 1 H) 6.74 6.84 (m, 2 H) 6.96 7.09 (m, 2 H) 7.33 (ddd, J=7.78, 4.93, 0.73 Hz, 1 H) 7.66 (dt, J=7.74, 1.90 Hz, 1 H) 8.52 (dd, J=4.89, 1.68 Hz, 1 H) 8.56 (dd, J=2.19, 0. 73 Hz, 1 H) 13 C NMR (75 MHz, CDCl 3 ) ppm 22.92 26.54 27.38 115.47 123.74 127.31 129.46, 130.77, 131.73 135.94 137.67 138.56 138.59 147.26 150.00 155.71. APCI MS: m/z 264.1388 [M+H H 2 ]+(cal: 264.1383). 1 C hlorocyclohex 1 ene 32. Phosphorus pe ntachloride (15 g, 0.072 mol) was stirred in 15 mL dry diethyl ether a 100 mL round bottom flask surmounted with an additional funnel and a reflux condenser. Dry cyclohexanone (6 g, 0.060 mol) in 5 mL diethyl ether was added dropwise via the addition funne l over 1 hour. Then the reaction mixture was refluxed for another hour and all of the phosphorus pentachloride solid dissolved. The reaction mixture was then cooled to room temperature and poured into 25 mL icy water slowly. After separation and extraction with diethyl ether (2 10 mL), the organic layer was washed with 30 mL deionized water and dried over sodium sulfate. The crude product was concentrated under reduced pressure and purified by fractional distillation. Compound 32 was obtained as a colorless liquid (4.1 g ) in 59 % yield (b.p. 120 C, 1 atm). 1 H NMR (300 MHz, CDCl 3 ) ppm 1.51 1.67 (m, 2 H) 1.68 1.87 (m, 2 H) 2.02 2.19 (m, 2 H) 2.22 2.41 (m, 2 H) 5.71 6.00 (m, 1 H) .This proton NMR spectrum is consistent with the literature. 269 1 (P yridin 3 yl)cyclohexanol 33. 260 n Butlyl lithium (8 mL; 2.5 M in hexane) was stirred in 22 mL dry diethyl ether under N 2 at 65 C. Then 2.00 g dry 3 bromopyridine

PAGE 185

185 (0.013 mol) was added drop by dropwise to the reaction mixture over 10 min while keeping the reaction mixture at 65 C. After 20 min, 1.86 g dry cyclohexanone (0.019 mol) was added to the reaction dropwise at 65 C. The reaction was then warmed to 5 C. After 3 hours, the reaction was quenched with 30 mL cold saturated NH 4 Cl and transferred to a separat ory funnel. The aqueous phase was extracted with diethyl ether (3 30 mL) and the combined organic phase was dried over MgSO 4 and concentrated in vacuo. After chromatography (silica, 2:1 ethyl acetate/hexanes), compound 33 was obtained as a faint yellow s olid (1.61 g) in 70% yield. Melting point: 89 90 C, identical with the literature. 260 1 H NMR (300 MHz, CDCl 3 ) d ppm 1.21 1.42 (m, 1 H) 1.54 1.90 (m, 9 H) 3.31 (br. s., 1 H) 7.23 (ddd, J=8.00, 4.78, 0.80 Hz, 1 H) 7.74 7.90 (m, 1 H) 8.38 (dd, J=4.82, 1.61 Hz, 1 H) 8.68 ( dd, J=2.48, 0.73 Hz, 1 H) 3 (cyclohex 1 en 1 yl)pyridine 34. To a solution of 33 (200 mg, 1.13 mmol), DMAP (47 mg, 0.38 mmol), triethylamine (1.16 mL, 6.67 mmol) in 8 mL dichloromethane, methanesulfonyl chloride (311 L, 4.07 mmol) was added dropwise whil e stirring under N 2 at room temperature. After half an hour, the reaction was quenched with 10 mL saturated sodium bicarbonate. The organic phase was separated and the aqueous phase was extracted with ethyl acetate (2 10 mL). The combined organic layers were washed with 20 mL brine, dried over Na 2 SO 4 and concentrated under reduced pressure. After chromatography (silica, 10:1 dichloromethane/ethyl acetate), compound 33 was obtained as a light yellow oil (117 mg ) in 65% yield. 1 H NMR (300 MHz, CDCl 3 ) ppm 1.62 1.72 (m, 2 H) 1.74 1.86 (m, 2 H) 2.11 2.29 (m, 2 H) 2.30 2.48 (m, 2 H) 6.16 (tt, J=3.91, 1.86 Hz, 1 H) 7.21 (ddd, J=7.96, 4.75, 0.88 Hz, 1 H) 7.63 (dt, J=8.00, 1.99 Hz, 1 H) 8.44 (dd, J=4.82, 1.61 Hz, 1 H) 8.63 (d, J=1.75

PAGE 186

186 Hz, 1 H) 13 C NMR (75 MHz, CDCl 3 ) ppm 22.08, 22.98, 26.02, 27.23, 123.14, 126.74, 132.20, 134.04, 137.98, 146.83, 147.81. DART MS: m/z 160.1124 [M+H]+(cal: 160.1121). N (pyridin 3 ylmethyl)benzamide 35. Benzoylchloride (10 mL, 0.086 mol) was stirred in 50 mL dry dichlorometh ane at room temperature. Triethylamine (14.5 mL, 0.103 mol) and 3 aminomethyl pyridine (10.5 mL, 0.103 mol) dissolved in 30 mL of dry dichloromethane were added in a dropwise fashion to the reaction mixture during which time a light yellow precipitate had formed. After the addition was completed, the reaction mixture was concentrated to dryness. The obtained solid was redissolved in hot absolute ethanol and the insoluble white solid was filtered off. The filtrated was concentrated to dryness and recrystalli zed in absolute ethanol. The obtained solid was redissolved in 80 mL sodium bicarbonate and 80 mL chloroform. The pH of the aqueous phase was adjusted to 6 7 using 4 M NaOH. Then the organic phase was separated and the aqueous phase was extracted with chlo roform (3 40 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to dryness. Compound 35 was obtained as a white solid (14.8 g ) in 81% yield. Melting point: 69.5 70.5 C, identical with the literature. 270 1 H NMR (300 MHz, CD 3 OD ) ppm 4.61 (s, 2 H) 7.33 7.63 (m, 4 H) 7.80 7.92 (m, 3 H) 8.43 (dd, J=4.92, 1.49 Hz, 1 H) 8.52 8.62 (m, 1 H) N (pyridin 3 ylmethyl) N (tert butoxycarbonyl amino) benzamide 36. Compound 35 (10.8 g, 0.05 mol) was stirred in 150 mL acetonitrile along with 0.1 equivalent DMAP (0.61 g) at room temperature. Then 14.4 g di tert butyl dicarbonate (0.066 mol) was added dropwise to the reaction mixture along with 100 mL acet onitrile.

PAGE 187

187 After 16 hours, the reaction was quenched with 100 mL deionized water and the organic phase was separated. After extraction of the aqueous phase (EtOAc, 2 150 mL), the combined organic layers were washed with 200 mL sodium bicarbonate and 200 m L brine, then dried over magnesium sulfate and concentrated to dryness. After chromatography (silica, 5:1 petroleum ether/ethyl acetate), compound 36 was obtained as a light yellow oil (17.2 g ) in 87% yield. 1 H NMR (300 MHz, CDCl 3 ) ppm 1.12 (s, 9 H) 4.99 (s, 2 H) 7.27 (ddd, J=7.52, 4.53, 0.66 Hz, 1 H) 7.33 7.42 (m, 2 H) 7.48 (m, 3 H) 7.80 (dt, J=7.85, 1.99 Hz, 1 H) 8.53 (dd, J=4.82, 1.61 Hz, 1 H) 8.72 (d, J=1.75 Hz, 1 H) 13 C NMR (75 MHz, CDCl 3 ) ppm 27.47 46.56 83.79 123.53 127.53 128.23 131.34 133.45, 136.28, 137.44 149.04 150.06 153.20, 173.05. 1 P henyl 2 (pyridin 3 yl) 2 ( tert butoxycarbonyl a mino) ethanone 37. 264 To a solution of n butyl lithium (45.6 m L; 2.5 M in hexane) in 75 mL dry THF was added 17 mL diisopropyl amine (0.121 mol) followed by 10.6 mL dry DMPU (10.6 mL 0.087 mol) at 75 C. After the addition was completed, the reaction mixture was warmed up to 50 C to facilitate formation of LDA, an d then cooled back down to 75 C. After half an hour, compound 36 (12 g, 0.038 mol) was added to the reaction mixture along with 170 mL dry THF dropwise over 1 hour. After another hour, the reaction was quenched with 200 mL cold saturated NH 4 Cl, separated and the aqueous phase was extracted with ethyl acetate (2 100 mL). Then the combined organic phase was washed with 150 mL saturated NH 4 Cl and 150 mL brine. After drying over magnesium sulfate and filtration, the filtrate was concentrated to dryness and purified via a flash column (silica, 2:1 petroleum ether/ethyl acetate). Compound 37 was obtained as a yellow white oily solid (9.8 g ) in 82% yield. Compound 37 was not absolutely pure due to decomposition after

PAGE 188

188 chromatography (Appendix A 50), but was jud ged good enough for the next step. 1 H NMR (300 MHz, CDCl 3 ) ppm 1.43 (s, 9 H) 6.33 (br. s., 1 H) 7.22 (dd, J=7.89, 4.82 Hz, 1 H) 7.35 7.46 (m, 2 H) 7.67 (d, J=7.74 Hz, 1 H) 7.84 8.03 (m, 2 H) 8.49 (dd, J=4.82, 1.61 Hz, 1 H) 8.69 (d, J=1.46 Hz, 1 H) 2 Amino 1 p henyl 2 (pyridin 3 yl)ethanone hydrochloric salt 38. A suspension of 9 g compound 37 (0.029 mol) in 240 mL of a mixture of 3N HCl in ethyl acetate (v/v of concentrated HCl and EtOAc is 1:3) was stirred vigorously at room temperature for 30 minute s. The mixture was evaporated to dryness and the brown white solid was redissolved in isopropanol. The undissolved white solid was filtered and collected, which produced compound 38 (5.9 g) in 82% yield (m.p. 214 216 C, decomposed at 173 C). 1 H NMR (30 0 MHz, D 2 O ) ppm 6.65 (s, 1 H) 7.46 7.58 (m, 1 H) 7.69 (m, 1 H) 7.97 (d, J=7.74 Hz, 2 H) 8.07 (dd, J=7.67, 6.50 Hz, 1 H) 8.64 (d, J=7.60 Hz, 1 H) 8.86 (d, J=5.84 Hz, 1 H) 9.07 (s, 1 H) 13 C NMR (75 MHz, D 2 O ) ppm 56.09 128.59 129.51 129.53 131.53 131.73 135.92 142.69 143.96 146.54 192.19 DART MS: m/z 213.1025 [M+H]+(cal: 213.1022). 4.4.2 Electrophysiology BHP ( 25 ), its two derivatives ( 27 and 31 ) and PyHexe ( 34 ) were acidified to transform them into their corresponding pyridium salt forms to increase their solubility in water for the electrophysiology tests. Although these salts were initially soluble, they tended to precipitate out over time. Therefore, 100 mM DMSO stocks were made monthly and diluted in Ringer buffer daily before testing. Th e 7 nAChR clones, site directed mutants and their expression in the X. laevis oocytes were prepared by Clare Stokes and Sara Copeland as described in Chapter 2.

PAGE 189

189 Shehd Abdullah Abbas Al Rubaiy, Sara Copeland, Mathew Kimbrell and Robin Rogers conducted elec trophysiology experiments using OpusXpress 6000A The conditions used were also described in Chapter 2. Data were collected at 50 Hz and filtered at 20Hz Flow rates were set at 2 mL/min for 7 and 4 mL/min for the other subtypes. Drug applications alterna ted between ACh controls and experimental applications Drug applications were 12 s in duration followed by 181 s washout periods with 7 receptors and 8 s in duration with 241 s washout periods for other subtypes. In competition tests, 100 M BHP was co a pplied with acetylcholine from 0.3 M to 3 mM. The concentration response curves were then generated from the Hill Equation as described in Chapter 3. In inhibition concentration response tests (iCRC), acetylcholine was co applied with various concentratio n of BHP ( 7 subtype: 0.03 M to 100 M; heteromeric subtypes: 1 to 1 mM). For inhibition concentration response relations, negative Hill slopes were applied for the calculation of IC 50 values associated with inhibition. Net charge responses were used fo r evaluating the 7 receptor because of its quick desensitization. 271 Although peak responses w ere generally used to compare for other receptor types, here we also used net charge response to report the iCRC analysis results of the heteromeric receptors to be consistent with the 7 type. There was no large discrepancy between the peak and net charge analysis. All of the responses were normalized to the ACh control applied prior to the experimental compounds. The concentration of the control is 300 M for the 7 subtype and 30 M for the muscle in the competition test. In the inhibition tests, the ACh control concentrations for the various receptor subtypes are as follows: 3 4, 100 M; 4 2, 30 M; muscle, 30 M; 7, 60 M.

PAGE 190

190 In the activation tests, each oocty es received 100 M BHP. Since the mutants tested did not always respond to ACh, 100 M 4 OH GTS 21 was applied after BHP to serve as a control. PAM PNU 120596 was co applied with 100 M BHP at 10 M concentration to test whether BHP can activate 7 recepto rs in the presence of a PAM.

PAGE 191

191 CHAPTER 5 5 CONCLUSIONS AND FU TURE WORK The dissertation work has identified that in the ligand and receptor complex, both the binding orientation of the ligands positively charged core pharmacore and the H bonding interactio n in the stability of the open and/or the desensitized states. Cysteine reactive agonist analogs or control molecules (SH reagents) have been tethered to different sites of the receptor to mimi c the different binding conformations of a ligand. H bonding interactions have been investigated by controlled variation of the hydrogen bonding properties of the ligand and/or the receptor via using different H s specific amino acid. The ability of a receptor treated with a covalent agonist analog to respond to ACh was used to report on the extent of covalent modification. The treatments of SH reagents inhibited the ACh responses of cysteine mutants of four amin o acids (S36C, L38C, W55C and L119C) to different degrees depending on the kind of SH reagents applied, but no such inhibition was observed in the I165C mutant regardless of the SH reagents being applied These results are consistent with the predicted ori entation of these residues in our 7 homology model, in which isoleucine 165 points away from the ligand binding site. This method may be also utilized to test other computational models of the ligand gated ion channels or G protein coupled receptors. The underlying basis for the loss of function of the covalently modified receptor was successfully revealed by PAM PNU 120596. If the modified receptor (W55C and L119C) puts the agonist like portion of the tether close to the predicted position of the core ph armacophore, the receptor will reside in the desensitized state that can be

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192 reactivated by PNU 120596. If the modified receptor (S36C and L38C) does occlude the agonist binding site but fails to mimic the binding pose of the ligand core pharmacophore, the receptor will enter into a non functional state insensitive to PNU 120596. In the future studies, a cysteine reactive group might be introduced into the 7 selective benzylidene motif such as shown in Figure 5 1 to identify functional states of different b inding poses. Figure 5 1. The structure of cysteine reactive 7 selective agonist analogs. Using non covalent probes such as arylidene anabaseines has provided a way to interpret ligand receptor interaction in the native receptor. It has been identified that a H bonding effect between the ligand and glutamine 57 in the receptor can vary the channel open probability, depending on the substitution pattern of the ligand. H bonding effects from an agonist have also been found to determine the rate that a rec eptor may enter and remain in the desensitized states. Among the two H bonding donating probes, 2FAB enters the PNU sensitive desensitized state quicker but is less stable in D s state than 3FAB. The experimental data with the Q57 mutants did support the id ea that desensitization profile differences in part arise from differences in H bonding orientation or pattern among the ligands. This H bonding analysis may be extended to the

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193 benzylidene quinuclidines to investigate whether such hydrogen bonding effects are common among the 7 selective agonists in the benzylidene family. The structural and functional relationship data obtained in the native receptor may guide drug development targeting the 7 nAChRs and a particular functional state. Finally, the positiv ely charged nitrogen has been revealed to be unnecessary for a ligand to bind underneath the C loop of the receptor, where an agonist binds. A benzylidene anabaseine analog was designed where the charged nitrogen was substituted with a carbon atom, i.e. ( E ) 3 (6 Benzylidenecyclohex 1 en 1 yl)pyridine ( BHP). The synthesis of BHP was successfully accomplished in a relatively good overall yield. The design of the synthetic route should also allow for the preparation of BHPs with a variety of side groups attach ed. With the help of its extended hydrophobic aryl acetylcholine with high selectivity. The IC 50 of BHP with the muscle type nAChRs is 9 2 M, which is twenty five times smalle r than the IC 50 of PyHexe. Ongoing work involves testing whether BHP and its derivative can activate the wild type 7 nAChRs or its mutants in the presence of PNU 120596.

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194 APPENDIX A NMR SPECTRA OF SYNTH ESIZED COMPOUNDS The NMR spectra of the synthesized c ompounds are shown in this Appendix. While the structure and number of the compound is shown in the spectra, the name of the molecule is described at the bottom of the page.

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195 Figure A 1. 4 B romo 1 methylpiperidine

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196 Figure A 2. 4 B romo 1,1 dimethyl piperidin 1 ium iodide

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197 Figure A 3. 1 Methylpiperidin 3 yl methanesulfonate

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198 Figure A 4. 1,1 Dimethyl 3 (( methylsulfonyl )oxy)piperidin 1 ium (The peaks for the desired compound are too broad to get a meaningful integration)

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199 Figure A 5. 1 Methyl piperidin 4 yl acetate

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200 Figure A 6. 4 Acetoxy 1 (bromomethyl) 1 methylpiperidin 1 ium bromide

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201 Figure A 7. Sodium methanesulfonate

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202 Figure A 8. Sodium methanethiosulfonate

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203 Figure A 9. 1 ((diethylamino) methyl )piperidin 2 one

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204 Figure A 10. So dium Salt of the Aminal of 3 Nicotinoyl 2 piperidone

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205 Figure A 11. Anabaseine dihydrochloride

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206 Figure A 12. 3 Pyrrolyl carboxaldehyde

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207 Figure A 13. 2 Pyrrolylmethylene anabaseine (2PyroAB)

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208 Figure A 14. 3 Pyrrolylmethylene anabaseine (3Pyr oAB)

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209 Figure A 15. 2 Furanylmethylene anabaseine (2FAB)

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210 Figure A 16. 3 Furanylmethylene anabaseine ( 3FAB )

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211 Figure A 17. 2 Thiophenylmethylene anabaseine (2TAB)

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212 Figure A 18. 3 Thiophenylmethylene anabaseine ( 3TAB )

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213 Figure A 19. 2 Pyrrid inylmethylene anabaseine ( 2PAB )

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214 Figure A 20. 3 Pyrridinylmethylene anabaseine (3PAB)

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215 Figure A 21. 4 Pyrridinylmethylene anabaseine ( 4PAB )

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216 Figure A 22. 2 Pyrrolylmethylene anabaseine (2PyroAB), NOESY

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217 Figure A 23. 3 Pyrrolylmethylene anabas eine (3PyroAB), 2D NOESY

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218 Figure A 24. 2 Pyrridinylmethylene anabaseine (2PAB), NOESY

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219 Figure A 25. 3 Pyrridinylmethylene anabaseine (3PAB), NOESY

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220 Figure A 25. 4 Pyrridinylmethylene anabaseine (4PAB), NOESY

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221 Figure A 27. 4 Pyrridinylmethyl ene anabaseine (4PAB), NOESY

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222 Figure A 28. 5 chloro 2,4 dimethoxyphenyl isocyanate

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223 Figure A 29. 1 (5 chloro 2,4 dimethoxyphenyl) 3 (5 methylisoxazol 3 yl)urea (PNU 120596)

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224 Figure A 30. 2 Chlorocyclohex 1 enecarbaldehyde

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225 Figure A 31. 2 (Pyrid in 3 yl)cyclohex 1 enecarbaldehyde

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226 Figure A 32. Phenyl(2 (pyridin 3 yl)cyclohex 1 en 1 yl) methanol

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227 Figure A 33. (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine ( BHP )

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228 Figure A 34. (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), COSY

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229 Figure A 35. (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), HMBC

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230 Figure A 36. (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), HMBC

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231 Figure A 37. (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), HMBC

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232 Figure A 38. (E) 6 Benzylidenecyclohex 1 en 1 yl)pyridine (BHP), NOESY spectrum (top) in comparison with proton spectrum (bottom)

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233 Figure A 39. (4 Methoxyphenyl)(2 (pyridin 3 yl)cyclohex 1 en 1 yl )methanol

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234 Figure A 40. ( E ) 3 (6 (4 Methoxybenzylidene)cyclohex 1 en 1 yl) pyridine (4 MeO BH P )

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235 Figure A 41. ( E ) 3 (6 (4 Methoxybenzylidene)cyclohex 1 en 1 yl)pyridine (4 MeO BHP), NOESY

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236 Figure A 42. (4 ((Tert butyldimethylsilyl)oxy)phenyl)(2 (pyridin 3 yl)cyclohex 1 en 1 yl)methanol

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237 Figure A 43. ( E ) 3 (6 (4 ((Tert butyldimethylsilyl) oxy)benzylidene)cyclohex 1 en 1 yl) pyridine

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238 Figure A 44. ( E ) 4 ((2 (Pyridin 3 yl)cyclohex 2 en 1 ylidene)methyl)phenol (4 OH BHP)

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239 Figure A 45. 1 Chlorocyclohex 1 ene

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240 Figure A 46. 1 (Pyridin 3 yl) cyclohexanol

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241 Figure A 47. 3 (C yclohex 1 en 1 yl)pyridine

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242 Figure A 48. N (pyridin 3 ylmethyl )benzamide

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243 Figure A 49. N (pyridin 3 ylmethyl) N (tert butoxycarbonyl amino )benzamide

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244 Figure A 50. 1 Phenyl 2 (pyridin 3 yl) 2 (tert butoxycarbonyl amino) ethanone

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245 Figure A 51. 2 Amino 1 p henyl 2 (pyridin 3 yl)ethanone hydrochloric salt

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246 APPENDIX B ELECTROPHYSIOLOGY AS SAY AND ANALYSIS The representative traces, additional analysis results and alternative mechanisms are presented in this section.

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247 Figure B 1. Representative traces and expressio n level test of the h 7 receptor and the glutamine 57 mutants. Figure B 2. The alternative mechanism for H bonding impact on the h entry and remain in the Ds state.

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248 Figure B 3. The relationship of the normalized PNU 120596 evoked peak response after 2FAB and the inhibition on ACh evoked response after 2FAB. The PNU 120596 evoked peak current after 2FAB (Figure 3 12 B) was normalized to the value of the PNU 120596 evoked peak response when co applied with 2FAB as shown in Figure 3 9 B. Figure B 4. PNU 120596 stimulated peak current on ACh applied after. Each cell was treated with two initial ACh controls, 300 M 2FAB, 300 M PNU 120596, and two following ACh controls. All of the responses are normalized to the average of the two initial ACh controls and the value of the last two ACh controls is displayed in the figure.

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249 Figure B 5. The energy landscape of the acetylcholine and arylidene anabaseines with different receptor types. These energy landscape represents experimental data obt ained using ligands at 300 M (high occupancy). Curves with experimental support are highlighted. The abbreviations for the states are as s 120596 sensitive i 120596 insensitiv e desensitized state.

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274 BIOGRAPHICAL SKETCH Jingyi Wang, daughter of Guangren Wang and Huazhen Dan, was born in Luoyang, China, in 1984. She graduated with a Bachelor of Science degree in chemistry from Nankai University, China, where she studied the binding affinity of the neocarzinostatin analogues with various bulge DNA structures, under the supervision of Dr. Zhen Xi. She then moved to Gainesville, Florida in August 2006 to pursue her PhD in chemistry at the University of Florida. She receive d her PhD in chemistry in December 2011, under the guidance of Dr. Nicole A. Horenstein. After completion of her studies, she will carry on her career in Pennsylvania.