Novel Nicotinic Acetylcholine Receptor Ligands based on Cytisine, Ferruginine, Anatoxin-a and Choline: In vitro Evaluation and Structure-Activity Relationships Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Maria Cristina Tilotta aus Trapani Bonn 2004 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Referent: PD. Dr. Daniela Gündisch 2. Referent: Prof. Dr. Christa E. Müller Tag der Promotion: 26.11.04 Die vorliegende Arbeit wurde in der Zeit von Januar 2001 bis November 2004 am Pharmazeutischen Institut der Rheinischen Friedrich-Wilhelms-Universität Bonn unter Leitung von Frau PD Dr. Daniela Gündisch durchgeführt. Ich danke herzlich Frau PD Dr. Daniela Gündisch für ihre Unterstützung und für die wissenschaftliche Leitung der Arbeit. Weiterhin möchte ich mich bei Frau Prof. Dr. Christa E. Müller für die freundliche Übernahme des Korreferats bedanken. To my parents, Enza and Bruno Ai miei genitori, Enza e Bruno Table of contents I Table of contents I. Introduction 1 I/1. Nicotinic Acetylcholine Receptors (nAChRs) ...........................................................1 I/1.1. Historical perspective of the receptor...........................................................1 I/1.2. The endogenous neurotransmitter acetylcholine........................................1 I/1.3. The architecture of the nAChR ......................................................................6 I/1.4. Ligand binding sites ........................................................................................9 I/1.5. Transition and functional states of the nAChR .........................................13 I/1.6. NAChRs in the central nervous system......................................................16 I/1.7. Distribution and function of nAChRs in non-neuronal cells ..................19 I/1.8. Knock-out (KO) mice.....................................................................................19 I/1.9. Pathophysiology ............................................................................................21 I/2. Modulators on nAChRs..............................................................................................29 I/2.1. Nicotine and its pharmacological action in the ANS and CNS...............29 I/2.2. Modulators on nAChRs (small ligand based) ...........................................31 I/2.3. Peptide toxins .................................................................................................46 I/3. Pharmacophore models for nAChR ligands ...........................................................49 I/4. Radioligand binding studies......................................................................................55 II. Objectives 71 III. Results 78 III/1. Radioligand binding studies for different nAChRs ............................................78 III/2. Characterization of α3β4* nAChRs........................................................................83 III/2.1. Introduction ....................................................................................................83 III/2.3. Results..............................................................................................................89 III/2.4. Discussion .......................................................................................................92 III/3. Cytisine as a lead compound for novel nAChR ligands.....................................94 III/3.1. Introduction ....................................................................................................94 III/3.2. Project ............................................................................................................100 III/3.3. Determination of affinities and structure-activity relationships (SAR)..... ........................................................................................................................101 III/3.4. Discussion .....................................................................................................118 III/4. Ferruginine as a lead compound for novel nAChRs ligands...........................123 III/4.1. Introduction ..................................................................................................123 III/4.2. Project ............................................................................................................126 III/4.3. Determination of affinities and structure-activity relationships (SAR)..... ........................................................................................................................128 III/4.4. Discussion .....................................................................................................143 III/5. Pinnamine variants.................................................................................................146 III/5.1. Introduction ..................................................................................................146 III/5.2. Project ............................................................................................................146 III/5.3. Determination of affinities and structure-activity relationships (SAR)..... ........................................................................................................................147 Table of contents II III/5.4. Discussion .....................................................................................................148 III/6. Anatoxin-a as a lead compound for novel nAChR ligands .............................149 III/6.1. Introduction..................................................................................................149 III/6.2. Project ............................................................................................................153 III/6.3. Determination of affinities and structure-activity relationships (SAR)..... ........................................................................................................................156 III/6.4. Discussion .....................................................................................................163 III/7. Quinuclidin-2-ene based derivatives as ligands for nicotinic acetylcholine receptors............................................................................................................................166 III/7.1. Introduction..................................................................................................166 III/7.2. Project ............................................................................................................167 III/7.3. Determination of affinities and structure-activity relationships (SAR)..... ........................................................................................................................167 III/7.4. Discussion .....................................................................................................168 III/8. Choline analogues as ligands for nicotinic acetylcholine receptors................170 III/8.1. Introduction..................................................................................................170 III/8.2. Project ............................................................................................................174 III/8.3. Determination of affinities and structure-activity relationships (SAR)..... ........................................................................................................................176 III/8.4. Discussion .....................................................................................................184 IV.Summary and Outlook 187 V. Experimental Section 199 V/1.1. General Information ....................................................................................199 V/1.2. Membrane Preparation ...............................................................................201 V/1.3. Protein Determination.................................................................................204 V/1.4. Radioligand binding studies for α4β2*nAChR .......................................206 V/1.5. Radioligand binding studies for α7* nAChR...........................................207 V/1.6. Radioligand binding studies for α3β4*nAChR .......................................209 V/1.7. Radioligand binding studies for (α1)2β1γδ nAChR................................211 V/1.8. Analysis of data............................................................................................212 VI. Abbreviations 213 VII. References 218 VIII. Publications 242 IX. Curriculum vitae 245 I. Introduction 1 nAChRs I. Introduction I/1. Nicotinic Acetylcholine Receptors (nAChRs) I/1.1. Historical perspective of the receptor Almost 100 years ago, John Langley of Cambridge University developed the idea of the "receptive substance" or "receptor" by using nicotine as an experimental tool 1. He suggested “pharmacological substances could possess the structure necessary for the combination with appropriate molecules on cells” 2. Between 1905 and 1907, Langley carried out a series of experiments on the somatic neuromuscular junction and postulated the existence of transmitter receptors 3. Later, Dale and his colleagues identified acetylcholine (ACh) 1 as the transmitter that acts on the receptors discovered by Langley (neuromuscular junction) as well as on receptors in the heart. In 1914, Dale distinguished the action of muscarine