The Action of Xanomeline on Human Muscarinic Receptors Expressed in Chinese Hamster Ovary Cells

The action of xanomeline on human muscarinic receptors expressed in Chinese hamster ovary cells A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY MEREDITH NOETZEL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor: Dr. Esam El-Fakahany JANUARY, 2009 © Meredith Noetzel, January 2009 Acknowledgements I would like to thank my advisor Dr. Esam El-Fakahany for his support and guidance. He kindly let me join his lab and be his last graduate student. He has also helped me to grow as a scientist. I would like to thank my committee members Dr. Virginia Seybold, Dr. William Engeland and Dr. Ping Law for their time and advice. Without their thoughtful input I would not have been able to complete my thesis as quickly. They have also been instrumental in helping me learn to present my work in a clear and concise manner. The El-Fakahany lab members that I have worked with have been great. I would especially like to thank Marianne Grant for all of her help and support. She was always there to teach me new techniques, answer questions and help with anything. Without her I would not have been able to get everything done. I would also like to thank Kayla De Lorme and Allison Cherry for the great conversions and making the lab a fun place to be. I would also like to acknowledge both Marianne Grant and Kayla De Lorme for providing some of the data presented in this thesis. I would also like to thank my former lab. Dr. Walter Low has been a great colleague over the years. I would specifically like to thank Terry Burns for his help and thoughtful discussions. I would also like to thank Aleta Rees, David Horn and Yuliya Perepelitsa for their hard work and helping me learn to be a better mentor. Lastly, I would like to thank my friends and family for supporting me through all of my schooling. They have always been there when I needed them, in both the good times and the bad. I would especially like to thank my parents for their support and encouragement over the years. I would like to thank my boyfriend, Mark Gramith, who has helped me through each stage and has had an incredible amount of patience. I i would also like to thank Thaddeus Brink for taking the time to read through my thesis and giving my feedback. ii This dissertation is dedicated to my grandfather David Noetzel, who got me interested in science at a very young age. iii Thesis abstract Muscarinic acetylcholine receptors are part of the G protein-coupled superfamily of receptors. There are five subtypes of muscarinic receptors (M1-M5). These receptors are therapeutic targets for in a number of diseases, including Alzheimer’s disease and schizophrenia. The orthosteric (primary) binding domain of muscarinic receptors is highly conserved across subtypes making it difficult to develop agonists or antagonists that bind with selectivity at a particular subtype. However, there are allosteric (secondary) binding sites on muscarinic receptors that are thought to be less well conserved between subtypes, which may lead to the development of drugs that bind in a subtype-selective manner. One potential drug of interest is xanomeline. Previous research has found that it binds in a unique manner to muscarinic receptors; it binds in a reversible manner at the orthosteric site and in a wash-resistant manner at an allosteric site. Furthermore, xanomeline is thought to be an M1/M4 selective agonist. The goal of the current research was to characterize the effects of prolonged exposure of the various receptor subtypes to xanomeline and determine the mechanisms through which these effects occur. To do this Chinese hamster ovary cells expressing the various individual muscarinic receptors were exposed to xanomeline for brief and long-term time periods and radioligand binding and functional assays were performed. Xanomeline was shown to bind in a reversible and wash-resistant manner at the M1-M4 receptor subtypes. Although the effects of acute xanomeline exposure were similar across subtypes, long- term treatment with xanomeline resulted in differential effects among subtypes. More detailed experiments were conducted at the M1 and M3 receptor subtypes to elucidate the mechanisms of the long-term effects of xanomeline. Wash-resistant xanomeline binding was able to modulate the functional properties of the receptor. The consequences of the long-term effects of wash-resistant xanomeline binding are iv dependent on activation of the receptor through the orthosteric binding site. It appears that these effects are a result of receptor internalization and allosteric modulation of the receptor. Thus, my research demonstrates that it is important to determine both the acute and long-term drug interactions with the receptor as part of the drug development process. v TABLE OF CONTENTS Chapter 1: Introduction – muscarinic receptors and xanomeline 1. Overview 2 2. G protein-coupled receptors 4 3. Subtypes, localization and function of muscarinic receptors 5 4. Structure and binding of muscarinic receptors 7 5. A model of receptor activation 9 6. Receptor trafficking 10 7. Drug-receptor interactions 10 8. Signaling pathways for muscarinic receptors 11 9. Consequences of prolonged exposure of muscarinic receptors to agonist 15 and antagonists 10. Influence of receptor mutations and expression levels on receptor activity 20 11. In vitro receptor expression systems 21 12. Allosteric interactions with the receptor 23 13. Pharmacological profile of allosteric binding 24 14. Allosteric sites on muscarinic receptors 25 15. Muscarinic receptors, allosteric sites and diseases 28 16. Alzheimer’s disease 28 17. Cholinergic hypothesis of Alzheimer’s disease 29 18. Cholinergic treatments for Alzheimer’s disease 31 19. Non-cholinergic treatments for Alzheimer’s disease 32 20. Schizophrenia 33 21. Treatment for schizophrenia 35 22. Binding and functional properties of xanomeline 35 23. Proposed mechanism of xanomeline binding 37 24. Wash-resistant xanomeline binding 38 25. Xanomeline as a treatment for Alzheimer’s disease and schizophrenia 39 26. Xanomeline treatment in Alzheimer’s disease models 40 27. Clinical trials with xanomeline for the treatment of Alzheimer’s disease 41 28. Xanomeline treatments in animal models of schizophrenia 42 29. Clinical trials with xanomeline for the treatment of Alzheimer’s disease 43 30. Summary 43 Chapter 2: Materials and methods 1. Materials and general treatment protocols 46 2. Radioligand binding assays 48 3. Functional assays 50 4. Variations on radioligand binding and functional assay paradigms 54 5. Data analysis 59 Chapter 3: Determination of the acute and long-term effects of xanomeline binding at M1-M4 muscarinic receptors 1. Introduction 61 2. Results 62 3. Discussion 72 4. Figures 80 5. Tables 115 vi Chapter 4: Determination of the acute and long-term functional effects of xanomeline at the M1 and M3 muscarinic receptors 1. Introduction 121 2. Results 122 3. Discussion 131 4. Figures 136 5. Tables 152 Chapter 5: Receptor activation is required for the development of the long-term effects of xanomeline at the M1 and M3 muscarinic receptors 1. Introduction 158 2. Results 159 3. Discussion 167 4. Figures 172 5. Tables 188 Chapter 6: Mechanisms of the long-term effects of xanomeline wash-resistant binding 1. Introduction 196 2. Results 197 3. Discussion 204 4. Figures 210 5. Tables 219 Chapter 7: Conclusions 224 References 230 Appendix A: Differences observed between suspension and monolayer protocols 1. Introduction 242 2. Results 242 3. Discussion 244 4. Figures 246 5. Table 248 vii LIST OF TABLES Chapter 3: Determination of the acute and long-term effects of xanomeline binding at M1-M4 muscarinic receptors Table 1. Short- and long-term effects of xanomeline, carbachol or pilocarpine 115 binding on [3H]NMS binding in CHO cells expressing hM1-hM4 receptor subtypes. Table 2. [3H]NMS saturation binding parameters for xanomeline, carbachol or 118 pilocarpine pretreatment and waiting periods in CHO cells expressing hM1-hM4 receptor subtypes. Chapter 4: Determination of the acute and long-term functional effects of xanomeline at the M1 and M3 muscarinic receptors Table 1. Effects of agonist treatment on PI hydrolysis in CHO cells expressing 152 hM1 and hM3 receptor subtypes. Table 2. Effects of carbachol, pilocarpine or xanomeline stimulation following 153 short- and long-term agonist pretreatment and washout on inositol phosphates production in CHO cells expressing hM3 receptors. Table 3. Effects of carbachol, pilocarpine or xanomeline stimulation following 155 short- and long-term agonist pretreatment and washout on inositol phosphates production in CHO cells expressing hM1 receptors. Chapter 5: Receptor activation is required for the development of the long-term effects of xanomeline at the M1 and M3 muscarinic receptors Table 1. Effects of xanomeline pretreatment and washout in the absence or 188 presence of atropine on [3H]NMS saturation binding and functional parameters of carbachol stimulation in CHO cells expressing hM1 or hM3 receptors. Table 2. Effects of xanomeline pretreatment on inhibition of NMS binding, 190 saturation binding and functional parameters in wild-type and mutant rM1 receptors expressed in CHO cells. Table 3. Effects of blocking receptor function using siRNA against Gq and G11 G 192 proteins on [3H]NMS saturation binding parameters in CHO cells expressing hM3 receptors assayed in suspension. Table 4. Effects of xanomeline analogs on inhibition of [3H]NMS binding and 193 functional parameters in CHO cells expressing hM3 receptors. Table 5. Effects of treatment with the propyl xanomeline analog on [3H]NMS 194 saturation binding parameters in CHO cells expressing hM3 receptors. viii Chapter 6: Mechanisms of the long-term effects of xanomeline wash-resistant binding Table 1. Inhibition of radioligand binding using a receptor-saturating concentration 219 of [3H]QNB or [3H]NMS following xanomeline pretreatment and various waiting periods in CHO cells expressing hM1 or hM3 receptor subtypes.

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