The Pennsylvania State University. The Graduate School. College of Medicine THE ALLOSTERIC MODULATING EFFECTS OF DRONEDARONE ON

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1 The Pennsylvania State University The Graduate School College of Medicine THE ALLOSTERIC MODULATING EFFECTS OF DRONEDARONE ON MUSCARINIC RECEPTOR SUBTYPES AT A NOVEL ALLOSTERIC BINDING SITE A Thesis in Cell and Molecular Biology by Gihan M. Jayasuriya 2013 Gihan M. Jayasuriya Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2013

2 The thesis of Gihan M. Jayasuriya was reviewed and approved* by the following: John Ellis Professor of Psychiatry and Pharmacology Thesis Advisor Richard Mailman Professor and College of Medicine Distinguished Senior Scholar of Pharmacology and Neurology Blaise Peterson Associate Professor of Cellular and Molecular Physiology Sarah K. Bronson Associate Professor of Cellular and Molecular Physiology Director, Cell and Molecular Biology Graduate Program * Signatures are on file in the Graduate School ii

3 Abstract: This thesis analyzes the allosteric modulating properties of the antiarrhythmic agent dronedarone across muscarinic receptor subtypes. The allosteric effects of dronedarone were examined because of the unique allosteric modulating properties exhibited by its parent compound amiodarone. One key finding was that amiodarone significantly enhances acetylcholine-stimulated arachidonic acid (AA) release without affecting potency at the M 3 receptor. This maximal response enhancement without a shift in potency is a novel allosteric modulating property. Furthermore, amiodarone does not interact competitively with gallamine, one of many muscarinic allosteric modulators binding at an extensively characterized common allosteric site. Analyzing amiodarone analogs such as dronedarone would further demonstrate the allosteric modulating properties at this novel allosteric site. Dronedarone exhibits significantly different allosteric effects on orthosteric ligand affinity, potency, and maximal response that depend on the orthosteric agonist, receptor subtype, and response type in M 1 and M 3 receptors. For example, dronedarone negatively modulates maximal acetylcholine-stimulated AA release at the M 1 receptor but neutrally modulates maximal acetylcholine-stimulated AA release at the M 3 receptor. The allosteric effects of dronedarone are mechanistically illustrated by Hall s allosteric two-state model simulations. Most importantly, dronedarone exhibits a range of allosteric effects across receptor subtypes that collectively have not been displayed by other ligands. Interaction studies were investigated to determine whether amiodarone and dronedarone are binding at a common site. Since amiodarone and dronedarone both significantly enhance maximal pilocarpine-stimulated AA release in M 3 receptors it would be difficult to characterize their interaction in a response interaction study. In this scenario, a large difference in efficacy modulation between amiodarone and dronedarone is optimal in determining if one ligand can reverse the efficacy modulating effects of the other ligand. Therefore, the interaction of dronedarone with the amiodarone analog N-ethylamiodarone (NEA) was investigated. NEA was used because previous laboratory findings showed that NEA reversed the amiodarone enhancement of maximal pilocarpine-stimulated AA release in M 3 receptors. Thus, the iii

4 interaction between NEA and amiodarone is consistent with a competitive interaction. We found that NEA also reverses the dronedarone enhancement of maximal pilocarpine-stimulated AA release which indicates an interaction consistent with competition. These two results help show that amiodarone and dronedarone would also interact in a competitive manner. Allosteric modulators can change the off-rate of an orthosteric ligand. Dronedarone slows the rate of [ 3 H] N-methylscopolamine (NMS) dissociation in both M 1 and M 3 receptors. At the M 3 receptor, dronedarone is able to completely reverse the effects of amiodarone on [ 3 H] NMS dissociation. These results are additional evidence that dronedarone is acting allosterically and that the interaction between amiodarone and dronedarone is consistent with competition. In summary, dronedarone exhibits a wide range of allosteric modulating properties that differ greatly from the allosteric modulating properties displayed by most other muscarinic allosteric modulators. The majority of muscarinic allosteric ligands (including those binding at the common site) modulate orthosteric ligand potency without changing maximal response. In contrast, dronedarone displays many different allosteric effects on binding and maximal response at a newly discovered allosteric site. The importance of targeting distinct allosteric sites has been shown through GABA A receptor allosteric modulation. Clinical examples of allosteric modulators binding at distinct GABA A receptor allosteric sites include benzodiazepines and general anesthetics which exhibit different pharmacological properties. Identifying new allosteric sites enables the development of more specific drugs that exhibit fewer side effects. Overall, the results presented in this thesis enhance knowledge about the allosteric modulating properties of muscarinic receptors at a novel allosteric binding site. iv

5 Table of Contents List of Figures... viii List of Tables... x Abbreviations... xi Acknowledgements... xiii CHAPTER 1. INTRODUCTION G Protein-Coupled Receptors Acetylcholine and Muscarinic Acetylcholine Receptors Acetylcholine Muscarinic acetylcholine receptors M 1, M 3, and M 5 signaling M 2 and M 4 signaling M 1 receptor expression and function M 2 receptor expression and function M 3 receptor expression and function M 4 receptor expression and function M 5 receptor expression and function Muscarinic Receptor Structure Allosteric Binding Sites and Types of Allosteric Modulators Allosteric Modulation of Binding and Response Hall s Allosteric Two-State Model Advantages of Allosteric Modulators Allosteric Modulation of the GABA A Receptor...17 v

6 1.9 Rationale...20 CHAPTER 2. METHODS Materials Cell Culture Membrane Preparation Radioligand Binding Assays Dissociation rate binding assays Response Assays [ 3 H] Arachidonic acid release [ 3 H] Inositol phosphate metabolism Data Analysis Response assay analysis Binding assay analysis Curve fitting, statistical tests, and simulations CHAPTER 3. RESULTS Introduction Effects of Dronedarone on Response in M 3 Receptors Effects of dronedarone on agonist-stimulated arachidonic acid release Effects of dronedarone on agonist-stimulated inositol phosphate metabolism Further significance of the M 3 receptor results Interaction of Dronedarone and N-ethylamiodarone in M 3 Receptors Effects of Dronedarone on Response in M 1 Receptors Effects of dronedarone on agonist-stimulated arachidonic acid release vi

7 3.4.2 Effects of dronedarone on agonist-stimulated inositol phosphate metabolism Further significance of the M 1 receptor results Effects of Dronedarone on [ 3 H] NMS Off-Rate in M 1 Receptors Interaction of Amiodarone and Dronedarone on [ 3 H] NMS Off-Rate in M 3 Receptors...50 CHAPTER 4. DISCUSSION CHAPTER 5. APPENDIX Equation 1: Log [Agonist] versus Response Variable Slope Equation Equation 2: Integrated Rate Law Equation of a First-Order Reaction Equation 3: Hall s Allosteric Two-State Model Equation for Response...61 REFERENCES vii

8 List of Figures GPCR signal transduction Structure of a machr Structures of allosteric modulators binding to the common allosteric site Mechanisms of allosteric modulation Examples of allosteric modulation A cubic depiction of Hall s allosteric two-state model An example of the inherent ceiling effect that allosteric modulators possess Allosteric modulation of the GABA A receptor Molecular structures of the allosteric modulators used in these studies The allosteric modulating effects of dronedarone on pilocarpine-stimulated AA release in M 3 receptors The allosteric modulating effects of dronedarone on ACh-stimulated AA release in M 3 receptors Simulations of Figure generated from Hall s ATSM The allosteric modulating effects of dronedarone on ACh-stimulated IP metabolism at the M 3 receptor Simulations of Figure generated from Hall s ATSM The allosteric modulating effects of dronedarone on pilocarpine-stimulated IP metabolism at the M 3 receptor Simulations of Figure generated from Hall s ATSM...38 viii

9 Interaction of amiodarone and NMS in pilocarpine-stimulated AA release at the M 3 receptor Interaction of amiodarone and NEA in pilocarpine-stimulated AA release at the M 3 receptor Interaction of dronedarone and NEA in pilocarpine-stimulated AA release at the M 3 receptor The allosteric modulating effects of dronedarone on ACh-stimulated AA release in M 1 receptors The allosteric modulating effects of dronedarone on pilocarpine-stimulated AA release in M 1 receptors The allosteric modulating effects of dronedarone on ACh-stimulated IP metabolism in M 1 receptors The allosteric modulating effects of dronedarone on pilocarpine-stimulated IP metabolism in M 1 receptors Amiodarone and dronedarone effects on [ 3 H] NMS dissociation at the M 1 receptor Interaction study between the common allosteric ligands gallamine and obidoxime Interaction of amiodarone and dronedarone on [ 3 H] NMS dissociation at the M 3 receptor Simulations portraying the allosteric modulating properties of the common site muscarinic allosteric ligands and most other muscarinic allosteric ligands ix

10 List of Tables Overview of machr subtype signal transduction, expression profiles, and disease relevance A summary of the allosteric modulating effects of 10 µm dronedarone on ACh-stimulated response at the M 1 and M 3 receptors A summary of the allosteric modulating effects of 10 µm dronedarone on pilocarpinestimulated response at the M 1 and M 3 receptors x

11 Abbreviations 4L-ATSM AA Aβ AC ACh AChEIs ATSM BQCA BZDs camp CHO CNS COPD DAG EC 50 EM-HEPES-BSA four-ligand allosteric two-state model arachidonic acid β-amyloid adenylyl cyclase acetylcholine acetylcholinesterase inhibitors allosteric two-state model benzyl quinolone carboxylic acid benzodiazepines cyclic adenosine monophosphate Chinese hamster ovary central nervous system chronic obstructive pulmonary disease 1,2-diacylglycerol half-maximal effective concentration Eagle s basal medium with 20 mm HEPES and 2 mg/ml fatty acid-free bovine serum albumin EM-HEPES-LiCl Eagle s basal medium with 20 mm HEPES and 10 mm lithium chloride FDA United States Food and Drug Administration xi

12 GABA GDP GEF GIRK GPCR GTP IP IP 3 K d KO machr nachr NAM NEA NMS PAM PB SAM SAR TM γ-aminobutyric acid guanosine diphosphate guanine nucleotide exchange factor G protein-coupled inwardly rectifying potassium G protein-coupled receptor guanosine triphosphate inositol phosphate inositol 1,4,5-triphosphate equilibrium dissociation constant knockout muscarinic acetylcholine receptor nicotinic acetylcholine receptor negative allosteric modulator N-ethylamiodarone N-methylscopolamine positive allosteric modulator phosphate buffer silent allosteric modulator structure activity relationship transmembrane xii

13 Acknowledgements I would like to begin this section by thanking my advisor Dr. John Ellis for providing me the opportunity to conduct research which made this thesis possible. He has introduced me to the intriguing field of receptor pharmacology and the importance of allosteric modulators as drug candidates. I was introduced to the works of prominent scientists such as Terry Kenakin. I also learned many new lab techniques in his lab. Overall, he has been a wonderful advisor and has provided me with valuable guidance. I would also like to thank the other members of my graduate committee: Drs. Bronson, Canfield, Mailman, and Peterson. Each member provided constructive criticism and excellent suggestions at committee meetings and seminars. Each member also gave me valuable career advice. I would next like to thank the other lab members Gwendolynne Elmslie, Margaret Seidenberg-Ellis and Dr. Edward Stahl. All of them were very helpful in assisting me with laboratory procedures, scientific writing, data analysis, and creating an enjoyable laboratory environment in general. Without their efforts this thesis would not be possible. I offer my gratitude to the Department of Psychiatry and the Cell and Molecular Biology Graduate Program. The Department of Psychiatry provided the venue for my research and I enjoyed meeting many of the members of this department. The Cell and Molecular Biology Graduate Program provided me with an opportunity to continue my education. Finally, I would like to thank my family and friends for their support while I was pursuing my degree. xiii

14 CHAPTER 1. INTRODUCTION 1.1 G Protein-Coupled Receptors G protein-coupled receptors (GPCRs) are a large protein family that is targeted by 50% of all drug candidates [1]. GPCRs can initiate signal transduction by activating different G proteins but non-g protein-mediated signaling pathways have been discovered. The G protein exists as a heterotrimeric complex consisting of α, β, and γ subunits. These subunits are bound when the receptor is unoccupied as shown in Figure Figure GPCR signal transduction. The four G α subunit classes are G i, G s, G q, and G 12. Within each class there are multiple subtypes. For example, the G 12 class is comprised of the G 12 and G 13 subtypes. Modified from [3]. In G protein-mediated signal transduction, agonist binding to GPCRs causes a receptor conformation change that activates its guanine nucleotide exchange factor (GEF) activity. GEFs enable guanosine diphosphate (GDP) to dissociate and guanosine triphosphate (GTP) to bind to the G α subunit. The GTP-bound G α subunit and the G βγ complex dissociate and activate downstream effectors such as adenylyl cyclase (AC) to initiate signal transduction. Conversely, 1

15 in non-g protein-mediated signaling pathways activation of signal transducers initiates downstream signal transduction. For example, agonist binding causes β arrestin to activate signaling molecules such as extracellular signal-regulated kinase 1/2 [2]. In both signal transduction pathways the effectors activate second messengers. Measurement of second messenger levels is often characterized as response. Examples of responses include arachidonic acid (AA) release, inositol phosphate (IP) metabolism, and cyclic adenosine monophosphate (camp) synthesis. These responses can be activated by different G α subunits in G proteinmediated signal transduction. There are four classes of G α subunits which are the G s, G i, G q, and G 12 classes and each class contains multiple subtypes [3]. The β and γ subunits also exist as multiple subtypes. Overall, agonist binding to GPCRs initiates many receptor-mediated signaling pathways. 1.2 Acetylcholine and Muscarinic Acetylcholine Receptors Acetylcholine Acetylcholine (ACh) is an agonist and a neurotransmitter that is distributed across the central nervous system (CNS) and the periphery. ACh binds to muscarinic acetylcholine receptors (machrs) and nicotinic acetylcholine receptors (nachrs). The machrs are GPCRs that consist of five subtypes which are designated as M 1 -M 5. Details about each subtype are provided in Sections The nachrs are ionotropic receptors that can also bind nicotine. nachr subtypes arise from numerous subunit compositions [4]. Most of the subunits are α and β subtypes and nachrs are assembled as pentameric complexes [4]. ACh is involved in many physiological roles such as inducing muscular contraction and lowering heart rate by binding to machrs and/or nachrs. In the CNS, ACh signaling across neuronal networks influences many brain functions such as cognition and motor control. ACh is synthesized in neurons and transported to the synaptic cleft by secretory vesicles where ACh binds to postsynaptic machrs and nachrs [5]. At high concentrations ACh is degraded by the enzyme acetylcholinesterase to prevent excessive ACh stimulation. ACh acts as a neuromodulator in regulating neural function which enables 2

16 control of large neuronal populations [5]. In the hippocampus, ACh regulates long-term potentiation whereas in the nucleus accumbens it regulates appetitive learning [6]. ACh release is modulated by the M 2 and M 4 machr subtypes which act as presynaptic autoreceptors in a negative feedback mechanism [7]. ACh neurotransmission impairments are clearly observed in many CNS disorders. For example, a progressive loss of ACh signaling occurs during Alzheimer s disease progression as β-amyloid (Aβ) plaque formation increasingly disrupts neuronal network function [6]. ACh affects many other CNS physiological processes. For instance, ACh levels are selectively enhanced during stress [8]. Only the hippocampus and prefrontal cortex experienced elevated ACh levels after stress inducement in rat models and stress relief caused a return to physiological ACh levels in these brain regions [8]. In another physiological role, decreased ACh levels reduce feeding behavior in the nucleus accumbens [9]. In fact, the hunger hormone ghrelin causes ACh release from the laterodorsal tegmental area of the brain [10]. These examples show that ACh plays an important role in CNS physiology. In the periphery, ACh is the primary neurotransmitter for the parasympathetic system which helps maintain bodily homeostasis [6]. For instance, ACh slows heart rate after binding to the M 2 machr, the predominant cardiac subtype. ACh binding inhibits the effector AC which decreases intracellular camp levels and leads to reduced Na + and Ca 2+ influx. The Na + influx decrease lengthens the time needed to attain the threshold potential whereas the Ca 2+ influx decrease extends the depolarization phase once threshold is reached [11]. Under both circumstances heart rate is reduced. Furthermore, ACh negatively modulates camp-dependent responses which include protein kinase A phosphorylation targets such as phospholamban [11]. All of these actions decrease cardiac contractility. ACh binding also slows heart rate through G βγ activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. The GIRK channels become more permeable to K + ions and the resulting K + efflux causes cell membrane hyperpolarization [12]. Cellular electrical excitability is decreased which slows heart rate [12]. ACh binds to machrs to induce smooth muscle contraction in regions such as the airways and the bladder. ACh binding releases Ca 2+ from the sarcoplasmic reticulum which then activates myosin light chain kinase activity to cause muscular contractions [13]. ACh also binds to nachrs in the somatic nervous system to stimulate skeletal muscle contraction [4]. Lastly, in 3

17 another physiological role, ACh binds to machrs to stimulate insulin and glucagon release in pancreatic β cells [14]. Overall, ACh is an important neurotransmitter that maintains bodily homeostasis through many physiological roles such as long-term potentiation. Table Overview of machr subtype signal transduction, expression profiles, and disease relevance. Modified from [6] Muscarinic acetylcholine receptors ACh is the endogenous ligand for machrs, GPCRs that are widely expressed in the CNS and periphery as shown in Table The machr family consists of five subtypes which are designated as M 1 -M 5. The physiological and behavioral roles of each subtype will be characterized in Sections The machrs are therapeutic targets for drug candidates treating diseases ranging from Alzheimer s disease to urinary incontinence. In Alzheimer s disease, machrs are targeted because ACh signaling is critical in physiological processes such as cognition, reward, and long-term potentiation [6]. In Alzheimer s disease treatment, the lack of subtype selectivity with acetylcholinesterase inhibitors (AChEIs) has led to machr agonists as the latest drug candidates [15]. Another example of machr disease relevance is in chronic obstructive pulmonary disease (COPD) treatment because ACh signaling induces airway smooth 4

18 muscle contractions. Muscarinic antagonists such as tiotropium bromide target M 3 machrs to decrease airway smooth muscle contractions [16]. The importance of drug candidates that selectively target individual machr subtypes in disease treatment will be shown throughout this thesis M 1, M 3, and M 5 signaling The M 1, M 3, and M 5 subtypes are preferentially G q -coupled receptors. Agonist binding to these receptor subtypes activates phospholipase C which results in the formation of the second messengers inositol 1,4,5-triphosphate (IP 3 ) and 1,2-diacylglycerol (DAG). IP 3 causes intracellular Ca 2+ release from the endoplasmic reticulum and DAG activates protein kinase C [3]. One common response assay in preferentially G q -coupled receptors is IP metabolism. This assay can track radiolabeled IP 3 as it is metabolized into other inositol compounds such as inositol diphosphate. Agonist binding also causes cytoplasmic phospholipase A 2 to release AA from the phospholipid bilayer. AA is an omega-6 fatty acid that is a lipid second messenger regulator [17]. AA release is another common response assay in preferentially G q -coupled receptors. Radiolabeled AA levels can be determined after agonist stimulation in [ 3 H] AA pretreated cells. Both AA release and IP metabolism assays are used in these studies to analyze receptor-mediated response in machr subtypes M 2 and M 4 signaling The M 2 and M 4 subtypes are preferentially G i -coupled receptors. Agonist binding causes G i to inhibit AC. Since AC catalyzes camp synthesis by converting adenosine triphosphate into camp a decrease in intracellular camp levels occurs. The second messenger camp regulates many physiological processes including lipid metabolism and ion channel interactions. In the heart, ACh binding at the M 2 subtype (the predominant machr subtype) reduces heart rate by 5

19 decreasing camp levels and activating GIRK channels. One common response assay used to analyze G i -coupled receptor response is camp synthesis. Lastly, note that all five machr subtypes are capable of coupling to multiple G α subunits. This occurrence has been termed promiscuous G protein-coupling and must be taken into consideration when analyzing machr subtypes [18]. For example, ACh stimulation has a biphasic effect on camp levels in M 2 receptors [19]. Low ACh concentrations cause the expected G i -mediated inhibitory phase which decreases camp levels due to AC inhibition [19]. However, at high ACh concentrations there is a G s -mediated stimulatory phase which increases camp levels due to AC activation [19] M 1 receptor expression and function The M 1 receptor is widely expressed in the CNS and the periphery as indicated in Table on page four. CNS M 1 receptors are highly expressed in forebrain regions such as the hippocampus and the cerebral cortex [6]. These two regions are associated with memory and cognition and are two of the many regions where multiple subtypes are expressed [6]. Due to a lack of subtype selective ligands knockout (KO) mice studies have provided most of the knowledge about the physiological and behavioral roles of each subtype. M 1 KO mice studies have shown why M 1 receptors are a common Alzheimer s disease drug target. For example, M 1 KO mice had trouble with memory consolidation but no trouble with spatial memory and contextual fear conditioning [20]. The results suggest that M 1 receptors are involved in memory processes that are dependent on hippocampus and cortex interactions [20]. M 1 KO mice have severely reduced machr agonist-mediated mitogen activated protein kinase activation and G q activation is virtually abolished [21, 22]. Thus, M 1 receptors are specifically involved in learning and memory. M 1 KO mice also have increased levels of Aβ plaques and neurofibrillary tangles which are the hallmarks of Alzheimer s disease pathology [23]. The importance of M 1 receptor function is evident because Aβ plaque formation primarily occurs in the hippocampus and the cortex [6]. In fact, M 1 agonists have been frequently tested as potential therapeutics in Alzheimer s disease treatment. 6

20 M 1 receptors also have important physiological roles in the periphery. For instance, the M 1 subtype is found in glandular tissue such as salivary glands where saliva secretion is controlled [24]. M 1 KO mice have reduced machr agonist-stimulated pepsinogen secretion which indicates that M 1 receptors are involved in gastric secretion [25] M 2 receptor expression and function M 2 receptors are distributed across the CNS and the periphery as shown in Table on page four. The M 2 subtype is the predominant cardiac receptor and ACh binding slows heart rate through decreased intracellular camp levels and increased GIRK channel activation. Administration of the muscarinic antagonist atropine is a common treatment in bradycardia (low resting heart rate) to increase heart rate [26]. M 2 KO mice studies have revealed additional functions for these receptors which include roles in smooth muscle contractions and antinociceptive responses [27]. In the CNS, M 2 receptors function in memory and behavior. M 2 KO mice have exhibited deficits in working memory and behavioral flexibility [28]. M 2 receptors also act as autoreceptors to regulate ACh levels through a negative feedback mechanism [18] M 3 receptor expression and function The M 3 receptor is highly expressed in the periphery as Table on page four indicates. The M 3 subtype is highly involved in smooth muscle contractions. For instance, M 3 KO mice exhibited minimal contractility in numerous smooth-muscle containing tissues [29]. Therefore, the M 3 subtype is a common drug target for muscular contraction disorders. For example, the muscarinic antagonists Spiriva and Darifenacin target M 3 receptors in treatment of COPD and urinary incontinence respectively [30, 31]. The M 3 subtype also plays a major role in regulating metabolic function. M 3 KO mice were actually protected against forms of experimentally and genetically induced obesity and displayed reduced obesity-affiliated metabolic deficits [32]. M 3 receptors are also found in the brain but are expressed at lower levels 7

21 than M 1 or M 2 receptors [6]. These receptors are found in brain regions such as the hypothalamus and help regulate insulin homeostasis [33]. The M 3 subtype is a potential drug target in treatment of obesity and other metabolic disorders M 4 receptor expression and function The M 4 receptor has limited expression in the forebrain in regions such as the hippocampus and striatum as evidenced by Table on page four. However, this limited expression profile makes the M 4 subtype an attractive therapeutic target. M 4 receptors (along with M 2 receptors) act as autoreceptors to prevent excessive ACh stimulation through a negative feedback mechanism [18]. The M 4 receptor is also significantly involved in stimulating striatal dopamine release. In fact, machr agonist-mediated potentiation of striatal dopamine release was completely abolished only in M 4 KO mice and not in M 1 KO mice, M 2 KO mice, or M 3 KO mice [34]. Locomotor control is dependent upon a balance between striatal ACh signaling and dopaminergic transmission [34]. A loss of striatal dopamine activity occurs in Parkinson s disease which results in well-documented symptoms such as rigidity and shaking. M 4 KO mice have displayed locomotor hyperactivity [35]. Therefore, muscarinic antagonists targeting the M 4 receptor are a common Parkinson s disease drug candidate in attempting to restore the balance between the cholinergic and dopaminergic systems in the CNS M 5 receptor expression and function M 5 receptor expression appears limited to the substantia nigra as seen in Table on page four which makes it an attractive therapeutic target. In fact, mrna studies have shown that M 5 receptors account for less than 2% of the machr population [36]. The M 5 subtype is deeply involved in the reward circuitry of the brain and is emerging as a popular therapeutic target in drug addiction treatment [18]. For example, M 5 KO mice have experienced reductions in cocaine self-administration [37]. Also, the severity of the cocaine withdrawal syndrome was greatly 8

22 reduced in M 5 KO mice [38]. The M 5 subtype also has vasodilatory effects because M 5 KO mice experienced a complete abolishment of ACh-mediated dilation in cerebral blood vessels [39]. 1.3 Muscarinic Receptor Structure The structure of a machr consists of an intracellular C-terminus, an extracellular N- terminus, and seven transmembrane (TM) helical bundles that are connected by three intracellular and three extracellular loops as depicted in Figure [40]. These structural regions have differences in sequence homology across the M 1 -M 5 subtypes. For example, the C- terminal and N-terminal domains display large differences in sequence homology [41]. Similarly, the third intracellular loop displays only about 40% sequence homology [41]. In contrast, the first and second intracellular loops exhibit about 90% sequence homology [41]. The binding site of ACh (the endogenous ligand) is defined as the orthosteric site and is represented as the lower oval in Figure The orthosteric site is located in the cavity within the highly conserved TM helical bundles [40]. Consequently, the orthosteric site exhibits a high degree of sequence homology among the machr subtypes that has resulted in a lack of subtype selective drug candidates. For example, no orthosteric muscarinic agonists have been clinically approved in treating Alzheimer s disease [6]. This failure is largely due to orthosteric agonists binding to multiple subtypes which have caused excessive side effects and a lack of clinical efficacy. Figure Structure of a machr. The orthosteric binding site (lower oval) and the common allosteric binding site (upper oval) are shown. Modified from [40]. 9

23 1.4 Allosteric Binding Sites and Types of Allosteric Modulators Due to extensive sequence homology at the orthosteric site, researchers have targeted topographically distinct allosteric sites on machrs. The allosteric sites exhibit a low degree of sequence homology across the subtypes. A common allosteric site has been identified on all five machr subtypes and is represented as the upper oval in Figure on the previous page [18]. Mutagenesis studies have identified the second extracellular loop and the interface between the third extracellular loop and the top of the seventh TM domain as contributing to the common allosteric site [18]. Ligands that bind to allosteric sites are known as allosteric modulators. Allosteric modulators that bind at the so-called common allosteric site include gallamine, brucine, and obidoxime and are shown in Figure [18]. Allosteric modulators can change orthosteric ligand binding and response as depicted in Figure on the next page. Common allosteric site modulators Figure Structures of allosteric modulators binding to the common allosteric site. Modified from [18]. 10

24 OA- Orthosteric agonism AM- Affinity modulation EM- Efficacy modulation AA- Allosteric agonism Figure Mechanisms of allosteric modulation. Interactions between the orthosteric and allosteric sites are depicted. An allosteric modulator can affect orthosteric ligand affinity and/or efficacy and/or exhibit allosteric agonism. Modified from [63]. Figure shows both the orthosteric and allosteric binding sites on a receptor and portrays how an allosteric modulator affects binding (affinity modulation) and maximal response (efficacy modulation). The intricacies of affinity modulation and efficacy modulation will be explained in the next section. Note that most allosteric modulators affect response only in the presence of an orthosteric ligand. Allosteric modulators that cause response in the absence of an orthosteric agonist are known as allosteric agonists. An allosteric modulator can act as a positive allosteric modulator (PAM), a negative allosteric modulator (NAM), or as a silent allosteric modulator (SAM). Numerous machr subtype selective allosteric ligands have been discovered. 11

25 Thus, machrs are a model system for studying allosteric modulation. Most machr allosteric modulators only exhibit affinity modulation which greatly contrasts with the allosteric modulating effects of dronedarone that are presented in Chapter Three. 1.5 Allosteric Modulation of Binding and Response Figure on the following page illustrates how allosteric modulators can affect orthosteric ligand binding and response. First, ligand binding to its receptor is characterized by affinity. Ligand affinity is defined by an equilibrium dissociation constant (K d ) which is the ligand concentration that causes 50% receptor binding. Lower K d values indicate higher affinity. An allosteric modulator can change orthosteric ligand affinity and this occurrence is known as affinity modulation. The K d values are changed in affinity modulation as shown in Figure A. The outer curves show examples of positive (left solid curve) and negative (right dotted curve) affinity modulation. Note that the middle curves in both graphs represent orthosteric ligand binding and response curves respectively in the absence of allosteric modulators. Second and finally, response is characterized by half-maximal effective concentration (EC 50 ) values which are the agonist concentrations that produce 50% response. The EC 50 values indicate the potency of an agonist; lower EC 50 values indicate higher potency. Response is also characterized by efficacy, the maximal level of receptor-mediated response. Figure B depicts how an allosteric ligand modulates response by changing orthosteric ligand efficacy without exhibiting allosteric agonism. The outer curves show instances of positive (left solid curve) and negative (right dotted curve) efficacy modulation which also change the EC 50 values. Note that allosteric modulators which change orthosteric agonist affinity without altering efficacy will change orthosteric agonist potency. For instance, consider an allosteric modulator that exhibits negative affinity modulation and neutral efficacy modulation. If this allosteric modulator causes a 100-fold rightward shift in the K d value there is also a 100-fold rightward shift in the EC 50 value without a change in efficacy. Further discussion about allosteric modulation properties will occur in the next section. 12

26 A. Affinity modulation B. Efficacy modulation Specific binding (%) K d values log [Orthosteric Agonist] Response (%) EC 50 values log [Orthosteric Agonist] Figure Examples of allosteric modulation. In both figures the middle curve is the orthosteric ligand binding or response curve in the absence of allosteric modulators. A. Simulations of how an allosteric modulator can affect binding. B. Simulations of how an allosteric modulator can affect maximal response without exhibiting allosteric agonism. Note that an allosteric modulator could only modulate binding, only modulate maximal response, modulate both binding and maximal response, or have neutral modulating effects on both binding and maximal response. All curves are simulations created from Equation 1 (a log [agonist] versus response variable slope equation). The entire equation is shown in Section

27 1.6 Hall s Allosteric Two-State Model Figure A cubic depiction of Hall s allosteric two-state model. All of the possible interactions between one orthosteric ligand (A) and one allosteric ligand (B) are shown. The receptor can exist in either active (R*) or inactive (R) states. All parameters are described in the text. Modified from [43]. Several models have been developed to simulate the interactions of orthosteric and allosteric ligands. Figure portrays Hall s allosteric two-state model (ATSM), a mechanistic model that has been described as the best model for simulating and analyzing allosterism [42]. The ATSM depicts allosteric modulating effects in the presence of one orthosteric ligand and one allosteric ligand (designated as A and B respectively in Figure although the model is perfectly symmetrical) [43]. The two states refer to the receptor existing in both active and inactive states (designated as R* and R respectively). Only the active receptor states can produce response. The receptor exists in both states in the absence of ligand binding and this ratio (R* to R) is denoted as the isomerization constant L. The affinities of A and B are designated by the respective equilibrium association constants K and M. The intrinsic efficacies of A and B are 14

28 represented as α and β respectively. Values greater than one indicate agonism (higher affinity for R*), values less than one indicate inverse agonism (higher affinity for R), and values of one indicate antagonism (equal affinity for R* and R). Finally, the net effect of the interaction between an orthosteric and allosteric ligand on binding or response is quantitatively represented as cooperativity. The ATSM quantifies cooperativity on binding and response through the respective parameters of binding cooperativity (γ) and activation cooperativity (δ). In both parameters values greater than one signify positive cooperativity, values less than one signify negative cooperativity, and values of one signify neutral cooperativity. The ATSM can classify an allosteric modulator as exhibiting positive modulation (increased affinity, efficacy, or both), negative modulation (decreased affinity, efficacy, or both), or neutral modulation (neutral changes to affinity and efficacy) [18]. An allosteric modulator can also have a mixture of positive and negative modulating effects (e.g. exhibit negative binding cooperativity and positive activation cooperativity). An allosteric ligand could also display agonism (β > 1) as shown previously in Figure on page 11. Moreover, previous work from this laboratory expanded upon Hall s ATSM to create a four-ligand allosteric two-state model (4L-ATSM). The 4L-ATSM can simulate the interactions of two orthosteric ligands with two allosteric ligands that are exclusively competing at a single binding site [44]. The types of parameters are the same as in the ATSM. The 4L-ATSM can be used to determine if the interaction between two allosteric ligands is consistent with competition. 1.7 Advantages of Allosteric Modulators Allosteric modulators have many advantages over orthosteric ligands. First, allosteric modulators often exhibit subtype selectivity due to allosteric sites exhibiting a low degree of sequence homology across receptor subtypes. Thus, several muscarinic allosteric modulators exhibit subtype selectivity. For instance, thiochrome exhibits M 4 receptor subtype selectivity [45]. In contrast, even high affinity orthosteric ligands (e.g. atropine) are not subtype selective. In extreme cases allosteric modulators only modulate binding and response at a single receptor 15

29 subtype, a phenomenon described as absolute subtype selectivity. For example, benzyl quinolone carboxylic acid (BQCA) is a PAM that enhances ACh affinity in only M 1 receptors [46]. Second, allosteric ligands can preserve physiological signaling patterns because modulation occurs only in the presence of an orthosteric ligand (except in cases of allosteric agonism). This advantage is critical since ACh is a neurotransmitter involved in numerous physiological processes. The importance of preserving ACh signaling patterns is clear with the usage of orthosteric M 1 agonists as drug candidates to increase ACh signaling in Alzheimer s disease treatment. Orthosteric M 1 agonists do not preserve ACh spatiotemporal signaling patterns and predictably none of these agonists have been clinically approved as Alzheimer s disease treatments [6]. Conversely, PAMs are an alternative treatment to orthosteric M 1 agonists that are activated only upon ACh binding to machrs. Third and finally, allosteric modulators have an inherent ceiling effect. This effect refers to a limit on the magnitude of binding and response cooperativity. Increasing the dosage of an allosteric modulator beyond a specific concentration will not further change binding and response. Figure on the next page provides an example of the ceiling effect. In this case, ACh-stimulation inhibits guinea pig left atrium contractions in the absence of any allosteric ligands ( ) [47]. Figure also shows the allosteric modulating effects of gallamine (which targets machrs) at five different concentrations in the presence of ACh. Gallamine negatively modulates potency but reaches a limit because there is no change in potency modulation from 300 µm ( ) to 500 µm ( ) [47]. Gallamine concentrations greater than 300 µm do not further decrease ACh potency. Another example shows why the ceiling effect improves the safety of allosteric modulators as potential therapeutics. In this case, PAMs targeting nicotinic receptors are being utilized to improve the therapeutic index (the ratio of a toxic dose to a therapeutic dose in humans) in pain treatment [48]. The inherent ceiling effect allows for overdose protection if an allosteric drug is administered. In contrast, high orthosteric ligand concentrations cause severe side effects from both off-target and on-target effects; the latter occurrence is described as targetbased toxicity [49]. 16

30 Figure An example of the inherent ceiling effect that allosteric modulators possess. There is no change in potency between 300 µm gallamine ( ) and 500 µm gallamine ( ). Modified from [47]. 1.8 Allosteric Modulation of the GABA A Receptor The importance of identifying and characterizing distinct muscarinic allosteric sites can be illustrated by analyzing allosteric modulation of the GABA A receptor. Allosteric modulation at this receptor is a precedent in portraying the clinical benefits of allosteric modulators and targeting distinct allosteric sites. The important point is that drugs binding at separate allosteric sites exhibit completely different therapeutic effects. At the GABA A receptor, benzodiazepines (BZDs) are anxiolytic/hypnotic agents that bind at an allosteric site [50]. Conversely, general anesthetics (e.g. propofol) bind at a separate allosteric site from the BZD site [50]. Neurosteroids are potential sedative treatments that bind to another distinct allosteric site [50]. Thus, each of these allosteric ligands exhibit different pharmacological properties through binding at distinct allosteric sites. In contrast, no GABA A orthosteric ligands have been approved as treatments due 17

31 to reasons such as having smaller therapeutic indices (due to lack of a ceiling effect) and greater side effects in comparison with GABA A allosteric modulators [51]. The orthosteric ligand for the GABA A receptor is γ-aminobutyric acid (GABA) which is the primary inhibitory neurotransmitter in the CNS. The GABA A receptor negatively regulates neurotransmission and is a target for drug candidates in treating conditions such as insomnia and epilepsy [52]. Figure displays a typical GABA A receptor that has multiple allosteric sites in addition to the orthosteric GABA binding sites. Figure Allosteric modulation of the GABA A receptor. The GABA sites are the orthosteric sites and the benzodiazepine, ethanol, neurosteroid, and barbiturate sites represent distinct allosteric sites. Adapted from [50]. BZDs are allosteric modulators that have experienced tremendous clinical success and have replaced barbiturates as safer therapeutics in treating anxiety, panic disorders, alcohol withdrawal syndrome, and insomnia [51-53]. The comparison of BZDs to barbiturates portrays 18

32 the importance of different allosteric sites exhibiting different clinical capabilities. Examples of BZDs include diazepam (marketed as Valium and one of the most commonly prescribed medications) and clonazepam (marketed as Klonopin) [52]. Most BZDs are PAMs that enhance the inhibitory effect of GABA on neuronal excitation. The GABA A receptor complex often consists of two α subunits, two β subunits, and one γ subunit. BZDs bind on the interface of the α and γ subunits. There are several BZD binding sites depending on the specific α and γ subunit isoforms that comprise the receptor complex. BZDs can exhibit subtype selectivity through higher affinity for specific α and γ subunit interfaces. For instance, BZDs with high affinity for the α 1 subtype have strong hypnotic effects while BZDs with high affinity for the α 2 and α 3 subtypes have strong anxiolytic effects [52, 53]. Thus, BZDs could be used to selectively treat insomnia and anxiety disorders, illustrating the therapeutic benefits of subtype selective allosteric modulators. Another advantage of allosteric modulators is shown by flumazenil administration. Flumazenil (marketed as Anexate) is a BZD that acts as a SAM since it neutrally modulates GABA A receptor binding and response [54]. Flumazenil is administered to reverse the effects of BZD overdose through competition at the allosteric BZD site. The key point is that flumazenil exhibits minimal intrinsic activity in the absence of another BZD [54]. Similarly, we utilized this concept of competition between two allosteric ligands where one ligand is neutral in efficacy modulation. We analyzed the interaction of dronedarone and its analog N-ethylamiodarone (NEA) in agonist-stimulated response at the M 3 receptor in Section 3.3 on page 39. The identification of new GABA A receptor allosteric modulators has further elucidated the clinical relevance of separately targeting allosteric sites. For example, the non-bzds are the latest allosteric modulators that bind at the allosteric BZD site. Unlike BZDs, the non-bzds lack a common core structure but provide better specificity in binding to specific GABA A receptor subunit compositions. The primary examples of non-bzds are the Z drugs which include zolpidem (marketed as Ambien) and eszopiclone (marketed as Lunesta) [55]. Both zolpidem and eszopiclone have high selectivity for GABA A receptors containing the α 1 subunit [55]. The Z drugs follow the general trend in drug development toward creating more specific drugs with fewer side effects. Overall, GABA A receptor allosteric modulation has shown the therapeutic benefits of targeting separate allosteric sites. 19

33 1.9 Rationale ACh is the endogenous ligand for the machr family, a receptor family that is widely expressed in the CNS and the periphery. The machrs are common drug targets in treating diseases such as COPD. Therapeutics often target the machr orthosteric site but often exhibit severe side effects due to the extensive sequence homology among the machr subtypes. As a result, researchers are targeting allosteric sites which display a lower degree of sequence homology among machr subtypes. This lower degree of sequence homology has enabled the development of subtype selective allosteric modulators. Allosteric modulators have additional advantages over orthosteric ligands such as preserving physiological signaling patterns and exhibiting an intrinsic ceiling effect. Distinct allosteric sites can exhibit different pharmacological properties as evidenced by GABA A receptor allosteric modulation. Therefore, identifying and targeting distinct muscarinic allosteric sites can lead to the development of more functionally selective drugs. The allosteric modulating effects of the antiarrhythmic agent dronedarone were analyzed in this thesis because of the unique allosteric modulating properties of its parent compound amiodarone. One important discovery was that amiodarone significantly enhances maximal ACh-stimulated AA release without affecting potency in M 3 receptors, a novel allosteric modulating property [44]. Furthermore, amiodarone does not interact competitively with the common site muscarinic allosteric ligand gallamine [56]. Accordingly, amiodarone displays new allosteric modulating capabilities at a distinct muscarinic allosteric site. Analyzing amiodarone analogs such as dronedarone might reveal additional allosteric modulating properties at this newly discovered allosteric site. Indeed, dronedarone exhibits a wide range of allosteric modulating capabilities that differ depending on subtype, orthosteric agonist, and response type. These results are summarized in Tables and on page 55. Unlike previous laboratory studies with amiodarone, dronedarone does not always enhance maximal response. Under specific circumstances dronedarone can increase, decrease, or neutrally modulate both potency and maximal response. ATSM simulations of the results illustrate the complexities in how dronedarone modulates orthosteric ligand binding and response. 20

34 To determine whether amiodarone and dronedarone are binding at the same site, an interaction study was conducted. In an interaction study, if one ligand at a sufficient concentration can reverse the effects of the other ligand on binding or response then the result is consistent with a competitive interaction. An interaction between amiodarone and dronedarone would be difficult to analyze because both compounds significantly increase maximal pilocarpine-stimulated AA release in M 3 receptors. Therefore, the interaction between dronedarone and NEA (an amiodarone analog that is neutral in efficacy modulation) was investigated. This interaction is consistent with competition because NEA reverses the dronedarone enhancement of maximal pilocarpine-stimulated AA release at the M 3 receptor. Given that prior findings revealed that amiodarone and NEA interact in a competitive manner it is concluded that amiodarone and dronedarone also interact competitively [44]. Overall, amiodarone and dronedarone bind at a novel allosteric site and exhibit a wide range of allosteric modulating properties that have not been discovered at the common allosteric site. Since a key advantage of allosteric modulators is their potential for subtype selectivity, it is important to characterize the modulating properties of a new allosteric ligand at the other machr subtypes. Thus, we analyzed the allosteric effects of dronedarone at the M 1 receptor. Dronedarone exhibits different allosteric effects in AA release and IP metabolism at the M 1 receptor when compared to the M 3 receptor results. Allosteric modulators are capable of altering the dissociation rate of an orthosteric ligand. Therefore, we examined the effects of dronedarone on [ 3 H] NMS dissociation to generate more evidence that dronedarone is acting allosterically. An interaction study with amiodarone and dronedarone on [ 3 H] NMS dissociation was conducted in M 3 receptors to identify a competitive interaction. The results support the earlier conclusion of a competitive interaction because dronedarone completely reverses the effects of amiodarone on [ 3 H] NMS off-rate. In conclusion, the identification of new allosteric sites can lead to the creation of safer and better targeted therapeutics. Previous laboratory studies showed that the unique allosteric modulating properties of the anti-arrhythmic agent amiodarone occur at a distinct allosteric site. Accordingly, the allosteric effects of the amiodarone analog dronedarone were analyzed to further determine the allosteric modulating capabilities at this binding site. Dronedarone exhibits a wide range of allosteric modulating properties which include the ability to increase maximal 21