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Structure Activity Relationships of Nicotine Analogs and Erythrina Alkaloids on the Alpha 4 Beta 2 Nicotinic Acetylcholi...

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STRUCTURE ACTIVITY RELATIONSHIPS OF NICOTINE ANALOGS AND Erythrina ALKALOIDS ON THE ALPHA 4 BETA 2 NICOTINIC ACETYLCHOLINE RECEPTOR By KRISTIN MARIE WILDEBOER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Kristin Marie Wildeboer

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To those I hold dearest: my parents

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ACKNOWLEDGMENTS At the completion of my graduate career I will have spent over three-fourths of my life in an academic environment. That amounts to many, many hours that teachers and professors have spent educating, guiding, listening, understanding and encouraging me both in and out of the classroom. I feel it is important to acknowledge and thank them all for their time and dedication to their students, and for helping to mold me into someone who possesses an intellectual curiosity that has driven me to where I am today. During my graduate studies I have turned to numerous people that for assistance, whether for experimental advice or for help with purchase orders. I would like to thank the Pharmacology Department staff: Donna Desmond-Kuhn, Barbara Reichert, Judy Adams, Lynn Rogers, Cookie Mundorff, Lynn Raynor, LaTrelle Davis, and Kari Bastow. I also thank Debbie Otero for lending binding assay supplies and advice over the years. I also need to thank BJ Streetman (in the Neuroscience Department) for help with registration and other such questions, which have been numerous. My family deserves much credit and recognition for being extremely supportive and understanding. I thank my father, Stanley; my mother, Judy; and my brothers, Todd and David. Although they may not understand what I have been studying, have always encouraged me. I also thank my dear friends who always provided laughter and compassion when needed. I especially thank Susan LeFrancois, my partner in crime during my years in the lab. She is one of the kindest, funniest, most caring people I have ever met and I am glad I had the opportunity to get to know her. iv

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I would like to thank current and past members of my supervisory committee: Dr. Stephen Baker, Dr. Thomas Vickroy, Dr. Laszlo Prokai, and Dr. Neil Rowland. I give special thanks to Dr. Roger Papke, who has gone above and beyond the role of a committee member, set aside many hours over the years to answer my questions (no matter how big or small), and always challenged me to better understand all that is membrane bound. I extend many thanks to Dr. Ferenc Soti for the isolation and syntheses of the many compounds for my study. I could always count on him to correct my chemical structures and names. I also thank Dr. Jon Lindstrom for graciously supplying the tsA201 cells used in functional assays. Finally, I would like to express my deep gratitude to the person who has guided and helped shape my research most significantly: my mentor, Dr. William Kem. I would like to thank him for giving me the opportunity to be a member of his lab. It has been an educational, enjoyable ride that I know I will miss when it ends. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ...........................................................................................................viii LIST OF FIGURES .............................................................................................................x ABSTRACT .......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 Superfamily of Ligand-Gated Ion Channels.................................................................1 Characterization of Nicotinic Acetylcholine Receptors (nAChRs)..............................2 Studies of the Muscle-type nAChR.......................................................................2 Muscle-type nAChR Stoichiometry......................................................................3 Using Electron Microscopy to Study the Two-Dimensional Structure.................4 Studies of Neuronal nAChRs................................................................................5 Structure of nAChRs.....................................................................................................7 Site-Directed Mutagenesis of the Receptor and Substituted Cysteine Accessibility Method.........................................................................................7 The ACh-Binding Protein......................................................................................8 Pharmacology and Function of nAChRs....................................................................11 Pharmacology of nAChRs...................................................................................11 Distribution and Physiology of nAChRs in the Mammalian CNS......................15 Function of nAChRs............................................................................................16 Functional states of the nAChR...................................................................19 Neuronal nAChR Involvement in Disease..........................................................21 Structure Activity Relationships of Neuronal nAChRs.......................................24 2 MATERIALS AND METHODS...............................................................................28 Radioligand Binding...................................................................................................28 Compound Syntheses..........................................................................................28 Rat Brain Membranes..........................................................................................29 Human Embryonic Kidney Subclone Cell (tsA201) Membranes.......................29 Binding Assay Protocol.......................................................................................30 Binding Assay Data Analysis..............................................................................31 vi

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Functional Measurements...........................................................................................32 Cell Culture.........................................................................................................32 Membrane Potential Dye, Cell Loading and Compound Plate Preperation........32 Membrane Potential Measurements....................................................................33 Functional Assay Data Analysis..........................................................................34 High Performance Liquid Chromatography (HPLC) Chiral Separation of Nicotine Analogs.............................................................................................34 3 RESULTS...................................................................................................................36 Nicotine Analogs........................................................................................................36 (S)-Nicotine and (S)-Nornicotine........................................................................40 5-Pyridyl Substituted Analogs.............................................................................44 5-Pyrrolidine Substituted Analogs.....................................................................46 1-N-Pyrrolidine Substituted Analogs.................................................................47 3-Pyrrolidine Substituted Analogs.....................................................................50 4 RESULTS...................................................................................................................54 Separation of Nicotine Analog Enantiomers..............................................................54 5 RESULTS...................................................................................................................61 Erythrina Alkaloids....................................................................................................61 -Erythroidines....................................................................................................63 Aromatic Alkaloids.............................................................................................70 6 DISCUSSION.............................................................................................................74 Substituents at Four Different Positions on Nicotine Decrease Affinity for the 42 nAChR and Confer Partial Agonist and Antagonist Properties....................75 Separated Nicotine Analog Enantiomers Display a Difference in Affinity for 42 Unlike Nicotine.............................................................................................83 Substituents on the D-ring of the Erythrina Alkaloids Allow for High Affinity Binding to the 42 nAChR and Inhibition of the 42 Acetylcholine Response.................................................................................................................84 Conclusions.................................................................................................................89 REFERENCES..................................................................................................................90 BIOGRAPHICAL SKETCH...........................................................................................107 vii

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LIST OF TABLES Table page 3-1 Inhibition of 125 I--bungarotoxin (btx) and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by (S)-nicotine or (S)-nornicotine.....................................41 3-2 Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 5-pyridyl substituted analogs...........................................................45 3-3 Inhibition of human 42 receptor acetylcholine (ACh) responses by 5-pyridyl substituted analogs...................................................................................................46 3-4 Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 5-pyrrolidine substituted analogs...................................................47 3-5 Inhibition of human 42 receptor ACh responses by 5-pyrrolidine substituted analogs......................................................................................................................47 3-6 Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 1-Npyrrolidine substituted analogs..............................................48 3-7 Inhibition of human 42 receptor ACh responses by 1-N-pyrrolidine substituted analogs...................................................................................................50 3-8 Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 3-pyrrolidine substituted analogs...................................................52 3-9 Inhibition of human 42 receptor ACh responses by 3-pyrrolidine substituted analogs......................................................................................................................53 4-1 Inhibition of 3 H-cytisine binding to rat brain or to human 42 expressing tsA201 cell membranes by nicotine enantiomers.....................................................58 5-1 Structures, nomenclature and binding results for -erythroidine, dihydro--erythroidine and tetrahydro--erythroidine............................................64 viii

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5-2 Structures, nomenclature and binding results for 3-desmethoxy--erythroidine, N-methyl--erythroidine and -erythroidinediol.....................................................65 5-3 Inhibition of human 42 receptor ACh responses by natural product and semi-synthetic -erythroidines..........................................................................................69 5-4 Structures, nomenclature and binding results for the aromatic alkaloids erysovine, erysodine and erythraline........................................................................71 5-5 Structures, nomenclature and binding results for the aromatic alkaloids erysotrine, and glucoerysodine.................................................................................72 5-6 Inhibition of human 42 receptor ACh responses by natural product aromatic Erythrina alkaloids...................................................................................................73 ix

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LIST OF FIGURES Figure page 3-1 Signal acquired for membrane potential fluorescence using tsA201 cells expressing human 42 nAChRs............................................................................43 3-2 Concentration response curves of nicotine, ACh and nornicotine for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential....................................................................................................................44 3-3 Average effects of the 1-N-pyrrolidine substituted analogs on human 42 receptors expressed in tsA201 cells as measured with membrane potential dye.....50 3-4 Average effects of the 3-pyrrolidine substituted analogs on human 42 nAChRs expressed in tsA201 cells as measured with membrane potential dye......53 4-1 HPLC chiral separation of 1-N-ethyl-(S,R)-nornicotine........................................57 4-2 Concentration response curves for the enantiomers of nicotine and nornicotine for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential..................................................................................................60 5-1 The common heterocyclic structure of Erythrina alkaloids.....................................62 5-2 Concentration response curve of dihydro--erythroidine (DHE) for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential....................................................................................................................66 5-3 Concentration response curve of ACh in the presence and absence of 1 M DHE for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential................................................................................67 5-4 Signal acquired for 42 nAChRs upon simultaneous application of DHE and ACh..........................................................................................................................68 5-5 Structure of a Phelline alkaloid, O-methylisophellibiline........................................70 x

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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 STRUCTURE ACTIVITY RELATIONSHIP OF NICOTINE ANALOGS AND Erythrina ALKALOIDS ON THE ALPHA 4 BETA 2 NICOTINIC ACETYLCHOLINE RECEPTOR By Kristin Marie Wildeboer December 2005 Chair: William R. Kem Major Department: Neuroscience Neuronal 42 nicotinic acetylcholine receptors (nAChRs) are the major high affinity nAChRs in the mammalian brain and have been implicated in mediating the dopamine-releasing and cognition-enhancing effects of nicotine. Nicotine, an alkaloid from the plant genus Nicotiana, is an agonist with a high affinity for the 42 receptor. Another high-affinity 42-nAChR ligand is dihydro--erythroidine (DHE), which is a competitive antagonist from the plant genus Erythrina. The goal of this study was to assess structural activity relationships of these prototypic plant alkaloids for 42 through characterization of the receptor binding and functional properties of several natural, semi-synthetic and synthetic analogs. Substituents were added at four different positions on nicotine: the 5-, 3and 1-N-positions on the pyrrolidine ring, and position 5 on the pyridine ring. We hypothesized that substituents at these positions would permit interaction with the 42 receptor yet xi

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influence the functional properties of these compounds. Modifications at any of these positions resulted in a decreased affinity for 42 relative to nicotine. Six of eleven analogs displayed partial agonist activity, three were antagonists and the remaining two were inactive on human 42. Increasing substituent size at the 1-N-pyrrolidine position greatly reduced affinity but had less influence on the maximum effects measured. The 5-trans-methyl-(S)-nicotine had over 90-fold greater affinity than the cis-isomer and was an antagonist. Separated enantiomer fractions of 1-ethyl-(S,R)-nornicotine and 5-phenyl-(S,R)-nicotine bound with 10-fold or greater difference in affinity to rat 42. Two groups of Erythrina compounds were studied: the D-ring lactones (-erythroidines) and the D-ring aromatic compounds. All alkaloids that had measurable affinity for the receptor also retained their full competitive antagonism. The aromatic compounds were the most active; erysovine displayed a higher affinity than its isomer erysodine. Opening the D-ring of -erythroidine did not result in loss of activity; major determinants for binding to 42 must also exist within the other three rings. Finally, dihydro--erythroidine was more active than either -erythroidine or tetrahydro--erythroidine. xii

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CHAPTER 1 INTRODUCTION The brain is a monstrous, beautiful mess. Its billions of nerve cellscalled neuronslie in a tangled web that displays cognitive powers far exceeding any of the silicon machines we have built to mimic it. -Allman, 1989 page 3 The brain remains a mystery of intricate circuits that allow for acquisition, integration, and retrieval of information. This tangled web of neurons forms synapses that allow for information transfer through specialized methods of signaling, both chemical and electrical. Neurotransmitters are chemical signals that, upon binding to specific target sites on neurons (known as receptors) can change the electrical properties of the target cell. Receptors, along with neurotransmitters, are critical for communication between neurons. The theory of receptors first originated in 1878, when the British physiologist John N. Langley was studying the actions of atropine and pilocarpine on salivary secretion of cats, and found that the inhibitory action of atropine could be overcome by increasing the concentration of pilocarpine. In 1905, Langley was studying the effects of nicotine on striated muscle in fowl when he first used the term receptive substance which (we now know) referred to the nicotinic acetylcholine receptor (nAChR). Superfamily of Ligand-Gated Ion Channels Nicotinic acetylcholine receptors belong to a superfamily of ligand-gated ion channels. The superfamily also includes 5-HT3, glycine and A and C type GABA (-amino butyric acid) receptors. Members of this superfamily of receptors all have a conserved sequence consisting of a pair of cysteines separated by 13 amino acids and 1

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2 linked by a disulfide bridge; thus they are also known as the Cys-loop receptors (Kao and Karlin, 1986). Each of these receptors has a central ion channel, around which are organized five homologous subunits, to which endogenous neurotransmitter may bind and cause a conformational change: allowing ions (including sodium, potassium, calcium and/or chloride) to flow through the channel. The vertebrate nAChRs are cationic ligand-gated ion channels that allow for the passage of positively charged ions (cations) through the channel (Takeuchi and Takeuchi, 1960), whereas glycine and GABA receptors permit the flow of anions. Characterization of Nicotinic Acetylcholine Receptors (nAChRs) The nicotinic acetylcholine receptor is the best-studied of the ligand-gated ion channels. Its name arises from the fact that nicotine, a plant alkaloid extracted from tobacco, binds to these receptors, as does the endogenous neurotransmitter acetylcholine. A primary reason that nAChRs are well characterized is due to the use of tissues derived from the electric organs of certain fishes, including the Torpedo, which are a rich source of nAChRs (Feldberg et al. 1940). The receptors in these electric organs were found to have a high degree of homology to those of the vertebrate embryonic muscle type nAChR. Studies of the Muscle-type nAChR Receptors from the Torpedo were first affinity-purified to determine the number, stoichiometry, and subunits involved (Olsen et al. 1972). Each receptor was determined to be a 250-kilo-Dalton protein consisting of , and -subunits (Reynolds and Karlin, 1978; Lindstrom et al. 1979). When covalently labeling the subunits, the -subunit was found to be twice as abundant as the others (Reiter et al. 1972; Weiland et

PAGE 15

3 al. 1979). This led to the theory that the receptor consisted of two -subunits in the formation of the pentameric receptor. Acetylcholine (ACh) produces its effects on a cell by binding to the nAChR. The location of and number of binding sites on the Torpedo (muscle-type) receptor were determined by using snake venom peptides (-toxins) that served as high-affinity labels for the ACh binding sites (Lee and Chang, 1966). The ability of ligands to bind to the receptor could now be studied using radiolabeled -toxins that would compete with unlabeled agonists and antagonists for the ACh-binding site on the receptor (Weber and Changeux, 1974). One of the snake -toxins, -bungarotoxin (-btx), bound to two sites per receptor oligomer, while the ratio of ACh to -btx binding was close to one (Neubig and Cohen, 1979). These data indicated that there were two ACh binding sites for the Torpedo receptor. It was initially presumed that ACh was binding to some extracellular portion of the receptor because the positive charge of the quaternary ammonium of ACh prevents it from crossing the cell membrane. More specifically, Silman and Karlin (1969) showed that the ACh binding sites were located near a disulfide bond by using bromoacetylcholine to form a covalent bond with the receptor after reduction of exposed cysteines. Muscle-type nAChR Stoichiometry The stoichiometry of the Torpedo receptor subunits was confirmed as (1) 2 (1) in the early 1980s by Raftery et al. (1980) who performed N-terminal microsequencing of the subunits. These N-terminal sequences allowed for creation of degenerate oligonucleotides to probe the Torpedo cDNA library (Ballivet et al. 1982; Claudio et al. 1983; Noda et al. 1982, 1983). The sequences showed homology among the subunits;

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4 and based on these sequences, hydrophobicity plots were created (Claudio et al. 1983) that led to models of how each of the subunits folds in the cell membrane. Hydrophobicity plots suggested that each subunit contains a large extracellular domain, followed by four hydrophobic transmembrane domains (known as M1 to M4): with a large cytoplasmic domain between M3 and M4 (Schofield et al. 1987; Unwin, 1993). The receptor is about 80 in diameter and extends 65 above the membrane, 35 below the membrane, and 40 across it. The central channel is about 30 in diameter, while the ACh-binding sites are located about 30 above the membrane (Unwin, 1993). Availability of cDNAs allowed the development of expression systems. Expression systems allowed for manipulation of subunits by omissions and mutations that permitted further exploration of the ligand-binding domain. The difference between the and pairs of subunits accounted for the difference in affinity of curare (a competitive antagonist) binding to the wild type receptor (Blount and Merlie, 1989). The binding sites in the case of vertebrate adult muscle exist between and subunits, because the -subunit is replaced by an -subunit in adults to give a stoichiometry of (1) 2 (1) (Mishina et al. 1986). It has been proposed, based on DNA sequences, that the subunits that compose the muscle-type nAChR are the most recent evolutionary type of nAChR (estimated to have developed 490-520 million years ago); and that the most ancient form of nicotinic receptor is the homomeric receptor (Le Novere and Changeux, 1995; Ortells and Lunt, 1995). Using Electron Microscopy to Study the Two-Dimensional Structure Through the use of cryoelectron microscopy, the two-dimensional structure of nAChRs began to take shape. Unwin and colleagues (1988) established conditions for

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5 forming ice crystals of nAChRs from Torpedo that showed the receptor to consist of five barrel staves in a circular arrangement surrounding a central channel at a resolution of 17 and 18 (Toyoshima and Unwin, 1988). As previously mentioned, each subunit is made up of four transmembrane domains known as M1 to M4. At a higher 9 resolution, it was possible for Unwin to study the electron densities of each subunit. Initial evidence (Wilson and Karlin, 1998) indicated that the M2 transmembrane domain of each subunit lined the central channel. It was also postulated that the four transmembrane domains were composed of -helices based on their amino-acid sequence (Claudio et al. 1983; Devillers-Thiery et al. 1983; Noda et al. 1983; Unwin, 1993). Unwin provided valuable data on the motion of nAChR activation by collecting images of the nAChR from Torpedo before and after a brief exposure to ACh at 9 Results showed that upon activation, the subunits rotate on an axis parallel to the central channel. The data also corroborated the proposed location of channel gate as determined by Wilson and Karlin (1998) at a position halfway in the membrane postulated to be formed by leucine residues from each subunit (Unwin, 1995). At a resolution of 4.6 the extracellular structures of the subunits appeared to be composed of -sheets. These higher-resolution images also showed that these -sheets form the extracellular entrance to the channel (which is lined by -helices) by connecting it to the ACh-binding pockets and that a central channel exists with lateral openings below the membrane that serve as a filter for ions (Miyazawa et al. 1999). Studies of Neuronal nAChRs In the early 1990s the 7 gene was cloned from both chick and rat brain (Couturier et al. 1990; Schoepfer et al. 1988; Seguela et al. 1993). These receptors were shown to

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6 have a very high affinity for -btx. They were determined to be homomeric, or consist of five identical subunits. There also existed a group of nAChRs that did not bind -btx with high affinity, but rather had a high affinity for nicotine or ACh and had a distinctly different labeling pattern from that of 7 (Clarke et al. 1985). This group of receptors was found to be composed of both and nonsubunits. Cloned subunits were classified as subunits if they contained vicinal cysteines in their N-terminal domain, homologous to positions 192 and 193 in the Torpedo. The nonsubunits that lacked these vicinal cysteines were designated as -subunits. There are currently 12 known neuronal nAChR subunits, 2 to 10 and 2 to 4. The 7-, 8-, and 9-subunits can form homomeric channels. The most commonly expressed homomeric nAChR in mammals is the 7-receptor. While 9 and 10 are also expressed in mammals, 8 nAChR expression has only been found in chickens (Gerzanich et al. 1993; Keyser et al. 1993). Heteromeric receptors contain combinations of and -subunits. The stoichiometry ratio of to -subunits was shown to depend on the expression system of the receptors and other factors, including temperature and pharmacological treatment (Nelson et al. 2003). The assembly composition of these heteromeric receptors is what gives rise to the diversity of nAChR properties, both binding and function. The location at which the nicotinic agonist ACh binds is known as the ligand-binding domain (LBD). The LBD is located between the interface of the -subunit and its adjacent subunit on the N-terminal domain. The N-terminal domain is about 210 amino-acid residues long. Heteromeric receptors contain two ACh-binding sites, while homomeric receptors might contain up to five ACh-binding sites. The LBDs

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7 are composed of six loops, three from the contributing -subunit and three from the non--subunit. In the case of homomeric receptors the adjacent -subunits each contribute three loops. The loops from the -subunits are considered the primary loops and are known as A, B and C (Galzi et al. 1991) while the loops from the non--subunits are the complementary loops D, E and F (Corringer et al. 1995). These loops and their residues were determined through numerous photoaffinity labeling, mutatgenesis, radioligand binding and functional assays. Structure of nAChRs Site-Directed Mutagenesis of the Receptor and Substituted Cysteine Accessibility Method Site-directed mutagenesis has been a valuable tool for investigating residues important in various aspects of the receptor. The receptor channel incorporates negatively charged amino acids that promote the passage of cations (Pascual and Karlin, 1998). Mutating these residues to uncharged amino-acids decreases the cationic conductance of the receptor (Imoto et al. 1998). Interestingly the cationic selectivity of the receptor can be converted to an anionic selectivity by mutating only three residues in the M2 domain and the M1-M2 loop, as was done with the chick 7 receptor (Galzi et al. 1992). Mutagenesis studies have determined that a threonine residue at position 59 within the 2-subunit is important for the interaction of the competitive antagonist dihydro--erythroidine with 32 receptors (Harvey and Luetje, 1996). The substituted cysteine accessibility method (SCAM) proved to be a valuable tool for initial identification of all the residues lining the M2 domains of the muscle type nAChR (Akabas et al. 1994; Zhang and Karlin, 1998); which (as previously determined) is the domain that lines the channel of the receptor and allows conduction of cations.

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8 SCAM has identified four important residues that exist in the middle of the M2 domain and are part of the channel gate for the active state of the receptor (Wilson and Karlin, 1998). The ACh-Binding Protein In 2001 there was a breakthrough in the structural analysis of the nAChR. A group in the Netherlands discovered a soluble protein from the fresh water snail Lymnaea stagnalis (Brejc et al. 2001; Smit et al. 2001). The protein had a sequence identity similar to subunits from the nAChR, as well as the other cys-loop ligand-gated ion channels. The discovery came about when experiments on preand postsynaptic cholinergic neurons from Lymnaea determined that excitatory postsynaptic potentials (EPSP) occurred only in the absence of glia cells. Further exploration found a protein secreted from the glia that bound the ACh released in the synapse. Thus it was named the acetylcholine-binding protein (AChBP). It has been proposed that Lymnaea can use the AChBP as a buffer to directly modulate cholinergic synaptic transmission (Smit et al. 2003). Purification and molecular cloning of the protein determined that it is made up of five subunits. Each subunit consists of 210 residues forming a homopentamer (Brejc et al. 2001). Each subunit has a ligand binding-domain between the N-terminals of adjacent subunits but lacks the pore-forming transmembrane domains. The majority of the residues that are conserved among the nAChRs are also conserved in the AChBP, which makes it a good model for the N-terminal domain of the nAChR including the LBD. The AChBP has the highest sequence homology to the nAChR -subunit extracellular domains with a 23.9% similarity to the 7 nAChR (Smit et al. 2001). Using radiolabeled

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9 -btx the IC 50 values were determined for several nicotinic agonists and antagonists including -btx (2.6 nM), nicotine (98 nM) and ACh (4.2 M), these values are more comparable to the pharmacology of homomeric receptors, specifically 7 and 9, over the high affinity heteromeric nAChRs (Smit et al. 2001). Brejc et al. (2001) crystalized this protein at 2.7 The measurements of the AChBP are 62 in height, a diameter of 80 and a pore size of 18 these measurements parallel Unwins data on the Torpedo nAChR. The residues important in the LBD were previously determined by biochemical and mutagenesis data. The crystal structure confirmed the involvement of these residues as well as elucidated how these residues are positioned in the binding domain. The principal side of the LBD consists of loops from the -subunit homolog known as loops A, B and C while the complementary side, the -subunit homolog in heteromeric receptors and -subunits in homopentameric receptors, consists of loops D, E and F made up of -sheets. The residues in the binding site (as crystallized with a HEPES molecule), which are primarily aromatic and hydrophobic, are as follows: loop A, tyrosine 89; loop B, tryptophan 143 and 145; loop C, tyrosine 185 and 192, and cysteines 187 and 188. Important LBD residues from the complementary side are: loop D, tryptophan 53 and glycine 55; loop E, arginine 104, valine 106, leucine 112, and methionine 114; loop F tyrosine 164 (Brejc et al. 2001; Corringer et al. 2000; Dougherty and Lester, 2001). AChBP has yet to be crystallized with an empty binding pocket. The original crystals contained a HEPES molecule in the ACh binding site. Recently nicotine and carbamylcholine, both nicotinic agonists have been crystallized in the AChBP (Celie et al. 2004). The data reaffirmed the proposed aromatic

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10 and hydrophobic residue contacts between these nicotinic agonists and the ACh binding site. The study by Celie et al. (2004) determined that hydrogen bonds exist between the receptor and the nitrogens on the pyridine and pyrrolidine rings of nicotine. Also, the carbonyl oxygen of W143 interacts with the positively charged nitrogens of nicotine and carbamylcholine. Interestingly they found that both ligands bind with their nitrogen atoms at nearly the same position in the binding pocket. A comparison between the LBD of the 42 nAChR and the AChBP reveal three residue substitutions, R104 (AChBP) is replaced by V109 (42), L112 by F117 and M114 by L119 (Celie et al. 2004). The utilization of the AChBP for molecular modeling has its limitations. One of the major limitations being that the AChBP is not a functioning channel so modeling ligand binding may not reflect molecular changes in movement that the nAChR undergo upon activation and desensitization. Unwin (2005) has helped to shed some light on the conformation of the receptor in the unliganded, or closed state. He has determined at a 4 resolution of the Torpedo nAChR in a closed conformation that the C loop of the subunits projects away from the receptor and that upon agonist binding the C loop moves in toward the receptors ACh binding site, which along with the movement of the B loop allow for a conformational change that would permit receptor activation and channel opening. Unwin (2005) terms this conformational state of the B and C loops of the subunits as distorted and that upon ligand binding both a salt bridge and hydrophobic interactions are broken, which allow for the loops to change to a conformationally relaxed state of the receptor.

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11 Pharmacology and Function of nAChRs The majority of the initial information about nAChRs came from ligand binding, photoaffinity-labeling, electrophysiological and mutagenesis experiments. Although the initial properties were determined by electrophysiological methods in the 1940s, it was only after the cloning of genes coding for nAChRs that electrophysiological experiments could be put to use for studying the physiology of specific subunit combinations. Much of the data up to that point relied upon radioligand-binding studies to elucidate the pharmacology of nAChRs expressed in various tissues and regions throughout the brain. Pharmacology of nAChRs The initial pharmacology of AChRs was largely based on the study of natural products and their ability to bind and affect nAChRs. Plant compounds including nicotine, muscarine, atropine, curare and physostigmine were all utilized due to the finding that they produced either activation or inhibition, depending upon concentrations used, of cholinergic (either nicotinic or muscarinic) transmission. The ability to radiolabel these compounds provided the evidence for the location of nAChRs in various tissues as well as provided the capability to measure the tightness with which these radiolabeled compounds bound to the receptor, known as affinity. A ligand that reversibly binds directly to the ACh binding site is called a competitive ligand. It will compete with ACh for the same binding site on the nAChR and with increasing concentrations of ligand it will displace ACh from the binding pocket. A ligand that does not bind to the ACh binding site is termed non-competitive and will not displace ACh with increasing concentrations. Non-competitive ligands bind to other sites on the receptor known as allosteric sites. Local anesthetics are ligands that bind to nAChRs at sites other than the ACh binding site (Papke and Oswald, 1989; Sine and Taylor, 1982).

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12 A group of alkaloids from the venom of Tetraponera ants have also been initially characterized as noncompetitive antagonists at several nAChR subtypes and are likely binding at a site within the channel (Kem et al. 2004b). There are several considerations when radiolabeling a compound that is to be used as a ligand in studying nAChRs. One concern is that the radiolabel must have a high enough specific radioactivity that binding of nanomolar (nM) concentrations of ligand can be reliably measured. It is also a requirement that the radiolabel not interfere with binding of the ligand to the receptor. Also, it is important that the label is not metabolized or exchanged during the experiment. Non-saturating sites on the membrane to which a radioligand binds are known as non-specific binding sites. It is important that the radioligand can be used to differentiate between non-specific binding and binding to only the receptors, known as specific binding. Obtaining specific binding is usually performed by adding a high concentration of a compound, such as nicotine, that binds specifically to the receptor sites in order to knock off the radioligand from the receptors, thus the radioligand is only bound to the non-specific sites. The counts due to non-specific binding (these are proportional to ligand concentration) can be subtracted from the total binding counts of radioligand bound to the cell membranes in order to determine specific binding. Receptor binding should be saturable, reversible, and over a period of time reach equilibrium binding. It is also ideal if the ligand is selective so that is binds with a high specificity to a specific subtype of receptor, or in the case of early development of nAChR radioligands specifically to nAChRs. One such compound that was initially employed for utilization in studying nAChRs was radiolabled muscarine (Waser, 1961). The problem with using muscarine, a toxin

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13 extracted from mushrooms, to study nAChRs is that it labeled two types of acetylcholine receptors, the ligand-gated nicotinic as well as the G-protein coupled muscarinic receptors. Acetylcholine also labels both types of ACh receptors and is readily hydrolyzed by acetylcholinesterase into acetate and choline, thus it is usually preferred to work with a more stable and selective ligand for nAChRs although the binding of radiolabeled ACh has been characterized in the presence of atropine, to prevent binding to muscarinic receptors (Schwartz et al. 1982). Several such ligands discussed earlier were the snake neurotoxins. -Bungarotoxin has been shown to be specific for certain nAChRs and has a low nanomolar affinity (Barrantes et al. 1995; Gopalakrishnan et al. 1995). The equilibrium dissociation constant K d (or K i ) is an inverse measure of this affinity. Several studies have compared the binding of ACh to nicotine and -btx in the rat and mouse brain (Clarke et al. 1985; Marks et al. 1986). These studies indicated that radiolabeled ACh and nicotine were binding to the same sites with high affinity while -btx was labeling different sites. Acetylcholine and nicotine were labeling sites in the cortex, cerebellum, ventral tegmental area and the thalamus while -btx was binding at higher densities in the thalamus and hippocampus. It was soon determined that the nAChR in the central nervous system to which -btx was binding was that of a homomeric 7 receptor (Couturier et al. 1990). Since its first utilization radiolabeled -btx has been a commonly used label for 7. A few years later a relatively selective label, as compared to nicotine or ACh, for the heteromeric 42 nAChR was discovered. Cytisine, a plant alkaloid from Laburnum anagyroides, is an autonomic ganglionic agonist. Exploration of its properties on neuronal cell membranes revealed that cytisine had a very high affinity for those sites

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14 labeled by radioactive nicotine and ACh. It had a measured K d value of less than 1 nM, with high levels of binding in the cortex, thalamus and striatum and represented 60-90% of total binding at the various concentrations examined (Pabreza et al. 1991). Flores et al. (1992) later determined that cytisine was primarily labeling the heteromeric 42 nAChR. Several ligands that also possess a high affinity for these receptors have been radiolabeled and studied since the development of cytisine. The compound known as ABT-418 is a relatively high affinity ligand for 42 that functions as an agonist, and like nicotine may function as a concentration dependent noncompetitive antagonist (Papke et al. 1997). Anderson et al. (1995) radiolabeled ABT-418 and measured a K d value of 2.9 nM for rat brain. Another ligand that has been radiolabeled for the study of 42 receptors is a semi-synthetic compound derived from the plant genus Erythrina. The alkaloid known as dihydro--erythroidine is commonly used in functional assays as a competitive nicotinic antagonist. Williams and Robinson (1984) investigated a tritiated this alkaloid and measured high and low affinity binding sites in rat brain membranes with respective K d values of 4 and 22 nM. They found that its distribution of binding in rat brain was highest in the thalamus and that cytisine was a more potent inhibitor than nicotine in displacing the radiolabeled alkaloid. Also, mecamylamine did not displace the radiolabeled dihydro--erythroidine at concentrations up to 100 M. Their data indicated that the tritiated alkaloid was preferentially interacting with 42 over the other neuronal subtypes of nAChRs. The 42-subtype has a high affinity for the agonist nicotine that induces a conformational change (desensitization), which displays this high affinity state (Changeux et al. 1984; Higgins and Berg, 1988; Buisson and Bertrand, 1998). Agonists

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15 for the 7 nAChR have not been found to induce such a high affinity state of this particular receptor. Thus, the 42-subtype is referred to as the high affinity nAChR, while the 7-subtype is known as a low affinity nicotine-binding receptor. Although there are numerous combinations of nAChR subtypes that form neuronal receptors, the 42 and 7 nAChRs are the two major subtypes in the mammalian central nervous system (CNS) (Clarke et al. 1985; Hogg et al. 2003; Pauly et al. 1989; Seguela et al. 1993; Wada et al. 1989; Whiting et al. 1991). Distribution and Physiology of nAChRs in the Mammalian CNS Nicotinic receptors in the CNS are known to facilitate synaptic transmission. There are several studies that indicate the existence of postsynaptic nAChRs in the mammalian CNS (Alkondon et al. 1998; Clarke, 1993; de la Garza et al. 1987; Frazier et al. 1998; Schroder et al. 1989) but the lack of evidence for the involvement of these receptors in EPSPs has lead to the theory that the primary location of nAChRs is presynaptic. Through autoradiography, immunolabeling, and co-localization with presynatpic markers, the existence of nAChRs on presynaptic neurons has been firmly established (Clarke and Pert, 1985; Lubin et al. 1999; Jones et al. 2001). The existence of these presynaptic nAChRs allows for the regulation of neurotransmitter release. Activation of these receptors has been shown to result in release of not only ACh (Wilkie et al. 1993), but also in dopamine (Rapier et al. 1990, Grady et al. 1992), glutamate (Alkondon et al. 1997; McGehee and Role, 1995) and GABA (Alkondon et al. 1999; Radcliffe et al. 1999; Yang et al. 1996). The release of neurotransmitter through activation of nAChRs on presynaptic terminals is due to the entry of calcium both through the nAChR channel and through voltage-gated calcium channels, which are activated upon depolarization of the

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16 cell (Soliakov et al. 1995). All nAChRs are permeable to calcium but the neuronal receptors have a higher permeability ratio as compared to the muscle subtype. The 7-homomeric receptors have the highest permeability to calcium, with a permeability ratio of about 6.6 or higher (P Ca /P Na ) (Bertrand et al. 1993; Fucile, 2004; Sands et al. 1993; Sequela et al. 1993). The activation of nAChRs and their subsequent ion permeability of is not the result of a static entity but rather a fluid movement in the conformation of the receptor. Function of nAChRs The prototypical site for the study of the mammalian nAChR is the neuromuscular junction. Postsynaptic cells of the muscle form large synapses, which contain millions of nAChRs. Large amounts of ACh are released from the presynaptic membranes directly onto the postsynaptic receptors in order to ensure that an action potential occurs in order to facilitate the movement of muscle. In the early 1950s, there were rapid developments in the understanding of synaptic transmission thanks in a large part to the study of the neuromuscular junction. In 1952 Hodgkin and Huxley revealed the mechanism of the action potential involved in electrical signaling in the giant squid axon. Shortly after Fatt and Katz (1950, 1951, 1952) provided a better understanding of neurotransmitter release and the resultant change in ion permeability by ACh. Hodgkin and Huxley employed the voltage clamp technique to study the permeability changes underlying the action potential in the giant squid axon. Through the use of a recording and current passing electrode, voltage clamp allows for the membrane potential of a cell to be held constant while measuring the change in ionic current flow required to keep the membrane potential at a constant voltage. This technique allowed for macroscopic measurements of receptor

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17 activation and inhibition. The voltage clamp technique known as patch clamp recording allowed for microscopic measurements and kinetic analysis of single receptors (Hamill et al. 1981; Neher and Sakmann, 1976). These techniques were major breakthroughs in the study of all ligand gated ion channels. In the past few years other methods of measuring functionality have been developed. These include both calcium and membrane potential dye measurements. Fitch et al. (2003) determined the potency and efficacy of nicotinic agonists on various cell lines expressing several subtypes of nAChRs using either a membrane potential or calcium fluorescent dye. Both dyes fluoresce due to either changes in membrane potential or increases in intracellular calcium reflecting activation of the expressed receptors. Changes in fluorescence can be calculated and graphed to determine either activation or inhibition concentration constants. They determined that the membrane potential dye was more sensitive than the calcium dye but that the overall results were comparable to those of the calcium dye as well as previously published radiolabeled rubidium efflux assays. The membrane potential dye was also useful in cell lines that have a low calcium conductance. One disadvantage to using these dyes is the inability to wash away compound after application. Compounds that function as agonists may produce a time dependent desensitization of the receptors. Another disadvantage in using the membrane potential dye is that agonists may appear more potent and antagonists less potent, as compared to electrophysiological data. This may be due to the phenomenon of spare receptors (fraction of receptors occupied that produces a maximum response). As discussed earlier, the ability and strength with which a ligand binds to the nAChR is known as affinity. It describes how a ligand binds but it does not characterize

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18 its capability to functionally activate the receptor. The term efficacy was coined to describe the ability of a ligand to activate a receptor (Colquhoun and Sakmann, 1998; Stephenson, 1956). A ligand can have varying degrees of efficacy based upon the subtype of receptor and concentration of ligand. The term agonist describes a ligand that activates a receptor. The endogenous neurotransmitter ACh is a full agonist for nAChRs. A ligands efficacy is often compared to that of a known full agonist, such as ACh, for the subtype of nAChR being studied. If a ligand activates a receptor with less than maximum activation as compared to ACh it is termed a partial agonist. For example, cytisine is a partial agonist for 42. It has high affinity for the receptor, a K d less than one nanomolar (Pabreza et al. 1991), but it causes only 15% of the maximum response at 1 mM as compared to ACh (Papke and Heinemann, 1994). Thus, cytisine is less efficacious for the 42 receptor than ACh. Ligands that bind to nAChRs but produce no activation are called antagonists. Antagonists exist as competitive or non-competitive ligands. Competitive antagonists bind to the ACh binding site and alone cause no change in the activity of the receptor. However, when present with an agonist that binds to the same site, the competitive antagonist will inhibit the receptor from activation at specific concentrations. -Bungarotoxin, as previously described, is a competitive antagonist for 7 receptors. It binds to the 7 ACh binding site with a K d of 0.16 nanomolar (Marks et al. 1986) and inhibits activation at higher nanomolar concentrations, such as 100nM (Frazier et al. 1998). Non-competitive antagonists can produce the same inhibition as competitive antagonists, but they do not bind to the ACh binding site. A non-competitive antagonist,

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19 such as the local anesthetics dibucaine or tetracaine, prevents activation of the muscle type nAChR by binding to one or possibly more allosteric sites on the receptor (Papke and Oswald, 1989; Sine and Taylor, 1982). Non-competitive antagonists may also inhibit activation without binding to an allosteric site but rather through blocking passage of ions through the channel, termed channel-block. Competitive agonists, such as nicotine, may become non-competitive antagonists at high concentrations due to the ability of nicotine to cause channel block. The capability of a compound to act as an agonist or antagonist depends upon the stoichiometry of the nAChR and the stoichiometry of the receptor determines the kinetics and ion permeability. Functional states of the nAChR The ability of nAChRs to successfully facilitate neurotransmission involves several different conformational states of the receptor. The transition from one state to another is dependent upon several factors including agonist concentration, time of exposure, and association and dissociation rate constants for binding. There exist at least three conformational states for nAChRs, closed, active and desensitized. Monod et al. (1965) produced their model of allosteric interactions to help explain the transition from one state to the next. The closed state is an unbound resting state of the receptor such that a net flow of ions is not passing through the channel. In the absence of agonist nAChRs exist in an equilibrium that greatly favors the closed state. Upon exposure to an agonist the receptors no longer exist in that resting equilibrium but rather have become active, opening their channels and allowing ions to flow through and thus producing a current across the membrane. The active state requires bound ligand, with at least two agonist molecules bound to the receptor. With two agonist molecules bound the receptor has a higher probability of opening than monoliganded receptors (Sine and Taylor, 1980,

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20 1981). The third state that exists is the desensitized state of the receptor. This state, which was originally studied at the motor end-plate by Katz and Thesleff (1957), is a nonconducting state that does not respond to additional application of agonist and must recover before subsequent activation or before returning to the resting state. Although this is a nonconducting state the conformation of the desensitized receptor is distinctly different from that of the resting state (Wilson and Karlin, 2001; Unwin, 1995). The properties of desensitization, such as onset and recovery, are dependent upon receptor stoichiometry. Desensitization can be induced by properties ranging from agonist concentration to disruption of the tissue (Whiteaker et al. 1998; Fenster et al. 1999), such as occurs during homogenization. The neuronal 7 nAChR are the most rapidly desensitizing of the nAChRs. The rapid desensitization of 7, by high concentrations of agonist (ACh), is so quick that it complicates data analysis due to the fact that by the time the agonist solution reaches its full concentration the peak response of the receptor has already occurred (Papke and Porter Papke, 2002). The fast desensitization of 7 is in part due to a leucine residue at position 247 in the second transmembrane domain. Through substitution of a threonine residue for the leucine (L247T) the rate of desensitization is slowed, but the pharmacology of the receptor is dramatically different from that of the wild type receptor (Revah et al. 1991; Bertrand et al. 1992). Placzek et al. (2005) created an 7 gain of function mutant that contained a serine residue in place of a threonine (as found in wild-type 7) in the second transmembrane domain. This mutant receptor has longer open times as compared to wild-type 7 with its pharmacology (for agonists and antagonists) similar to that of the wild-type receptor, unlike the confused pharmacology of the L247T mutant.

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21 Neuronal nAChR Involvement in Disease Since nAChRs do facilitate neurotransmission, it is not surprising that they are also involved in neuronal dysfunction of several disease states. A downregulation of nAChRs has been found in brains of persons diagnosed with Alzheimers disease (AD) (Norberg and Winblad, 1986; Schroder et al. 1991; Court et al. 2000). The primary target for downregulation is the 42-subtype (Warpman and Nordberg, 1995) although 7 may also be affected (Perry et al. 2000). The exact involvement of nAChRs in the impairment of memory and cognition in AD patients is not known, but treatment with nicotinic receptor agonists and acetylcholine esterase inhibitors have been found to alleviate some of the clinical symptoms of AD in the early stages of the disease. Nicotinic acetylcholine receptors have also been found to be downregulated in Parkinsons disease based upon 3 H-nicotine binding assays (Rinne et al. 1991; Perry et al. 1995; Court et al. 2000). The pathology of Parkinsons disease is the loss of dopaminergic neurons in the nigro-striatal and ventral tegmentum area-mesocortical pathways in the brain. Several subtypes of nAChRs have been found to play a role in the release of dopamine from these neurons. Both 42 and 632 nAChR subtypes are located on dopaminergic neurons (Champtiaux et al. 2002, 2003; Cui et al. 2003; Zoli et al. 2002). There exist at least two neuronal disorders that involve mutations of the gene encoding for specific nAChR subunits, schizophrenia and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The chromosome encoding the 7 gene has been found to be partially duplicated in schizophrenic families (Chini et al. 1994; Leonard et al. 1996; Freedman et al. 2001; Xu et al. 2001). Interestingly, 90% of schizophrenic

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22 patients self-administer some form of nicotine (Lohr and Flynn, 1992; Poirier et al. 2002). Mutations on genes that encode both the 4 and 2 nAChR subunits are found in ADNFLE patients. The manifestation of these mutations can be traced to residue mutations of both the 4 and 2 membrane-spanning domains M2 (Phillips et al. 1995, 2001; De Fusco et al. 2000; Hirose et al. 1999; Steinlein et al. 1995, 1997). The culmination of these mutations is an increased sensitivity of the receptors to ACh, which has been proposed to generate seizures resulting from a synchronization of spontaneous oscillations in the thalamo-cortical circuits (Raggenbass and Bertrand, 2002). It has been established that nicotinic receptors are involved in the reinforcing properties of nicotine, in part due to activation of nAChRs on dopaminergic neurons in the nucleus accumbens and ventral tegmental area (Corrigall et al. 1994; Picciotto and Corrigall, 2002). Nicotine dependence is often established and sustained through smoking cigarettes. The chronic and prolonged use of tobacco products often results in negative health effects including cancer and cardiovascular disease. Although the detrimental effects of tobacco dependence are produced not through nicotine but the many other ingredients in cigarettes, it is the nicotine that has been determined to produce the reinforcement. Nicotine itself has been found to produce the enhancement of cognition and memory (Levin, 1992; Arendash et al. 1995a). It also has the ability to both activate and inhibit nAChRs based upon concentration and length of exposure. The development of knock out mice in which a gene for a specific nAChR subunit has been mutated to prevent expression have allowed for investigation of subunits involved in nicotine reinforcement. Picciotto et al. (1995) were the first to develop a 2-knock out mouse. They showed that these knock out mice lacked the majority of high

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23 affinity nAChRs while the levels of -btx binding remained the same as in wild-type mice. Most importantly, these mice were shown to have decreased dopamine release in response to nicotine exposure and do not self-administer nicotine (Picciotto et al. 1998). Although it has recently been shown that 6-subunits associate with the 2-subunit in the CNS, the majority of 2 are associated with 4 (Champtiaux et al. 2003). The involvement of 42 nAChRs in addiction has lead these receptors to become targets for the design of pharmacological therapeutics. Over the years several approaches and therapeutic agents have been tested for their ability to aid in smoking cessation. In the 1960s cytisine, a partial agonist for 42, was examined but lacked the ability to readily cross the blood brain barrier. The combination of mecamylamine, a nAChR antagonist, and nicotine administered through a nicotine skin patch has been shown to be somewhat beneficial in aiding cessation (Rose et al. 1994). Bupropion, a compound used to treat depression, has also been determined to aid smoking cessation. The precise mechanism of action is unknown but it is though to prevent the reinforcing properties of nicotine (Cryan et al. 2003). More recently a compound known as varenicline, a partial 42 agonist, has begun clinical trials for smoking cessation (Coe et al. 2005). Based on these studies it may be expected that a partial agonist would be a useful smoking cessation agent due to its ability to result in a release of dopamine, yet block additional dopamine release upon stimulation with a full agonist such as nicotine. The competitive antagonist, DHE, has been shown to prevent nicotine self-administration when applied directly to the ventral tegmental area (Corrigall et al. 1994). Thus, it is also possible that an antagonist may also function as a smoking cessation agent.

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24 The development of either partial agonists or antagonists may be useful not only for smoking cessation, but also as in vivo probes for the involvement of 42 in neuronal processes. Although knock out mice have been created, the involvement of 42 receptors in cognition and nicotine addiction may be masked by compensatory mechanisms from other nAChR subunits. In order to develop ligands that display partial agonist or antagonist activity on the 42 receptor, it is necessary to better understand the interaction of a compound with the receptor. Specifically, what features of the compound confer affinity and influence functional properties. Structure activity relationship studies involve analysis of a group of similar compounds, such as nicotine analogs, where slight to large modification are made to the structure of the compound. The ability of these modified compounds to bind and activate or inhibit the receptors provides information useful in the design of 42 specific ligands. Structure Activity Relationships of Neuronal nAChRs Nature has provided various molecules, including cytisine and -btx, which have allowed for elucidation of structure, location and function of nAChRs expressed in various tissues. With increased understanding of these receptors and their involvement in several diseases the possibility of alleviating symptoms or correcting deficiencies often leads to the re-visitation of natural products. The design of pharmacological agents often starts with a lead compound, one that has been found to interact with the receptor of interest. One lead compound that has proved useful in the development of clinically based therapeutics is a natural product from a marine worm, the toxin known as anabaseine. Anabaseine itself stimulates all nAChRs (Kem, 1971; Kem et al. 1997) but is most potent on muscle-type and neuronal 7 receptors (Kem et al. 1997). Through the

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25 addition of a substituted benzylidene moiety, anabaseine becomes a selective partial agonist for 7 and an antagonist for 42. This compound known as DMXBA (GTS-21), whose chemical name is 3-(2,4-benzylidene)-anabaseine, has been shown to be neuroprotective in a PC12 neuronal growth factor deprived model of cytotoxicity (Li et al. 1999). It also has been found to enhance cognition in various behavioral tasks performed by aged rats (Arendash et al. 1995b), and therefore may prove a useful therapeutic for Alzheimers disease (Kem, 2000). DMXBA is currently in clinical trials for correcting the auditory gating deficiency experienced by schizophrenic patients (Stevens et al. 1998; Simosky et al. 2001). Similar to the development of DMXBA from the natural product anabaseine, nicotine may serve as a good lead compound for the design of smoking cessation drugs. Several previous studies have determined structure activity relationships of nicotine for the 42 nAChR. Substituents at various positions on both the pyridine and pyrrolidine rings of nicotine have been determined to affect affinity. Fewer studies have addressed the functional consequences of these substitutions on the 42 receptor. Including Beers and Reichs (1957) well-known structure activity relationship for agonists and antagonists with cholinergic binding sites, other findings about the interaction of ligands with nicotinic receptors have since been discussed. One theory proposed by several groups is that a cationinteraction exists between Trp149, of the -subunit of the Torpedo receptor as well as the AChBP, with the nitrogen atom in an interacting ligand (such as nicotine) (Beene et al. 2002; Tnder and Olesen, 2001). Structure activity relationships also exist for other ligands and the 42 nAChR. Epibatidine, the most potent nAChR agonist, has 7-azabicyclo[2.2.1]heptane ring attached to an

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26 exo-5-(2-chloropyridinyl) substituent. Although it is exceptionally potent, epibatidine is not selective for particular nAChRs. Carroll et al. (2001) reported that a phenyl substituent at the 3-position of epibatidine changed it into an 42 antagonistic in vitro. Nicotine is a tertiary amine with a chiral carbon at the 2-pyrrolidine position. In 1957 Beers and Reich proposed that the ability of the naturally occurring (S)-nicotine to interact with the nAChR existed due a hydrogen bond formed between the nitrogen on the pyridine ring and through electrostatic interactions by the nitrogen on the pyrrolidine ring. They measured a minimum distance of 5.9 from the nitrogen group to the hydrogen bond. They found this measurement existed for both nicotinic agonists (ACh, cytisine, nicotine) and nicotinic antagonists (strychnine, -erythroidine). This initial analysis of nicotinic ligands was performed in order to determine structural basis for the interaction with nAChRs. Although nicotine has a higher affinity for neuronal 42 receptors over 7, it is not a selective molecule. Thus, the structure of nicotine may be used as a template for the design of other compounds. This method of study, structure activity relationship, involves determining the affinity (K i value) and EC 50 or IC 50 values of a group of compounds. This dissertation characterizes the SAR relationship for two groups of plant alkaloids on the high affinity neuronal 42 nAChR. The first group of compounds are nicotine analogs, which as we have determined act as weak partial agonists and antagonists on the 42 receptor. The other group of compounds are Erythrina alkaloids, which like the commonly used dihydro-beta erythroidine, also act as antagonists on the neuronal 42 receptor. Like nicotine they too are tertiary amines. In Beers and Reichs classic study from 1970

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27 they examined the structure of -erythroidine as compared to ACh in order to determine structural elements required for binding to the ACh receptor. -Erythroidine with its fused ring system is a more rigid molecule than nicotine. Beers and Reich (1970) determined that the oxygen atoms on rings D and A can both form hydrogen bonds that measure 5.9 from the nitrogen between rings C and B. They proposed that although -erythroidine is a rigid molecule its ability to bind and inhibit receptor function might be due to dual hydrogen binding sites. The goal of this dissertation was to create structure activity relationships for each of these two groups of plant alkaloids that may be useful in designing partial agonists and antagonists for the possible treatment of nicotine dependence. These structure activity relationships may also be useful for designing in vitro and in vivo probes for studying the function of 42 nAChRs.

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CHAPTER 2 MATERIALS AND METHODS Radioligand Binding Compound Syntheses Dr. Ferenc Soti synthesized all nicotine analogs; purity was determined by reversed phase HPLC and NMR confirmed compound structure. The nicotine analogs were free bases. All were suspended in high performance liquid chromatography (HPLC) grade methanol to create stock solutions of 10 to 100 mM. These stock solutions were further diluted in 50 mM Tris binding saline containing 2 mg/ml of bovine serum albumin (Sigma, St. Louis, MO) to create desired concentrations used in binding or functional assays. The extraction and semi-synthetic preparation of the Erythrina alkaloids were also performed by Dr. Ferenc Soti according to traditional methods. Following extraction of several natural aromatic Erythrina compounds, separation was performed by high performance liquid chromatography (HPLC) using a C-18 column (Beckman Coulter, Fullerton, CA) as well as by silica gel chromatography. Erysotrine was the only Erythrina alkaloid that was isolated as a free base. All the other alkaloids were acetate or hydrochloride salts. The Erythrina alkaloids also were suspended in HPLC grade methanol to create stock solutions and then further diluted in 50 mM Tris binding saline containing 2 mg/ml bovine serum albumin. The (R)-form of nicotine was obtained from Research Biochemicals Inc. (Natick, MA) as a di-p-toluoyl tartrate salt. The (S)-form of nicotine was obtained from Sigma (St. Louis, MO) as a tartrate salt. The (R)and (S)-nornicotines were obtained from 28

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29 Peyton Jacobs III (San Francisco General Hospital, San Francisco, CA) and both existed as dicamsylate and camsylate salts, respectively. Rat Brain Membranes Whole male Sprague-Dawley rat brains obtained from Pel-Freeze Biologicals (Rogers, AZ) were prepared according to Marks and Collins (1982). Whole rat brains were homogenized with a 30 ml Wheaton (location) glass homogenizing tube and pestle attached to a motor source. The tissue was homogenized in Tris binding saline consisting of 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 50 mM Trizma HCl Buffer pH=7.4. After homogenization the tissue was centrifuged at 11,000 rpm for a ten-minute period. After centrifugation the pellet was resuspended in fresh binding saline and again homogenized. A BCA protein assay (Pierce, Rockford, IL) was then performed to obtain the protein concentration of the rat brain membranes. Homogenized tissue was stored at -80C until use. Rat brain membranes at a concentration of 200 g protein were used per tube for radioligand binding. Human Embryonic Kidney Subclone Cell (tsA201) Membranes A subclone of the human embryonic kidney cell line (tsA201 cell line) was graciously provided by John Lindstrom (University of Pennsylvania, Philadelphia, PA). These cells stably express human 42 nAChRs on their membranes. These cells were prepared according to Xiao and Kellar (2004). Cells at about 80 to 90% confluence were collected after removing the culturing media from the flask (75 cm 2 ) and adding 6-10 mls of ice-cold Tris binding saline (pH = 7.4) with a disposable cell scraper. The dislodged cells were then collected in centrifuge tubes and spun down at 1000 rpm for five minutes. The loose pellet was then collected and homogenized in the same method as the rat brain

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30 membranes. After performing a protein assay the homogenized membranes were stored at -80C until use. Homogenized tsA201 cell membranes at concentrations of 50 to 125 g were used per tube for radioligand binding. Binding Assay Protocol The radioligands used in the competition and saturation binding assays were obtained from Perkin Elmer Life and Analytical Sciences (Boston, MA). 3 H-Cytisine (34 Ci/mmol) experiments were performed according to Flores et al. (1992) with a few minor alterations, specifically that the incubation time was increased to four hours at 4C to allow for the binding to reach equilibrium. The 125 I--bungarotoxin (-btx) (136 Ci/mmol) experiments were incubated at 37C for three hours. Both radioligands were used at a final concentration of 1 nM in competition binding assays. Homogenized tissue at the above mentioned concentrations were suspended in 50 mM Tris binding saline containing 2 mg/ml of bovine serum albumin (Sigma, St. Louis, MO). The tissue along with the radioligand and compound of interest were incubated in 13x100 mm disposable glass culture tubes at a final volume of 0.5 ml. For each radioligand nonspecific binding was measured in the presence of a final concentration of 1 mM (S)-nicotine hydrogen tartrate salt (Sigma, St. Louis, MO). After incubation radioligand bound membranes were collected by vacuum filtration with a Brandel cell harvester (Gaithersburg, MD) onto Whatman GF/C glass fiber filters that had been pre-soaked in 0.5% polyethylenimine for 45 minutes. The radiolabeled membranes were washed three times with 3 mls of ice-cold 50 mM Tris binding saline. Filters containing 3 H-cytisine bound membranes were collected in 20 ml scintillation tubes and suspended in 8 mls of 30% Scintisafe scintillation fluid (Fisher), then counted in a Beckman LS-6500 liquid

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31 scintillation counter (Fullerton, CA). Filters containing 125 I--btx bound membranes were placed in 4 ml scintillation vials and counted in a Beckman 5500B gamma counter (Fullerton, CA). Whereas each concentration used for saturation and competition binding assays with rat brain membranes was performed in quadruplicate, each concentration for assays involving tsA201 membranes was performed in triplicate. Binding Assay Data Analysis Binding assay data were analyzed using GraphPad Prism software (San Diego, CA). The count per minute values for each concentration were averaged and normalized to the specific binding value obtained within each experiment. Saturation assay data were analyzed by fitting the data to a one site binding hyperbola model (Y=B max *X/(K d +X)) in order to determine the K d and Bmax value for the respective radioligand and tissue. The data could be transformed into a Scatchard plot for visualization of the respective slopes and X-axis intercepts using the Prism software. Competition assay data were analyzed using a sigmoidal dose response with variable slope (Y=Bottom+((Top-Bottom)/(1+10 ((LogIC50-X)*Hillslope) ))) from which a hillslope and IC 50 value were determined; Top = Y value at the top plateau of the curve; Bottom = Y value at the bottom plateau of the curve. The IC 50 value along with the pre-determined K d value for the radioligand and nAChR-containing membrane of interest were then used in the Cheng Prusoff equation (K i =IC 50 /(1+(Ligand)/K d )) to calculate the K i value. Significant differences were determined using an unpaired, two-tailed T-test in GraphPad Prism.

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32 Functional Measurements Cell Culture Cells were cultured essentially according to Nelson et al. (2003). The tsA201 cells expressing 42 nAChRs were maintained in a culture medium consisting of Dulbeccos modified Eagles medium (Gibco, Carlsbad, CA) supplemented with 10% FBS (MediaTech Inc., Herdon, VA), 100 units/ml penicillin and 100 g/ml streptomycin (Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Irvine Scientific, Santa Ana, CA), 0.5 mg/ml Zeocin (Invitrogen, Carlsbad, CA), and 0.6 mg/ml G-418. Cells were grown in 75 cm 2 culture flasks which were housed in a humidified incubator at 37C in an atmosphere of 5% CO 2 Cells were grown to around 80-90% confluence before being split with 0.25% Trypsin (Gibco, Carlsbad, CA) at a subcultivation ratio of between 1:6 and 1:10 weekly. Membrane Potential Dye, Cell Loading and Compound Plate Preperation Cells were seeded at a density of roughly 510 4 cells/well to 1010 4 cells/well onto 96-well flat-bottom black-wall culture plates that had been coated with 50 l per well of 50 g/ml poly-D-lysine (70-150kDa). Cells were then grown overnight in 100 l of culture medium in order to achieve a single layer of cells on the bottom of each well. The membrane potential dye, obtained from Molecular Devices, was prepared by dissolving one bottle of the dye into 30 mls of 1X Hanks balanced salt solution (HBSS) supplemented with 20 mM Hepes (pH=7.4). The dye was then added (100 l to each well) on top of the existing 100 l of media. Finally, the cells were incubated in the dark with the dye at 37C for 30 minutes before reading. Serial dilutions of a compound were prepared in a separate clear-walled 96-well V-bottom plate by evaporation of the correct

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33 volume of a methanol stock solution. The evaporated compounds were then reconstituted in 250 l of HBSS/Hepes (pH=7.4) containing 1 M atropine. Membrane Potential Measurements Flexstation protocol was performed essentially as described by Fitch et al. (2003) with several modifications. Fluid transfer and readings were performed by a Flexstation fluorometer (Molecular Devices, Sunnyvale, CA). The dye (composition withheld for proprietary reasons) is a lipophilic, anionic, bis-oxonol dye. When the cells are depolarized the dye enters and binds to cytosolic proteins, causing an increase in fluorescence signal. During hyperpolarization the dye exits the cells and there is a decrease in fluorescence signal. Excitation and emission wavelengths were set to 530 nm and 565 nm with a cutoff of 550 nm. A reading was taken every 1.44 seconds over about a three minute period for a total of 139 readings per well. The first 17 seconds were used for a basal read. At 18 seconds the addition of 50 l of a test compound is added (to assess possible agonist activity, EC 50 ), followed by a 25 l addition of KCl (40 mM final concentration) at 160 seconds to measure the maximum possible signal and to correct for differences in dye loading and cell count (only the KCl response of the drug-nave cells was used for normalization). Compound applications were done at about 78 l per second. Compounds that had no measurable intrinsic activation properties upon the tsA201 cells were then examined to determine their IC 50 values. The compound of interest was applied to the cells as above except that they were simultaneously applied with 5 M acetylcholine (ACh) in order to measure inhibition of the ACh response. 5 M ACh was slightly above the measured EC 50 but well below the dose required to

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34 produce a maximal response, for ACh upon the 42-expressing tsA201 cells as measured with the Flexstation. Functional Assay Data Analysis Responses of experimental compounds were measured as the maximum RFU (relative fluorescent unit) subtracted from the averaged basal read for each individual well. Data was collected with SOFTmax Pro software from Molecular Devices (Sunnyvale, CA). These raw compound values were then divided by the average maximum response of cells in control wells to 40 mM KCl (compound nave cells run in parallel). It was necessary to use values from control wells because it was determined that KCl response larger for cells previously exposed to agonist. The compound values were then graphed using the sigmoidal dose response with variable slope equation in Prism (Y=Bottom+((Top-Bottom)/(1+10 ((LogEC50-X)*Hillslope) ))) to calculate either an EC 50 or IC 50 value as well as the hillslope for each compound. The EC 50 responses were normalized to the maximum response of ACh. High Performance Liquid Chromatography (HPLC) Chiral Separation of Nicotine Analogs Racemic nicotine analogs were separated on a Daicel chiral OJ-H semi-preparative column (250 x10 mm i.d.) from Chiral Technologies, Inc. (West Chester, PA) using a Beckman System Gold 126 solvent module attached to a System Gold 168 photodiode array detector (Fullerton, CA). The column was eluted with an increasing gradient (0-94% buffer B) over a 35-minute period where buffer A was n-hexane/ethanol/diethylamine/trifluoroacetic acid (97:3:0.1:0.1 v/v/v/v) and buffer B consisted of the same solvents at a volume ratio of 80:20:0.1:0.1 v/v/v/v for 1-ethyl-nornicotine-(S,R)-nicotine. The pyridyl and phenyl nicotine compounds were

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35 separated using an isocratic method (90:10:0.1:0.1 v/v/v/v for pyridyl nicotine and 94:6:0.1:0.1 for phenyl nicotine v/v/v/v). The separated compounds were collected with a Foxy Jr. Fraction collector using PeakTrak software (Isco Inc., Lincoln, NE). The amount of collected compound was determined by absorbance spectrum measurements in 95% ethanol using a Beckman DU 650 spectrophotometer (Fullerton, CA) and published molecular extinction coefficients (in 95% ethanol) for unionized forms of nicotine and nornicotine (Swain et al., 1949).

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CHAPTER 3 RESULTS Nicotine Analogs The involvement of nicotinic receptors in nicotine addiction has led to the synthesis of a variety of nicotine analogs in order to determine relationships between structure and function. Numerous studies have characterized the binding of analogs with substituents at various positions on either the pyridine or pyrrolidine ring of nicotine. However, most of these studies provided binding data for only the high-affinity (42) receptor and lacked functional data. In order to determine the 42 versus 7 selectivity for several previously published as well as novel nicotine analogs, binding assays were performed to measure the ability to displace either 3 H-cytisine (42) or 125 I--btx (7) in rat brain membranes. The nicotine analogs were then tested in functional assays utilizing tsA201 cells expressing human 42 receptors and a membrane potential dye to measure agonistic activity (EC 50 ) and inhibition of the ACh response (IC 50 ). (S)-Nicotine has a high affinity for the neuronal 42 nAChR; it binds at low nanomolar (2.3 nM) concentrations (Dukat et al. 1996). The naturally occurring (S)-nornicotine, which is also a metabolite of (S)-nicotine, has been reported to have a 16to 18-fold lower affinity for the 42 receptor (Copeland et al. 1991; Glassco et al. 1994). The functional effects of (S)-nicotine have also been well characterized. Much of the functional data for 42 has been provided by electrophysiological experiments, radioactive rubidium efflux and radiolabeled-dopamine release assays. (S)-Nicotine acts 36

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37 as a concentration and time-dependent agonist on 42 nAChRs with measured EC 50 values ranging from 1 to 10 M. At increasing concentrations and times after application nicotine may behave as an antagonist, primarily due to receptor desensitization. The functional effects of (S)-nornicotine are less well characterized than (S)-nicotine. In dopamine release assays from rat striatal slices, (S)-nornicotine at concentrations up to 100 M has been found to cause nAChR mediated 3 H-dopamine release (Teng et al. 1997). Of the published nicotine analogs, only the 5-pyridyl substituents have been electrophysiologically characterized to obtain IC 50 values for 42. Dukat et al. (2002) found that adding smaller substituents at the 5-pyridyl position of nicotine did not greatly reduce the affinity for the 42 receptor. They measured the K i values for 5-bromonicotine and 5-methoxynicotine for displacing 3 H-nicotine as 6.9 2.6 nM and 14.3 1.5 nM, respectively, as compared to the K i value of 2.4 0.4 nM for (S)-nicotine. However, they found that substituents at this position even more significantly influenced their functional properties. Dukat et al. (2002) determined that although the K i values for both compounds were similar to that of nicotine, the 5-bromo compound functioned as a partial agonist on rat 42 receptors expressed in Xenopus oocytes whereas the 5-methoxy compound had no agonistic activity. Substituents at the 6-position on the pyridine ring were characterized for binding to 42 as well as for in vivo effects on mice (Dukat et al. 1996). They determined that a chlorine at the 6-position of nicotine produced an analog with 3.8-fold greater affinity than (S)-nicotine for 42. Other analogs with small substituents, including 6-bromo and 6-fluoronicotine had affinities that were respectively 1.4and 1.6-fold lower than that

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38 of (S)-nicotine. However, a larger methoxy substituent at this position decreased affinity 35 times that of nicotine for 42. Both the 6-bromo and 6-fluoro analogs were as equipotent as (S)-nicotine in antinociceptive measurements on mice whereas the 6-methoxy analog was much less active. Dukat et al. (1996) determined that both lipophilicity and size of the substituent at the 6-pyridyl position influence binding properties as well as in vivo effects. A group of compounds that have been characterized for their ability to inhibit 3 H-dopamine release from striatal slices contain substituents (increasing chain length alkyl) at the 1-pyridyl position. Crooks et al. (2004) found that adding alkyl substituents with chain lengths ranging from 1 to 4 carbons resulted in low potency antagonists (IC 50 >10 M). Interestingly, the most potent compound had a 1-pyridyl-chain length of 12 carbons. This potent antagonist, known as NDDNI, had an IC 50 value of 0.009 M and a K i value for 3 H-nicotine displacement of 0.14 M (Crooks et al. 2004). The binding affinities of substituents at the 3-, 4-, and 5-pyrrolidine positions have been well characterized by Lin et al. (1994) in rat brain. They found that increasing substituent size at the 3-position resulted in decreased affinity, indicating steric effects of the ligand-receptor interaction. A methyl group at the 4-position had an affinity that was only 3.7-fold lower than that of (S)-nicotine. However, when larger nonpolar or polar substituents were added at the 4-position, the affinity for 42 decreased. Lin et al. (1994) noted that electronic along with steric effects may be influencing the binding affinities for the 42 receptor. Unlike at the 4-position, a methyl substituent at the 5-position was not well tolerated; it decreased affinity over 30-fold as compared to (S)-nicotine. Also, they found that there exists a stereoselectivity for substituents at the

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39 5-position. The trans-5-methyl analog had a 34.5-fold greater affinity for the receptor relative to the cis-5-methyl nicotine. Thus, steric influences as well as stereochemistry produce effects upon binding at the 3-, 4and 5-positions on (S)-nicotine. Damaj et al. (1996) found that an ethyl at the 1-N-position reduced affinity for rat 42 35-fold as compared to (S)-nicotine. Glassco et al. (1994) measured a trend of decreasing affinity for 42 in rat brain with increasing 1-N-substituent size. They found about a 20-fold decrease in affinity (as compared to (S)-nicotine) when the size of the substituent at the 1-N-position was increased from a methyl to an ethyl, a 369-fold decrease (as compared to (S)-nicotine) when substituting a propyl group and greater than a 7,000-fold decrease when substituting a cyclopropyl group. Like the other pyrrolidine substituents, bulky groups at the 1-N-position result in steric hindrance for binding with a high affinity to the 42 receptor. Because these previous studies lacked data on efficacy and nAChR selectivity, we have further characterized several of these compounds and additionally, other novel nicotine analogs on 7 as well as 42 nAChRs. Our goal was to create a structure activity relationship for nicotine binding to the 42 receptor to assist in reaching our ultimate goal of designing a partial agonist for smoking cessation. Based upon hypotheses structured from previously published data (primarily binding) of nicotine analogs and the 42 receptor, we created nicotine analogs at four different positions on the nicotine molecule: the 5-pyridyl and 5-, 1-N-, and 3-pyrrolidine positions.

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40 Hypotheses: 1) Relatively bulky substituents at the 5-pyridyl position would permit effective binding but reduce efficacy. 2) A trans-methyl group at the 5-pyrrolidine position would reduce efficacy with retention of acceptable binding affinity and receptor selectivity. 3) Bulky alkyl groups at the 1-N-position would decrease the 42 affinity but relatively strong partial agonism would be retained. 4) Bulky alkyl groups at the 3-position would decrease the affinity for rat and human 42 but enhance the affinity for 7 (based on previously published DMXBA data). (S)-Nicotine and (S)-Nornicotine Using 3 H-cytisine as a label for 42, nicotine displaced the radiolabel from rat brain with a K i value of 9.2 nM. As a comparison for selectivity we report selectivity ratios (SR = K i value for 7/ K i value for 42). The selectivity ratio of (S)-nicotine was 83. (S)-Nornicotine binds with a much lower affinity to rat 42 (K i = 0.11 M) than (S)-nicotine. It is also less selective for rat 42 over rat 7, with a SR of 16 (Table 3-1).

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41 Table 3-1. Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by (S)-nicotine or (S)-nornicotine. 0.11 0.0290.0092 0.0020Ki (M)3H-Cytisine (42)1.7 0.29(S)-Nornicotine0.76 0.11(S)-NicotineRat125I--Btx (7)Compound Name and Structure 0.11 0.0290.0092 0.0020Ki (M)3H-Cytisine (42)1.7 0.29(S)-Nornicotine0.76 0.11(S)-NicotineRat125I--Btx (7)Compound Name and Structure 0.48 0.260.0089 0.0039Human 3H-Cytisine(42) 0.48 0.260.0089 0.0039Human 3H-Cytisine(42) N N CH3 N N H K i values were calculated according to the Cheng Prusoff equation. The concentration of each radioligand was 1 nM. Each value represents the mean SEM of three separate experiments unless otherwise indicated. Concentrations for each experiment were done in triplicate. The K d values for 125 I--btx and 3 H-cytisine binding to rat brain membranes were 0.32 nM 0.04 and 0.92 0.1 nM respectively. The K d value for 3 H-cytisine binding to tsA201 cell membranes was 0.48 nM 0.2. Because the functional assays were performed using cells (tsA201) that express human 42, I also measured the binding properties of the nicotine analogs on homogenized tsA201 cell membranes. Statistical T-tests were performed to determine any significant differences between rat brain 42 and human 42 K i values. I also performed a sequence alignment with ClustalW (on the European Bioinformatics website) for human and rat 4 and 2 subunits loops A through F in order to determine any residue differences. The alignment revealed two residue differences between the species as follows:

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42 Loop F Loop E Human 2 EVASLDDF Human 4 LTKAHLFHDGRVQWT Rat 2 DVASLDDF Rat 4 LTKAHLFYDGRVQWT The F loop of the human 2 subunit contains a glutamic acid at position 190 whereas the rat 2 subunit contains an aspartic acid at position the homologous position (189). The 4 human subunit loop E contains a histidine at position 145 and in its place in the rat 4-receptor subunit is a tyrosine residue at homologous position 147. Experiments were done to determine the agonist activity of each nicotine analog on 42 receptors. These measurements were performed by first adding the nicotine analog alone at various concentrations followed by the addition of KCl (40 mM final concentration), which was used as a calibrant (Figure 3-1). Fitch et al. (2003) previously published results using KCl as a calibrant on various cell lines expressing different subtypes of nAChRs, thus we also chose to use KCl. The addition of KCl results in a large depolarization (as measured by changes in fluorescence) that was used to normalize for cell count, dye loading and possible differences in resting membrane potential between passages of cells. After performing a concentration response curve for the tsA201 cells and KCl, it was determined that 40 mM KCl produces about 85% of the maximum KCl response. Measurable changes in fluorescence were relatively rapid, starting within three seconds, after application of agonist. There was no decay of fluorescence after application of (S)-nicotine, thus calibration calculations were performed with the KCl response as measured on agonist nave cells (those that received only HEPES/HBSS).

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43 40 m M KCl Nicotine Figure 3-1. Signal acquired for membrane potential fluorescence using tsA201 cells expressing human 42 nAChRs. During the first 17 seconds baseline fluorescence was measured for each well. At 18 seconds well A10 () received 50 M nicotine, well B11 () received 0.16 M nicotine and well B7 () received only 20 mM HEPES/HBSS. All three wells received a final concentration of 40 mM KCl at 120 seconds. A fluorescence reading was taken every 1.44 seconds for a total of 139 reads over a three minute period. The functional responses of the nicotine analogs as measured with membrane potential dye indicated that several analogs, along with the naturally occurring (S)-nicotine and (S)-nornicotine, had agonistic activity on human 42 receptors. The average EC 50 values for (S)-nicotine and ACh were 1.1 0.15 M and 3.6 0.9 M respectively (Figure 3-2). (S)-Nornicotine displayed partial agonist activity: it was half as efficacious as compared to ACh, with an EC 50 value of 8.9 0.67 M (Figure 3-2). Several experiments were done to determine the maximum effect of ACh on the human 42 expressing tsA201 cells by increasing the concentration of ACh from 2,000 M to

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44 10,000 M. The results from those separate experiments (not shown) produced the same EC 50 and maximum response as the response seen in Figure 3-2. -10 -9 -8 -7 -6 -5 -4 -3 -2 0 20 40 60 80 100 120 140ACh (S)-Nicotine (S)-Nornicotine Log [Compound], MPercent Activation(% relative to maximumACh response) Figure 3-2. Concentration response curves of nicotine, ACh and nornicotine for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential. Responses were normalized to the maximum ACh response. Each curve represents an average of 20 wells for nicotine, 14 wells for ACh and 4 wells for nornicotine (averaged from 2 to 10 separate experiments). 5-Pyridyl Substituted Analogs The 5-pyridyl substituted analogs, 5-phe-nic and 5-pyr-nic, both bound with low micromolar affinities to rat 42: K i s = 0.066 and 0.14 M, respectively (Table 3-2). Interestingly, these compounds showed no interaction with rat 7 at concentrations up to 20 M. Thus, although both compounds bound with lower affinities to rat 42 than (S)-nicotine, they had higher selectivity ratios (SR): SR of 5-phe-nic was 303 while the SR of 5-pyr-nic was 143. These values are greater than the SR of nicotine, which was 83.

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45 Table 3-2. Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 5-pyridyl substituted analogs. 0.14 0.0 21 0.066 Ki (M)3H-Cytisine (42)>205-(3-Pyridyl)-(S,R)-Nicotine5-Pyr-Nic*>205-Phenyl-(S,R)-Nicotine5-Phe-Nic*Rat125I--Btx (7)Compound Name and Structure 0.013 0.14 0.00.066 Ki (M)3H-Cytisine (42)>205-(3-Pyridyl)-(S,R)-Nicotine5-Pyr-Nic*>205-Phenyl-(S,R)-Nicotine5-Phe-Nic*Rat125I--Btx (7)Compound Name and Structure 21 0.013 0.053 0.0170.020 0.0037Human 3H-Cytisine (42) 0.053 0.0170.020 0.0037Human 3H-Cytisine (42) N N CH3 N N N CH3 ** ** Parameters were the same as in Table 3-1. Diamonds indicate a significant difference between the K i values for rat and human with 3 H-cytisine. Abbreviations for the analogs are indicated by an asterisk. The 5-pyridyl substituted analogs were tested for their ability to activate human 42 receptors expressed in tsA201 cells. There was no measurable activation produced by either compound. The 5-pyridyl substituted analogs were then tested for their ability to inhibit the ACh response as measured by changes in membrane potential. ACh was chosen over (S)-nicotine as the standard agonist primarily because the occurrence of channel block by ACh is expected to be less than for (S)-nicotine. A concentration of 5 M ACh was applied to the cells simultaneously with the nicotine analog of interest. A concentration of 5 M ACh was chosen because it was slightly above the EC 50 (3.6 M) and well below the concentration required to produce a maximum response (500 M). The 5-phe-nic and 5-pyr-nic compounds had the highest affinities (K i values = 0.02 M and 0.053 M, respectively) for human 42 receptors as compared to the other nicotine

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46 analogs. These two compounds produced measurable IC 50 values of 14.3 (5-phe-nic) and 11.6 M (5-pyr-nic) for human 42 (Table 3-3). Table 3-3. Inhibition of human 42 receptor ACh responses by 5-pyridyl substituted analogs. Compound Name or Abbreviation IC 50 (M) 5-Pyridyl substituted analogs 5-Phe-Nic 14.3 1.3 5-Pyr-Nic 11.6 0.41 The compounds and ACh (5 M) were administered simultaneously. Each value represents the average SEM of four to six separate wells. 5-Pyrrolidine Substituted Analogs The results for the 5-pyrrolidine substituted analogs indicate that the receptor possesses a strong conformational preference for ligand binding: the 5-trans-met-nic analog bound with an affinity of 0.150 M to rat 42 whereas 5-cis-met-nic displayed no interaction with this receptor at concentrations up to 20 M (Table 3-4). While 5-trans-met-nic did bind to rat 7 with a K i of 2.1 M, 5-cis-met-nic did not interact with rat 7 at concentrations up to 20 M. The SR for 5-trans-met-nic was 14, thus, the methyl substituent at the 5-trans position dramatically reduced the selectivity for rat 42 as compared to nicotine. There was no significant difference between the binding of either 5-substituted analog to rat 42 or human 42. Like the 5-pyridyl substituted analogs, the 5-pyrrolidine analogs produced no activation of human 42 receptors expressed in tsA201 cells. However, the 5-trans-met-nic analog did inhibit the human 42 ACh response (Table 3-5). Full concentration response curves were not obtained at the concentrations tested, therefore

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47 percent of inhibition values were reported. At the highest concentration of 5-trans-met-nic used (50 M), the human 42 ACh response was inhibited by 50%. Table 3-4. Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 5-pyrrolidine substituted analogs. >200.15 0.013Ki (M)3H-Cytisine (42)>205-Cis-Methyl-(S,R)-Nicotine5-Cis-Met-Nic*2.1 0.0335-Trans-Methyl-(S,R)-Nicotine5-Trans-Met-Nic*Rat125I--Btx (7)Compound Name and Structure >200.15 0.013Ki (M)3H-Cytisine (42)>205-Cis-Methyl-(S,R)-Nicotine5-Cis-Met-Nic*2.1 0.0335-Trans-Methyl-(S,R)-Nicotine5-Trans-Met-Nic*Rat125I--Btx (7)Compound Name and Structure >200.22 0.038Human 3H-Cytisine(42) >200.22 0.038Human 3H-Cytisine(42) N N CH3 CH3 N N CH3 CH3 Parameters were the same as in Figure 3-1. Abbreviations for the analogs are indicated by an asterisk. Table 3-5. Inhibition of human 42 receptor ACh responses by 5-pyrrolidine substituted analogs. Compound Name or Abbreviation Percent Inhibition (I) 5-Pyrrolidine substituted analogs 5-Trans-Nic 50% I (50 M) 5-Cis-Nic 0% I (50 M) The compounds and ACh (5 M) were administered simultaneously. The concentrations in parenthesis indicate the highest concentration tested. Each value represents the average SEM of four to six separate wells. 1-N-Pyrrolidine Substituted Analogs The ability of the 1-N-pyrrolidine substituted analogs to bind to either rat 42 or rat 7 nAChRs decreased as the size of the substituent increased (Table 3-6). The

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48 smallest substituent, an ethyl group, at the 1-N-position bound with an affinity (K i = 0.14 M) similar to that of 5-trans-met-nic (0.15 M). As the substituent sizes increased to a propyl and cyclopropyl group, there was no measurable interaction with either rat 42 or rat 7 up to 20 M. The SR of 1-N-ethyl-nor (SR = 17.9) was less than that of (S)-nicotine (SR = 83). Initial data on the binding of 1-N-ethyl-nor to human 42 indicates a K i value very similar to that for rat 42. Table 3-6. Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 1-Npyrrolidine substituted analogs. >20>201-N-Propyl-(S,R)-Nornicotine1-N-Propyl-Nor*>200.14 0.04Ki (M)3H-Cytisine (42)>201-N-Cyclopropylmethyl-(S,R)-Nornicotine1-N-Cyclopropyl-Nor*2.5 0.091-N-Ethyl-(S)-Nornicotine1-N-Ethyl-Nor*Rat125I--Btx(7)Compound Name and Structure >20>201-N-Propyl-(S,R)-Nornicotine1-N-Propyl-Nor*>200.14 0.04Ki (M)3H-Cytisine (42)>201-N-Cyclopropylmethyl-(S,R)-Nornicotine1-N-Cyclopropyl-Nor*2.5 0.091-N-Ethyl-(S)-Nornicotine1-N-Ethyl-Nor*Rat125I--Btx(7)Compound Name and Structure N N N N N N >20>200.14 (n=2)Human 3H-Cytisine(42) >20>200.14 (n=2)Human 3H-Cytisine(42) Parameters were the same as in Figure 3-1. Abbreviations for the analogs are indicated by an asterisk. Within the four classes of nicotine analogs, two of them (the 1-N-pyrrolidine and 3-pyrrolidine substituted analogs) produced activation. The highest concentrations of analogs utilized in these studies did not produce full concentration response curves

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49 (maximal responses) to allow for EC 50 values to be determined, therefore only percent activations at these high concentrations are reported (Figure 3-3). The 1-N-ethyl-nor compound was the only 1-N-pyrrolidine substituted analog with a measurable affinity for the rat and human receptors and it produced about 35% activation of human 42 (at 500 M) as compared to the maximum ACh response. Both 1-N-propyl-nor and 1-N-cyclopropyl-nor activated the human receptor about 40% (at 2,000 M) and 75% (at 2,000 M) respectively. Although neither of these compounds had a measurable affinity for the human 42 receptor as determined with the radioligand binding assays, which utilized lower concentrations than in the functional assays, the concentrations used for the membrane potential measurements were several-fold higher. Therefore, at concentrations above 20 M these compounds are binding to the receptor and causing activation. All three 1-N-substituted analogs were also found to inhibit the ACh response of human 42. The 1-N-cyclopropyl-nor analog was the most potent at activating human 42 (at the concentrations tested) as well as the most potent inhibitor (Table 3-7). The 1-N-ethyl-nor and 1-N-cyclopropyl-nor both produced less than 50% inhibition of the human 42 ACh response.

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50 01020304050607080901001'-N-Ethyl-Nor1'-N-Propyl-Nor 1'-N-Cyclopropyl-NorPercent of Activation (normalized to maximum ACh response) 500 M2,000 M2,000 M N N N N N N 01020304050607080901001'-N-Ethyl-Nor1'-N-Propyl-Nor 1'-N-Cyclopropyl-NorPercent of Activation (normalized to maximum ACh response) 500 M2,000 M2,000 M N N N N N N Figure 3-3. Average effects of the 1-N-pyrrolidine substituted analogs on human 42 receptors expressed in tsA201 cells as measured with membrane potential dye. Effects by each analog were produced by the concentration listed above the corresponding bar. Each bar represents 4 separate wells. Responses were normalized to the maximum ACh response (500 M). Table 3-7. Inhibition of human 42 receptor ACh responses by 1-N-pyrrolidine substituted analogs. Compound Name or Abbreviation Percent Inhibition (I) 1-N-Pyrrolidine substituted analogs 1-N-Ethyl-Nor 35% I (500 M) 1-N-Propyl-Nor 45% I (500 M) 1-N-Cyclopropyl-Nor 80% I (50 M) The compounds and ACh (5 M) were administered simultaneously. The concentrations in parenthesis indicate the highest concentration tested. Each value represents the average SEM of four to six separate wells. 3-Pyrrolidine Substituted Analogs The same trend of decreasing affinity with increasing substituent size was observed for the 3-pyrrolidine substituted analogs (Table 3-8). The 3-met-nic analog, which has only a methyl group at the 3-position, bound to rat 42 with a decreased affinity

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51 (Ki=0.37 M) as compared to (S)-nicotine. 3-Dimethoxybenzyl-nornicotine (DMXBN) has a large substituent, a benzene ring with two methoxy groups attached, yet it still bound to the rat 42 receptor with a K i value of 2.8 M and had no interaction with rat 7 at up to 20 M (SR > 7.1). The differences between DMXBM and DMXBN are related to the presence of two additional double bonds in DMXBM, allowing for all these rings of the compound to be electronically conjugated. DMXHMN has an additional hydroxymethyl group attached to the benzyl ring, which is attached at (but not electronically conjugated to) the 3-pyrrolidine position of nicotine. DMXHMN displayed no interaction with either rat 42 or rat 7 at up to 20 M. The 3-pyrrolidine substituted analogs also had very low or no measurable affinity for human 42 at concentrations up to 20 M. They are however activating human 42 receptors at higher concentrations (Figure 3-4). DMXBN produced 80% activation (at 500 M) whereas DMXBM produced only 20% activation (at 500 M). The conjugated structure of DMXBM makes it more rigid and the ionizable nitrogen less basic. 3-Met-nic, the 3-pyrrolidine substituted analog with the smallest substituent, produced 40% activation at 500 M.

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52 Table 3-8. Inhibition of 125 I--btx and 3 H-cytisine binding to rat brain membranes or inhibition of 3 H-cytisine binding to human 42 expressing tsA201 cell membranes by 3-pyrrolidine substituted analogs. >20>203-(2,4-Dimethoxybenzylidene)-MyosmineDMXBM*2.8 0.58Ki (M)3H-Cytisine (42)>203-Dimethoxybenzyl-(S,R)-NornicotineDMXBN*Rat125I--Btx(7)Compound Name and Structure >20>203-(2,4-Dimethoxybenzylidene)-MyosmineDMXBM*2.8 0.58Ki (M)3H-Cytisine (42)>203-Dimethoxybenzyl-(S,R)-NornicotineDMXBN*Rat125I--Btx(7)Compound Name and Structure >20Not DeterminedHuman 3H-Cytisine(42) >20Not DeterminedHuman 3H-Cytisine(42) OMeMeO N N OMeMeO N N 0.37 0.000154.2 n = 23-Methyl-(S,R)-Nicotine3-Met-Nic*>20>203-(2,4-Dimethoxy-5-hydroxymethyl)benzyl-(S,R)-NicotineDMXHMN* 0.37 0.000154.2 n = 23-Methyl-(S,R)-Nicotine3-Met-Nic*>20>203-(2,4-Dimethoxy-5-hydroxymethyl)benzyl-(S,R)-NicotineDMXHMN* 0.63 0.094>20 0.63 0.094>20 N N CH2OH OMe MeOCH3 N N CH3 CH3 Parameters were the same as in Figure 3-1. Abbreviations for the analogs are indicated by an asterisk.

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53 0102030405060708090100DMXBN DMXBM DMXHMN 3'-Methyl-NicPercent of Activation (normalized to maximum ACh response) OMeMeO N N OMeMeO N N N N CH2OH OMe MeOCH3 N N CH3 CH3 500 M500 M500 M500 M 0102030405060708090100DMXBN DMXBM DMXHMN 3'-Methyl-NicPercent of Activation (normalized to maximum ACh response) OMeMeO N N OMeMeO N N N N CH2OH OMe MeOCH3 N N CH3 CH3 500 M500 M500 M500 M Figure 3-4. Average effects of the 3-pyrrolidine substituted analogs on human 42 nAChRs expressed in tsA201 cells as measured with membrane potential dye. Effects by each analog were produced by the concentration listed within or above the corresponding bar. Each bar represents 4 separate wells. Responses were normalized to the maximum ACh response (500 M). Three out of four of the 3-pyrrolidine substituted analogs produced varying degrees of human 42 nAChR activation. They did not however produce much if any inhibition of the ACh response (Table 3-9). The DMXBN, DMXBM and DMXHMN analogs had no inhibitory action at concentrations up to 50 M (500 M for DMXBN). 3-Met-nic did produce a 20% inhibition at 50 M. Table 3-9. Inhibition of human 42 receptor ACh responses by 3-pyrrolidine substituted analogs. Compound Name or Abbreviation Percent Inhibition (I) DMXBN 0% I (500 M) DMXBM 0% I (50 M) DMXHMN 0% I (50 M) 3-Met-Nic 20% I (50 M) The compounds and ACh (5 M) were administered simultaneously. The concentrations in parenthesis indicate the highest concentration tested. Each value represents the average SEM of four to six separate wells.

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CHAPTER 4 RESULTS Separation of Nicotine Analog Enantiomers Naturally occurring (S)-nicotine (approximately 99% S-form, about 1% R-form as an impurity) has been reported as more potent than its enantiomer in binding to high affinity nicotinic receptors as measured with 3 H-nicotine (Copeland et al. 1991; Zhang and Nordberg, 1993). Copeland et al. (1991) measured a 7-fold difference in the binding of the enantiomers to rat cortex; (S)-nicotine had the higher affinity (K i = 0.014 M) relative to (R)-nicotine (K i = 0.102 M). Zhang and Nordberg (1993) reported a 3-fold difference in the binding of the stereoisomers to rat cortex but an 11-fold difference in binding to rat cerebellum. Both studies found no significant differences in the binding affinities of the enantiomers of nornicotine. Zhang and Nordberg (1993) determined a 1.6-fold difference in the binding of the nornicotine enantiomers to rat cerebellum whereas Copeland et al. (1991) reported a 1.1-fold difference. The existence of nicotinic receptors on dopaminergic terminals along with evidence of nAChR involvement in nicotine addiction has lead several groups to study the effects of nicotine and nornicotine in dopamine release assays using synaptosomes and rat brain slices. (S)-Nornicotine, like (S)-nicotine is a naturally occurring alkaloid found in tobacco plant (Saitoh et al. 1985). It is also an active metabolite of nicotine and has a half-life three times longer than that of (S)-nicotine (Crooks et al. 1997; Green et al. 2001). Whiteaker et al. (1995) measured an EC 50 of 0.5 M for (S)-nicotine stimulated 3 H-dopamine release from striatal synaptosomes. Based upon the reported decrease in 54

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55 affinity of (R)-nicotine, it has yet to be characterized in dopamine release assays. The enantiomers of nornicotine however have been investigated. Green et al. (2001) determined that (R)-nornicotine (EC 50 = 0.48 M) was 6.3 times more potent than (S)-nornicotine (EC 50 = 3.0 M) at evoking radiolabeled dopamine release from rat nucleus accumbens slices. It has also been determined that (S)-nornicotine desensitizes nAChRs with 12-fold lower potency than (R)-nornicotine as measured with dopamine release assays from rat striatum (Dwoskin et al. 2001). Several of the nicotine analogs in this dissertation were initially studied as racemic compounds because the enantiomeric species were difficult to synthesize. Given that previous binding data indicates a difference in affinity for the enantiomers of nicotine, it was important to try and separate the (R)and (S)-forms of the racemic nicotine analogs and determine the affinity and functional properties of these chiral compounds. Our hypothesis was that like (S)-nicotine, the (S)-forms of the racemic analogs would have a higher affinity and functional potency for 42. Three of the racemic nicotine analogs, 5-phe-nic, 5-pyr-nic and 1N-ethyl-nor, were successfully separated by chiral HPLC (Figure 4-1). The 5-phe-nic and 1-N-ethyl-nor compounds were tested for their ability to bind to rat 42 nAChRs as measured by 3 H-cytisine displacement (Table 4-1). The affinities of the nicotine and nornicotine enantiomers were measured for human 42 and then further tested for potency and efficacy (Figure 4-2) on the human 42 receptor in tsA201 cells The affinities of the racemic 5-phe-nic and 5-pyr-nic analogs were similar as measured for rat and human 42 (Table 3-2) as may be expected based on their similar structures. Thus, only the enantiomers of the 5-phe-nic were tested for their ability to

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56 bind to rat 42. The enantiomers of both the 5-phe-nic and 1-N-ethyl-nor have yet to be identified as to which peak corresponds to the (R) or (S)-form, therefore the enantiomers are labeled as peak 1 or 2. It may be expected that peak 1 is the (S)-enantiomer based upon data from the chiral column indicating that (S)-nicotine elutes before its (R)-enantiomer (Armstrong et al. 1998). Peak 1 of 5-phe-nic had a K i value of 0.17 M as compared to the K i value of 3.6 M for peak 2. The difference in affinities of the enantiomers was significantly different for rat 42 (P = 0.0104). The enantiomeric selectivity ratio (ESR), calculated by dividing the K i of peak 2 by the K i of peak 1, gives a value of 21. The K i value for peak 1 of 1-N-ethyl-nor was 0.24 M and 2.3 M for peak 2 was also significantly different (P =0.0015). The higher affinity of peak 1 for rat 42 of both analogs was anticipated based upon previous findings (Copeland et al. 1991; Zhang and Nordberg 1993).

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57 A B C Figure 4-1. HPLC chiral separation of 1-N-ethyl-(S,R)-nornicotine. A) The chromatogram with retention times for the separation of presumed 1-N-ethyl-(S)-nor (peak 1) and presumed 1-N-ethyl(R)-nor (peak 2). B) The spectra of peaks one and two indicate an absorbance at 260 nm.

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58 Table 4-1. Inhibition of 3 H-cytisine binding to rat brain or to human 42 expressing tsA201 cell membranes by nicotine enantiomers. Compound Name or Abbreviation K i for 42 (M) (S)-Nicotine 0.0089 0.0039 (human) (R)-Nicotine 0.011 0.0013 (human) (S)-Nornicotine 0.48 0.26 (human) (R)-Nornicotine 0.042 0.025 (human) 1-N-Ethyl-Nor (peak 1) 0.24 0.060 (rat) 1-N-Ethyl-Nor (peak 2) 2.3 0.26 (rat) 5-Phe-Nic (peak 1) 0.17 0.023 (rat) 5-Phe-Nic (peak 2) 3.6 0.76 (rat) K i values were calculated according to the Cheng Prusoff equation. The concentration of radioligand was 1 nM. Each value represents the mean SEM of three separate experiments. Concentrations for each experiment were done in triplicate. The K d values for 3 H-cytisine binding to rat brain membranes was 0.92 0.1 nM and 0.48 nM 0.2 for tsA201 membranes. The binding of the enantiomers of either nicotine or nornicotine to human 42 nAChRs interestingly did not differ significantly as measured by 3 H-cytisine displacement. The ESR for the two nicotine enantiomers on human 42 was 1.2. Since previously published data indicates that a stereospecificity exists for nicotine and the high-affinity nAChR, this result was unexpected. The K i value for (R)-nornicotine (0.042 M) was also not significantly different (P value = 0.1619) from the (S)-enantiomer (0.48 M) for human 42. The large difference in standard error may account for the lack of significance; further replicates will decrease the standard error and may make apparent a significant difference. The nornicotine data agrees with previous binding data obtained with rat brain in which there was no significant difference between (S)and (R)-nornicotine (Copeland et al. 1991; Zhang and Nordberg, 1993).

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59 The binding results for the enantiomers of nicotine and nornicotine are made more complex when compared to the activity of these compounds on human 42 receptors as measured by changes in membrane potential (Figure 4-2). Both (S)and (R)-nicotine are full agonists as compared to the maximum ACh response of human 42 nAChRs. Although there was no significant difference in the measured K i values for the nicotine enantiomers as measured by binding experiments, there was however a difference in potency, although it was not statistically significant (P = 0.16). The EC 50 value of (S)-nicotine (2.3 1.2 M) was 7.0 fold lower than that of (R)-nicotine (16 8.8 M). The calculated EC 50 for (S)-nornicotine was 8.5 1.8 M as compared to an EC 50 of 39 5.4 M for (R)-nornicotine. The difference between the EC 50 values of the nornicotine enantiomers was significant (P = 0.0016). Both forms of nornicotine appear to be functioning as partial agonists as compared to nicotine at the concentrations tested. (R)-Nornicotine appears to be more efficacious than (S)-nornicotine. Further replicates at higher concentrations would be needed to determine if the maximum effects and EC 50 values obtained by fitting the concentration response curve are consistent.

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60 -9 -8 -7 -6 -5 -4 -3 0 20 40 60 80 100(S)-Nicotine (R)-Nicotine (S)-Nornicotine (R)-Nornicotine Log [Compound], MPercent Activation(normalized to max AChresponse) Figure 4-2. Concentration response curves for the enantiomers of nicotine and nornicotine for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential. Responses were normalized to the maximum ACh response (500 M). Each point represents the average SEM of four separate wells. The (S)-nornicotine data is also in Figure 3-3.

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CHAPTER 5 RESULTS Erythrina Alkaloids Plant alkaloids extracted from the genus Erythrina share a common heterocyclic ring system (Figure 5-1). Sheridan et al. (1986) determined that the essential aromatic groups of dihydro--erythroidine (DHE) superimpose with those of nicotine indicating that they may share a similar conformation. The affinities and inhibition of the Erythrina alkaloids for nAChRs have been less well characterized in vitro than the nicotine analogs. The only published literature on these alkaloids and their interaction with nicotinic receptors pertains primarily to DHE (Williams and Robinson, 1984; Anderson and Arneric, 1994; Harvey and Luetje, 1996; Harvey et al. 1996). Erysodine is the only other alkaloid that has been studied for its interaction with the 42 nAChR (Decker et al. 1995). Aromatic erysodine was determined to have a higher apparent affinity for the receptor (5 nM) than DHE (35 nM) in rat brain membranes as measured by 3 H-cytisine displacement (Decker et al. 1995). Erysodine also had a selectivity 1800-fold greater for rat 42 than rat 7 whereas DHE had a selectivity only 114-fold greater for rat 42 than rat 7 (Decker et al. 1995). Since the early 1940s it has been known that curare-like effects result upon administration of Erythrina alkaloids in vivo (Lehman, 1936; Folkers and Major, 1937). The alkaloids were apparently producing their primary effects through blockade of neuromuscular transmission, but ganglionic blocking effects were also observed. 61

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62 17 11 16 10 14 8 4 7 1 2 Figure 5-1. The common heterocyclic structure of Erythrina alkaloids. The rings are designated A through D and atoms numbered accordingly. In 1984 Williams and Robinson first studied the effects of DHE on nAChRs. Their results indicated that it was binding with high affinity (2 nM) to a neuronal nicotinic receptor in rat brain and that its distribution of binding was similar to the binding of 3 H-nicotine. Since then DHE has been referred to and used as a competitive antagonist for selective inhibition of 2-containing nAChRs. Decker et al. (1995) determined that DHE was inhibiting (S)-nicotine-evoked 3 H-dopamine release from slices of rat striatum with an IC 50 of 58 nM. DHE also interacts with other 2-containing receptors. Electrophysiological measurements on oocytes expressing human 42 indicate that DHE is 14.7-fold less potent of an inhibitor for the 32-subtype (Chavez-Noriega et al. 1997). In order to further understand the interaction of antagonists with 42 nAChRs our goal was to create a structure activity relationship for Erythrina alkaloids and the 42 receptor with the ultimate goal of designing a selective probe for use in vivo and in vitro to study the resting state of the receptor as well as a possible smoking cessation drug. To accomplish this goal several natural and semi-synthetic alkaloids were studied for their ability to displace 125 I--btx and 3 H-cytisine from rat brain membranes and 3 H-cytisine

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63 from tsA201 cells expressing human 42 nAChRs. The inhibitory properties of these alkaloids were then characterized using tsA201 cells expressing human 42 nAChR and a membrane potential dye. Our hypothesis was that alterations to the D-ring of the Erythrina alkaloids would have a large affect on affinity and potency for 42 and that the nitrogen is critical for binding. -Erythroidines The -erythroidine compounds contain an unconjugated lactone group in their D-ring and thus lack D-ring aromaticity. -Erythroidine contains two conjugated double bonds, one in the A ring and another in the B ring. Its K i value as measured for rat 42 was 1.1 M and its selectivity ratio (SR = K i 7/K i 42) was 45 (Table 5-1). The two double bonds in -E have been reduced to one in DHE so the resulting double bond is now between the 1and 6-positions, which still maintains approximate co-planarity of the A and B rings. Interestingly, this reduction resulted in an increased affinity for rat 42. The affinity of DHE for rat 42 (K i = 0.14 M) was eight times greater than that of -E. Reducing the remaining double bond of DHE produces THE. The absence of double bonds in the A and B rings greatly reduced the affinity of this compound for rat 42. THE had an affinity (K i = 7.4 M) that was about 7-fold less than that of -E. Neither DHE nor THE displaced -btx at concentrations up to 50 M (SR = 377 and 6.76 respectively), indicating that they have a selectivity for rat 42 over rat 7. The affinity of -erythroidine for human 42 was not determined. The decrease in affinity of THE as compared to DHE was also observed in binding to human 42. There was

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64 a 4.4-fold difference in the affinity of DHE for rat versus human 42. THE had a 2.7-fold difference in affinity between rat and human 42. Table 5-1. Structures, nomenclature and binding results for -Erythroidine, dihydro--erythroidine and tetrahydro--erythroidine. >207.4 4.2>50Tetrahydro--Erythroidine0.62 0.170.14 0.018>50Dihydro--ErythroidineNot Determined1.1 0.84>50-ErythroidineHuman 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name >207.4 4.2>50Tetrahydro--Erythroidine0.62 0.170.14 0.018>50Dihydro--ErythroidineNot Determined1.1 0.84>50-ErythroidineHuman 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name O N O MeO O O N MeO O N O MeOH SSTHE*SSDHE*NP-E* K i values were calculated according to the Cheng Prusoff equation. The concentration of each radioligand was 1 nM. Each value represents the mean SEM of three separate experiments unless otherwise indicated. Concentrations for each experiment were done in triplicate or quadruplicate. The K d values for 125 I--btx and 3 H-cytisine binding to rat brain membranes were 0.32 nM 0.04 and 0.92 0.1 nM respectively. The K d value for 3 H-cytisine binding to tsA201 cell membranes was 0.48 nM 0.2. NP indicates a natural product. SS indicates a semi-synthetic compound. Abbreviations for the analogs are indicated by an asterisk. 3-Des-Met-E differs from -E in that it contains two double bonds in the A ring and one in the B ring resulting in an even more extensive conjugation between these two rings (but this is accompanied by movement of the C and D rings with respect to the A and B rings). As may be predicted from the results of -E and DHE, the addition of the

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65 third double bond was accompanied by a decrease in affinity for rat 42 (Table 5-2). This reduction in affinity also may be due to the loss of the methoxy group at the 3-position. 3-Des-Met-E did not displace either 3 H-cytisine or 125 I--btx at concentrations up to 20 M. Table 5-2. Structures, nomenclature and binding results for 3-Desmethoxy--Erythroidine, N-Methyl--erythroidine and -erythroidinediol. 0.24 0.0150.31 0.12>50-ErythroidinediolNot Determined>20>20N-Methyl--ErythroidineNot Determined>20>203-Desmethoxy--ErythroidineHuman 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name 0.24 0.0150.31 0.12>50-ErythroidinediolNot Determined>20>20N-Methyl--ErythroidineNot Determined>20>203-Desmethoxy--ErythroidineHuman 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name O N O O N O MeOCH3 + N MeO OH OH SSMe-E*SSE-Diol*SS3-Des-Met-E* Parameters were the same as in Table 5-1. Abbreviations for the analogs are indicated by an asterisk. Me-E has a quaternary ammonium in place of the tertiary amine in -E. The presence of this positively charged nitrogen decreased the affinity of Me-E (K i > 20 M) for rat 42 at least 20-fold as compared to -E. Me-E had no interaction with 7 at concentrations up to 20 M. The final -erythroidine studied, E-Diol, contains an

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66 open D-ring. It may be expected that opening the D-ring would decrease the affinity of E-Diol as compared to -E. The results however are just the opposite. The K i value of E-Diol for rat 42 was 0.31 M, 3.5-fold greater than that of -E. E-Diol had a minimum selectivity ratio (SR) of 161. To ensure that DHE was not producing any activation of the human 42 receptors expressed by tsA201 cells as measured using the membrane potential fluorescent dye, it was administered in the absence of agonist and as expected there was no measurable activation (Figure 5-2). The ability of the other Erythrina alkaloids to produce activation was also measured and the results indicated that all the alkaloids were functioning only as antagonists. -9 -8 -7 -6 -5 -4 -3 0 20 40 60 80 100DHBE Log [DHBE], MPercent Activation(normalized to maximumACh response) Figure 5-2. Concentration response curve of DHE for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential. The response was normalized to the maximum ACh response. The result represents an average of four wells. To verify that DHE was acting as a competitive antagonist in our system, measurements were made to determine EC 50 values for ACh in the presence and absence of 1 M DHE. A competitive antagonist would shift the concentration response curve

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67 to the right and as the results indicate DHE is acting as a competitive antagonist (Figure 5-3). The EC 50 of ACh in the presence of 1 M DHE was 298 29 M versus the EC 50 of ACh alone, 2.33 0.94 M. -10 -9 -8 -7 -6 -5 -4 -3 -2 0 20 40 60 80 100 120ACh + 1uM DHE ACh Log [ACh], MPercent Activation(normalized to maximumACh response) Figure 5-3. Concentration response curve of ACh in the presence and absence of 1 M DHE for human 42 receptors expressed in tsA201 cells as measured by changes in membrane potential. The responses were normalized to the maximum ACh response. The result represents an average of 3 experiments for ACh and 8 experiments for ACh + 1 M DHE. Figure 5-4 represents the signals of simultaneous applications of 5 M ACh with increasing concentrations of DHE as recorded using membrane potential fluorescent dye and human 42 expressing tsA201 cells. The signals of the 5 M ACh response decrease with increasing concentrations of DHE. The 50 M concentration of DHE produces a response that is identical to that of the blank (which did not receive 5 M ACh), indicating that at 50 M DHE the ACh response was completely inhibited.

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68 40 m M KCl DHE and 5 M ACh Figure 5-4. Signal acquired for 42 nAChRs upon simultaneous application of DHE and ACh. During the first 17 seconds baseline fluorescence was measured for each well. At 18 seconds well H8 () received 0.0005 M DHE + 5 M ACh, well F12 () received 0.05 M DHE + 5 M ACh, well G12 () received 50 M DHE + 5 M ACh and well H7 () received only 20 mM HEPES/HBSS. All wells received a final concentration of 40 mM KCl at 160 seconds All of the -erythroidine alkaloids except for 3-Des-Met-E and Me-E were able to fully inhibit cell response to 5 M ACh (Table 5-3). Me-E had no interaction with rat 42 receptors at concentrations up to 20 M as measured by 3 H-cytisine displacement and it also did not inhibit the human 42 ACh response at concentrations up to 50 M. 3-Des-Met-E also had no measured binding to rat 42 at concentrations up to 20 M but it did inhibit the human 42 ACh response by 50% at the highest concentration tested (50 M). The potency of DHE to inhibit the human 42 ACh response is 5.5-fold greater than that for -E as indicated by their IC 50 values of 0.067 M and 0.37 M respectively. THE, with all double bonds in the A and B rings reduced, is the least potent of the -erythroidines on human 42 with a measured IC 50

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69 of 2.5 M. After DHE, E-Diol had the second highest affinity (for both rat and human 42) of the -erythroidine compounds. E-Diol was also the second most potent of the erythroidines on human 42 with an IC 50 value of 0.065 M. Table 5-3. Inhibition of human 42 receptor ACh responses by natural product and semi-synthetic -erythroidines. Compound Name or Abbreviation IC 50 (M)/Percent Inhibition for Human 42 Erythroidine compounds -E 0.37 0.055 DHE 0.067 0.0035 THE 2.5 0.20 3-Des-Met-E 50% Inhibition at 50 M Me-E No inhibition up to 50 M E-Diol 0.065 0.012 The compounds and ACh (5 M) were administered simultaneously. Each value represents the mean SEM of four separate wells. To determine the importance of the C-ring for binding to rat 42 a homoerythrinan compound from another plant genus, Phelline, was studied for its ability to displace 3 H-cytisine. This compound, named O-methylisophellibiline, is similar in structure to DHE except that it has a seven membered C-ring, which increases the size of the molecule and alters the 3-dimensional position of the lactone carbonyl oxygen (Figure 5-5). Enlarging the C-ring resulted in a decreased affinity for rat 42. There was over 50% displacement of 3 H-cytisine from rat 42.

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70 O O N MeO Figure 5-5. Structure of a Phelline alkaloid, O-methylisophellibiline. This alkaloid is similar in structure to DHE except for its seven-membered C-ring. Aromatic Alkaloids Erythraline (ELA), an aromatic alkaloid, lacks the D-ring lactone moiety of the erythroidines and instead has a benzenoid ring (Table 5-4). ELA has an additional (1,3-dioxolane) ring fused to the D-ring. The measured affinity (K i value = 0.056 M) of ELA is greater than that of DHE (K i value = 0.14 M) for rat 42. ELA does have an affinity, although low, for the rat 7 receptor (SR = 46). The aromatic alkaloids, erysovine and erysodine (ERV and ERD), are isomers (Table 5-4). ERV contains a hydroxy group at position 15 and a methoxy group at position 16 on its D-ring. In ERD the hydroxy and methoxy attached to the D-ring are reversed. The reversal of those two substituents has a large influence upon the affinity for rat 42. ERV has a high affinity for rat 42 (12 nM), which is 18 times greater than the affinity of DHE for rat 42. ERD, with the hydroxy and methoxy groups reversed, had a much lower affinity than ERV, 23-fold less, for rat 42 and 8.9-fold less for human 42. ERV also exhibited an increased affinity for rat 7, with a selectivity ratio of 50.8. ERD had no measurable interaction with rat 7 at up to 20 M.

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71 Table 5-4. Structures, nomenclature and binding results for the aromatic alkaloids erysovine, erysodine and erythraline. Human 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name0.081 0.0180.056 0.00352.6 1.4Erythraline0.24 0.0740.28 0.087>20Erysodine0.027 0.00320.012 0.00320.61 0.13Erysovine Human 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name0.081 0.0180.056 0.00352.6 1.4Erythraline0.24 0.0740.28 0.087>20Erysodine0.027 0.00320.012 0.00320.61 0.13Erysovine NPERD*NPELA*NPERV* N MeO OH MeO N MeO OH MeO N MeO OO Parameters were the same as in Table 5-1. Abbreviations for the analogs are indicated by an asterisk. ERT has a methoxy group at both position 15 and 16 on the D-ring (Table 5-5). The presence of the two methoxy groups resulted in an affinity of 0.33 M for rat 42, which was similar to that of ERD for rat 42. It did however have a minimum of 26.6-fold greater affinity for rat 7 as compared to ERD. Gluco-ERD is ERD with a glucose molecule attached. Gluco-ERD is a natural product and like other phenolic D-ring Erythrina compounds is attached to large sugar molecules until they are liberated by application of an acid. Even with this large substituent Gluco-ERD had an affinity (K i value = 0.032 M) 4.4-fold greater than that of DHE for rat 42. It had no measurable affinity for rat 7 at concentrations up to 20 M (SR = a minimum of 625).

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72 Table 5-5. Structures, nomenclature and binding results for the aromatic alkaloids erysotrine, and glucoerysodine. Human 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name2.2 0.740.032 0.0085>20Glucoerysodine0.86 0.220.33 0.0700.75 0.20Erysotrine Human 3H-Cytisine(42)Ki (M)3H-Cytisine (42)Rat125I--Btx(7)Compound Name2.2 0.740.032 0.0085>20Glucoerysodine0.86 0.220.33 0.0700.75 0.20Erysotrine N MeO MeOMeO N MeO OMeO O OH OH OHCH2 OH NPERT*NP Gluco-ERD** Parameters were the same as in Table 5-1. Abbreviations for the analogs are indicated by an asterisk. The diamond indicates a significant difference between the K i values for rat and human with 3 H-cytisine. Interestingly, the only alkaloid that had a significantly different K i value (P< 0.043) between rat (K i value = 0.032 M) and human (K i value = 2.2 M) 42 nAChR was Gluco-ERD, which is a natural product. The other aromatic alkaloids had similar rat and human 42 affinities.

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73 Table 5-6. Inhibition of human 42 receptor ACh responses by natural product aromatic Erythrina alkaloids. Compound Name or Abbreviation IC 50 (M) for Human 42 Aromatic compounds ERV 0.054 0.00049 ERD 0.092 0.0035 ELA 0.13 0.0049 ERT 1.6 0.52 Gluco-ERD 0.43 0.079 The compounds and ACh (5 M) were administered simultaneously. Each value represents the mean SEM of four separate wells. Several of the aromatic compounds were as potent as DHE in inhibiting human 42 response to ACh. ERV was the most potent of all the alkaloids, it had an IC 50 value of 0.054 M for human 42 (Table 5-6). As indicated earlier, ERV also had the highest affinity for both the rat and human 42-receptor. Although ERD had a 8.9-fold decrease in affinity (for human 42) as compared to ERV, it was the second most potent aromatic compound with an IC 50 of 0.092 M on human 42. ELA was the third most potent of the aromatic compounds (on human 42) with and IC 50 value of 0.13 M for human 42. ERT was the least potent (IC 50 = 1.6 M) of the aromatic alkaloids on human 42. The final aromatic compound, Gluco-ERD was the second to last potent with an IC 50 value of 0.43 M.

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CHAPTER 6 DISCUSSION Nicotine and dihydro--erythroidine (DHE) have both been used extensively as tools for studying nAChRs. The reason being, that nicotine binds to and activates various subtypes of nAChRs with its highest affinity and potency for 42 nAChRs, while DHE is one of the only antagonists with a relative selectivity for 2-containing receptors. Although their functional effects for the receptor differ, both are competing for the acetylcholine (ACh) binding site. The role of the 42 receptor in nicotine addiction as well as its involvement in several neurological dysfunctions warrants further investigation of the interactions of these compounds with 2-subunit containing receptors. There are various methods that have been employed to study the interaction of the 42 nAChR with specific compounds in order to elucidate properties of the binding site. The 42 receptor has undergone mutagenesis and chimeric studies in order to determine residues important in allowing for activation or inhibition. Another approach to understanding ligand and receptor interaction is to study the ligand directly. Through alteration of the ligands structure the consequential effects upon binding and function tell a story about requirements of a molecule for interacting with 42 (structure activity relationship approach). The goal of this dissertation was to determine structure activity relationships to understand what aspects of their structures allow for nicotine and DHE to bind and either activate or inhibit the 42 receptor. Determination of ligand 74

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75 structural properties important for receptor interaction will provide a basis for further manipulation of these molecules to develop higher affinity selective probes for in vitro and in vivo studies of the 42 nAChR as well as possible drugs to treat nicotine addiction. Substituents at Four Different Positions on Nicotine Decrease Affinity for the 42 nAChR and Confer Partial Agonist and Antagonist Properties This structure activity relationship study of nicotine analogs built on previously published data of several substituted nicotine analogs as well as explored several novel nicotine compounds. The previous studies (Damaj et al. 1996; Dukat et al. 1996; Glassco et al. 1994; Kim et al. 1996; Lin et al. 1994) however only examined the ability of the nicotine analogs to bind to the 42 nAChR as measured with either 3 H-cytisine or 3 H-nicotine. The studies conducted in this dissertation further explore the ability of nicotine analogs to bind to the other high affinity neuronal nAChR, 7, for the first time. We also investigate the effects of these compounds on the human 42 receptor expressed in a mammalian cell line. Substitutions were made at the 5-, 1-, and 3-pyrrolidine positions as well as at the 5-pyridyl position on nicotine. The substituents at the 5-pyridyl position, either a phenyl or pyridyl ring, were large. They decreased the affinity of these compounds for rat and human 42 receptors as compared to that of (S)-nicotine. The presence of the tertiary nitrogen in the 5-pyridyl ring did not increase the affinity of this compound for either the rat or human 42 receptor as compared to the nitrogen lacking phenyl ring. It may have been expected that the tertiary nitrogen would provide an additional group for possible hydrogen bonding or other additional electrostatic interactions with the binding site and thus increase the affinity, but this was not the case. The K i values for the

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76 interactions of these two compounds with the rat 7 receptor were greater than 20 M. This was an important finding because the selectivity ratios (K i 7/ K i 42) for both compounds were greater than that of (S)-nicotine. Thus, the large substituents added at the 5-position resulted in an enhanced selectivity for the high affinity receptor (as seen for the rat 7 and 42 receptors). The 5-pyridyl substituted analogs were the only nicotine analogs with significantly different K i values for rat and human 42 receptors (P = 0.0326 for 5-phe-nic and P = 0.0334 for 5-pyr-nic). The compound DMXBA displays a variation in potency between human and animal (rat) forms of a nicotinic receptor, specifically 7 (Meyer et al. 1998; Papke and Porter-Papke, 2002). Stokes et al. (2004) determined by mutational analysis that this variation in potency between human and rat was due to differences in residues within the C loop (ser184 in human to asn184 in rat; arg186 in human to lys186 in rat) and the F loop (gly167 in human to ser167 in rat) with some influence from the E loop. Although there are two residue differences between the rat and human 42 receptors, only one of them is likely to impact binding. The 4 subunit is the primary, or principal subunit, thus loops A, B and C from this subunit interact with loops D, E and F from the 2, or complimentary, subunit. The only residue within the binding loops that might be anticipated to differently influence ligand interaction between rat and human 42 is a glutamic acid in the human receptor in place of an aspartic acid in the rat. This residue difference seems small; there is an additional methyl group on glutamic acid, but both residues are polar and charged. There is also the possibility that supporting residues outside of the six binding loops may be influencing (restricting) the ability of the 5-pyridyl analogs to enter the binding site of the human 42 receptor as compared to

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77 rat. Another possible explanation of the difference between rat and human K i values is that in the CNS there exists a heterogenous population of 42 receptors. It has been shown that 5-subunits are localized with 42 receptors in the chick brain (Conroy and Berg, 1998) as well as in the cortex of rat (Mao et al. 2005). Therefore, if 3 H-cytisine is labeling both populations of receptors it may explain the differential interaction of several of the nicotine analogs (as well as Erythrina alkaloids) between rat brain 42 and human 42 receptors expressed in the tsA201 cell line. Both 5-position substituents prevented the ligands from producing any activation of the human 42 receptor. However, these compounds displayed antagonistic properties with similar potencies to each other as measured using a membrane potential fluorescent dye. Dukat et al. (2002) also observed the influence of smaller substituents at the 5-pyridyl position on the functional properties of rat 42 receptors and determined using electrophysiological recordings from Xenopus oocytes that modifications resulted in partial agonist and antagonist properties. Also, Carroll et al. (2001) found that a phenyl group added at the 3-position on epibatidine produced similar effects (antagonism). The method we used to study receptor activation or inhibition by these nicotine analogs involves the measurement of changes in membrane potential using a fluorescent dye. The dye fluoresces upon membrane depolarization resultant from receptor activation. Fitch et al. (2003) published data on the effects of nicotinic receptors with membrane potential and calcium dye and measurements from various cell lines expressing nAChRs. They measured an EC 50 for nicotine of 0.86 M on the human 42 nAChR expressing cell line they utilized (K-177 cells which are a HEK-293

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78 derived cell line). The EC 50 values measured in their experiments for agonists were 2 to 3-fold less than those reported in the literature for radiolabeled rubidium efflux assays whereas the IC 50 values for antagonists were higher (5 to 20-fold or greater) for 34 expressing cells. They note that these estimates may possibly be affected by spare receptors, only a sub-maximal occupation of receptors may be required in order to depolarize the membrane resulting in an apparent increase in potency. The cells used for this study are tsA201 cells, a human embryonic kidney cell subclone (HEK-293) obtained from Jon Lindstrom (Kuryatov et al. 2005). Since HEK-293 cells endogenously express M1 muscarinic acetylcholine receptors (Mundell and Benovic, 2000), 1 mM atropine (a muscarinic antagonist) was used in the Flexstation assays. Nelson et al. (2003) determined that a population of the cells had nicotine and ACh potencies similar to oocytes expressing human 42 receptors. They also determined that the partial agonist cytisine had an efficacy too low to correctly measure potency. Overall, Nelson et al. (2003) concluded that the receptors expressed in the tsA201 cells exist in two stoichiometries: the majority exist with three 4-and two 2-subunits, while the minority population possesses two 4-subunits and three 2-subunits. The substituents at the 5-pyrrolidine position were methyl groups in either the trans or cis configuration. Kim et al. (1996) had determined that the addition of the methyl at the cis-position resulted in a 35-fold decrease in affinity for rat 42 as compared to that of the methyl group in the trans configuration. The results in this dissertation indicated at least a 90-fold difference in affinity between the trans and cis conformations for both the rat and human 42 with the 5-cis-met-nic having no interaction with the 42 (human and rat) receptor at concentrations up to 20 M.

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79 5-Trans-met-nic had a decreased selectivity ratio of 14 as compared to the selectivity ratio of nicotine (SR = 83). 5-Cis-met-nic also had no interaction with the 7 receptor at concentrations up to 20 M. The functional measurements of these two analogs corroborate the binding results for human 42 in indicating preferential interaction of the trans analog with the receptor and the relative lack of interaction between the receptor and the cis analog. Trans-5-met-nic produced 50% inhibition (at 50 M) of the human 42 ACh response. The 1-N-pyrrolidine analogs provided information on the influence of substituent size on binding and function. Increasing the size of substituents on the nitrogen of the pyrrolidine ring resulted in a decreased affinity for the rat 42 receptor. 1-N-Ethyl-nor had a measured affinity (K i = 0.14 M) comparable to (S)-nornicotine (K i = 0.12 M) for rat 42. It also had a selectivity ratio of 18, which is less than that of (S)-nicotine (SR = 83). There was an inverse relationship between the size of the substituent on the pyrrolidine nitrogen and affinity. As the size of the substituent increased from an ethyl to propyl and cyclopropyl, the affinity for 42 decreased. Neither 1-N-propyl-nor or 1-N-cyclopropyl-met-nor had measurable binding to rat or human 42 or rat 7 receptors at concentrations up to 20 M. Glassco et al. (1994) also measured this trend of decreasing affinity for rat 42 with increasing 1-N-substituent size. They found a 20-fold decrease in affinity (as compared to (S)-nicotine) when the size of the substituent at the 1-N-position was increased from a methyl to an ethyl, a 369-fold decrease (as compared to (S)-nicotine) when substituting a propyl-group and greater than 7,000-fold decrease when substituting a cyclopropyl-group. Ferretti et al. (2003) determined that a

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80 methyl group was optimal for binding to rat 42 such that nornicotine or the addition of a methyl or benzyl group to the pyrrolidine nitrogen resulted in reduced affinity. Interestingly, although the 1-N-pyrrolidine substituted analogs displayed decreased affinity for the rat 42 receptor as compared to (S)-nicotine, they were found (in separate experiments) to both activate and inhibit the human 42 receptor. Analogs containing the ethyl or propyl substituent produced about a 40% activation of the receptor as compared to the ACh maximum response for human 42. However, it is possible that a higher concentration would produce greater activation. The cyclopropyl-methyl substituent produced about a 75% activation of the receptor as compared to ACh. Upon simultaneous application with ACh, each of the three of the analogs produced an inhibition ranging from 35-80% (1-N-ethyl-nor = 35%; 1-N-propyl-nor = 45%; 1-N-ethyl-nor = 80%). Channel block at high concentrations of these analogs might explain the resultant inhibition, but it does not account for the ability of these compounds to activate the receptor. Also, the greater percent inhibition of human 42 as compared to percent activation by the 1-N-cyclopropyl-nor indicates the involvement of spare receptors. The possible involvement of spare receptors would increase the apparent potency of these compounds resulting in a weak partial agonist appearing more potent and efficacious than if its effects were measured under voltage-clamp conditions. Based upon their sub-maximal activation at high concentrations as well as the predicted influence of spare receptors, these nicotine analogs are most likely functioning as partial agonists. The results from the final group of nicotine compounds, the 3-pyrrolidine substituted analogs, paralleled those of the 1-N-pyrrolidine substituted analogs. With

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81 increasing substituent size there was a decrease in affinity for both the rat 42 and rat 7 receptors. Adding a small substituent (methyl) at the 3-position resulted in an affinity for the rat 42 receptor that was 40-fold less than that of (S)-nicotine for the receptor. This analog also interacted with rat 7 with a K i value of 4.2 M (SR for 3-met-nic = 11 for rat 42 and 6.7 for human 42). The only other 3-substitued analog that had measurable binding interaction with rat 42 was DMXBN, which had a K i value of 2.8 M. The other two 3-position analogs, like DMXBN, had large benzene ring-containing substituents. The structures of these two compounds (DMXBN and DMXBM) resemble that of DMXBA except that the benzyl or benzylidene moieties are attached to nornicotine and myosmine, respectively, instead of anabaseine. Whereas DMXBA has an affinity of 0.13 M for rat 7 and 0.25 M for rat 42 (Kem et al. 2004a), neither DMXBN nor DMXBM had measurable K i values for rat 7. The low activity of DMXBM was probably due to its small degree of ionization at physiological pH. Myosmine has a pKa of 5.5, which is well below the pKa (8.05) of nicotine (pKa of nornicotine is 9.12) (Fujita et al. 1971); Glennon and Dukat, 2000). DMXBM is predicted to have a pKa no higher than 6.0 (Kem, personal communication), so it would be largely unionized at physiological pH (7.4). The functional properties of the 3-substituted analogs upon human 42 measured with membrane potential fluorescent dye varied with the compound. Three of the 3-pyrrolidine substituted analogs produced activation of human 42 receptors; DMXBN produced 80% activation, DMXBM produced 20% activation and 3-met-nic produced 40% activation. The only compound that produced an inhibition of the human 42 ACh response was 3-met-nic (20% inhibition).

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82 Overall, my results for the nicotine analogs indicate that substituents at any of the four positions examined in this dissertation result in a decreased affinity for the human and rat 42 nAChR as compared to (S)-nicotine. However, the selectivity ratios for the 5-pyridyl substituted analogs were greater than that of (S)-nicotine. Therefore, substituents at this position confer a greater selectivity for rat 42 and merit further investigation for possible use as smoking cessation drug. The substituted nicotine analogs in this study also greatly influence the function of these compounds by converting those that interact with the receptor into partial agonists and antagonists. Rowland et al. (2003) also found that 5-trans-met-nic produced a dose-dependent inhibition of nicotine self-administration in rats. Previous studies have addressed the influence of pKa for both the pyridyl and pyrrolidine ring nitrogens. Their results indicate that differences in the basicity of these nitrogens could not alone account for variation in affinities of nicotine analogs for the 42 receptor (Dukat et al. 1998; Copeland et al. 1991). To further understand the interaction of nicotine with the ACh binding site, Celie et al. (2004) crystallized nicotine bound to the AChBP. Their results indicate that nicotine is primarily in contact with the principal side of the binding site (loops A, B, and C). Also, both nitrogens form hydrogen bonds with residues within these loops. Specifically, the pyrrolidine nitrogen forms a hydrogen bond with tryptophan 143 within the B-loop and the pyridyl nitrogen forms its hydrogen bond with both methionine 114 and leucine 102 within the E-loop. Their results also indicate that the primary differences in the binding site of the AChBP versus the 42 receptor are three amino acids. The three residue differences in 42 may provide for better contact with nicotine. The nicotine substitutions reported in this

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83 dissertation may be preventing the proper orientation of the analogs within the binding site for establishing the hydrogen bonds necessary for the tight binding. The 5-pyridyl substituted nicotine analogs likely fit the binding site because the large phenyl or pyridyl-ring does not interfere with the primary interactions between the analog and the receptor. This may explain the ability of these analogs to retain an affinity for rat and human 42. Separated Nicotine Analog Enantiomers Display a Difference in Affinity for 42 Unlike Nicotine Interestingly we found that unlike previous studies, there was no significant difference between (S)and (R)-nicotine in binding to 42 (Copeland et al. 1991; Zhang and Nordberg, 1993). We measured the affinity for human 42 whereas the previous publications were performed using rat brain. It is possible that in a heterogenous population of receptors, as in rat brain, these previous studies were not measuring 3 H-nicotine displacement from only 42 receptors but that their results were influenced by other subtypes of nAChRs. Our results for the enantiomers of nornicotine agree with those published by Copeland et al. (1991) in that the binding affinities for each enantiomer were very similar; their results were done with rat brain tissue whereas our results were measured for the human 42-subtype. The higher affinity for the enantiomers of nicotine over those of nornicotine is most likely not due to the differences in pKa of each molecule. Copeland et al. (1991) discuss that the difference in the basicity (pKa) of the nornicotine pyrrolidine nitrogen relative to that of nicotine does not produce a difference significant enough to result in the differing affinities of the two compounds. Nicotine is however more lipophilic than nornicotine and the intimate

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84 interaction of nicotines methyl group may enhance the cationinteraction with the binding site and account for nicotines higher affinity. The results of the nornicotine enantiomers on the activation of human 42 receptors expressed in tsA201 cells indicated that they are less efficacious than the nicotine enantiomers. The EC 50 values for the nornicotine enantiomers indicated that (S)-nornicotine is more potent than its (R)-enantiomer. This contradicts previously published data acquired from dopamine release assays on the nucleus accumbens indicating that (R)-nornicotine is more potent than its enantiomer (Green et al. 2001). The results of the separated nicotine analogs, 1-N-ethyl-nor and 5-phe-nic are in better agreement with the published literature (Copeland et al. 1991; Zhang and Nordberg, 1993) on nicotine for the increased affinity of the (S)-enantiomer (presumably peaks 1 for both analogs). It is important to note however, that both nicotine analogs were studied for their affinities to rat brain membranes like the previously reported findings for nicotine and nornicotine. Our results for the separated enantiomers do indicate that the selectivity of the (S)-enantiomer (presumed peak 1) of 5-phe-nic for rat 42 over 7 would be even greater than that of racemic 5-phe-nic (SR = 303). Substituents on the D-ring of the Erythrina Alkaloids Allow for High Affinity Binding to the 42 nAChR and Inhibition of the 42 Acetylcholine Response In the literature that has been published on DHE, it is often referred to as a competitive antagonist selective for the 42 receptor (Williams and Robinson, 1984; Anderson and Arneric, 1994; Harvey and Luetje, 1996; Harvey et al. 1996). The characterization of DHE along with several other natural product and semi-synthetic Erythrina alkaloids as conducted in these studies have provided a structure activity relationship between the 42 receptor and these competitive antagonists. Only one

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85 other Erythrina alkaloid has been studied for its effects upon nicotinic receptors. Decker et al. (1995) published that the natural product erysodine has a seven-fold higher affinity for rat 42 than DHE. Although our results vary from Decker et al. (1995) in that we found DHE to have a higher affinity (as compared to erysodine) for rat 42, it was determined that several structural trends exist among the alkaloids, which result in an increased affinity for the rat and human 42 receptors. The Erythrina alkaloids were divided into two groups containing different D-rings: the lactone-bearing erythroidines and the aromatic D-ring compounds. The importance of the double bonds between the A and B rings of the erythroidine compounds was determined from the binding data. By partial reduction, the two double bonds in the A and B rings of -E are converted to the one double bond of DHE; there was about an eight-fold increase in affinity for the rat 42 receptor. Reduction of the remaining double bond between the A and B ring results in the structure of THE. This compound had an affinity for the rat 42 receptor that was less than that of -E. These results indicate that the optimal number of bonds between the A and B ring is one. Neither -E, nor DHE nor THE had any binding interaction with rat 7 receptors at concentrations up to 50 M with DHE having the highest selectivity ratio (357) of these three -erythroidine alkaloids. The possible influence of three double bonds among the A and B rings also was studied using the 3-Des-Met-E compound. Consistent with the idea that a single double bond among these two rings is optimal for binding, the three double bonds in 3-Des-Met-E (along with the lack of the 3-methoxy) abolished measurable binding to the rat 42 receptor at concentrations up to 20 M. This also caused a movement of the

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86 C and D rings with respect to the A and B rings. 3-Des-Met-E also had no measurable binding to the rat 7 receptors at concentrations up to 20 M. Me-E, unlike the other -erythroidine compounds, has a charged quaternary ammonium group. Me-E had no interaction with rat 42 or 7 at concentrations up to 20 M. The addition of the methyl group may be preventing the nitrogen from forming the necessary hydrogen bonds required for binding. The remaining -erythroidine compound, E-Diol, allowed the importance of the D-ring in binding to be investigated. Interestingly, the opening of the D-ring still allowed for an interaction with rat 42 that is comparable to that of DHE. The opening of the D-ring may allow for additional hydrogen bonding and possibly increase the flexibility of the molecule, thus resulting in a greater affinity than that of -E. Like the other erythroidine compounds E-Diol also had no interaction with rat 7 at concentrations up to 50 M and its selectivity ratio (161) was less than that of DHE. Alteration in the size of the C-ring (increasing from a 6 to 7-membered ring), as determined with O-methylisophellibiline, resulted in a decreased affinity for rat 42. This indicates the importance of the smaller C-ring size for binding to rat 42. All of the aromatic compounds were obtained as natural products. Their D-rings lack the lactone moiety of the erythroidine compounds, and are replaced by a substituted phenyl ring. Several of these alkaloids had measured affinities greater than that of DHE. ELA and Gluco-ERD were similar in that they both had large substituents on their D-ring. With the increase in affinity for rat 42, there was also an increased affinity for the rat 7 receptor. Gluco-ERD, which has a large sugar molecule attached to

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87 the D-ring, had an affinity greater than that of ELA or DHE for rat 42. It did not however have any interaction with the rat 7 receptor at concentrations up to 20 M. The importance of a hydroxyl group at position 15 was determined from the binding results of ERV, ERD and EST. ERV had the highest affinity for the rat 42 receptor of all the Erythrina compounds studied. ERD is identical in structure to ERV except that the hydroxy and methoxy groups are reversed. This is important because there is a large decrease in affinity, 23 times less, of ERD for rat 42 as compared to ERV. The four fused rings of these alkaloids create rigidity in the 3-dimensional structures. It is possible that when the methoxy group is attached at position 15 there may be repulsion between the methyl of the methoxy group and a side chain of the receptor. Also, it is possible that the 15-hydroxy group is a H-bond donor (methoxy group is not a H-bond donor) that enhances overall binding. The results from the rat membrane binding studies indicate that one double bond between the A and B ring is optimal for nAChR binding. Various manipulations and substituents to the D-ring still allow for high affinity binding to 42. Finally, the aromatic compounds on average have a higher affinity for both the 42 and 7 nAChRs. All of the Erythrina alkaloids that had a measurable binding affinity for the 42-receptor functioned as antagonists according to measurements performed with the membrane potential fluorescent dye. ERV, which had the highest affinity of the Erythrina alkaloids for rat and human 42, also had the highest potency for inhibiting the human 42 ACh response. Although there was a significant difference (P value = 0.0186) in binding (for human 42) between ERV and ERD as measured by 3 H-cytisine

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88 displacement, there was less than a two-fold difference in inhibitory potency (for human 42) between the two alkaloids. There exist several published reports on the administration of several Erythrina alkaloids in vivo. In 1955, Megirian et al. found that -E, DHE, and 3-Des-Met-E produced ganglionic and neuromuscular blocking action on mice, rats and cats. Several groups determined the effects of DHE in the CNS. Corrigall et al. (1994) administered DHE directly to the ventral tegmental area of rats and found that DHE attenuated self-administration (intravenous) of nicotine. Systemic administration of DHE can antagonize the locomotor activating effect of nicotine in rats (Stolerman et al. 1997). When injected intraventricularly it was determined that DHE disrupted spatial memory and inhibited nicotines stimulatory effects (Curzon et al. 1996). As an herbal medicine, Erythrina leaves and bark (which contain alkaloids) have been found to produce anxiolytic effects (Onusic et al. 2003). The discovery of the acetylcholine binding protein (AChBP) has allowed for the crystallization of ligands bound within the ACh binding sites. Until recently only agonists were crystallized in the binding site. In 2005, two groups crystallized antagonists, an -conotoxin (PnIA) and an -neurotoxin (-cobratoxin), within the binding sites (Celie et al. 2005; Bourne et al. 2005). Both studies found that the C-loop was in a conformation distinctly different (projects away from the adjacent subunit) from that of the HEPES or nicotine bound AChBP. Both groups suggest that these antagonists are binding to the resting state as opposed to the desensitized or open state of the receptor.

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89 Conclusions In summary, this dissertation has presented several nicotine analogs and Erythrina alkaloids that have varying degrees of affinity for rat and human 42 as well as potency for human 42 nAChRs. Through alterations of structure we have determined properties important for binding and activation or inhibition, as well as differences in affinity between rat and human 42 nAChRs. Whereas the alterations to four different positions on the structure of nicotine result in nicotine analogs with a decreased affinity for 42, alterations to the D-ring of the Erythrina alkaloids continue to allow tight binding to 42. Modifications to the structure of nicotine decrease binding and convert the compounds to partial agonists and antagonists. All Erythrina alkaloids that had a measurable affinity for the 42 receptor acted as competitive antagonists, like DHE. This is the first study to provide affinities of several nicotine analogs and Erythrina alkaloids for 7 as well as measure the functional effects on human 42 nAChRs. These results provide data on several compounds that may be used as leads for the design of partial agonists and antagonists. Partial agonists and antagonists would be useful as smoking cessation drugs. Selective antagonists would be useful as probes to study the resting state of the 42 receptor. Future directions, based upon the results of this study, may include; electrophysiological analysis of the effects of nicotine analogs and Erythrina alkaloids, comparison of the properties of enantiomers from racemic nicotine analogs, and molecular modeling of several of these compounds to the AChBP and 42 human and rat receptors. Some of these compounds may also have quantitatively different binding affinities and functional effects on other naturally-occuring nAChRs.

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REFERENCES Akabas, M.H., Kaufmann, C., Archdeacon, P., and Karlin, A. (1994). Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron 13, 919-927. Alkondon, M., Pereira, E.F., Albuquerque, E.X. (1998). Alpha-bungarotoxinand methyllycaconitine-sensitive nicotinic receptors mediate fast synaptic transmission in interneurons of rat hippocampal slices. Brain Res. 810, 257-263. Alkondon, M., Pereira, E.F., Barbosa, C.T., Albuquerque, E.X. (1997). Neuronal nicotinic acetylcholine receptor activation modulates gamma-aminobutyric acid release from CA1 neurons of rat hippocampal slices. J. Pharmacol. Exp. Ther. 283, 1396-1411. Alkondon, M., Pereira, E., and Eisenberg, H. (1999). Choline and selective antagonists identify two subtypes of nicotinic acetylcholine receptors that modulate GABA release from CA1 interneurons in rat hippocampal slices. J. Neurosci. 19, 2693-2705. Allman, W.F. (1989). Apprentices of Wonder. Inside the Neural Network Revolution Bantam Books, New York, pp 1-211. Anderson, D.J., and Arneric, S.P. (1994). Nicotinic receptor binding of [3H] cytisine, [3H] nicotine and [3H] methylcarbamylcholine in rat brain. Eur. J. Pharmacol. 253, 261-267. Anderson, D.J., Williams, M., Pauly, J.R., Raszkiewicz, J.L., Campbell, J.E., Rotert, G., Surber, B., Thomas, S.B., Wasicak, J., Arneric, S.P., and Sullivan, J.P. (1995). Characterization of [3H]ABT-418: a novel cholinergic channel ligand. J. Pharmacol. Exp. Ther. 273, 1434-1441. Arendash, G.W., Sanberg, P.R., and Sengstock, G.J. (1995a). Nicotine enhances the learning and memory of aged rats. Pharmacol. Biochem. Behav. 52, 517-523. Arendash, G.W., Sengstock, G.J., Sanberg, P.R., and Kem, W.R. (1995b). Improved learning and memory in aged rats with chronic administration of the nicotinic receptor agonist GTS-21. Brain Res. 674, 252-259. Armstrong, D.W., Wang, X., and Ercal, N. (1998). Enantiomeric composition of nicotine in smokeless tobacco, medicinal products, and commercial reagents. Chirality 10, 587-591. 90

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91 Ballivet, M., Patrick, J., Lee, J., and Heinemann, S. (1982). Molecular cloning of cDNA coding for the gamma subunit of Torpedo acetylcholine receptor. Proc. Natl. Acad. Sci. U.S.A. 79, 4466-4470. Barrantes, G.E., Rogers, A.T., Lindstrom, J., and Wonnacott, S. (1995). alpha-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterization, colocalisation with alpha 7 subunits and up-regulation by chronic nicotine treatment. Brain Res. 672,228-236. Beers, W.H., and Reich, E. (1970). Structure and activity of acetylcholine. Nature 228, 917-922. Beene, D.L., Brandt, G.S., Zhong, W., Zacharias, N.M., Lester, H.A., and Dougherty, D.A. (2002). Cationinteractions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine. Biochem. 41, 10262-10269. Bencherif, M., and Lukas, R.J. (1991). Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line. J. Pharmacol. Exp. Ther. 257, 946-953. Bertrand, D., Devillers-Thiery, A., Revah, F., Galzi, J.L., Hussy, N., Mulle, C., Bertrand, S., Balliver, M., and Changeux, J.P. (1992). Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain. Proc. Natl. Acad. Sci. U.S.A. 89, 1261-1265. Bertrand, D., Galzi, J.L., Devillers-Thiery, A., Bertrand, S., and Changeux, J.P. (1993). Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal 7 nicotinic receptor. Proc. Natl. Acad. Sci. U.S.A. 90, 6971-6975. Blount, P., and Merlie, J.P. (1989). Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron 3, 349-357. Bourne, Y., Talley, T.T., Hansen, S.B., Taylor, P., and Marchot, P. (2005). Crystal structure of a Cbtx-AchBP complex reveals essential interactions between snake -neurotoxins and nicotinic receptors. EMBO J. 24, 1512-1522. Brejc, K., van Dijk, W., Klassen R., Schuurmans, M., van der Oost J., Smit, A., and Sixma, T. (2001). Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269-276. Buisson, B., and Bertrand, D. (1998). Allosteric modulation of neuronal nicotinic acetylcholine receptors. J. Physiol. Paris 92, 89-100.

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92 Carroll, F.I., Lee, J.R., Navarro, H.A., Brieaddy, L.E., Abraham, P., Damaj, M.I., and Martin, B.R. (2001). Synthesis, nicotine acetylcholine receptor binding, and antinociceptive properties of 2-exo-2-(2-substituted-3-phenyl-5-pyridinyl)-7-azabicyclo[2.2.1]-heptanes. Novel nicotinic antagonist. J. Med. Chem. 44, 4039-4041. Celie, P.H., van Rossum-Fikkert, S.E., van Dijk, W.J., Brejc, K., Smit, A.B., and Sixma, T.K. (2004). Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied AChBP crystal structure. Neuron 41, 907-914. Celie, P.H., Kasheverov, I.E., Mordvintsev, D.Y., Hogg, R.C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S.E., Zhmak, M.N., Bertrand, D., Tsetlin, V., Sixma, T.K., and Smit, A.B. (2005). Crystal structure of nicotinic acetylcholine receptor homolog AchBP in complex with an -conotoxin PnIA variant. Nat. Struc. Mol. Biol. 582-588. Champtiaux, N., Gotti, C., Cordero-Erausquin, M., David, D.J., Przybylski, C., Lena, C., Clementi, F., Moretti, M., Rossi, F.M., Le Novere, N., McIntosh, J.M., Gardier, A.M., and Changeux, J.P. (2003). Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. J. Neurosci. 23, 7820-7829. Champtiaux, N., Han, Z.Y., Bessis, A., Rossi, F.M., Zoli, M., Marubio, L., McIntosh, J.M., and Changeux, J.P. (2002). Distribution and pharmacology of 6-containing nicotinic acetylcholine receptors analyzed with mutant mice. J. Neurosci. 22, 1208-1217. Changeux, J.P., Devillers-Thiery, A., and Chemouilli, P. (1984). Acetylcholine receptor: an allosteric protein. Science 225, 1335-1345. Chavez-Noriega, L.E., Crona, J.H., Washburn, M.S., Urrutia, A., Elliott, K.J., and Johnson, E.C. (1997). Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes. J. Pharmacol. Exp. Ther. 280, 346-356. Chini, B., Raimondi, E., Elgoyhen, A., Moralli, D., Balzaretti, M. and Heinemann, S. (1994). Molecular cloning and chromosomal localization of the human 7-nicotinic receptor subunit gene. Genomics 19, 379-381. Clarke, P.B. (1993). Nicotinic receptors in mammalian brain: localization and relation to cholinergic innervation. Prog. Brain Res. 98, 77-83. Clarke, P.B., and Pert, A. (1985). Autoradiographic evidence for nicotine receptors on nigrostriatal and mesolimbic dopaminergic neurons. Bran Res. 348, 355-358.

PAGE 105

93 Clarke, P.B.S., Schwartz, R.D., Paul, S.M., Pert, C.B., and Pert, A. (1985). Nicotinic binding in rat brain: autoradiographic comparison of [ 3 H] acetylcholine [ 3 H] nicotine and [ 125 I] alpha-bungarotoxin. J. Neurosci. 5, 1307-1315. Claudio, T., Ballivet, M., Patrick, J., and Heinemann, S. (1983). Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor gamma subunit. Proc. Natl. Acad. Sci. U.S.A. 80, 1111-1115. Coe, J.W., Brooks, P.R., Vetelino, M.G., Wirtz, M.C., Arnold, E.P., Huang, J., Sands, S.B., Davis, T.I., Lebel, L.A., Fox, C.B., Shrikhande, A., Heym, J.H., Schaeffer, E., Rollema, H., Lu, Y., Mansbach, R.S., Chambers, L.S., Rovetti, C.C., Schulz, D.W., Tingley, D., and ONeill, B.T. (2005). Varencline: an 42 nicotinic receptor partial agonist for smoking cessation. J. Med. Chem. 48, 3474-3477. Colquhoun, D., and Sakmann, B. (1998). From muscle endplate to brain synapses: a short history of synapses and agonist-activated ion channels. Neuron 20, 381-387. Conroy, W.G., and Berg, D.K. (1998). Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the alpha4, beta2, and alpha5 gene products. Mol. Pharmacol. 53, 392-401. Copeland, J.R., Adem, A., Jacob III, P., Nordberg, A. (1991). A comparison of the binding of nicotine and nornicotine stereoisomers to nicotinic binding sites in rat brain cortex. Naunyn-Schmiedebergs Arch. Pharmacol. 343, 123-127. Corrigall, W.A., Coen, K.M., Adamson, K.L. (1994). Self-administered nicotine activates the mesolimbic dopamine system through the ventral tegmental area. Brain Res. 653, 278-284. Corringer, P.J., Galzi, J.L., Eisele, J.L., Bertrand, S., Changeux, J.P., and Bertrand, D. (1995). Identification of a new component of the agonist bidning site of the nicotinic alpha 7 homooligomeric receptor. J. Biol. Chem. 270, 11749-11752. Corringer, P.J., Le Novere, N., and Changeux, J.P. (2000). Nicotinic receptors at the amino acid level. Annu. Rev. Pharmacol. Toxicol. 40, 431-458. Court, J.A., Piggott, M.A., Lloyd, S., Cookson, N., Ballard, C.G., McKeith, I.G., Perry, R.H., and Perry, E.K. (2000). Nicotine binding in human striatum: elevation in schizophrenia and reductions in dementia with Lewy bodies, Parkinsons disease and Alzheimers disease and in relation to neuroleptic medication. Neuroscience 98, 79-87. Couturier, S., Bertrand, D., Matter, J.M., Hernandez, M.C., Bertrand, S., Millar, N., Valera, S., Barkas, T., and Ballivet, M. (1990). A neuronal nicotinic acetylcholine receptor subunit (7) is developmentally regulated and forms a homo-oligomeric channel blocked by -btx. Neuron 5, 847-856.

PAGE 106

94 Crooks, P.A., Ayers, J.T., Xu, R., Sumithran, S.P., Grinevich, V.P., Wilkins, L.H., Deaciuc, A.G., Allen, D.D., and Dwoskin, L.P. (2004). Development of subtype-selective ligands as antagonists at nicotinic receptors mediating nicotine-evoked dopamine release. Bioorg. Med. Chem. Lett. 14, 1869-1874. Crooks, P.A., Li, M., and Dwoskin, L.P. (1997). Metabolites of nicotine in rat brain after peripheral nicotine administration. Drug Metab. Dispos. 25, 47-54. Cryan, J.F., Bruijnzeel, A.W., Skjei, K.L., and Markou, A. (2003). Bupropion enhances brain reward function and reverses the affective and somatic aspects of nicotine withdrawl in the rat. Psychopharmacology (Berl.) 168, 347-358. Cui, C., Booker, T.K., Allen, R.S., Grady, S.R., Whiteaker, P., Marks, M.J., Salminen, O., Tritto, T., Butt, C.M., Allen, W.R., Stitzel, J.A., McIntosh, J.M., Boulter, J., Collins, A.C., Heinemann, S.F. (2003). The 3 nicotinic receptor subunit: a component of -conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors. J. Neurosci. 23, 11045-11053. Curzon, P., Brioni, J.D., and Decker, M.W. (1996). Effect of intraventricular injections of dihydro-beta-erythroidine (DHE) on spatial memory in the rat. Brain Res. 714, 185-191. Damaj, M.I., Glassco, W., Dukat, M., May, E.L., Glennon, R.A., Martin, B.R. (1996). Pharmacology of novel nicotine analogs. Drug Dev. Res. 38, 177-187. Decker, M.W., Anderson, D.J., Brioni, J.D., Donnelly-Roberts, D.L., Kang, C.H., ONeill, A.B., Piattoni-Kaplan, M., Swanson, S., and Sullivan, J.P. (1995). Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine receptors. Eur. J. Pharmacol. 280, 79-89. De Fusco, M., Becchetti, A., Patrignani, A., Annesi, G., Gambardells, A., Quattrone, A., Ballabio, A., Wanke, E., and Casari, G. (2000). The nicotinic receptor beta 2 subunit is mutant in nocturnal frontal lobe epilepsy. Nat. Genet. 26, 275-276. de la Garza, R., Bickford-Wimer, P.C., Hoffer, B.J., and Freedman, R. (1987). Heterogeneity of nicotine actions in the rat cerebellum: an in vivo electrophysiologic study. J. Pharmacol. Exp. Ther. 240, 689-695. Devillers-Thiery, A., Giraudat, J., Bentaboulet, M., Changeux, J.P. (1983). Complete mRNA coding sequence of the actylcholine binding alpha subunit of Torpedo marmorata acetylcholine receptor: a model for the transmembrane organization of the polypeptide chain. Proc. Natl. Acad. Sci. U.S.A. 80, 2067-2071. Dougherty, D.A., and Lester, H.A. (2001). Neurobiology. Snails, synapses and smokers. Nature 411, 252-253, 255.

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95 Dukat, M., Damaj, I.M., Young, R., Vann, R., Collins, A.C., Marks, M.J., Martin, B.R., and Glennon, R.A. (2002). Functional diversity among 5-substituted nicotine analogs; in vitro and in vivo investigations. Eur. J. Pharmacol. 435, 171-180. Dukat, M., Dowd, M., Damaj, I., Martin, B., El-Zahabi, M.A., and Glennon, R.A. (1998). Synthesis, receptor binding and QSAR studies on 6-substituted nicotine derivatives as cholinergic ligands. Eur. J. Med. Chem. 33, 1-10. Dukat, M., Fiedler, W., Dumas, D., Damaj, I., Martin, B.R., Rosecrans, J.A., James, J.R., and Glennon, R.A. (1996). Pyrrolidine-modified and 6-substituted analogs of nicotine: a structure-affinity investigation. Eur. J. Med. Chem. 31, 875-888. Dwoskin, L.P., Teng, L.H., and Crooks, P.A. (2001). Nornicotine, a nicotine metabolite and tobacco alkaloid: desensitization of nicotinic receptor-stimulated dopamine release from rat striatum. Eur. J. Pharmacol. 428, 69-79. Fatt, P., and Katz, B. (1950). Some observations on biological noise. Nature 166, 597. Fatt, P., and Katz, B. (1951). An analysis of the endplate potential recorded with and inta-cellular electrode. J. Physiol. (Lond.) 115, 320-370. Fatt, P., and Katz, B., (1952). Spontaneous subthreshold activity at motor nerve endings. J. Physiol. (Lond.) 117, 109-128. Feldberg, W., Fessard, A., and Nachmansohn, D. (1940). The cholinergic nature of the nervous supply to the electric organ of the Torpedo (Torpedo marmorata). J. Physiol. 97, 3-4. Fenster, C.P., Whitworth, T.L., Sheffield, E.B., Quick, M.W., and Lester, R.A.J. (1999). Upregulation of surface 42 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine. J. Neurosci. 19, 4804-4814. Ferretti, G., Dukat, M., Giannella, M., Piergentili, A., Pigini, M., Quaglia, W., Damaj, M.I., Martin, B.R., and Glennon, R.A. (2003). Binding of Nicotine and Homoazanicotine analogues at neuronal nicotinic acetylcholinergic (nACh) receptors. Bioorg. Med. Chem. Lett. 13, 733-735. Fitch R.W., Xiao, Y., Kellar, K.J., and Daly, J.W. (2003). Membrane potential fluorsecene: a rapid and highly sensitive assay for nicotinic receptor channel function. Proc. Natl. Acad. Sci. U.S.A. 15, 4909-4914. Flores, C.M., Rogers, S.W., Pabreza, L.A., Wolfe, B.B., and Kellar, K.J. (1992). A subtype of nicotinic cholinergic receptor in rat brain is composed of 4 and 2 subunits and is up-regulated by chronic nicotine treatment. Mol. Pharm. 41, 31-37. Folkers, K., and Major, R. (1937). Isolation of an Erythrina alkaloid of curare action from E. Americana. J. Am. Chem. Soc. 59, 1580-1581.

PAGE 108

96 Frazier, C.J., Buhler, A.V., Weiner, J.L., and Dunwiddie, T.V. (1998). Synaptic potentials mediated via alpha-bungarotoxin-sensitive nicotinic acetylcholline receptors in rat hippocampal interneurons. J. Neurosci. 18, 8228-8235. Freedman, R., Leonard, S., Gault, J.M., Hopkins, J., Cloninger, C.R., Kaufmann, C.A., Tsuang, M.R., Farone, S.V., Malaspina, D., Svrakic, D.M., Sanders, A., and Gejman, P. (2001). Linkage disequilibrium for schizophrenia at the chromosome 15q13-14 locus of the alpha7-nicotinic acetylcholine receptor subunit gene (CHRNA7). Am. J. Med. Genet. 105, 20-22. Fucile, S. (2004). Ca2+ permeability of nicotinic acetylcholine receptors. Cell Caclium 35, 1-8. Fujita, T., Nakajima, M., Soeda, Y., Yamaoto, I. (1971). Physicochemical properties of biological interest of nicotine and its related compounds. Pestic. Biochem. Physiol. 1, 151-162. Galzi, J.L., Bertrand, D., Devillers-Thiery, A., Revah, F., Bertrand, S., Changeux, J.P. (1991). Functional significance of aromatic amino acids from three peptide loops of the alpha 7 neuronal nicotinic receptor site investigated by site-directed mutagenesis. FEBS Lett. 294, 198-202. Galzi, J.L., Devillers-Thiery, A., Hussy, N., Bertrand, S., Changeux, J.P., and Bertrand, D. (1992). Mutations in the channel domain of a neuronal nicotinic receptor convert ion-selectivity from cationic to anionic. Nature 359, 500-505. Gerzanich, V., Anand, R., and Lindstrom, J. (1993). Homomers of 8 and 7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties. Mol. Pharm. 45, 212-220. Glassco, W., May, E.L., Damaj, M. I., and Martin, B.R. (1994). In vivo and in vitro activity of some N-substituted ()-nornicotine analogs. Med. Chem. Res. 4, 273-282. Glennon, R.A., and Dukat, M. (2000). Central nicotinic receptor ligands and pharmacophores. Pharm. Acta. Helv. 74, 103-114. Gopalkrishnan, M., Buisson, B., Touma, E., Giordanao, T., Campbell, J.E., Hu, I.C., Donnelly-Roberts, D., Arneric, S.P., Bertrand, D., and Sullivan, J.P. (1995). Stable expression and pharmacological properties of the human alpha 7nicotinic acetylcholine receptor. Eur. J. Pharmacol. 290, 237-246. Grady, S., Marks, M.J., Wonnacott, S., and Collins, A.C. (1992). Characterization of nicotinic receptor-mediated [3H]dopamine release from synaptosomes prepared from mouse striatum. J. Neurochem. 59, 848-856.

PAGE 109

97 Green, T.A., Crooks, P.A., Bardo, M.T., and Dwoskin, L.P. (2001). Contributory role for nornicotine in nicotine pharmacology: nornicotine-evoked [ 3 H]dopamine overflow from rat nucleus accumbens slices. Biochem. Pharmacol. 62, 1597-1603. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. (1981). Improved patch clamp techniques for high-resolution current recording from cells and cell-free membranes. Pflgers Arch. 391, 85-100. Harvey, S.C., and Luetje, C.W. (1996). Determinants of competitive antagonist sensitivity on neuronal nicotinic receptor beta subunits. J. Neurosci. 16, 3798-3806. Harvey, S.C., Maddox, F.N., and Luetje, C.W. (1996). Multiple determinants of dihydro--erythroidine sensitivity on rat neuronal nicotinic receptor subunits. J. Neurochem. 67, 1953-1959. Higgins, L.S., and Berg, D.K. (1988). A desensitized form of neuronal acetylcholine receptor detected by 3H-nicotine binding on bovine chromaffin cells. J. Neurosci. 8, 1436-1446. Hirose, S., Iwata, H., Akiyoshi, H., Kobayashi, K., Ito, M., Wada, K., Kaneko, S., and Mitsudome, A. (1999). A novel mutation of CHRNA4 responsible for autosomal dominant nocturnal frontal lobe epilepsy. Neurology 53, 1749-1753. Hodgkin, A.L., and Huxley, A.F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. (Lond.) 117, 500-554. Hogg, R.C., Raggenbass, M. and Bertrand, D. (2003). Nicotinic acetylcholine receptors: from structure to brain function. Rev. Physiol. Biochem. Pharmacol. 147, 1-46. Imoto, K., Busch, C., Sakmann, B., Mishina, M., Konno, T., Nakai, J., Bujo, H., Mori, Y., Fukuda, K., and Numa, S. (1998) Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature 335, 645-648. Jones, I.W., Bolam, J.P., and Wonnacott, S. (2001). Presynaptic localixation of the nicotinic acetylcholine receptor beta2 subunit immunoreactivity in rat nigrostriatal dopaminergic neurons. J. Comp. Neurol. 439, 235-247. Kao, P. N., and Karlin, A. (1986). Acetylcholine receptor binding site contains a disulphide cross-link between adjacent half-cystinyl residues. J. Biol. Chem. 261, 8085-8088. Katz, B., and Thesleff, S. (1957). A study of the desensitization produced by acetylcholine at the motor end-plate. J. Physiol. (Lond.) 138, 63-80. Kem, W.R. (1971). A study of the occurrence of anabaseine in Paranemertes and other nemertines. Toxicon 9, 23-32.

PAGE 110

98 Kem W.R. (2000). The brain alpha7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimer's disease: studies with DMXBA (GTS-21). Behav. Brain Res. 113, 169-181. Kem, W.R., Mahnir, V.M, Papke, R.L., and Lingle, C.J. (1997). Anabaseine is a potent agonist on muscle and neuronal alpha-bungarotoxin-sensitive nicotinic receptors. J. Pharmacol. Exp. Ther. 283, 979-992. Kem, W.R., Mahnir, V.M., Prokai, L., Papke, R.L., Cao, X., LeFrancois, S., Wildeboer, K., Prokai-Tatrai, K., Porter-Papke, J., and Soti, F. (2004a). Hydroxy metabolites of the Alzheimers drug candidate 3-[2,4-dimethoxy)benzylidene]-anabaseine dihydrochloride (GTS-21): their molecular properties, interactions with brain nicotinic receptors, and brain penetration. Mol. Pharmacol. 65, 56-67. Kem, W.R., Wildeboer, K.M., LeFrancois, S.E., Raja, M., Marszalec, W., and Brakeman, J.C. (2004b). Nicotinic receptor inhibition by Tetraponera ant alkaloids. Cell. Mol. Neurobiol. 24, 535-551. Keyser, K.T., Britto, L.R., Schoepher, R., Whiting, P., Cooper, J., Conroy, W., Brozozowska-Prechtl, A., Karten, H.J., and Lindstrom, J. (1993). Three subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors are expressed in chick retina. J. Neurosci. 13, 442-454. Kim, K.H., Lin, N.-H., and Anderson, D.J. (1996). Quantitative structure-activity relationships of nicotine analogues as neuronal nicotinic acetylcholine receptor ligands. Bioorg. Med. Chem. 4, 2211-2217. Kuryatov, A., Luo, J., Cooper, J., and Lindstrom, J. (2005). Nicotine acts as a pharmacological chaperone to upregulate human alpha4 beta2 acetylcholine receptors. Mol. Pharmacol. 68, 1839-1851. Langley, J.N. (1878). On the physiology of salivary secretion. Part II. On the mutual antagonism of atropin and pilocarpin, having expecial reference to their relations in the sub-maxillary gland of the cat. J. Physiol. (Lond.) 1. 339-369. Langley, J.N. (1905). On the reaction of cells and of nerve-endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and curare. J. Physiol. (Lond.) 33, 374-413. Lee, C.Y., and Chang, L.F. (1966). Modes of actions of purifed toxins from venoms on neuromuscular transmission. Mem. Inst. Butantan. 33, 555-572. Lehman, A.J. (1936). Curare-actions of Erythrina americana. Proc. Soc. Exp. Biol. Med. 33, 501-503. Le Novere, N., and Changeux, J.P. (1995). Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells. J. Mol. Evol. 40, 155-172.

PAGE 111

99 Leonard, S., Adams, C., Breese, C.R., Adler, L.E., Bickford, P., Byerley, W., Coon, H., Griffith, J.M., Miller, C., Myles-Worsley, M., Nagamoto, H.T., Rollins, Y., Stevens, K.E., Waldo, M., and Freedman, R. (1996). Nicotinic receptor function in schizophrenia. Schizophr. Bull. 22, 431-445. Levin, E. (1992). Nicotinic systems and cognitive function. Psychopharmacology (Berl.) 108, 417-431. Li, Y., Papke, R.L., He, Y.J., Millard, W.J., and Meyer, E.M. (1999). Characterization of the neuroprotective and toxic effects of alpha7 nicotinic receptor activation in PC12 cells. Brain. Res. 830, 218-225. Lin N-H, Carrera Jr, G.M., Anderson, D.J. (1994). Synthesis and evalution of nicotine analogs as neuronal nicotinic acetylcholine receptor ligands. J. Med. Chem. 37, 3542-3553. Lindstrom, J., Walter, B., Einarson, B. (1979). Immunochemical similarities between subunits of acetylcholine receptors from Torpedo, Electrophorus, and mammalian muscle. Biochem. 21, 2210-2217. Lohr, J.B., and Flynn, K. (1992). Smoking and schizophrenia. Schizophr. Res. 8, 93-102. Lubin, M., Erisir, A., and Aoki, C. (1999). Ultrastructural immunolocalization of the alpha 7 nAChR subunit in guinea pig medial prefrontal cortex. Ann. N.Y. Acad. Sci. 868, 628-632. Mao, D., Yasuda, R.P., Wolfe, B.B., and Kellar, K.J. (2005). Association of the 5 subunit with different nicotinic receptor subtypes in the rat peripheral and central nervous system. Program number 722.7 Abstract Viewer/Itinerary Planner, Washington, D.C.: Society for Neuroscience. Marks, M.J., and Collins, A.C. (1982). Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate. Mol. Pharm. 22, 554-564. Marks, M.J., Stitzel, J.A., Romm, E., Wehner, J.M., and Collins, A.C. (1986). Nicotinic binding sites in rat and mouse brain: comparison of acetylcholine, nicotine and -bungarotoxin. Mol. Pharm. 30, 427-436. McGehee, D.S., and Role, L.W. (1995). Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu. Rev. Physiol. 57, 521-546. Megirian, D., Leary, D.E., and Slater, I.H. (1955). The action of some derivatives of beta-erythroidine on peripheral neuro-effector systems. J. Pharmacol. Exp. Ther. 113, 212-227.

PAGE 112

100 Meyer, E.M., Kuryatov, A., Gerzanich, V., Lindstrom, J., and Papke, R.L. (1998). Analysis of 3-(4-hydroxy, 2-Methoxybenzylidene) anabaseine selectivity and activity at human and rat alpha-7 nicotinic receptors. J. Pharmacol. Exp. Ther. 287, 918-925. Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfessel, C., and Sakmann, B. (1986). Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. 321, 406-411. Miyazawa, A., Fujiyoshi, Y., Stowell, M., and Unwin, N. (1999). Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall. J. Mol. Biol. 288, 765-786. Monod, J., Wyman, J., and Changeux, J.P. (1965). On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 3, 318-356. Mundell, S.J., and Benovic, J.L. (2000). Selective regulation of endogenous G protein-coupled receptors by arrestins in HEK293 cells. J. Biol. Chem. 275, 12900-12908. Neher, E., and Sakmann, B. (1976). Single channel currents recorded from membrane of denervated frog muscle fibers. Nature 260, 799-802. Nelson, M.E., Kuryatov, A., Choi, C.H., Zhou, Y., and Lindstrom, J. (2003). Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors. Mol. Pharm. 63, 332-341. Neubig, R.R., and Cohen, J.B. (1979). Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand intereactions. Biochem. 18, 5464-5475. Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982). Primary structure of alpha-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nature. 299, 793-797. Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Furutani, T., Hirose, T., Takashima, H., Inayama, S., Miyata. T., Numa, S. (1983). Structural homology of Torpedo californica acetylcholine receptor subunits. Nature. 302, 528-532. Olsen, R., Meunier, J.C., and Changeux, J.P. (1972). Progress in purification of the cholinergic receptor protein from Electrophorous electricus by affinity chromatography. FEBS Lett. 28, 96-100. Onusic, G.M., Nogueira, R.L., Pereira, A.M.S., Flausino Jr., O.A., and Viana, M.B. (2003). Effects of chronic treatment with a water-alcohol extract from Erythrina mulungu on anxiety-related responses in rats. Biol. Pharm. Bull. 26, 1538-1542.

PAGE 113

101 Ortells, M.O., and Lunt, G.G. (1995). Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends. Neurosci. 18, 121-127. Pabreza, L.A., Dhawan, S., and Kellar, K.J. (1991). [ 3 H]Cytisine binding to nicotinic cholinergic receptors in brain. Mol. Pharm. 39, 9-12. Papke, R.L., and Heinemann, S.F. (1994). Partial agonist properties of cytisine on neuronal nicotinic receptors containing the 2 subunit. Mol. Pharm. 45, 142-149. Papke, R.L., and Oswald, R.E. (1989). Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents. J. Gen. Physiol. 93, 785-811. Papke, R.L., and Porter Papke, J.K. (2002). Comparative pharmacology of rat and human 7 nAChR conducted with net charge analysis. Br. J. Pharmacol. 137, 49-61. Papke, R.L., Thinschmidt, J.S., Moulton, B.A., Meyer, E.M., and Poirier, A. (1997). Activation and inhibition of rat neuronal nicotinic receptors by ABT-418. Br. J. Pharmacol. 120, 429-438. Pascual, J.M., and Karlin, A. (1998). State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit. J. Gen. Physiol. 111, 717-739. Perry, E., Martin-Ruiz, C., Lee, M., Griffiths, M., Johnson, M., Piggott, M., Haroutunian, V., Buxbaum, J.D., Nasland, J., Davis, K., Gotti, C., Clementi, F., Tzartos, S., Cohen, O., Soreq, H., Jaros, E., Perry, R., Ballard, C., McKeith, I., and Court, J. (2000). Nicotinic receptor subtypes in human brain and ageing, Alzheimer and Lewy body diseases. Eur. J. Pharmacol. 393, 215-222. Perry, E.K., Morris, C.M., Court, J.A., Cheng, A., Fairbairn, A.F., McKeith, I.G., Irving, D., Brown, B., and Perry, R.H. (1995). Alteration in nicotine binding sites in Parkinsons disease, Lewy body dementia and Alzheimers disease: possible index of early neuropathology. Neuroscience 64, 385-395. Phillips, H.A., Scheffer, I.E., Berkovic, S.F., Hollway, G.E., Sutherland, G.R., and Mulley, J.C. (1995). Localization of a gene for autosomal dominant nocturnal frontal lobe epilepsy to chromosome 20q 13.2. Nat. Genet. 10, 117-118. Picciotto, M.R., and Corrigall, W.A. (2002). Neuronal systems underlying behaviors related to nicotine addiction: neural circuits and molecular genetics. J. Neurosci. 22, 3338-3341. Picciotto, M.R., Zoli, M., Lena, C., Bessis, A., Lallemand, Y., Le Novere, N., Vincent, P., Pich, E.M., Brulet, P., and Changeux, J.P. (1995). Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain. Nature 374, 65-67.

PAGE 114

102 Picciotto, M.R., Zoli, M., Rimondini, R., Lena, C., Marubio, L.M., Pich, E.M., Fuxe, K., Changeux, J.P. (1998). Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature 391, 173-177. Placzek, A.N., Grassi, F., Meyer, E.M., and Papke, R.L. (2005). An alpha 7 nicotinic acetylcholine receptor gain-of-function mutant that retains pharmacological fidelity. Mol. Pharmacol. 68, 1863-1876. Poirier, M.F., Canceil, O., Bayle, F., Millet, B., Bourdel., M.C., Moatti, C., Olie, J.P., and Attar-Levy, D. (2002). Prevalence of smoking in psychiatric patients. Prog. Neuropsychopharmacol. Biol. Psychiatr. 26, 529-537. Radcliffe, K.A., Fisher, J.L., Gray, R., and Dani, J.A. (1999). Nicotinic modulation of glutamate and GABA synaptic transmission of hippocampal neurons. Ann. N.Y. Acad. Sci. 868, 561-610. Raftery, M.A., Hunkapiller, M.W., Strader, C.D., and Hood, L.E. (1980). Acetylcholine receptor: complex of homologous subunits. Science. 208, 1454-1456. Raggenbass, M., and Bertrand, D. (2002). Nicotinic receptors in circuit excitability and epilepsy. J. Neurobiol. 53, 580-589. Rapier, C., Lunt, G.G., and Wonnacott, S. (1990). Nicotinic modulation of [ 3 H] dopamine release from striatal synaptosomes: pharmacological characterization. J. Neurochem. 54, 937-945. Reiter, M.J., Cowburn, D.A., Prives, J.M., and Karlin, A. (1972). Affinity labeling of the acetylcholine receptor in the electroplax: electrophoretic separation in sodium dodecyl sulfate. Proc. Natl. Acad. Sci. U.S.A. 69, 1168-1172. Revah, F., Bertrand, D., Galzi, J.L., Devillers-Thiery, A., Mulle, C., Hussy, N., Bertrand, S., Ballivet, M., and Changeux, J.P. (1991). Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 353, 846-849. Reynolds, J.A., and Karlin, A. (1978). Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica. Biochemistry 17, 2035-2038. Rinne, J.O., Myllykyla, T., Lonnberg, P., and Marjamaki, P. (1991). A postmortem study of brain nicotinic receptors in Parkinsons and Alzheimers disease. Brain Res. 547, 167-170. Rose, M.E., Behn, F.M., Westman, E.C. (1994). Mecamylamine combined with nicotine skin patch treatment facilitates smoking cessation beyond nicotine patch treatment alone. Clin. Pharmacol. Ther. 56, 86-99.

PAGE 115

103 Rowland, N.E., Vaughan, C.H., Bastian, J.R., Robertson, Soti, F.R., and Kem, W.R. (2003). Antagonism of behavioral effects of nicotine in rats by partial agonist. Program number 247.14 Abstract Viewer/Itinerary Planner, Washington, D.C.: Society for Neuroscience. Saitoh, F., Noma, M., Kawashima, N. (1985). The alkaloid contents of sixty Nicotiana species. Phytochemistry 24, 477-480. Sands, S.B., Costa, A.C.S, and Patrick, J.W. (1993). Barium permeability of neuronal nicotinic receptor 7 expressed in Xenopus oocytes. Biophys. J. 65, 2614-2621. Schoepfer, R., Whiting, P., Esch, F., Blacher, R., Shimasaki, S., Lindstrom, J. (1988). cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor. Neuron 1, 241-248. Schofield, P.R., Darlison, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodriguez, H., Rhee, L.M., Ramachandran, J., Reale, V., Glencorse, T.A. Seeburg P.H., and Barnard, E.A. (1987). Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature 328, 221-227. Schroder, H., Giacobini, E., Struble, R.G., Zilles, K., Maelicke, A., Luiten, P.G., and Strosberg, A.D. (1991). Cellular distribution and expression of cortical acetylcholine receptors in aging and Alzheimers disease. Ann. N.Y. Acad. Sci. 640, 189-192. Schroder, H., Zilles, K., Maelicke, A., Hajos, F. (1989). Immunohistoand cytochemical localization of cortical nicotinic cholinoceptors in rat and man. Brain Res. 502, 287-295. Schwartz, R.D., McGee, R., and Kellar, K.J. (1982). Nicotinic cholinergic receptors labeled by [3H]acetylcholine in rat brain. Mol. Pharm. 22, 56-62. Seguela, P., Wadiche, J., Dinely-Miller, K., Dani, J.A., and Patrick, J.W. (1993). Molecular cloning, functional properties and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J. Neurosci. 13, 596-604. Sheridan, R.P., Nilakantan, R., Dixon, J.S., and Venkataraghavan, R. (1986). The ensemble approach to distance geometry: application to the nicotinic pharmacophore. J. Med. Chem. 29, 899-906. Silman, I., and Karlin A. (1969). Acetylcholine receptor: covalent attachment of depolarizing groups at the active site. Science. 164, 1420-1421. Simosky, J.K., Stevens, K.E., Kem, W.R., and Freedman, R. (2001). Intragastric DMXB-A an alpha7 nicotinic agonist, improves deficient sensory inhibition in DBA/2 mice. Biol. Psychiatry 50, 493-500.

PAGE 116

104 Sine, S.M., and Taylor, P. (1982). Local anesthetics and histrionicotoxin are allosteric inhibitors of the acetylcholine receptor. Studies of clonal muscle cells. J. Biol. Chem. 257, 8106-8114. Sine, S.M., and Taylor, P. (1981). Relationships between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. J. Biol. Chem. 256, 6692-6699. Sine, S.M., and Taylor, P. (1980). Relationship between agonist occupation and permeability response of the cholinergic receptor revealed by bound cobra-toxin. J. Biol. Chem. 225, 10144-10156. Smit, A.B., Brejc, K., Syed, N., and Sixma, T.K. (2003). Structure and function of AChBP, homologue of the ligand-binding domain of the nicotinic acetylcholine receptor. Ann. N.Y. Acad. Sci. 998, 81-92. Smit, A.B., Syed, N.I., Schaap, D., van Minnen, J., Klumperman, J., Kits, K.S., Lodder, H., van der Schors, R.C., van Elk R., Sorgedrager, B., Brejc, K., Sixma, T. K., and Geraerts, W.P.M. (2001). A glia-derived acetylcholine-binding protein that modulates synaptic transmission. Nature. 411, 261-268. Soliakov, L., Gallagher, T., and Wonnacott, S. (1995). Anatoxin-a-evoked [3H]dopamine release from rat striatal synaptosomes. Neuropharmacology 34, 1535-1541. Steinlein, O.K., Magnusson, A., Stoodt, J., Bertrand, S., Weiland, S., Berkovic, S.F., Nakken, K.O., Propping, P., and Bertrand, D. (1997). An insertion mutation of the CHRNA4 gene in a family with autosomal dominant nocturnal frontal lobe epilepsy. Hum. Mol. Genet. 6, 943-947. Steinlein, O.K., Mulley, J.C., Propping, P., Wallace, R.H., Phillips, H.A., Sutherland, G.R., Scheffer, I.E., and Berkovic, S.F. (1995). A missense mutation in the neuronal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet. 11, 201-203. Stephenson, R.P. (1956). A modification of receptor theory. Br. J. Pharmacol. 11, 379-393. Stevens, K.E., Kem, W.R., Mahnir, V.M., and Freedman, R. (1998). Selective alpha7-nicoitnic agonists normalize inhibition of auditory response in DBA mice. Psychopharmacology (Berl.) 136, 320-327. Stokes, C., Papke, J.K., Horenstein, N.A., Kem, W.R., McCormack, T.J., and Papke, R.L. (2004). The structural basis for GTS-21 selectivity between human and rat nicotinic alpha7 receptors. Mol. Pharmacol. 66, 14-24. Stolerman, I.P., Chandler, C.J., Garcha, H.S., and Newton, J.M. (1997). Selective antagonism of behavioral effects of nicotine by dihydro--erythroidine in rats. Psychopharmacology (Berl.) 129, 390-397.

PAGE 117

105 Swain, M.L., Eisner, A., Woodward, C.F., and Brice, B.A. (1949). Ultraviolet absorption spectra of nicotine, nornicotine and some of their derivatives. J. Am. Chem. Soc. 71, 1341-1345. Takeuchi, A., and Takeuchi, N. (1960). On the permeability of the endplate membrane during the action of transmitter. J. Physiol. (Lond.) 154, 52-67. Teng, L., Crooks, P.A., Buxton, S.T., and Dwoskin, L.P. (1997). Nicotinic-receptor mediation of S(-)nornicotine-evoked [3H]overflow from rat striatal slices preloaded with [3H]dopamine. J. Pharmacol. Exp. Ther. 283, 778-787. Tnder, J.E., and Olesen, P.H. (2001). Agonists at the 42 nicotinic acetylcholine receptors: structure relationships and molecular modeling. Curr. Med. Chem. 8, 651-674. Toyoshima, C., and Unwin, N. (1988). Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes. Nature 336, 247-250. Unwin, N. (2005). Refined structure of the nicotinic acetylcholine receptor at 4 resolution. J. Mol. Biol. 346, 967-989. Unwin, N. (1995). Acetylcholine receptor channel imaged in the open state. Nature 373, 37-43. Unwin, N. (1993). Nicotinic acetylcholine receptor at 9 resolution. J. Mol. Biol. 229, 1101-1124. Unwin, N., Toyoshima, C., Kublaek, E. (1988). Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes. J. Cell. Biol. 107, 1123-1138. Warpman, U., and Nordberg, A. (1995). Epibatidine and ABT-418 reveal selective losses of alpha 4 beta 2 nicotinic receptors in Alzheimer brains. Neuroreport. 6, 2419-2423. Waser, P. (1961). Strukturabhangigkeit der wirkung muscarinahnlicher verbindungen. Experientia. 17, 300-302. Weber, M., and Changeux, J.P. (1974). Binding of Naja nigricollis [3H] alpha-toxin to membrane fragments from Electrophorus and Torpedo electric organs. II. Effect of cholinergic agonists and anatagonists on the binding of the tritiated alpha-neurotoxin. Mol. Pharm. 10, 15-34. Weiland, G., Frisman, D., and Taylor, P. (1979). Affinity labeling of the subunits of the membrane associated cholinergic receptor. Mol. Pharm. 15, 213-226.

PAGE 118

106 Whiteaker, P., Garcha, H.S., Wonnacott, S., Stolerman, I.P. (1995). Locomotor activation and dopamine release produced by nicotine and isoarecolone in rats. Br. J. Pharmacol. 116, 2097-2105. Whiteaker, P., Sharples, C., and Wonnacott, S. (1998). Agonist-induced upregulation of 42 nicotine acetylcholine receptors in M10 cells: pharmacological and spatial definition. Mol. Pharm. 53, 950-962. Wilkie, G.I., Hutson, P.H., Stephens, M.W., Whiting, P., and Wonnacott, S. (1993). Hippocampal nicotinic autoreceptors modulate acetylcholine release. Biochem. Soc. Trans. 21, 429-431. Williams, M. and Robinson, J. (1984). Binding of the nicotinic cholinergic antagonist, dihydro--erythroidine, to rat brain tissue. J. Neurosci. 4, 2906-2911. Wilson, G.G., and Karlin, A. (1998). The location of the gate in the acetylcholine receptor channel. Neuron 20, 1269-1281. Wilson, G., and Karlin, A. (2001). Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method. Proc. Natl. Acad. Sci. U.S.A. 98, 1241-1248. Xiao, Y and Kellar, K.J. (2004). The comparative pharmacology and up-regulation of rat neuronal nicotinic receptor subtype binding sites stably expressed in transfected mammalian cells. J. Pharmacol. Exp. Ther. 310, 98-107. Xu, J., Pato, M.T., Torre, C.D., Medeiros, H., Carvalho, C., Basile, V.S., Bauer, A., Dourado, A., Valente, J., Soares, M.J., Macedo, A.A., Coelho, I., Ferreira, C.P., Azevedo, M.H., Macciardi, F., Kennedy, J.L., and Pato, C.N. (2001). Evidence for linkage disequilibrium between the alpha 7-nicotinic receptor gene (CHRNA7) locus and schizophrenia in Azorean families. Am. J. Med. Genet. 105, 669-674. Yang, X., Criswell, H.E., and Breese, G.R. (1996). Nicotine-induced inhibition in medial septum involves activation of presynaptic nicotinic cholinergic receptors on gamma-aminobutyric acid-containing neurons. J. Pharmacol. Exp. Ther. 276, 482-489. Zhang, H., and Karlin, A. (1998). Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor. Biochem. 37, 7952-7964. Zhang, X., and Nordberg, A. (1993). The competition of ()-[ 3 H]nicotine binding by the enantiomers of nicotine, nornicotine and anatoxin-a in membranes and solubilized preparations of different brain regions of rat. Naunyn-Schmiedebergs Arch. Pharmacol. 348, 28-34. Zoli, M., Moretti, M., Zanardi, A., McIntosh, J.M., Clementi, F., and Gotti, C. (2002). Idnetification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum. J. Neurosci. 22, 8785-8789.

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BIOGRAPHICAL SKETCH Kristin Marie Wildeboer was born February 23, 1978, in Grand Haven, Michigan, to Stanley and Judith Wildeboer. She was born 6 minutes after the birth of her twin brother, Todd. Kristin also has a brother, David who is 4 years her junior. When Kristin was 4 years old, her family moved to Ft. Wayne, Indiana where she grew up and attended Snider High School for a year before her family moved to Tampa, Florida. Kristin finished her high school education at Gaither High School, in Tampa. For her college education, Kristin attended Stetson University in Deland, Florida. In Spring 2000, she received her Bachelor of Science degree with a major in biology and a minor in psychology. In the fall, she began her graduate career in the Interdisciplinary Program at the University of Florida. In Spring 2001 Kristin joined the laboratory of Dr. William Kem, to study binding and functional properties of nicotinic acetylcholine receptors. 107


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STRUCTURE ACTIVITY RELATIONSHIPS OF NICOTINE ANALOGS AND
Erythrina ALKALOIDS ON THE ALPHA 4 BETA 2 NICOTINIC ACETYLCHOLINE
RECEPTOR















By

KRISTIN MARIE WILDEBOER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005





























Copyright 2005

by

Kristin Marie Wildeboer

































To those I hold dearest: my parents















ACKNOWLEDGMENTS

At the completion of my graduate career I will have spent over three-fourths of my

life in an academic environment. That amounts to many, many hours that teachers and

professors have spent educating, guiding, listening, understanding and encouraging me

both in and out of the classroom. I feel it is important to acknowledge and thank them all

for their time and dedication to their students, and for helping to mold me into someone

who possesses an intellectual curiosity that has driven me to where I am today.

During my graduate studies I have turned to numerous people that for assistance,

whether for experimental advice or for help with purchase orders. I would like to thank

the Pharmacology Department staff: Donna Desmond-Kuhn, Barbara Reichert, Judy

Adams, Lynn Rogers, Cookie Mundorff, Lynn Raynor, LaTrelle Davis, and Kari Bastow.

I also thank Debbie Otero for lending binding assay supplies and advice over the years. I

also need to thank BJ Streetman (in the Neuroscience Department) for help with

registration and other such questions, which have been numerous.

My family deserves much credit and recognition for being extremely supportive

and understanding. I thank my father, Stanley; my mother, Judy; and my brothers, Todd

and David. Although they may not understand what I have been studying, have always

encouraged me. I also thank my dear friends who always provided laughter and

compassion when needed. I especially thank Susan LeFrancois, my partner in crime

during my years in the lab. She is one of the kindest, funniest, most caring people I have

ever met and I am glad I had the opportunity to get to know her.









I would like to thank current and past members of my supervisory committee: Dr.

Stephen Baker, Dr. Thomas Vickroy, Dr. Laszlo Prokai, and Dr. Neil Rowland. I give

special thanks to Dr. Roger Papke, who has gone above and beyond the role of a

committee member, set aside many hours over the years to answer my questions (no

matter how big or small), and always challenged me to better understand all that is

membrane bound. I extend many thanks to Dr. Ferenc Soti for the isolation and

syntheses of the many compounds for my study. I could always count on him to correct

my chemical structures and names. I also thank Dr. Jon Lindstrom for graciously

supplying the tsA201 cells used in functional assays.

Finally, I would like to express my deep gratitude to the person who has guided and

helped shape my research most significantly: my mentor, Dr. William Kem. I would like

to thank him for giving me the opportunity to be a member of his lab. It has been an

educational, enjoyable ride that I know I will miss when it ends.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST O F TA B LE S .................................................. .............. .. viii

LIST OF FIGURES ................................... ...... ... ................. .x

ABSTRACT ........ .............. ............. ...... ...................... xi

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Superfamily of Ligand-Gated Ion Channels.........................................................
Characterization of Nicotinic Acetylcholine Receptors (nAChRs)..............................2
Studies of the M uscle-type nAChR ................ ............. ................ 2
M uscle-type nA ChR Stoichiom etry ........................... .......... ................ ... 3
Using Electron Microscopy to Study the Two-Dimensional Structure .............4
Studies of Neuronal nAChRs .................. ........ ........ ...................5
Structure of nA C hR s ................... ................ .. ............ ........ ........... .. .. ........... ... .7
Site-Directed Mutagenesis of the Receptor and Substituted Cysteine
A accessibility M ethod ............................................. .............. ........ 7
The A C h-B finding Protein........................................................... ............... 8
Pharmacology and Function of nAChRs ...... .................................11
Pharm acology of nA ChR s .......... ....... ........ ... ...... ........... ..................... 1
Distribution and Physiology of nAChRs in the Mammalian CNS...................15
F unction of nA C hR s ..................... ..................... .............. .. ................ .. 16
Functional states of the nAChR ....................................... ............... 19
Neuronal nAChR Involvement in Disease .................. ................... ...............21
Structure Activity Relationships of Neuronal nAChRs.................. ............24

2 M A TERIAL S AN D M ETH OD S .................................................... .....................28

R adioligand B finding .......... .......... .. .. ...... .. ...................... .. ................ .. 28
C om pound Syntheses ................................................ .............................. 28
Rat Brain M em branes...... ...................................... ................. 29
Human Embryonic Kidney Subclone Cell (tsA201) Membranes .....................29
Binding Assay Protocol ............................ ....... .. .. ...... ............. 30
B finding A ssay D ata A nalysis........... ................. ........................ ....... ........ 31









F unctional M easurem ents ................................................................ .....................32
C ell C culture ................3....... ............... ... ............. ................ 32
Membrane Potential Dye, Cell Loading and Compound Plate Preperation........32
M membrane Potential M easurem ents ....................................... ............... 33
Functional Assay Data Analysis.................................. .. ... ............... 34
High Performance Liquid Chromatography (HPLC) Chiral Separation of
N icotin e A n alo g s ................................................................... ................ .. 3 4

3 R E S U L T S .............................................................................3 6

N icotine A nalogs .............. ..............................................................36
(S)-Nicotine and (S)-Nornicotine ......... ......... .... ................. ............... 40
5-Pyridyl Substituted A nalogs....................................... .......................... 44
5'-Pyrrolidine Substituted Analogs ...................................... ......................... 46
1'-N -Pyrrolidine Substituted A nalogs.............................................................. 47
3'-Pyrrolidine Substituted Analogs ...................................... ......................... 50

4 R E S U L T S .............................................................................5 4

Separation of Nicotine Analog Enantiomers ............................................................54

5 R E S U L T S .............................................................................6 1

E rythrina A lkaloids .......................... .............. ................. .... ....... 61
-Erythroidines .................................. .......................... .. ......... 63
A rom atic A lkaloids ....................... .. .... ........................... ...... ............70

6 DISCUSSION .............. ................................ ............... 74

Substituents at Four Different Positions on Nicotine Decrease Affinity for the
a4P2 nAChR and Confer Partial Agonist and Antagonist Properties ..................75
Separated Nicotine Analog Enantiomers Display a Difference in Affinity for
a 4P2 U like N nicotine .......... ............... ..... .. ...... .. ......... ....................83
Substituents on the D-ring of the Erythrina Alkaloids Allow for High Affinity
Binding to the a432 nAChR and Inhibition of the a432 Acetylcholine
R e sp o n se ......................................................................... 8 4
C o n clu sio n s..................................................... ................ 8 9

REFERENCES ..................... .... ......... ................. ........ 90

BIOGRAPHICAL SKETCH ............. ................................... .................................. 107















LIST OF TABLES


Table page

3-1 Inhibition of 125I-a-bungarotoxin (btx) and 3H-cytisine binding to rat brain
membranes or inhibition of 3H-cytisine binding to human a432 expressing
tsA201 cell membranes by (S)-nicotine or (S)-nornicotine ...............................41

3-2 Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by 5-pyridyl substituted analogs. .......................................................... 45

3-3 Inhibition of human a432 receptor acetylcholine (ACh) responses by 5-pyridyl
sub stituted analogs. ......................... .......................... .. ....... .... ........... 46

3-4 Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by 5'-pyrrolidine substituted analogs. .............................................47

3-5 Inhibition of human a432 receptor ACh responses by 5'-pyrrolidine substituted
a n a lo g s ................................. ........................................................... ............... 4 7

3-6 Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by I'-N- pyrrolidine substituted analogs. ...........................................48

3-7 Inhibition of human a432 receptor ACh responses by l'-N-pyrrolidine
sub stituted analogs. ......................... .......................... .. ....... .... ........... 50

3-8 Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by 3'-pyrrolidine substituted analogs. .............................................52

3-9 Inhibition of human a432 receptor ACh responses by 3'-pyrrolidine substituted
a n a lo g s ................................. ........................................................... ............... 5 3

4-1 Inhibition of 3H-cytisine binding to rat brain or to human a432 expressing
tsA201 cell membranes by nicotine enantiomers ................................................58

5-1 Structures, nomenclature and binding results for P-erythroidine,
dihydro-P-erythroidine and tetrahydro-P-erythroidine. ........................................64









5-2 Structures, nomenclature and binding results for 3-desmethoxy-P-erythroidine,
N-methyl-P-erythroidine and P-erythroidinediol. ..................................................65

5-3 Inhibition of human a432 receptor ACh responses by natural product and semi-
sy nth etic P -erythroidin es ........................................... ......................................... 69

5-4 Structures, nomenclature and binding results for the aromatic alkaloids
erysovine, erysodine and erythraline ............................ ............... ............... 71

5-5 Structures, nomenclature and binding results for the aromatic alkaloids
erysotrine, and glucoerysodine................................ ......................72

5-6 Inhibition of human a432 receptor ACh responses by natural product aromatic
E rythrina alkaloids ....................... ...... ............ .............................73















LIST OF FIGURES


Figure page

3-1 Signal acquired for membrane potential fluorescence using tsA201 cells
expressing human 4 2 nAChRs. ........................... ...................................... 43

3-2 Concentration response curves of nicotine, ACh and nomicotine for human
a432 receptors expressed in tsA201 cells as measured by changes in membrane
p potential ......... ..... .......................... .....................................................44

3-3 Average effects of the l'-N-pyrrolidine substituted analogs on human a432
receptors expressed in tsA201 cells as measured with membrane potential dye.....50

3-4 Average effects of the 3'-pyrrolidine substituted analogs on human a432
nAChRs expressed in tsA201 cells as measured with membrane potential dye......53

4-1 HPLC chiral separation of l'-N-ethyl-(S,R)-nornicotine. ....................................57

4-2 Concentration response curves for the enantiomers of nicotine and nornicotine
for human a432 receptors expressed in tsA201 cells as measured by changes in
m em brane potential. .......................... ................ .................. ........ 60

5-1 The common heterocyclic structure of Erythrina alkaloids................................62

5-2 Concentration response curve of dihydro-P-erythroidine (DH3E) for human
a4P2 receptors expressed in tsA201 cells as measured by changes in membrane
p o te n tia l ...................................... ............ ................... ................ 6 6

5-3 Concentration response curve of ACh in the presence and absence of 1 [tM
DHPE for human a432 receptors expressed in tsA201 cells as measured by
changes in m em brane potential. ......................................................................... 67

5-4 Signal acquired for a432 nAChRs upon simultaneous application of DHPE and
A C h ............................................................. ................ 6 8

5-5 Structure of a Phelline alkaloid, O-methylisophellibiline................... ............70















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

STRUCTURE ACTIVITY RELATIONSHIP OF NICOTINE ANALOGS AND
Erythrina ALKALOIDS ON THE ALPHA 4 BETA 2 NICOTINIC ACETYLCHOLINE
RECEPTOR

By

Kristin Marie Wildeboer

December 2005

Chair: William R. Kem
Major Department: Neuroscience

Neuronal a432 nicotinic acetylcholine receptors (nAChRs) are the major high

affinity nAChRs in the mammalian brain and have been implicated in mediating the

dopamine-releasing and cognition-enhancing effects of nicotine. Nicotine, an alkaloid

from the plant genus Nicotiana, is an agonist with a high affinity for the a432 receptor.

Another high-affinity ca432-nAChR ligand is dihydro-P-erythroidine (DH3E), which is a

competitive antagonist from the plant genus Erythrina. The goal of this study was to

assess structural activity relationships of these prototypic plant alkaloids for a432

through characterization of the receptor binding and functional properties of several

natural, semi-synthetic and synthetic analogs.

Substituents were added at four different positions on nicotine: the 5'-, 3'- and 1'-

N-positions on the pyrrolidine ring, and position 5 on the pyridine ring. We hypothesized

that substituents at these positions would permit interaction with the a432 receptor yet









influence the functional properties of these compounds. Modifications at any of these

positions resulted in a decreased affinity for a432 relative to nicotine. Six of eleven

analogs displayed partial agonist activity, three were antagonists and the remaining two

were inactive on human a432. Increasing substituent size at the l'-N-pyrrolidine

position greatly reduced affinity but had less influence on the maximum effects

measured. The 5'-trans-methyl-(S)-nicotine had over 90-fold greater affinity than the cis-

isomer and was an antagonist. Separated enantiomer fractions of 1'-ethyl-(S,R)-

nornicotine and 5-phenyl-(S,R)-nicotine bound with 10-fold or greater difference in

affinity to rat a432.

Two groups of Erythrina compounds were studied: the D-ring lactones (P-

erythroidines) and the D-ring aromatic compounds. All alkaloids that had measurable

affinity for the receptor also retained their full competitive antagonism. The aromatic

compounds were the most active; erysovine displayed a higher affinity than its isomer

erysodine. Opening the D-ring of P-erythroidine did not result in loss of activity; major

determinants for binding to a432 must also exist within the other three rings. Finally,

dihydro-P-erythroidine was more active than either P-erythroidine or tetrahydro-P-

erythroidine.














CHAPTER 1
INTRODUCTION

The brain is a monstrous, beautiful mess. Its billions of nerve cells-called
neurons-lie in a tangled web that displays cognitive powers far exceeding any of
the silicon machines we have built to mimic it. -Allman, 1989 page 3

The brain remains a mystery of intricate circuits that allow for acquisition,

integration, and retrieval of information. This "tangled web" of neurons forms synapses

that allow for information transfer through specialized methods of signaling, both

chemical and electrical. Neurotransmitters are chemical signals that, upon binding to

specific target sites on neurons (known as receptors) can change the electrical properties

of the target cell. Receptors, along with neurotransmitters, are critical for communication

between neurons.

The theory of receptors first originated in 1878, when the British physiologist John

N. Langley was studying the actions of atropine and pilocarpine on salivary secretion of

cats, and found that the inhibitory action of atropine could be overcome by increasing the

concentration of pilocarpine. In 1905, Langley was studying the effects of nicotine on

striated muscle in fowl when he first used the term "receptive substance" which (we now

know) referred to the nicotinic acetylcholine receptor (nAChR).

Superfamily of Ligand-Gated Ion Channels

Nicotinic acetylcholine receptors belong to a superfamily of ligand-gated ion

channels. The superfamily also includes 5-HT3, glycine and A and C type GABA

(y-amino butyric acid) receptors. Members of this superfamily of receptors all have a

conserved sequence consisting of a pair of cysteines separated by 13 amino acids and









linked by a disulfide bridge; thus they are also known as the Cys-loop receptors (Kao and

Karlin, 1986). Each of these receptors has a central ion channel, around which are

organized five homologous subunits, to which endogenous neurotransmitter may bind

and cause a conformational change: allowing ions (including sodium, potassium, calcium

and/or chloride) to flow through the channel. The vertebrate nAChRs are cationic

ligand-gated ion channels that allow for the passage of positively charged ions cationss)

through the channel (Takeuchi and Takeuchi, 1960), whereas glycine and GABA

receptors permit the flow of anions.

Characterization of Nicotinic Acetylcholine Receptors (nAChRs)

The nicotinic acetylcholine receptor is the best-studied of the ligand-gated ion

channels. Its name arises from the fact that nicotine, a plant alkaloid extracted from

tobacco, binds to these receptors, as does the endogenous neurotransmitter acetylcholine.

A primary reason that nAChRs are well characterized is due to the use of tissues derived

from the electric organs of certain fishes, including the Torpedo, which are a rich source

of nAChRs (Feldberg et al. 1940). The receptors in these electric organs were found to

have a high degree of homology to those of the vertebrate embryonic muscle type

nAChR.

Studies of the Muscle-type nAChR

Receptors from the Torpedo were first affinity-purified to determine the number,

stoichiometry, and subunits involved (Olsen et al. 1972). Each receptor was determined

to be a 250-kilo-Dalton protein consisting of a, 3, 6, and y-subunits (Reynolds and

Karlin, 1978; Lindstrom et al. 1979). When covalently labeling the subunits, the

a-subunit was found to be twice as abundant as the others (Reiter et al. 1972; Weiland et









al. 1979). This led to the theory that the receptor consisted of two a-subunits in the

formation of the pentameric receptor.

Acetylcholine (ACh) produces its effects on a cell by binding to the nAChR. The

location of and number of binding sites on the Torpedo (muscle-type) receptor were

determined by using snake venom peptides (a-toxins) that served as high-affinity labels

for the ACh binding sites (Lee and Chang, 1966). The ability of ligands to bind to the

receptor could now be studied using radiolabeled a-toxins that would compete with

unlabeled agonists and antagonists for the ACh-binding site on the receptor (Weber and

Changeux, 1974). One of the snake a-toxins, a-bungarotoxin (a-btx), bound to two sites

per receptor oligomer, while the ratio of ACh to a-btx binding was close to one (Neubig

and Cohen, 1979). These data indicated that there were two ACh binding sites for the

Torpedo receptor. It was initially presumed that ACh was binding to some extracellular

portion of the receptor because the positive charge of the quaternary ammonium of ACh

prevents it from crossing the cell membrane. More specifically, Silman and Karlin

(1969) showed that the ACh binding sites were located near a disulfide bond by using

bromoacetylcholine to form a covalent bond with the receptor after reduction of exposed

cysteines.

Muscle-type nAChR Stoichiometry

The stoichiometry of the Torpedo receptor subunits was confirmed as (al)2(P31)Y6

in the early 1980s by Raftery et al. (1980) who performed N-terminal microsequencing of

the subunits. These N-terminal sequences allowed for creation of degenerate

oligonucleotides to probe the Torpedo cDNA library (Ballivet et al. 1982; Claudio et al.

1983; Noda et al. 1982, 1983). The sequences showed homology among the subunits;









and based on these sequences, hydrophobicity plots were created (Claudio et al. 1983)

that led to models of how each of the subunits folds in the cell membrane.

Hydrophobicity plots suggested that each subunit contains a large extracellular domain,

followed by four hydrophobic transmembrane domains (known as Ml to M4): with a

large cytoplasmic domain between M3 and M4 (Schofield et al. 1987; Unwin, 1993).

The receptor is about 80 A in diameter and extends 65 A above the membrane, 35 A

below the membrane, and 40 A across it. The central channel is about 30 A in diameter,

while the ACh-binding sites are located about 30 A above the membrane (Unwin, 1993).

Availability of cDNAs allowed the development of expression systems.

Expression systems allowed for manipulation of subunits by omissions and mutations

that permitted further exploration of the ligand-binding domain. The difference between

the a-6 and a-y pairs of subunits accounted for the difference in affinity of curare (a

competitive antagonist) binding to the wild type receptor (Blount and Merlie, 1989). The

binding sites in the case of vertebrate adult muscle exist between a-6 and ca- subunits,

because the y-subunit is replaced by an e-subunit in adults to give a stoichiometry of

(alc)2(pl)s6 (Mishina et al. 1986). It has been proposed, based on DNA sequences, that

the subunits that compose the muscle-type nAChR are the most recent evolutionary type

of nAChR (estimated to have developed 490-520 million years ago); and that the most

ancient form of nicotinic receptor is the homomeric receptor (Le Novere and Changeux,

1995; Ortells and Lunt, 1995).

Using Electron Microscopy to Study the Two-Dimensional Structure

Through the use of cryoelectron microscopy, the two-dimensional structure of

nAChRs began to take shape. Unwin and colleagues (1988) established conditions for









forming ice crystals of nAChRs from Torpedo that showed the receptor to consist of five

"barrel staves" in a circular arrangement surrounding a central channel at a resolution of

17 and 18 A (Toyoshima and Unwin, 1988). As previously mentioned, each subunit is

made up of four transmembrane domains known as Ml to M4. At a higher 9 A

resolution, it was possible for Unwin to study the electron densities of each subunit.

Initial evidence (Wilson and Karlin, 1998) indicated that the M2 transmembrane domain

of each subunit lined the central channel. It was also postulated that the four

transmembrane domains were composed of a-helices based on their amino-acid sequence

(Claudio et al. 1983; Devillers-Thiery et al. 1983; Noda et al. 1983; Unwin, 1993).

Unwin provided valuable data on the motion of nAChR activation by collecting

images of the nAChR from Torpedo before and after a brief exposure to ACh at 9 A.

Results showed that upon activation, the subunits rotate on an axis parallel to the central

channel. The data also corroborated the proposed location of channel gate as determined

by Wilson and Karlin (1998) at a position halfway in the membrane postulated to be

formed by leucine residues from each subunit (Unwin, 1995). At a resolution of 4.6 A,

the extracellular structures of the subunits appeared to be composed of P-sheets. These

higher-resolution images also showed that these P-sheets form the extracellular entrance

to the channel (which is lined by a-helices) by connecting it to the ACh-binding pockets

and that a central channel exists with lateral openings below the membrane that serve as a

filter for ions (Miyazawa et al. 1999).

Studies of Neuronal nAChRs

In the early 1990s the a7 gene was cloned from both chick and rat brain (Couturier

et al. 1990; Schoepfer et al. 1988; Seguela et al. 1993). These receptors were shown to









have a very high affinity for a-btx. They were determined to be homomeric, or consist of

five identical subunits. There also existed a group of nAChRs that did not bind a-btx

with high affinity, but rather had a high affinity for nicotine or ACh and had a distinctly

different labeling pattern from that of c7 (Clarke et al. 1985). This group of receptors

was found to be composed of both a- and non-a subunits. Cloned subunits were

classified as a subunits if they contained vicinal cysteines in their N-terminal domain,

homologous to positions 192 and 193 in the Torpedo. The non-a subunits that lacked

these vicinal cysteines were designated as P-subunits.

There are currently 12 known neuronal nAChR subunits, c2 to a10 and 32 to 34.

The c7-, c8-, and c9-subunits can form homomeric channels. The most commonly

expressed homomeric nAChR in mammals is the a7-receptor. While c9 and a10 are

also expressed in mammals, a8 nAChR expression has only been found in chickens

(Gerzanich et al. 1993; Keyser et al. 1993). Heteromeric receptors contain combinations

of a- and P-subunits. The stoichiometry ratio of a- to P-subunits was shown to depend

on the expression system of the receptors and other factors, including temperature and

pharmacological treatment (Nelson et al. 2003). The assembly composition of these

heteromeric receptors is what gives rise to the diversity of nAChR properties, both

binding and function.

The location at which the nicotinic agonist ACh binds is known as the

ligand-binding domain (LBD). The LBD is located between the interface of the a-

subunit and its adjacent subunit on the N-terminal domain. The N-terminal domain is

about 210 amino-acid residues long. Heteromeric receptors contain two ACh-binding

sites, while homomeric receptors might contain up to five ACh-binding sites. The LBDs









are composed of six loops, three from the contributing a-subunit and three from the

non-a-subunit. In the case of homomeric receptors the adjacent a-subunits each

contribute three loops. The loops from the a-subunits are considered the primary loops

and are known as A, B and C (Galzi et al. 1991) while the loops from the non-a-subunits

are the complementary loops D, E and F (Corringer et al. 1995). These loops and their

residues were determined through numerous photoaffinity labeling, mutatgenesis,

radioligand binding and functional assays.

Structure of nAChRs

Site-Directed Mutagenesis of the Receptor and Substituted Cysteine Accessibility
Method

Site-directed mutagenesis has been a valuable tool for investigating residues

important in various aspects of the receptor. The receptor channel incorporates

negatively charged amino acids that promote the passage of cations (Pascual and Karlin,

1998). Mutating these residues to uncharged amino-acids decreases the cationic

conductance of the receptor (Imoto et al. 1998). Interestingly the cationic selectivity of

the receptor can be converted to an anionic selectivity by mutating only three residues in

the M2 domain and the M1-M2 loop, as was done with the chick a7 receptor (Galzi et al.

1992). Mutagenesis studies have determined that a threonine residue at position 59

within the 32-subunit is important for the interaction of the competitive antagonist

dihydro-P-erythroidine with a332 receptors (Harvey and Luetje, 1996).

The substituted cysteine accessibility method (SCAM) proved to be a valuable tool

for initial identification of all the residues lining the M2 domains of the muscle type

nAChR (Akabas et al. 1994; Zhang and Karlin, 1998); which (as previously determined)

is the domain that lines the channel of the receptor and allows conduction of cations.









SCAM has identified four important residues that exist in the middle of the M2 domain

and are part of the channel gate for the active state of the receptor (Wilson and Karlin,

1998).

The ACh-Binding Protein

In 2001 there was a breakthrough in the structural analysis of the nAChR. A group

in the Netherlands discovered a soluble protein from the fresh water snail Lymnaea

stagnalis (Brejc et al. 2001; Smit et al. 2001). The protein had a sequence identity

similar to subunits from the nAChR, as well as the other cys-loop ligand-gated ion

channels. The discovery came about when experiments on pre- and postsynaptic

cholinergic neurons from Lymnaea determined that excitatory postsynaptic potentials

(EPSP) occurred only in the absence of glia cells. Further exploration found a protein

secreted from the glia that bound the ACh released in the synapse. Thus it was named the

acetylcholine-binding protein (AChBP). It has been proposed that Lymnaea can use the

AChBP as a buffer to directly modulate cholinergic synaptic transmission (Smit et al.

2003).

Purification and molecular cloning of the protein determined that it is made up of

five subunits. Each subunit consists of 210 residues forming a homopentamer (Brejc et

al. 2001). Each subunit has a ligand binding-domain between the N-terminals of adjacent

subunits but lacks the pore-forming transmembrane domains. The majority of the

residues that are conserved among the nAChRs are also conserved in the AChBP, which

makes it a good model for the N-terminal domain of the nAChR including the LBD. The

AChBP has the highest sequence homology to the nAChR a-subunit extracellular

domains with a 23.9% similarity to the a7 nAChR (Smit et al. 2001). Using radiolabeled









a-btx the IC5o values were determined for several nicotinic agonists and antagonists

including a-btx (2.6 nM), nicotine (98 nM) and ACh (4.2 [LM), these values are more

comparable to the pharmacology of homomeric receptors, specifically a7 and c9, over

the high affinity heteromeric nAChRs (Smit et al. 2001).

Brejc et al. (2001) crystalized this protein at 2.7 A. The measurements of the

AChBP are 62 A in height, a diameter of 80 A and a pore size of 18 A, these

measurements parallel Unwin's data on the Torpedo nAChR. The residues important in

the LBD were previously determined by biochemical and mutagenesis data. The crystal

structure confirmed the involvement of these residues as well as elucidated how these

residues are positioned in the binding domain. The principal side of the LBD consists of

loops from the a-subunit homolog known as loops A, B and C while the complementary

side, the P-subunit homolog in heteromeric receptors and a-subunits in homopentameric

receptors, consists of loops D, E and F made up of P-sheets. The residues in the binding

site (as crystallized with a HEPES molecule), which are primarily aromatic and

hydrophobic, are as follows: loop A, tyrosine 89; loop B, tryptophan 143 and 145; loop

C, tyrosine 185 and 192, and cysteines 187 and 188. Important LBD residues from the

complementary side are: loop D, tryptophan 53 and glycine 55; loop E, arginine 104,

valine 106, leucine 112, and methionine 114; loop F tyrosine 164 (Brejc et al. 2001;

Corringer et al. 2000; Dougherty and Lester, 2001). AChBP has yet to be crystallized

with an empty binding pocket. The original crystals contained a HEPES molecule in the

ACh binding site.

Recently nicotine and carbamylcholine, both nicotinic agonists have been

crystallized in the AChBP (Celie et al. 2004). The data reaffirmed the proposed aromatic









and hydrophobic residue contacts between these nicotinic agonists and the ACh binding

site. The study by Celie et al. (2004) determined that hydrogen bonds exist between the

receptor and the nitrogens on the pyridine and pyrrolidine rings of nicotine. Also, the

carbonyl oxygen of W143 interacts with the positively charged nitrogens of nicotine and

carbamylcholine. Interestingly they found that both ligands bind with their nitrogen

atoms at nearly the same position in the binding pocket. A comparison between the LBD

of the a432 nAChR and the AChBP reveal three residue substitutions, R104 (AChBP) is

replaced by V109 (ca42), L112 by F117 and M114 by L119 (Celie et al. 2004).

The utilization of the AChBP for molecular modeling has its limitations. One of

the major limitations being that the AChBP is not a functioning channel so modeling

ligand binding may not reflect molecular changes in movement that the nAChR undergo

upon activation and desensitization. Unwin (2005) has helped to shed some light on the

conformation of the receptor in the unliganded, or closed state. He has determined at a

4A resolution of the Torpedo nAChR in a closed conformation that the C loop of the a

subunits projects away from the receptor and that upon agonist binding the C loop moves

in toward the receptor's ACh binding site, which along with the movement of the B loop

allow for a conformational change that would permit receptor activation and channel

opening. Unwin (2005) terms this conformational state of the B and C loops of the a

subunits as "distorted" and that upon ligand binding both a salt bridge and hydrophobic

interactions are broken, which allow for the loops to change to a conformationally

"relaxed" state of the receptor.









Pharmacology and Function of nAChRs

The majority of the initial information about nAChRs came from ligand binding,

photoaffinity-labeling, electrophysiological and mutagenesis experiments. Although the

initial properties were determined by electrophysiological methods in the 1940s, it was

only after the cloning of genes coding for nAChRs that electrophysiological experiments

could be put to use for studying the physiology of specific subunit combinations. Much

of the data up to that point relied upon radioligand-binding studies to elucidate the

pharmacology of nAChRs expressed in various tissues and regions throughout the brain.

Pharmacology of nAChRs

The initial pharmacology of AChRs was largely based on the study of natural

products and their ability to bind and affect nAChRs. Plant compounds including

nicotine, muscarine, atropine, curare and physostigmine were all utilized due to the

finding that they produced either activation or inhibition, depending upon concentrations

used, of cholinergic (either nicotinic or muscarinic) transmission. The ability to

radiolabel these compounds provided the evidence for the location of nAChRs in various

tissues as well as provided the capability to measure the tightness with which these

radiolabeled compounds bound to the receptor, known as affinity. A ligand that

reversibly binds directly to the ACh binding site is called a competitive ligand. It will

compete with ACh for the same binding site on the nAChR and with increasing

concentrations of ligand it will displace ACh from the binding pocket. A ligand that does

not bind to the ACh binding site is termed non-competitive and will not displace ACh

with increasing concentrations. Non-competitive ligands bind to other sites on the

receptor known as allosteric sites. Local anesthetics are ligands that bind to nAChRs at

sites other than the ACh binding site (Papke and Oswald, 1989; Sine and Taylor, 1982).









A group of alkaloids from the venom of Tetraponera ants have also been initially

characterized as noncompetitive antagonists at several nAChR subtypes and are likely

binding at a site within the channel (Kem et al. 2004b).

There are several considerations when radiolabeling a compound that is to be used

as a ligand in studying nAChRs. One concern is that the radiolabel must have a high

enough specific radioactivity that binding of nanomolar (nM) concentrations of ligand

can be reliably measured. It is also a requirement that the radiolabel not interfere with

binding of the ligand to the receptor. Also, it is important that the label is not

metabolized or exchanged during the experiment. Non-saturating sites on the membrane

to which a radioligand binds are known as non-specific binding sites. It is important that

the radioligand can be used to differentiate between non-specific binding and binding to

only the receptors, known as specific binding. Obtaining specific binding is usually

performed by adding a high concentration of a compound, such as nicotine, that binds

specifically to the receptor sites in order to knock off the radioligand from the receptors,

thus the radioligand is only bound to the non-specific sites. The counts due to

non-specific binding (these are proportional to ligand concentration) can be subtracted

from the total binding counts of radioligand bound to the cell membranes in order to

determine specific binding. Receptor binding should be saturable, reversible, and over a

period of time reach equilibrium binding. It is also ideal if the ligand is selective so that

is binds with a high specificity to a specific subtype of receptor, or in the case of early

development of nAChR radioligands specifically to nAChRs.

One such compound that was initially employed for utilization in studying nAChRs

was radiolabled muscarine (Waser, 1961). The problem with using muscarine, a toxin









extracted from mushrooms, to study nAChRs is that it labeled two types of acetylcholine

receptors, the ligand-gated nicotinic as well as the G-protein coupled muscarinic

receptors. Acetylcholine also labels both types of ACh receptors and is readily

hydrolyzed by acetylcholinesterase into acetate and choline, thus it is usually preferred to

work with a more stable and selective ligand for nAChRs although the binding of

radiolabeled ACh has been characterized in the presence of atropine, to prevent binding

to muscarinic receptors (Schwartz et al. 1982). Several such ligands discussed earlier

were the snake neurotoxins. a-Bungarotoxin has been shown to be specific for certain

nAChRs and has a low nanomolar affinity (Barrantes et al. 1995; Gopalakrishnan et al.

1995). The equilibrium dissociation constant Kd (or Ki) is an inverse measure of this

affinity. Several studies have compared the binding of ACh to nicotine and a-btx in the

rat and mouse brain (Clarke et al. 1985; Marks et al. 1986). These studies indicated that

radiolabeled ACh and nicotine were binding to the same sites with high affinity while

a-btx was labeling different sites. Acetylcholine and nicotine were labeling sites in the

cortex, cerebellum, ventral tegmental area and the thalamus while a-btx was binding at

higher densities in the thalamus and hippocampus. It was soon determined that the

nAChR in the central nervous system to which a-btx was binding was that of a

homomeric a7 receptor (Couturier et al. 1990). Since its first utilization radiolabeled

a-btx has been a commonly used label for c7.

A few years later a relatively selective label, as compared to nicotine or ACh, for

the heteromeric a432 nAChR was discovered. Cytisine, a plant alkaloid from Laburnum

anagyroides, is an autonomic ganglionic agonist. Exploration of its properties on

neuronal cell membranes revealed that cytisine had a very high affinity for those sites









labeled by radioactive nicotine and ACh. It had a measured Kd value of less than 1 nM,

with high levels of binding in the cortex, thalamus and striatum and represented 60-90%

of total binding at the various concentrations examined (Pabreza et al. 1991). Flores et

al. (1992) later determined that cytisine was primarily labeling the heteromeric a432

nAChR. Several ligands that also possess a high affinity for these receptors have been

radiolabeled and studied since the development of cytisine. The compound known as

ABT-418 is a relatively high affinity ligand for a432 that functions as an agonist, and

like nicotine may function as a concentration dependent noncompetitive antagonist

(Papke et al. 1997). Anderson et al. (1995) radiolabeled ABT-418 and measured a Kd

value of 2.9 nM for rat brain. Another ligand that has been radiolabeled for the study of

a4P2 receptors is a semi-synthetic compound derived from the plant genus Erythrina.

The alkaloid known as dihydro-P-erythroidine is commonly used in functional assays as a

competitive nicotinic antagonist. Williams and Robinson (1984) investigated a tritiated

this alkaloid and measured high and low affinity binding sites in rat brain membranes

with respective Kd values of 4 and 22 nM. They found that its distribution of binding in

rat brain was highest in the thalamus and that cytisine was a more potent inhibitor than

nicotine in displacing the radiolabeled alkaloid. Also, mecamylamine did not displace

the radiolabeled dihydro-P-erythroidine at concentrations up to 100 pM. Their data

indicated that the tritiated alkaloid was preferentially interacting with a432 over the other

neuronal subtypes of nAChRs.

The a4P2-subtype has a high affinity for the agonist nicotine that induces a

conformational change (desensitization), which displays this high affinity state

(Changeux et al. 1984; Higgins and Berg, 1988; Buisson and Bertrand, 1998). Agonists









for the a7 nAChR have not been found to induce such a high affinity state of this

particular receptor. Thus, the a432-subtype is referred to as the high affinity nAChR,

while the u7-subtype is known as a low affinity nicotine-binding receptor. Although

there are numerous combinations of nAChR subtypes that form neuronal receptors, the

a432 and a7 nAChRs are the two major subtypes in the mammalian central nervous

system (CNS) (Clarke et al. 1985; Hogg et al. 2003; Pauly et al. 1989; Seguela et al.

1993; Wada et al. 1989; Whiting et al. 1991).

Distribution and Physiology of nAChRs in the Mammalian CNS

Nicotinic receptors in the CNS are known to facilitate synaptic transmission. There

are several studies that indicate the existence of postsynaptic nAChRs in the mammalian

CNS (Alkondon etal. 1998; Clarke, 1993; de la Garza etal. 1987; Frazier etal. 1998;

Schroder et al. 1989) but the lack of evidence for the involvement of these receptors in

EPSPs has lead to the theory that the primary location of nAChRs is presynaptic.

Through autoradiography, immunolabeling, and co-localization with presynatpic

markers, the existence of nAChRs on presynaptic neurons has been firmly established

(Clarke and Pert, 1985; Lubin et al. 1999; Jones et al. 2001). The existence of these

presynaptic nAChRs allows for the regulation of neurotransmitter release. Activation of

these receptors has been shown to result in release of not only ACh (Wilkie et al. 1993),

but also in dopamine (Rapier et al. 1990, Grady et al. 1992), glutamate (Alkondon et al.

1997; McGehee and Role, 1995) and GABA (Alkondon et al. 1999; Radcliffe et al. 1999;

Yang et al. 1996). The release of neurotransmitter through activation of nAChRs on

presynaptic terminals is due to the entry of calcium both through the nAChR channel and

through voltage-gated calcium channels, which are activated upon depolarization of the









cell (Soliakov et al. 1995). All nAChRs are permeable to calcium but the neuronal

receptors have a higher permeability ratio as compared to the muscle subtype. The

c&7-homomeric receptors have the highest permeability to calcium, with a permeability

ratio of about 6.6 or higher (Pca/PNa) (Bertrand et al. 1993; Fucile, 2004; Sands et al.

1993; Sequela et al. 1993). The activation of nAChRs and their subsequent ion

permeability of is not the result of a static entity but rather a fluid movement in the

conformation of the receptor.

Function of nAChRs

The prototypical site for the study of the mammalian nAChR is the neuromuscular

junction. Postsynaptic cells of the muscle form large synapses, which contain millions of

nAChRs. Large amounts of ACh are released from the presynaptic membranes directly

onto the postsynaptic receptors in order to ensure that an action potential occurs in order

to facilitate the movement of muscle. In the early 1950s, there were rapid developments

in the understanding of synaptic transmission thanks in a large part to the study of the

neuromuscular junction. In 1952 Hodgkin and Huxley revealed the mechanism of the

action potential involved in electrical signaling in the giant squid axon. Shortly after Fatt

and Katz (1950, 1951, 1952) provided a better understanding of neurotransmitter release

and the resultant change in ion permeability by ACh. Hodgkin and Huxley employed the

voltage clamp technique to study the permeability changes underlying the action potential

in the giant squid axon. Through the use of a recording and current passing electrode,

voltage clamp allows for the membrane potential of a cell to be held constant while

measuring the change in ionic current flow required to keep the membrane potential at a

constant voltage. This technique allowed for macroscopic measurements of receptor









activation and inhibition. The voltage clamp technique known as patch clamp recording

allowed for microscopic measurements and kinetic analysis of single receptors (Hamill et

al. 1981; Neher and Sakmann, 1976). These techniques were major breakthroughs in the

study of all ligand gated ion channels.

In the past few years other methods of measuring functionality have been

developed. These include both calcium and membrane potential dye measurements.

Fitch et al. (2003) determined the potency and efficacy of nicotinic agonists on various

cell lines expressing several subtypes of nAChRs using either a membrane potential or

calcium fluorescent dye. Both dyes fluoresce due to either changes in membrane

potential or increases in intracellular calcium reflecting activation of the expressed

receptors. Changes in fluorescence can be calculated and graphed to determine either

activation or inhibition concentration constants. They determined that the membrane

potential dye was more sensitive than the calcium dye but that the overall results were

comparable to those of the calcium dye as well as previously published radiolabeled

rubidium efflux assays. The membrane potential dye was also useful in cell lines that

have a low calcium conductance. One disadvantage to using these dyes is the inability to

wash away compound after application. Compounds that function as agonists may

produce a time dependent desensitization of the receptors. Another disadvantage in using

the membrane potential dye is that agonists may appear more potent and antagonists less

potent, as compared to electrophysiological data. This may be due to the phenomenon of

spare receptors (fraction of receptors occupied that produces a maximum response).

As discussed earlier, the ability and strength with which a ligand binds to the

nAChR is known as affinity. It describes how a ligand binds but it does not characterize









its capability to functionally activate the receptor. The term efficacy was coined to

describe the ability of a ligand to activate a receptor (Colquhoun and Sakmann, 1998;

Stephenson, 1956).

A ligand can have varying degrees of efficacy based upon the subtype of receptor

and concentration of ligand. The term agonist describes a ligand that activates a receptor.

The endogenous neurotransmitter ACh is a full agonist for nAChRs. A ligand's efficacy

is often compared to that of a known full agonist, such as ACh, for the subtype of nAChR

being studied. If a ligand activates a receptor with less than maximum activation as

compared to ACh it is termed a partial agonist. For example, cytisine is a partial agonist

for a4P2. It has high affinity for the receptor, a Kd less than one nanomolar (Pabreza et

al. 1991), but it causes only 15% of the maximum response at 1 mM as compared to ACh

(Papke and Heinemann, 1994). Thus, cytisine is less efficacious for the a432 receptor

than ACh.

Ligands that bind to nAChRs but produce no activation are called antagonists.

Antagonists exist as competitive or non-competitive ligands. Competitive antagonists

bind to the ACh binding site and alone cause no change in the activity of the receptor.

However, when present with an agonist that binds to the same site, the competitive

antagonist will inhibit the receptor from activation at specific concentrations.

a-Bungarotoxin, as previously described, is a competitive antagonist for c7 receptors. It

binds to the u7 ACh binding site with a Kd of 0.16 nanomolar (Marks et al. 1986) and

inhibits activation at higher nanomolar concentrations, such as 100nM (Frazier et al.

1998). Non-competitive antagonists can produce the same inhibition as competitive

antagonists, but they do not bind to the ACh binding site. A non-competitive antagonist,









such as the local anesthetics dibucaine or tetracaine, prevents activation of the muscle

type nAChR by binding to one or possibly more allosteric sites on the receptor (Papke

and Oswald, 1989; Sine and Taylor, 1982). Non-competitive antagonists may also inhibit

activation without binding to an allosteric site but rather through blocking passage of ions

through the channel, termed channel-block. Competitive agonists, such as nicotine, may

become non-competitive antagonists at high concentrations due to the ability of nicotine

to cause channel block. The capability of a compound to act as an agonist or antagonist

depends upon the stoichiometry of the nAChR and the stoichiometry of the receptor

determines the kinetics and ion permeability.

Functional states of the nAChR

The ability of nAChRs to successfully facilitate neurotransmission involves several

different conformational states of the receptor. The transition from one state to another is

dependent upon several factors including agonist concentration, time of exposure, and

association and dissociation rate constants for binding. There exist at least three

conformational states for nAChRs, closed, active and desensitized. Monod et al. (1965)

produced their model of allosteric interactions to help explain the transition from one

state to the next. The closed state is an unbound resting state of the receptor such that a

net flow of ions is not passing through the channel. In the absence of agonist nAChRs

exist in an equilibrium that greatly favors the closed state. Upon exposure to an agonist

the receptors no longer exist in that resting equilibrium but rather have become active,

opening their channels and allowing ions to flow through and thus producing a current

across the membrane. The active state requires bound ligand, with at least two agonist

molecules bound to the receptor. With two agonist molecules bound the receptor has a

higher probability of opening than monoliganded receptors (Sine and Taylor, 1980,









1981). The third state that exists is the desensitized state of the receptor. This state,

which was originally studied at the motor end-plate by Katz and Thesleff (1957), is a

nonconducting state that does not respond to additional application of agonist and must

recover before subsequent activation or before returning to the resting state. Although

this is a nonconducting state the conformation of the desensitized receptor is distinctly

different from that of the resting state (Wilson and Karlin, 2001; Unwin, 1995). The

properties of desensitization, such as onset and recovery, are dependent upon receptor

stoichiometry. Desensitization can be induced by properties ranging from agonist

concentration to disruption of the tissue (Whiteaker et al. 1998; Fenster et al. 1999), such

as occurs during homogenization. The neuronal a7 nAChR are the most rapidly

desensitizing of the nAChRs. The rapid desensitization of c7, by high concentrations of

agonist (ACh), is so quick that it complicates data analysis due to the fact that by the time

the agonist solution reaches its full concentration the peak response of the receptor has

already occurred (Papke and Porter Papke, 2002). The fast desensitization of c7 is in

part due to a leucine residue at position 247 in the second transmembrane domain.

Through substitution of a threonine residue for the leucine (L247T) the rate of

desensitization is slowed, but the pharmacology of the receptor is dramatically different

from that of the wild type receptor (Revah et al. 1991; Bertrand et al. 1992). Placzek et

al. (2005) created an a7 gain of function mutant that contained a serine residue in place

of a threonine (as found in wild-type a7) in the second transmembrane domain. This

mutant receptor has longer open times as compared to wild-type a7 with its

pharmacology (for agonists and antagonists) similar to that of the wild-type receptor,

unlike the confused pharmacology of the L247T mutant.









Neuronal nAChR Involvement in Disease

Since nAChRs do facilitate neurotransmission, it is not surprising that they are

also involved in neuronal dysfunction of several disease states. A downregulation of

nAChRs has been found in brains of persons diagnosed with Alzheimer's disease (AD)

(Norberg and Winblad, 1986; Schroder et al. 1991; Court et al. 2000). The primary

target for downregulation is the a432-subtype (Warpman and Nordberg, 1995) although

a7 may also be affected (Perry et al. 2000). The exact involvement of nAChRs in the

impairment of memory and cognition in AD patients is not known, but treatment with

nicotinic receptor agonists and acetylcholine esterase inhibitors have been found to

alleviate some of the clinical symptoms of AD in the early stages of the disease.

Nicotinic acetylcholine receptors have also been found to be downregulated in

Parkinson's disease based upon 3H-nicotine binding assays (Rinne et al. 1991; Perry et

al. 1995; Court et al. 2000). The pathology of Parkinson's disease is the loss of

dopaminergic neurons in the nigro-striatal and ventral tegmentum area-mesocortical

pathways in the brain. Several subtypes of nAChRs have been found to play a role in the

release of dopamine from these neurons. Both a432 and a6f3332 nAChR subtypes are

located on dopaminergic neurons (Champtiaux et al. 2002, 2003; Cui et al. 2003; Zoli et

al. 2002).

There exist at least two neuronal disorders that involve mutations of the gene

encoding for specific nAChR subunits, schizophrenia and autosomal dominant nocturnal

frontal lobe epilepsy (ADNFLE). The chromosome encoding the a7 gene has been

found to be partially duplicated in schizophrenic families (Chini et al. 1994; Leonard et

al. 1996; Freedman et al. 2001; Xu et al. 2001). Interestingly, 90% of schizophrenic









patients self-administer some form of nicotine (Lohr and Flynn, 1992; Poirier et al.

2002). Mutations on genes that encode both the a4 and 32 nAChR subunits are found in

ADNFLE patients. The manifestation of these mutations can be traced to residue

mutations of both the a4 and 32 membrane-spanning domains M2 (Phillips et al. 1995,

2001; De Fusco etal. 2000; Hirose et al. 1999; Steinlein et al. 1995, 1997). The

culmination of these mutations is an increased sensitivity of the receptors to ACh, which

has been proposed to generate seizures resulting from a synchronization of spontaneous

oscillations in the thalamo-cortical circuits (Raggenbass and Bertrand, 2002).

It has been established that nicotinic receptors are involved in the reinforcing

properties of nicotine, in part due to activation of nAChRs on dopaminergic neurons in

the nucleus accumbens and ventral tegmental area (Corrigall et al. 1994; Picciotto and

Corrigall, 2002). Nicotine dependence is often established and sustained through

smoking cigarettes. The chronic and prolonged use of tobacco products often results in

negative health effects including cancer and cardiovascular disease. Although the

detrimental effects of tobacco dependence are produced not through nicotine but the

many other ingredients in cigarettes, it is the nicotine that has been determined to produce

the reinforcement. Nicotine itself has been found to produce the enhancement of

cognition and memory (Levin, 1992; Arendash et al. 1995a). It also has the ability to

both activate and inhibit nAChRs based upon concentration and length of exposure.

The development of knock out mice in which a gene for a specific nAChR subunit

has been mutated to prevent expression have allowed for investigation of subunits

involved in nicotine reinforcement. Picciotto et al. (1995) were the first to develop a

32-knock out mouse. They showed that these knock out mice lacked the majority of high









affinity nAChRs while the levels of a-btx binding remained the same as in wild-type

mice. Most importantly, these mice were shown to have decreased dopamine release in

response to nicotine exposure and do not self-administer nicotine (Picciotto et al. 1998).

Although it has recently been shown that ca6-subunits associate with the 32-subunit in the

CNS, the majority of 32 are associated with a4 (Champtiaux et al. 2003).

The involvement of c432 nAChRs in addiction has lead these receptors to become

targets for the design of pharmacological therapeutics. Over the years several approaches

and therapeutic agents have been tested for their ability to aid in smoking cessation. In

the 1960's cytisine, a partial agonist for a432, was examined but lacked the ability to

readily cross the blood brain barrier. The combination of mecamylamine, a nAChR

antagonist, and nicotine administered through a nicotine skin patch has been shown to be

somewhat beneficial in aiding cessation (Rose et al. 1994). Bupropion, a compound used

to treat depression, has also been determined to aid smoking cessation. The precise

mechanism of action is unknown but it is though to prevent the reinforcing properties of

nicotine (Cryan et al. 2003). More recently a compound known as varenicline, a partial

a432 agonist, has begun clinical trials for smoking cessation (Coe et al. 2005). Based on

these studies it may be expected that a partial agonist would be a useful smoking

cessation agent due to its ability to result in a release of dopamine, yet block additional

dopamine release upon stimulation with a full agonist such as nicotine. The competitive

antagonist, DH3E, has been shown to prevent nicotine self-administration when applied

directly to the ventral tegmental area (Corrigall et al. 1994). Thus, it is also possible that

an antagonist may also function as a smoking cessation agent.









The development of either partial agonists or antagonists may be useful not only for

smoking cessation, but also as in vivo probes for the involvement of c432 in neuronal

processes. Although knock out mice have been created, the involvement of c432

receptors in cognition and nicotine addiction may be masked by compensatory

mechanisms from other nAChR subunits. In order to develop ligands that display partial

agonist or antagonist activity on the a432 receptor, it is necessary to better understand

the interaction of a compound with the receptor. Specifically, what features of the

compound confer affinity and influence functional properties. Structure activity

relationship studies involve analysis of a group of similar compounds, such as nicotine

analogs, where slight to large modification are made to the structure of the compound.

The ability of these modified compounds to bind and activate or inhibit the receptors

provides information useful in the design of a432 specific ligands.

Structure Activity Relationships of Neuronal nAChRs

Nature has provided various molecules, including cytisine and a-btx, which have

allowed for elucidation of structure, location and function of nAChRs expressed in

various tissues. With increased understanding of these receptors and their involvement in

several diseases the possibility of alleviating symptoms or correcting deficiencies often

leads to the re-visitation of natural products. The design of pharmacological agents often

starts with a lead compound, one that has been found to interact with the receptor of

interest. One lead compound that has proved useful in the development of clinically

based therapeutics is a natural product from a marine worm, the toxin known as

anabaseine. Anabaseine itself stimulates all nAChRs (Kem, 1971; Kem et al. 1997) but

is most potent on muscle-type and neuronal a7 receptors (Kem et al. 1997). Through the









addition of a substituted benzylidene moiety, anabaseine becomes a selective partial

agonist for a7 and an antagonist for a432. This compound known as DMXBA

(GTS-21), whose chemical name is 3-(2,4-benzylidene)-anabaseine, has been shown to

be neuroprotective in a PC 12 neuronal growth factor deprived model of cytotoxicity (Li

et al. 1999). It also has been found to enhance cognition in various behavioral tasks

performed by aged rats (Arendash et al. 1995b), and therefore may prove a useful

therapeutic for Alzheimer's disease (Kem, 2000). DMXBA is currently in clinical trials

for correcting the auditory gating deficiency experienced by schizophrenic patients

(Stevens et al. 1998; Simosky et al. 2001).

Similar to the development of DMXBA from the natural product anabaseine,

nicotine may serve as a good lead compound for the design of smoking cessation drugs.

Several previous studies have determined structure activity relationships of nicotine for

the a4P2 nAChR. Substituents at various positions on both the pyridine and pyrrolidine

rings of nicotine have been determined to affect affinity. Fewer studies have addressed

the functional consequences of these substitutions on the a432 receptor. Including Beers

and Reich's (1957) well-known structure activity relationship for agonists and

antagonists with cholinergic binding sites, other findings about the interaction ofligands

with nicotinic receptors have since been discussed. One theory proposed by several

groups is that a cation-7T interaction exists between Trpl49, of the a-subunit of the

Torpedo receptor as well as the AChBP, with the nitrogen atom in an interacting ligand

(such as nicotine) (Beene et al. 2002; T0nder and Olesen, 2001). Structure activity

relationships also exist for other ligands and the a432 nAChR. Epibatidine, the most

potent nAChR agonist, has 7-azabicyclo[2.2.1 ]heptane ring attached to an









exo-5-(2'-chloropyridinyl) substituent. Although it is exceptionally potent, epibatidine is

not selective for particular nAChRs. Carroll et al. (2001) reported that a phenyl

substituent at the 3'-position of epibatidine changed it into an a432 antagonistic in vitro.

Nicotine is a tertiary amine with a chiral carbon at the 2'-pyrrolidine position. In

1957 Beers and Reich proposed that the ability of the naturally occurring (S)-nicotine to

interact with the nAChR existed due a hydrogen bond formed between the nitrogen on

the pyridine ring and through electrostatic interactions by the nitrogen on the pyrrolidine

ring. They measured a minimum distance of 5.9 A from the nitrogen group to the

hydrogen bond. They found this measurement existed for both nicotinic agonists (ACh,

cytisine, nicotine) and nicotinic antagonists (strychnine, P-erythroidine). This initial

analysis of nicotinic ligands was performed in order to determine structural basis for the

interaction with nAChRs.

Although nicotine has a higher affinity for neuronal a432 receptors over U7, it is

not a selective molecule. Thus, the structure of nicotine may be used as a template for

the design of other compounds. This method of study, structure activity relationship,

involves determining the affinity (Ki value) and EC50 or IC50 values of a group of

compounds. This dissertation characterizes the SAR relationship for two groups of plant

alkaloids on the high affinity neuronal a432 nAChR. The first group of compounds are

nicotine analogs, which as we have determined act as weak partial agonists and

antagonists on the a432 receptor.

The other group of compounds are Erythrina alkaloids, which like the commonly

used dihydro-beta erythroidine, also act as antagonists on the neuronal a432 receptor.

Like nicotine they too are tertiary amines. In Beers and Reich's classic study from 1970









they examined the structure of P-erythroidine as compared to ACh in order to determine

structural elements required for binding to the ACh receptor. P-Erythroidine with its

fused ring system is a more rigid molecule than nicotine. Beers and Reich (1970)

determined that the oxygen atoms on rings D and A can both form hydrogen bonds that

measure 5.9 A from the nitrogen between rings C and B. They proposed that although

P-erythroidine is a rigid molecule its ability to bind and inhibit receptor function might be

due to dual hydrogen binding sites.

The goal of this dissertation was to create structure activity relationships for each of

these two groups of plant alkaloids that may be useful in designing partial agonists and

antagonists for the possible treatment of nicotine dependence. These structure activity

relationships may also be useful for designing in vitro and in vivo probes for studying the

function of ca42 nAChRs.














CHAPTER 2
MATERIALS AND METHODS

Radioligand Binding

Compound Syntheses

Dr. Ferenc Soti synthesized all nicotine analogs; purity was determined by reversed

phase HPLC and NMR confirmed compound structure. The nicotine analogs were free

bases. All were suspended in high performance liquid chromatography (HPLC) grade

methanol to create stock solutions of 10 to 100 mM. These stock solutions were further

diluted in 50 mM Tris binding saline containing 2 mg/ml of bovine serum albumin

(Sigma, St. Louis, MO) to create desired concentrations used in binding or functional

assays. The extraction and semi-synthetic preparation of the Erythrina alkaloids were

also performed by Dr. Ferenc Soti according to traditional methods. Following extraction

of several natural aromatic Erythrina compounds, separation was performed by high

performance liquid chromatography (HPLC) using a C-18 column (Beckman Coulter,

Fullerton, CA) as well as by silica gel chromatography. Erysotrine was the only

Erythrina alkaloid that was isolated as a free base. All the other alkaloids were acetate or

hydrochloride salts. The Erythrina alkaloids also were suspended in HPLC grade

methanol to create stock solutions and then further diluted in 50 mM Tris binding saline

containing 2 mg/ml bovine serum albumin.

The (R)-form of nicotine was obtained from Research Biochemicals Inc. (Natick,

MA) as a di-p-toluoyl tartrate salt. The (S)-form of nicotine was obtained from Sigma

(St. Louis, MO) as a tartrate salt. The (R)- and (S)-nornicotines were obtained from









Peyton Jacobs III (San Francisco General Hospital, San Francisco, CA) and both existed

as dicamsylate and camsylate salts, respectively.

Rat Brain Membranes

Whole male Sprague-Dawley rat brains obtained from Pel-Freeze Biologicals

(Rogers, AZ) were prepared according to Marks and Collins (1982). Whole rat brains

were homogenized with a 30 ml Wheaton (location) glass homogenizing tube and pestle

attached to a motor source. The tissue was homogenized in Tris binding saline consisting

of 120 mM NaC1, 5 mM KC1, 2 mM CaC12, 1 mM MgC12 and 50 mM Trizma HC1

Buffer pH=7.4. After homogenization the tissue was centrifuged at 11,000 rpm for a

ten-minute period. After centrifugation the pellet was resuspended in fresh binding saline

and again homogenized. A BCA protein assay (Pierce, Rockford, IL) was then

performed to obtain the protein concentration of the rat brain membranes. Homogenized

tissue was stored at -800C until use. Rat brain membranes at a concentration of 200 |tg

protein were used per tube for radioligand binding.

Human Embryonic Kidney Subclone Cell (tsA201) Membranes

A subclone of the human embryonic kidney cell line (tsA201 cell line) was

graciously provided by John Lindstrom (University of Pennsylvania, Philadelphia, PA).

These cells stably express human ca432 nAChRs on their membranes. These cells were

prepared according to Xiao and Kellar (2004). Cells at about 80 to 90% confluence were

collected after removing the culturing media from the flask (75 cm2) and adding 6-10 mls

of ice-cold Tris binding saline (pH = 7.4) with a disposable cell scraper. The dislodged

cells were then collected in centrifuge tubes and spun down at 1000 rpm for five minutes.

The loose pellet was then collected and homogenized in the same method as the rat brain









membranes. After performing a protein assay the homogenized membranes were stored

at -800C until use. Homogenized tsA201 cell membranes at concentrations of 50 to 125

|tg were used per tube for radioligand binding.

Binding Assay Protocol

The radioligands used in the competition and saturation binding assays were

obtained from Perkin Elmer Life and Analytical Sciences (Boston, MA). 3H-Cytisine (34

Ci/mmol) experiments were performed according to Flores et al. (1992) with a few minor

alterations, specifically that the incubation time was increased to four hours at 40C to

allow for the binding to reach equilibrium. The 125I-a-bungarotoxin (c-btx) (136

Ci/mmol) experiments were incubated at 370C for three hours. Both radioligands were

used at a final concentration of 1 nM in competition binding assays. Homogenized tissue

at the above mentioned concentrations were suspended in 50 mM Tris binding saline

containing 2 mg/ml of bovine serum albumin (Sigma, St. Louis, MO). The tissue along

with the radioligand and compound of interest were incubated in 13x100 mm disposable

glass culture tubes at a final volume of 0.5 ml. For each radioligand nonspecific binding

was measured in the presence of a final concentration of 1 mM (S)-nicotine hydrogen

tartrate salt (Sigma, St. Louis, MO). After incubation radioligand bound membranes

were collected by vacuum filtration with a Brandel cell harvester (Gaithersburg, MD)

onto Whatman GF/C glass fiber filters that had been pre-soaked in 0.5%

polyethylenimine for 45 minutes. The radiolabeled membranes were washed three times

with 3 mls of ice-cold 50 mM Tris binding saline. Filters containing 3H-cytisine bound

membranes were collected in 20 ml scintillation tubes and suspended in 8 mls of 30%

Scintisafe scintillation fluid (Fisher), then counted in a Beckman LS-6500 liquid









scintillation counter (Fullerton, CA). Filters containing 125I-ac-btx bound membranes

were placed in 4 ml scintillation vials and counted in a Beckman 5500B gamma counter

(Fullerton, CA). Whereas each concentration used for saturation and competition binding

assays with rat brain membranes was performed in quadruplicate, each concentration for

assays involving tsA201 membranes was performed in triplicate.

Binding Assay Data Analysis

Binding assay data were analyzed using GraphPad Prism software (San Diego,

CA). The count per minute values for each concentration were averaged and normalized

to the specific binding value obtained within each experiment. Saturation assay data

were analyzed by fitting the data to a one site binding hyperbola model

(Y=Bmax*X/(Kd+X)) in order to determine the Kd and Bmax value for the respective

radioligand and tissue. The data could be transformed into a Scatchard plot for

visualization of the respective slopes and X-axis intercepts using the Prism software.

Competition assay data were analyzed using a sigmoidal dose response with variable

slope (Y=Bottom+((Top-Bottom)/( +10((LogIC50-X)*Hillslope)))) from which a hillslope and

IC50 value were determined; Top = Y value at the top plateau of the curve; Bottom = Y

value at the bottom plateau of the curve. The IC50 value along with the pre-determined

Kd value for the radioligand and nAChR-containing membrane of interest were then used

in the Cheng Prusoff equation (Ki=ICso/(l+(Ligand)/Kd)) to calculate the Ki value.

Significant differences were determined using an unpaired, two-tailed T-test in GraphPad

Prism.









Functional Measurements

Cell Culture

Cells were cultured essentially according to Nelson et al. (2003). The tsA201 cells

expressing ca432 nAChRs were maintained in a culture medium consisting of Dulbecco's

modified Eagle's medium (Gibco, Carlsbad, CA) supplemented with 10% FBS

(MediaTech Inc., Herdon, VA), 100 units/ml penicillin and 100 [tg/ml streptomycin

(Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Irvine Scientific, Santa Ana, CA), 0.5

mg/ml Zeocin (Invitrogen, Carlsbad, CA), and 0.6 mg/ml G-418. Cells were grown in 75

cm2 culture flasks which were housed in a humidified incubator at 370C in an atmosphere

of 5% CO2. Cells were grown to around 80-90% confluence before being split with

0.25% Trypsin (Gibco, Carlsbad, CA) at a subcultivation ratio of between 1:6 and 1:10

weekly.

Membrane Potential Dye, Cell Loading and Compound Plate Preperation

Cells were seeded at a density of roughly 5x104 cells/well to 10x104 cells/well

onto 96-well flat-bottom black-wall culture plates that had been coated with 50 dtl per

well of 50 [tg/ml poly-D-lysine (70-150kDa). Cells were then grown overnight in 100 [tl

of culture medium in order to achieve a single layer of cells on the bottom of each well.

The membrane potential dye, obtained from Molecular Devices, was prepared by

dissolving one bottle of the dye into 30 mls of 1X Hanks' balanced salt solution (HBSS)

supplemented with 20 mM Hepes (pH=7.4). The dye was then added (100 [tl to each

well) on top of the existing 100 [tl of media. Finally, the cells were incubated in the dark

with the dye at 37C for 30 minutes before reading. Serial dilutions of a compound were

prepared in a separate clear-walled 96-well V-bottom plate by evaporation of the correct









volume of a methanol stock solution. The evaporated compounds were then reconstituted

in 250 [tl ofHBSS/Hepes (pH=7.4) containing 1 LM atropine.

Membrane Potential Measurements

Flexstation protocol was performed essentially as described by Fitch et al. (2003)

with several modifications. Fluid transfer and readings were performed by a Flexstation

fluorometer (Molecular Devices, Sunnyvale, CA). The dye (composition withheld for

proprietary reasons) is a lipophilic, anionic, bis-oxonol dye. When the cells are

depolarized the dye enters and binds to cytosolic proteins, causing an increase in

fluorescence signal. During hyperpolarization the dye exits the cells and there is a

decrease in fluorescence signal. Excitation and emission wavelengths were set to 530 nm

and 565 nm with a cutoff of 550 nm. A reading was taken every 1.44 seconds over about

a three minute period for a total of 139 readings per well. The first 17 seconds were used

for a basal read. At 18 seconds the addition of 50 [tl of a test compound is added (to

assess possible agonist activity, EC5o), followed by a 25 [tl addition of KCl (40 mM final

concentration) at 160 seconds to measure the maximum possible signal and to correct for

differences in dye loading and cell count (only the KC1 response of the drug-naive cells

was used for normalization). Compound applications were done at about 78 [tl per

second. Compounds that had no measurable intrinsic activation properties upon the

tsA201 cells were then examined to determine their IC50 values. The compound of

interest was applied to the cells as above except that they were simultaneously applied

with 5 [tM acetylcholine (ACh) in order to measure inhibition of the ACh response. 5

[tM ACh was slightly above the measured EC5o, but well below the dose required to









produce a maximal response, for ACh upon the ca432-expressing tsA201 cells as

measured with the Flexstation.

Functional Assay Data Analysis

Responses of experimental compounds were measured as the maximum RFU

(relative fluorescent unit) subtracted from the averaged basal read for each individual

well. Data was collected with SOFTmax Pro software from Molecular Devices

(Sunnyvale, CA). These raw compound values were then divided by the average

maximum response of cells in control wells to 40 mM KC1 (compound naive cells run in

parallel). It was necessary to use values from control wells because it was determined

that KC1 response larger for cells previously exposed to agonist. The compound values

were then graphed using the sigmoidal dose response with variable slope equation in

Prism (Y=Bottom+((Top-Bottom)/( +10((LogEC50-X)*Hillslope)))) to calculate either an ECso

or IC50 value as well as the hillslope for each compound. The ECso responses were

normalized to the maximum response of ACh.

High Performance Liquid Chromatography (HPLC) Chiral Separation of Nicotine
Analogs

Racemic nicotine analogs were separated on a Daicel chiral OJ-H semi-preparative

column (250 x10 mm i.d.) from Chiral Technologies, Inc. (West Chester, PA) using a

Beckman System Gold 126 solvent module attached to a System Gold 168 photodiode

array detector (Fullerton, CA). The column was eluted with an increasing gradient

(0-94% buffer B) over a 35-minute period where buffer A was

n-hexane/ethanol/diethylamine/trifluoroacetic acid (97:3:0.1:0.1 v/v/v/v) and buffer B

consisted of the same solvents at a volume ratio of 80:20:0.1:0.1 v/v/v/v for

l'-ethyl-nornicotine-(S,R)-nicotine. The pyridyl and phenyl nicotine compounds were






35


separated using an isocratic method (90:10:0.1:0.1 v/v/v/v for pyridyl nicotine and

94:6:0.1:0.1 for phenyl nicotine v/v/v/v). The separated compounds were collected with

a Foxy Jr. Fraction collector using PeakTrak software (Isco Inc., Lincoln, NE). The

amount of collected compound was determined by absorbance spectrum measurements in

95% ethanol using a Beckman DU 650 spectrophotometer (Fullerton, CA) and published

molecular extinction coefficients (in 95% ethanol) for unionized forms of nicotine and

nornicotine (Swain et al., 1949).














CHAPTER 3
RESULTS

Nicotine Analogs

The involvement of nicotinic receptors in nicotine addiction has led to the synthesis

of a variety of nicotine analogs in order to determine relationships between structure and

function. Numerous studies have characterized the binding of analogs with substituents

at various positions on either the pyridine or pyrrolidine ring of nicotine. However, most

of these studies provided binding data for only the high-affinity (Ua432) receptor and

lacked functional data. In order to determine the a432 versus a7 selectivity for several

previously published as well as novel nicotine analogs, binding assays were performed to

measure the ability to displace either 3H-cytisine (a432) or 125I-a-btx (a7) in rat brain

membranes. The nicotine analogs were then tested in functional assays utilizing tsA201

cells expressing human a432 receptors and a membrane potential dye to measure

agonistic activity (EC5o) and inhibition of the ACh response (IC50).

(S)-Nicotine has a high affinity for the neuronal a432 nAChR; it binds at low

nanomolar (2.3 nM) concentrations (Dukat et al. 1996). The naturally occurring

(S)-nornicotine, which is also a metabolite of (S)-nicotine, has been reported to have a

16- to 18-fold lower affinity for the a432 receptor (Copeland et al. 1991; Glassco et al.

1994). The functional effects of (S)-nicotine have also been well characterized. Much of

the functional data for a432 has been provided by electrophysiological experiments,

radioactive rubidium efflux and radiolabeled-dopamine release assays. (S)-Nicotine acts









as a concentration and time-dependent agonist on a432 nAChRs with measured ECso

values ranging from 1 to 10 pM. At increasing concentrations and times after application

nicotine may behave as an antagonist, primarily due to receptor desensitization. The

functional effects of (S)-nomicotine are less well characterized than (S)-nicotine. In

dopamine release assays from rat striatal slices, (S)-nornicotine at concentrations up to

100 pM has been found to cause nAChR mediated 3H-dopamine release (Teng et al.

1997).

Of the published nicotine analogs, only the 5-pyridyl substituents have been

electrophysiologically characterized to obtain IC50 values for a432. Dukat et al. (2002)

found that adding smaller substituents at the 5-pyridyl position of nicotine did not greatly

reduce the affinity for the a432 receptor. They measured the Ki values for

5-bromonicotine and 5-methoxynicotine for displacing 3H-nicotine as 6.9 + 2.6 nM and

14.3 + 1.5 nM, respectively, as compared to the Kivalue of 2.4 0.4 nM for (S)-nicotine.

However, they found that substituents at this position even more significantly influenced

their functional properties. Dukat et al. (2002) determined that although the Ki values for

both compounds were similar to that of nicotine, the 5-bromo compound functioned as a

partial agonist on rat a432 receptors expressed in Xenopus oocytes whereas the

5-methoxy compound had no agonistic activity.

Substituents at the 6-position on the pyridine ring were characterized for binding to

a4P2 as well as for in vivo effects on mice (Dukat et al. 1996). They determined that a

chlorine at the 6-position of nicotine produced an analog with 3.8-fold greater affinity

than (S)-nicotine for a432. Other analogs with small substituents, including 6-bromo

and 6-fluoronicotine had affinities that were respectively 1.4- and 1.6-fold lower than that









of (S)-nicotine. However, a larger methoxy substituent at this position decreased affinity

35 times that of nicotine for a432. Both the 6-bromo and 6-fluoro analogs were as

equipotent as (S)-nicotine in antinociceptive measurements on mice whereas the

6-methoxy analog was much less active. Dukat et al. (1996) determined that both

lipophilicity and size of the substituent at the 6-pyridyl position influence binding

properties as well as in vivo effects.

A group of compounds that have been characterized for their ability to inhibit

3H-dopamine release from striatal slices contain substituents (increasing chain length

alkyl) at the 1-pyridyl position. Crooks et al. (2004) found that adding alkyl substituents

with chain lengths ranging from 1 to 4 carbons resulted in low potency antagonists (IC50

>10 [LM). Interestingly, the most potent compound had a 1-pyridyl-chain length of 12

carbons. This potent antagonist, known as NDDNI, had an IC5o value of 0.009 [LM and a

Ki value for 3H-nicotine displacement of 0.14 [LM (Crooks et al. 2004).

The binding affinities of substituents at the 3'-, 4'-, and 5'-pyrrolidine positions have

been well characterized by Lin et al. (1994) in rat brain. They found that increasing

substituent size at the 3'-position resulted in decreased affinity, indicating steric effects of

the ligand-receptor interaction. A methyl group at the 4'-position had an affinity that was

only 3.7-fold lower than that of (S)-nicotine. However, when larger nonpolar or polar

substituents were added at the 4'-position, the affinity for a432 decreased. Lin et al.

(1994) noted that electronic along with steric effects may be influencing the binding

affinities for the a432 receptor. Unlike at the 4'-position, a methyl substituent at the

5'-position was not well tolerated; it decreased affinity over 30-fold as compared to

(S)-nicotine. Also, they found that there exists a stereoselectivity for substituents at the









5'-position. The trans-5'-methyl analog had a 34.5-fold greater affinity for the receptor

relative to the cis-5'-methyl nicotine. Thus, steric influences as well as stereochemistry

produce effects upon binding at the 3'-, 4'- and 5'-positions on (S)-nicotine.

Damaj et al. (1996) found that an ethyl at the 1 '-N-position reduced affinity for rat

a432 35-fold as compared to (S)-nicotine. Glassco et al. (1994) measured a trend of

decreasing affinity for a432 in rat brain with increasing 1'-N-substituent size. They

found about a 20-fold decrease in affinity (as compared to (S)-nicotine) when the size of

the substituent at the 1'-N-position was increased from a methyl to an ethyl, a 369-fold

decrease (as compared to (S)-nicotine) when substituting a propyl group and greater than

a 7,000-fold decrease when substituting a cyclopropyl group. Like the other pyrrolidine

substituents, bulky groups at the 1 '-N-position result in steric hindrance for binding with

a high affinity to the a432 receptor.

Because these previous studies lacked data on efficacy and nAChR selectivity, we

have further characterized several of these compounds and additionally, other novel

nicotine analogs on a7 as well as a432 nAChRs. Our goal was to create a structure

activity relationship for nicotine binding to the a432 receptor to assist in reaching our

ultimate goal of designing a partial agonist for smoking cessation. Based upon

hypotheses structured from previously published data (primarily binding) of nicotine

analogs and the a432 receptor, we created nicotine analogs at four different positions on

the nicotine molecule: the 5-pyridyl and 5'-, 1'-N-, and 3'-pyrrolidine positions.









Hypotheses:

1) Relatively bulky substituents at the 5-pyridyl position would permit effective

binding but reduce efficacy.

2) A trans-methyl group at the 5'-pyrrolidine position would reduce efficacy with

retention of acceptable binding affinity and receptor selectivity.

3) Bulky alkyl groups at the l'-N-position would decrease the a432 affinity but

relatively strong partial agonism would be retained.

4) Bulky alkyl groups at the 3'-position would decrease the affinity for rat and

human a432 but enhance the affinity for a7 (based on previously published

DMXBA data).

(S)-Nicotine and (S)-Nornicotine

Using 3H-cytisine as a label for a432, nicotine displaced the radiolabel from rat

brain with a Ki value of 9.2 nM. As a comparison for selectivity we report selectivity

ratios (SR = Ki value for a7/ Ki value for a432). The selectivity ratio of (S)-nicotine was

83. (S)-Nomicotine binds with a much lower affinity to rat a432 (Ki = 0.11 [tM) than

(S)-nicotine. It is also less selective for rat a432 over rat c7, with a SR of 16 (Table

3-1).









Table 3-1. Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by (S)-nicotine or (S)-nornicotine.
Ki (iM)
Rat Human
Compound Name and 125I-a-Btx (a7) 3H-Cytisine 3H-Cytisine
Structure
(a4p2) (a4p2)

(S)-Nicotine

-(S)N 0.76 0.11 0.0092 0.0020 0.0089 + 0.0039
S CH3
N 3
(S)-Nornicotine

(-ro N 1.7 0.29 0.11 0.029 0.48 + 0.26
N H
N

Ki values were calculated according to the Cheng Prusoff equation. The concentration of
each radioligand was 1 nM. Each value represents the mean SEM of > three separate
experiments unless otherwise indicated. Concentrations for each experiment were done
in triplicate. The Kd values for 125I-a-btx and 3H-cytisine binding to rat brain membranes
were 0.32 nM 0.04 and 0.92 0.1 nM respectively. The Kd value for 3H-cytisine
binding to tsA201 cell membranes was 0.48 nM 0.2.

Because the functional assays were performed using cells (tsA201) that express

human a4p2, I also measured the binding properties of the nicotine analogs on

homogenized tsA201 cell membranes. Statistical T-tests were performed to determine

any significant differences between rat brain a4p2 and human a4p2 Ki values. I also

performed a sequence alignment with ClustalW (on the European Bioinformatics

website) for human and rat a4 and 32 subunits loops A through F in order to determine

any residue differences. The alignment revealed two residue differences between the

species as follows:









Loop F Loop E *
Human 32 EVASLDDF Human a4 LTKAHLFHDGRVQWT
Rat 32 DVASLDDF Rat a4 LTKAHLFYDGRVQWT


The F loop of the human 32 subunit contains a glutamic acid at position 190

whereas the rat 32 subunit contains an aspartic acid at position the homologous position

(189). The a4 human subunit loop E contains a histidine at position 145 and in its place

in the rat oa4-receptor subunit is a tyrosine residue at homologous position 147.

Experiments were done to determine the agonist activity of each nicotine analog on

a432 receptors. These measurements were performed by first adding the nicotine analog

alone at various concentrations followed by the addition of KCl (40 mM final

concentration), which was used as a calibrant (Figure 3-1). Fitch et al. (2003) previously

published results using KC1 as a calibrant on various cell lines expressing different

subtypes of nAChRs, thus we also chose to use KC1. The addition of KC1 results in a

large depolarization (as measured by changes in fluorescence) that was used to normalize

for cell count, dye loading and possible differences in resting membrane potential

between passages of cells. After performing a concentration response curve for the

tsA201 cells and KC1, it was determined that 40 mM KC1 produces about 85% of the

maximum KC1 response. Measurable changes in fluorescence were relatively rapid,

starting within three seconds, after application of agonist. There was no decay of

fluorescence after application of (S)-nicotine, thus calibration calculations were

performed with the KC1 response as measured on agonist naive cells (those that received

only HEPES/HBSS).






















'..


120000 .. I I
0 20 40 60 80 100 120 140 160 180 200
Time secss)
Well 0 A10 D B7 A B11


Figure 3-1. Signal acquired for membrane potential fluorescence using tsA201 cells
expressing human a432 nAChRs. During the first 17 seconds baseline
fluorescence was measured for each well. At 18 seconds well A10 (o)
received 50 [LM nicotine, well B11 (A) received 0.16 [LM nicotine and well B7
(o) received only 20 mM HEPES/HBSS. All three wells received a final
concentration of 40 mM KC1 at 120 seconds. A fluorescence reading was
taken every 1.44 seconds for a total of 139 reads over a three minute period.

The functional responses of the nicotine analogs as measured with membrane

potential dye indicated that several analogs, along with the naturally occurring

(S)-nicotine and (S)-nomicotine, had agonistic activity on human a432 receptors. The

average EC5o values for (S)-nicotine and ACh were 1.1 + 0.15 [LM and 3.6 0.9 [LM

respectively (Figure 3-2). (S)-Nornicotine displayed partial agonist activity: it was half

as efficacious as compared to ACh, with an EC50 value of 8.9 0.67 [LM (Figure 3-2).

Several experiments were done to determine the maximum effect of ACh on the human

a4P2 expressing tsA201 cells by increasing the concentration of ACh from 2,000 |tM to









10,000 [LM. The results from those separate experiments (not shown) produced the same

EC50 and maximum response as the response seen in Figure 3-2.


S 140-
M 120- t
0l o 100- (S)-Nicotine
t o 80- ACh
60 g (S)-Nornicotine

M-< 40
S 20-

-10 -9 -8 -7 -6 -5 -4 -3 -2
Log [Compound], M

Figure 3-2. Concentration response curves of nicotine, ACh and nornicotine for human
a432 receptors expressed in tsA201 cells as measured by changes in
membrane potential. Responses were normalized to the maximum ACh
response. Each curve represents an average of 20 wells for nicotine, 14 wells
for ACh and 4 wells for nornicotine (averaged from 2 to 10 separate
experiments).

5-Pyridyl Substituted Analogs

The 5-pyridyl substituted analogs, 5-phe-nic and 5-pyr-nic, both bound with low

micromolar affinities to rat a432: Kis = 0.066 and 0.14 [tM, respectively (Table 3-2).

Interestingly, these compounds showed no interaction with rat a7 at concentrations up to

20 [LM. Thus, although both compounds bound with lower affinities to rat a432 than

(S)-nicotine, they had higher selectivity ratios (SR): SR of 5-phe-nic was 303 while the

SR of 5-pyr-nic was 143. These values are greater than the SR of nicotine, which was 83.









Table 3-2. Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by 5-pyridyl substituted analogs.

Ki (,M)
Rat Human
Compound Name and 1251-a-Btx 3H-Cytisine 3H-Cytisine
Structure
(a7) (a4p2) (a4p2)

5-Phenyl-(S,R)-Nicotine
5-Phe-Nic* 1
5-P >20 0.066 0.013 0.020 0.0037
\ N
CH3
N
5-(3-Pyridyl)-(S,R)-Nicotine
5-Pyr-Nic* 1
N J N >20 0.14 +0.021 0.053 +0.017
CH3
N
Parameters were the same as in Table 3-1. Diamonds indicate a significant difference
between the Ki values for rat and human with 3H-cytisine. Abbreviations for the analogs
are indicated by an asterisk.

The 5-pyridyl substituted analogs were tested for their ability to activate human

a4p2 receptors expressed in tsA201 cells. There was no measurable activation produced

by either compound. The 5-pyridyl substituted analogs were then tested for their ability

to inhibit the ACh response as measured by changes in membrane potential. ACh was

chosen over (S)-nicotine as the standard agonist primarily because the occurrence of

channel block by ACh is expected to be less than for (S)-nicotine. A concentration of 5

[tM ACh was applied to the cells simultaneously with the nicotine analog of interest. A

concentration of 5 [tM ACh was chosen because it was slightly above the EC50 (3.6 [tM)

and well below the concentration required to produce a maximum response (500 [LM).

The 5-phe-nic and 5-pyr-nic compounds had the highest affinities (Ki values = 0.02 [tM

and 0.053 [tM, respectively) for human a4p2 receptors as compared to the other nicotine









analogs. These two compounds produced measurable IC50 values of 14.3 (5-phe-nic) and

11.6 [M (5-pyr-nic) for human a432 (Table 3-3).

Table 3-3. Inhibition of human a432 receptor ACh responses by 5-pyridyl substituted
analogs.
Compound Name or Abbreviation ICso (rIM)


5-Pyridyl substituted analogs
5-Phe-Nic 14.3 1.3
5-Pyr-Nic 11.6 0.41
The compounds and ACh (5 [M) were administered simultaneously. Each value
represents the average SEM of four to six separate wells.

5'-Pyrrolidine Substituted Analogs

The results for the 5'-pyrrolidine substituted analogs indicate that the receptor

possesses a strong conformational preference for ligand binding: the 5'-trans-met-nic

analog bound with an affinity of 0.150 [LM to rat a432 whereas 5'-cis-met-nic displayed

no interaction with this receptor at concentrations up to 20 [LM (Table 3-4). While

5'-trans-met-nic did bind to rat a7 with a Ki of 2.1 [LM, 5'-cis-met-nic did not interact

with rat a7 at concentrations up to 20 [LM. The SR for 5'-trans-met-nic was 14, thus, the

methyl substituent at the 5'-trans position dramatically reduced the selectivity for rat

a4P2 as compared to nicotine. There was no significant difference between the binding

of either 5'-substituted analog to rat a432 or human a432.

Like the 5-pyridyl substituted analogs, the 5'-pyrrolidine analogs produced no

activation of human a432 receptors expressed in tsA201 cells. However, the

5'-trans-met-nic analog did inhibit the human a432 ACh response (Table 3-5). Full

concentration response curves were not obtained at the concentrations tested, therefore









percent of inhibition values were reported. At the highest concentration of 5'-trans-

met-nic used (50 [LM), the human a432 ACh response was inhibited by 50%.

Table 3-4. Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by 5'-pyrrolidine substituted analogs.
Ki (IM)
Rat Human
Compound Name and 12I-a-Btx (a7) 3H-Cytisine 3H-Cytisine
Structure
(a4p2) (a4p2)

5'-Trans-Methyl-(S,R)-
Nicotine
5'-Trans-Met-Nic* >- CH3 2.1 0.033 0.15 0.013 0.22 0.038

CH3

5'-Cis-Methyl-(S,R)-
Nicotine
I -"'""CH
5'-Cis-Met-Nic* *N 3' >20 >20 >20
CH3

Parameters were the same as in Figure 3-1. Abbreviations for the analogs are indicated by
an asterisk.

Table 3-5. Inhibition of human a4p2 receptor ACh responses by 5'-pyrrolidine
substituted analogs.
Compound Name or Abbreviation Percent Inhibition (I)


5'-Pyrrolidine substituted analogs
5'-Trans-Nic 50% I (50 [LM)
5'-Cis-Nic 0% I (50 [tM)
The compounds and ACh (5 [LM) were administered simultaneously. The concentrations
in parenthesis indicate the highest concentration tested. Each value represents the
average SEM of four to six separate wells.

l'-N-Pyrrolidine Substituted Analogs

The ability of the 1'-N-pyrrolidine substituted analogs to bind to either rat ca42 or

rat a7 nAChRs decreased as the size of the substituent increased (Table 3-6). The









smallest substituent, an ethyl group, at the 1'-N-position bound with an affinity (Ki = 0.14

[LM) similar to that of 5'-trans-met-nic (0.15 [LM). As the substituent sizes increased to a

propyl and cyclopropyl group, there was no measurable interaction with either rat a432

or rat a7 up to 20 [LM. The SR of l'-N-ethyl-nor (SR = 17.9) was less than that of

(S)-nicotine (SR = 83). Initial data on the binding of l'-N-ethyl-nor to human a432

indicates a Ki value very similar to that for rat a432.

Table 3-6. Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by 1'-N- pyrrolidine substituted analogs.


Ki (LM)
Rat Human
Compound Name and 125I-a-Btx 3H-Cytisine 3H-Cytisine (a402)
Structure (a7) (a4p2)

1 '-N-Ethyl-(S)-Nornicotine
l'-N-Ethyl-Nor* 2.5 + 0.09 0.14 + 0.04 0.14 (n=2)
"'^ ^N

l'-N-Propyl-(S,R)-
Nornicotine
NornicotieN >20 >20 >20
l'-N-Propyl-Nor* I
N

1'-N-Cyclopropylmethyl-
(S,R)-Nornicotine i
1'-N-Cyclopropyl-Nor* N >20 >20 >20

N


Parameters were the same as in Figure 3-1. Abbreviations for the analogs are indicated by
an asterisk.

Within the four classes of nicotine analogs, two of them (the 1'-N-pyrrolidine and

3'-pyrrolidine substituted analogs) produced activation. The highest concentrations of

analogs utilized in these studies did not produce full concentration response curves









(maximal responses) to allow for ECso values to be determined, therefore only percent

activations at these high concentrations are reported (Figure 3-3). The l'-N-ethyl-nor

compound was the only l'-N-pyrrolidine substituted analog with a measurable affinity

for the rat and human receptors and it produced about 35% activation of human a432 (at

500 [LM) as compared to the maximum ACh response. Both 1'-N-propyl-nor and

l'-N-cyclopropyl-nor activated the human receptor about 40% (at 2,000 [LM) and 75% (at

2,000 LM) respectively. Although neither of these compounds had a measurable affinity

for the human a432 receptor as determined with the radioligand binding assays, which

utilized lower concentrations than in the functional assays, the concentrations used for the

membrane potential measurements were several-fold higher. Therefore, at concentrations

above 20 |JM these compounds are binding to the receptor and causing activation.

All three 1'-N-substituted analogs were also found to inhibit the ACh response of

human a432. The l'-N-cyclopropyl-nor analog was the most potent at activating human

a4P2 (at the concentrations tested) as well as the most potent inhibitor (Table 3-7). The

l'-N-ethyl-nor and 1'-N-cyclopropyl-nor both produced less than 50% inhibition of the

human a432 ACh response.






50



100
U 90
0 K 2.0 pNI
Q 80 .Th
080
0 60

E 500 50 T 1
4 E 40 T
0
-" (U 30 v

& o 20
a- 10
N
co 0
1 '-N-Ethyl-Nor 1 '-N-Propyl-N o r 1'-N-C yclopropyl-
Nor


Figure 3-3. Average effects of the l'-N-pyrrolidine substituted analogs on human a432
receptors expressed in tsA201 cells as measured with membrane potential dye.
Effects by each analog were produced by the concentration listed above the
corresponding bar. Each bar represents 4 separate wells. Responses were
normalized to the maximum ACh response (500 [LM).

Table 3-7. Inhibition of human a432 receptor ACh responses by 1'-N-pyrrolidine
substituted analogs.
Compound Name or Abbreviation Percent Inhibition (I)


l'-N-Pyrrolidine substituted analogs
1'-N-Ethyl-Nor 35% I (500 [LM)
l'-N-Propyl-Nor 45% I (500 [LM)
1'-N-Cyclopropyl-Nor 80% I (50 LM)
The compounds and ACh (5 [LM) were administered simultaneously. The
concentrations in parenthesis indicate the highest concentration tested. Each value
represents the average + SEM of four to six separate wells.

3'-Pyrrolidine Substituted Analogs

The same trend of decreasing affinity with increasing substituent size was observed

for the 3'-pyrrolidine substituted analogs (Table 3-8). The 3'-met-nic analog, which has

only a methyl group at the 3'-position, bound to rat a432 with a decreased affinity









(Ki=0.37 [LM) as compared to (S)-nicotine. 3'-Dimethoxybenzyl-nornicotine (DMXBN)

has a large substituent, a benzene ring with two methoxy groups attached, yet it still

bound to the rat a432 receptor with a Ki value of 2.8 pM and had no interaction with rat

a7 at up to 20 [tM (SR > 7.1). The differences between DMXBM and DMXBN are

related to the presence of two additional double bonds in DMXBM, allowing for all these

rings of the compound to be electronically conjugated. DMXHMN has an additional

hydroxymethyl group attached to the benzyl ring, which is attached at (but not

electronically conjugated to) the 3'-pyrrolidine position of nicotine. DMXHMN

displayed no interaction with either rat a432 or rat a7 at up to 20 pM.

The 3'-pyrrolidine substituted analogs also had very low or no measurable affinity

for human a432 at concentrations up to 20 pM. They are however activating human

a432 receptors at higher concentrations (Figure 3-4). DMXBN produced 80% activation

(at 500 [tM) whereas DMXBM produced only 20% activation (at 500 [tM). The

conjugated structure of DMXBM makes it more rigid and the ionizable nitrogen less

basic. 3'-Met-nic, the 3'-pyrrolidine substituted analog with the smallest substituent,

produced 40% activation at 500 pM.









Table 3-8. Inhibition of 125I-a-btx and 3H-cytisine binding to rat brain membranes or
inhibition of 3H-cytisine binding to human a432 expressing tsA201 cell
membranes by 3'-pyrrolidine substituted analogs.

Ki (9M)
Rat Human
Compound Name and 1251-a-Btx (a7) 3H-Cytisine 3H-Cytisine
Structure_ (a4p2) (a4p2)


3'-Dimethoxy
benzyl-(S,R)-
Nornicotine
DMXBN*


OMe
3'-(2,4-Dimethoxy
benzylidene)- MeO
Myosmine
DMXBM*

NN

3'-(2,4-Dimethoxy-5- OMe
hydroxymethyl) CH2C
benzyl-(S,R)- MeO
Nicotine
DMXHMN*


>20


2.8 + 0.58


>20


>20


>20


>20


Not Determined


>20







>20


3'-Methyl-(S,R)-
Nicotine
3'-Met-Nic*


H3C,,,


ri~~V H3
N


4.2 n= 2


0.37 + 0.00015


0.63 + 0.094


Parameters were the same as in Figure 3-1. Abbreviations for the analogs are indicated by
an asterisk.












Cr.le


MeO



I-
Ii
f^ r "


100
90
80
70
60
50
- 40
30
20
10
0


DMXBM DMXHMN


3'-Methyl-
Nic


Figure 3-4. Average effects of the 3'-pyrrolidine substituted analogs on human a432
nAChRs expressed in tsA201 cells as measured with membrane potential dye.
Effects by each analog were produced by the concentration listed within or
above the corresponding bar. Each bar represents 4 separate wells.
Responses were normalized to the maximum ACh response (500 [LM).

Three out of four of the 3'-pyrrolidine substituted analogs produced varying

degrees of human a432 nAChR activation. They did not however produce much if any

inhibition of the ACh response (Table 3-9). The DMXBN, DMXBM and DMXHMN

analogs had no inhibitory action at concentrations up to 50 [LM (500 [LM for DMXBN).

3'-Met-nic did produce a 20% inhibition at 50 LM.

Table 3-9. Inhibition of human a432 receptor ACh responses by 3'-pyrrolidine
substituted analogs.
Compound Name or Abbreviation Percent Inhibition (I)


DMXBN 0% I (500 [LM)
DMXBM 0% I (50 [LM)
DMXHMN 0% I (50 [LM)
3'-Met-Nic 20% I (50 [LM)
The compounds and ACh (5 pLM) were administered simultaneously. The concentrations
in parenthesis indicate the highest concentration tested. Each value represents the
average SEM of four to six separate wells.


5uIu 1L IM


OMe
,CHMOH






Sr i
5ii t .j l


SH.
51-1 i\l


DMXBN














CHAPTER 4
RESULTS

Separation of Nicotine Analog Enantiomers

Naturally occurring (S)-nicotine (approximately 99% S-form, about 1% R-form as

an impurity) has been reported as more potent than its enantiomer in binding to high

affinity nicotinic receptors as measured with 3H-nicotine (Copeland et al. 1991; Zhang

and Nordberg, 1993). Copeland et al. (1991) measured a 7-fold difference in the binding

of the enantiomers to rat cortex; (S)-nicotine had the higher affinity (Ki = 0.014 [tM)

relative to (R)-nicotine (Ki = 0.102 [M). Zhang and Nordberg (1993) reported a 3-fold

difference in the binding of the stereoisomers to rat cortex but an 11-fold difference in

binding to rat cerebellum. Both studies found no significant differences in the binding

affinities of the enantiomers of nomicotine. Zhang and Nordberg (1993) determined a

1.6-fold difference in the binding of the nornicotine enantiomers to rat cerebellum

whereas Copeland et al. (1991) reported a 1.1-fold difference.

The existence of nicotinic receptors on dopaminergic terminals along with evidence

of nAChR involvement in nicotine addiction has lead several groups to study the effects

of nicotine and nornicotine in dopamine release assays using synaptosomes and rat brain

slices. (S)-Nornicotine, like (S)-nicotine is a naturally occurring alkaloid found in

tobacco plant (Saitoh et al. 1985). It is also an active metabolite of nicotine and has a

half-life three times longer than that of (S)-nicotine (Crooks et al. 1997; Green et al.

2001). Whiteaker et al. (1995) measured an EC50 of 0.5 [tM for (S)-nicotine stimulated

3H-dopamine release from striatal synaptosomes. Based upon the reported decrease in









affinity of (R)-nicotine, it has yet to be characterized in dopamine release assays. The

enantiomers of nornicotine however have been investigated. Green et al. (2001)

determined that (R)-nornicotine (EC5o = 0.48 [LM) was 6.3 times more potent than

(S)-nornicotine (EC5o = 3.0 [LM) at evoking radiolabeled dopamine release from rat

nucleus accumbens slices. It has also been determined that (S)-nornicotine desensitizes

nAChRs with 12-fold lower potency than (R)-nornicotine as measured with dopamine

release assays from rat striatum (Dwoskin et al. 2001).

Several of the nicotine analogs in this dissertation were initially studied as racemic

compounds because the enantiomeric species were difficult to synthesize. Given that

previous binding data indicates a difference in affinity for the enantiomers of nicotine, it

was important to try and separate the (R)- and (S)-forms of the racemic nicotine analogs

and determine the affinity and functional properties of these chiral compounds. Our

hypothesis was that like (S)-nicotine, the (S)-forms of the racemic analogs would have a

higher affinity and functional potency for a432.

Three of the racemic nicotine analogs, 5-phe-nic, 5-pyr-nic and 1'N-ethyl-nor, were

successfully separated by chiral HPLC (Figure 4-1). The 5-phe-nic and 1'-N-ethyl-nor

compounds were tested for their ability to bind to rat a432 nAChRs as measured by

3H-cytisine displacement (Table 4-1). The affinities of the nicotine and nornicotine

enantiomers were measured for human a432 and then further tested for potency and

efficacy (Figure 4-2) on the human a432 receptor in tsA201 cells

The affinities of the racemic 5-phe-nic and 5-pyr-nic analogs were similar as

measured for rat and human a432 (Table 3-2) as may be expected based on their similar

structures. Thus, only the enantiomers of the 5-phe-nic were tested for their ability to









bind to rat a432. The enantiomers of both the 5-phe-nic and 1'-N-ethyl-nor have yet to

be identified as to which peak corresponds to the (R) or (S)-form, therefore the

enantiomers are labeled as peak 1 or 2. It may be expected that peak 1 is the

(S)-enantiomer based upon data from the chiral column indicating that (S)-nicotine elutes

before its (R)-enantiomer (Armstrong et al. 1998). Peak 1 of 5-phe-nic had a Ki value of

0.17 [tM as compared to the Ki value of 3.6 [tM for peak 2. The difference in affinities of

the enantiomers was significantly different for rat a432 (P = 0.0104). The enantiomeric

selectivity ratio (ESR), calculated by dividing the Ki of peak 2 by the Ki of peak 1, gives

a value of 21. The Kivalue for peak 1 of 1'-N-ethyl-nor was 0.24 [tM and 2.3 [tM for

peak 2 was also significantly different (P =0.0015). The higher affinity of peak 1 for rat

a432 of both analogs was anticipated based upon previous findings (Copeland et al.

1991; Zhang and Nordberg 1993).





D O 168-258 nm Pump Gradient B
421 05 NEthyNorNic 1mg per ml 100ul
Retention To. .





















A Nm m NN 00 N NO ON





5 10 5 20 25 30 35 40 45 50 55
Mnutes


nm



Figure 4-1. HPLC chiral separation of l'-N-ethyl-(S,R)-noricotine. A) The

chromatogram with retention times for the separation of presumed

l'-N-ethyl-(S)-nor (peak 1) and presumed l'-N-ethyl(R)-nor (peak 2). B) The

spectra of peaks one and two indicate an absorbance at 260 nm.


400


-200


1000-







0-
0-


91.32 Min
4 21 05 NEthnl Nor Nic 1mg per ml 100ul


a .









Table 4-1. Inhibition of 3H-cytisine binding to rat brain or to human a432 expressing
tsA201 cell membranes by nicotine enantiomers.
Compound Name or Ki for a432 (gM)
Abbreviation
(S)-Nicotine 0.0089 0.0039 (human)
(R)-Nicotine 0.011 + 0.0013 (human)

(S)-Nornicotine 0.48 0.26 (human)
(R)-Nomicotine 0.042 0.025 (human)

l'-N-Ethyl-Nor (peak 1) 0.24 0.060 (rat)
1'-N-Ethyl-Nor (peak 2) 2.3 0.26 (rat)

5-Phe-Nic (peak 1) 0.17 0.023 (rat)
5-Phe-Nic (peak 2) 3.6 + 0.76 (rat)
Ki values were calculated according to the Cheng Prusoff equation. The concentration of
radioligand was 1 nM. Each value represents the mean SEM of three separate
experiments. Concentrations for each experiment were done in triplicate. The Kd values
for 3H-cytisine binding to rat brain membranes was 0.92 + 0.1 nM and 0.48 nM 0.2 for
tsA201 membranes.


The binding of the enantiomers of either nicotine or nomicotine to human a432

nAChRs interestingly did not differ significantly as measured by 3H-cytisine

displacement. The ESR for the two nicotine enantiomers on human a432 was 1.2. Since

previously published data indicates that a stereospecificity exists for nicotine and the

high-affinity nAChR, this result was unexpected. The Ki value for (R)-nornicotine (0.042

pM) was also not significantly different (P value = 0.1619) from the (S)-enantiomer (0.48

pM) for human a432. The large difference in standard error may account for the lack of

significance; further replicates will decrease the standard error and may make apparent a

significant difference. The nornicotine data agrees with previous binding data obtained

with rat brain in which there was no significant difference between (S)- and

(R)-nomicotine (Copeland et al. 1991; Zhang and Nordberg, 1993).









The binding results for the enantiomers of nicotine and nornicotine are made more

complex when compared to the activity of these compounds on human a432 receptors as

measured by changes in membrane potential (Figure 4-2). Both (S)- and (R)-nicotine are

full agonists as compared to the maximum ACh response of human a432 nAChRs.

Although there was no significant difference in the measured Ki values for the nicotine

enantiomers as measured by binding experiments, there was however a difference in

potency, although it was not statistically significant (P = 0.16). The EC5o value of

(S)-nicotine (2.3 1.2 [tM) was 7.0 fold lower than that of (R)-nicotine (16 8.8 [tM).

The calculated EC50 for (S)-nornicotine was 8.5 + 1.8 [tM as compared to an EC50 of 39 +

5.4 [LM for (R)-nornicotine. The difference between the EC5o values of the nornicotine

enantiomers was significant (P = 0.0016). Both forms of nornicotine appear to be

functioning as partial agonists as compared to nicotine at the concentrations tested.

(R)-Nomicotine appears to be more efficacious than (S)-nornicotine. Further replicates at

higher concentrations would be needed to determine if the maximum effects and EC50

values obtained by fitting the concentration response curve are consistent.












o (S)-Nicotine
* (R)-Nicotine
* (S)-Nornicotine
o (R)-Nornicotine


-9 -8 -7 -6 -5 -4 -3
Log [Compound], M


Figure 4-2. Concentration response curves for the enantiomers of nicotine and
nornicotine for human ca432 receptors expressed in tsA201 cells as measured
by changes in membrane potential. Responses were normalized to the
maximum ACh response (500 [tM). Each point represents the average + SEM
of four separate wells. The (S)-nornicotine data is also in Figure 3-3.


.2
m>
U,,
t50
0.
NNQ

E
0


100-

80-

60-

40-

20-

0-














CHAPTER 5
RESULTS

Erythrina Alkaloids

Plant alkaloids extracted from the genus Erythrina share a common heterocyclic

ring system (Figure 5-1). Sheridan et al. (1986) determined that the essential aromatic

groups of dihydro-P-erythroidine (DH3E) superimpose with those of nicotine indicating

that they may share a similar conformation. The affinities and inhibition of the Erythrina

alkaloids for nAChRs have been less well characterized in vitro than the nicotine analogs.

The only published literature on these alkaloids and their interaction with nicotinic

receptors pertains primarily to DH3E (Williams and Robinson, 1984; Anderson and

Arneric, 1994; Harvey and Luetje, 1996; Harvey et al. 1996). Erysodine is the only other

alkaloid that has been studied for its interaction with the a432 nAChR (Decker et al.

1995). Aromatic erysodine was determined to have a higher apparent affinity for the

receptor (5 nM) than DH3E (35 nM) in rat brain membranes as measured by 3H-cytisine

displacement (Decker et al. 1995). Erysodine also had a selectivity 1800-fold greater for

rat a432 than rat a7 whereas DH3E had a selectivity only 114-fold greater for rat a432

than rat a7 (Decker et al. 1995).

Since the early 1940s it has been known that curare-like effects result upon

administration of Erythrina alkaloids in vivo (Lehman, 1936; Folkers and Major, 1937).

The alkaloids were apparently producing their primary effects through blockade of

neuromuscular transmission, but ganglionic blocking effects were also observed.














0-' B"
14 4

MeO" 1
2
3-Erythroidine

Figure 5-1. The common heterocyclic structure of Erythrina alkaloids. The rings are
designated A through D and atoms numbered accordingly.

In 1984 Williams and Robinson first studied the effects of DHPE on nAChRs.

Their results indicated that it was binding with high affinity (2 nM) to a neuronal

nicotinic receptor in rat brain and that its distribution of binding was similar to the

binding of 3H-nicotine. Since then DH3E has been referred to and used as a competitive

antagonist for selective inhibition of 32-containing nAChRs. Decker et al. (1995)

determined that DH3E was inhibiting (S)-nicotine-evoked 3H-dopamine release from

slices of rat striatum with an IC50 of 58 nM. DH3E also interacts with other

P2-containing receptors. Electrophysiological measurements on oocytes expressing

human a432 indicate that DH3E is 14.7-fold less potent of an inhibitor for the

ca32-subtype (Chavez-Noriega et al. 1997).

In order to further understand the interaction of antagonists with a432 nAChRs our

goal was to create a structure activity relationship for Erythrina alkaloids and the a432

receptor with the ultimate goal of designing a selective probe for use in vivo and in vitro

to study the resting state of the receptor as well as a possible smoking cessation drug. To

accomplish this goal several natural and semi-synthetic alkaloids were studied for their

ability to displace 125I-a-btx and 3H-cytisine from rat brain membranes and 3H-cytisine









from tsA201 cells expressing human a432 nAChRs. The inhibitory properties of these

alkaloids were then characterized using tsA201 cells expressing human a432 nAChR and

a membrane potential dye. Our hypothesis was that alterations to the D-ring of the

Erythrina alkaloids would have a large affect on affinity and potency for a432 and that

the nitrogen is critical for binding.

P-Erythroidines

The P-erythroidine compounds contain an unconjugated lactone group in their

D-ring and thus lack D-ring aromaticity. P-Erythroidine contains two conjugated double

bonds, one in the A ring and another in the B ring. Its Ki value as measured for rat a432

was 1.1 [tM and its selectivity ratio (SR = Ki u7/Ki a432) was 45 (Table 5-1). The two

double bonds in 3-E have been reduced to one in DH3E so the resulting double bond is

now between the 1- and 6-positions, which still maintains approximate co-planarity of the

A and B rings. Interestingly, this reduction resulted in an increased affinity for rat a432.

The affinity of DH3E for rat a432 (Ki = 0.14 [tM) was eight times greater than that of

P-E. Reducing the remaining double bond of DHPE produces TH3E. The absence of

double bonds in the A and B rings greatly reduced the affinity of this compound for rat

a4p2. THPE had an affinity (Ki = 7.4 [tM) that was about 7-fold less than that of 3-E.

Neither DH3E nor TH3E displaced a-btx at concentrations up to 50 [LM (SR = 377 and

6.76 respectively), indicating that they have a selectivity for rat a432 over rat a7. The

affinity of P-erythroidine for human a432 was not determined. The decrease in affinity

of THPE as compared to DHPE was also observed in binding to human a4p2. There was









a 4.4-fold difference in the affinity of DH3E for rat versus human a432. TH3E had a

2.7-fold difference in affinity between rat and human a432.

Table 5-1. Structures, nomenclature and binding results for P-Erythroidine,
dihydro- P-erythroidine and tetrahydro- P-erythroidine.
Ki (gM)
Rat Human
Compound 125I-a-Btx (a7) 3H-Cytisine (a4p2) 3H-Cytisine (a4p2)
Name


P-Erythroidine 0 I N
NP o
-E* MeO"'J >50 1.1 + 0.84 Not Determined


Dihydro-P- 0 1
Erythroidine o
Sy >50 0.14 0.018 0.62 0.17

DHPE* MeO"'

Tetrahydro-p- 0
Erythroidine N
SS o y n >50 7.4 + 4.2 >20
THPE* MeO H
MeO"'

Ki values were calculated according to the Cheng Prusoff equation. The concentration of
each radioligand was 1 nM. Each value represents the mean SEM of > three separate
experiments unless otherwise indicated. Concentrations for each experiment were done
in triplicate or quadruplicate. The Kd values for 125I-a-btx and 3H-cytisine binding to rat
brain membranes were 0.32 nM + 0.04 and 0.92 + 0.1 nM respectively. The Kd value for
3H-cytisine binding to tsA201 cell membranes was 0.48 nM 0.2. NP indicates a natural
product. SS indicates a semi-synthetic compound. Abbreviations for the analogs are
indicated by an asterisk.

3-Des-Met-pE differs from P-E in that it contains two double bonds in the A ring

and one in the B ring resulting in an even more extensive conjugation between these two

rings (but this is accompanied by movement of the C and D rings with respect to the A

and B rings). As may be predicted from the results of P-E and DHPE, the addition of the









third double bond was accompanied by a decrease in affinity for rat a432 (Table 5-2).

This reduction in affinity also may be due to the loss of the methoxy group at the

3-position. 3-Des-Met-pE did not displace either 3H-cytisine or 125I-a-btx at

concentrations up to 20 pM.

Table 5-2. Structures, nomenclature and binding results for
3-Desmethoxy-3P-Erythroidine, N-Methyl-P-erythroidine and
P-erythroidinediol.
Ki (pM)
Rat Human
Compound Name 125I-a-Btx 3H-Cytisine 3H-Cytisine
(a7) (a4p2) (a4p2)


3-Desmethoxy-P-
Erythroidine
SS >20 >20 Not Determined
3-Des-Met-pE*

o CH3
N-Methyl-P- +/
Erythroidine 0 >20 >20 Not Determined
SS
aS MeO "
Me-pE*
HO
P-Erythroidinediol N
HO" vtNe >50 0.31 0.12 0.24 0.015
SS
PE-Diol* MeO "

Parameters were the same as in Table 5-1. Abbreviations for the analogs are indicated by
an asterisk.

Me-pE has a quaternary ammonium in place of the tertiary amine in 3-E. The

presence of this positively charged nitrogen decreased the affinity of Me-pE (Ki > 20

pM) for rat a4p2 at least 20-fold as compared to P-E. Me-pE had no interaction with a7

at concentrations up to 20 LM. The final P-erythroidine studied, 3E-Diol, contains an









open D-ring. It may be expected that opening the D-ring would decrease the affinity of

3E-Diol as compared to 3-E. The results however are just the opposite. The Ki value of

PE-Diol for rat a432 was 0.31 [iM, 3.5-fold greater than that of 3-E. pE-Diol had a

minimum selectivity ratio (SR) of 161.

To ensure that DH3E was not producing any activation of the human a432

receptors expressed by tsA201 cells as measured using the membrane potential

fluorescent dye, it was administered in the absence of agonist and as expected there was

no measurable activation (Figure 5-2). The ability of the other Erythrina alkaloids to

produce activation was also measured and the results indicated that all the alkaloids were

functioning only as antagonists.



E 100
cE DHBE
0--- 80-
SE O 60
0 6CL
40-

20
0 I j .


-9 -8 -7 -6 -5 -4 -3
Log [DHBE], M

Figure 5-2. Concentration response curve of DH3E for human a432 receptors expressed
in tsA201 cells as measured by changes in membrane potential. The response
was normalized to the maximum ACh response. The result represents an
average of four wells.

To verify that DH3E was acting as a competitive antagonist in our system,

measurements were made to determine EC5o values for ACh in the presence and absence

of 1 [tM DHpE. A competitive antagonist would shift the concentration response curve










to the right and as the results indicate DH3E is acting as a competitive antagonist (Figure

5-3). The EC50 of ACh in the presence of 1 LM DH3E was 298 29 LM versus the EC50

of ACh alone, 2.33 + 0.94 [tM.


E 120
E 100
I
C CO
0 80- ACh
to ..
W 60- ACh + luM DHpE
N. 40-
o
S20-
0- .
I I I I
-10 -9 -8 -7 -6 -5 -4 -3 -2
Log [ACh], M


Figure 5-3. Concentration response curve of ACh in the presence and absence of 1 JM
DHPE for human a432 receptors expressed in tsA201 cells as measured by
changes in membrane potential. The responses were normalized to the
maximum ACh response. The result represents an average of 3 experiments
for ACh and 8 experiments for ACh + 1 [tM DH3E.

Figure 5-4 represents the signals of simultaneous applications of 5 |JM ACh with

increasing concentrations of DHPE as recorded using membrane potential fluorescent dye

and human a432 expressing tsA201 cells. The signals of the 5 [tM ACh response

decrease with increasing concentrations of DHpE. The 50 [tM concentration of DH3E

produces a response that is identical to that of the blank (which did not receive 5 JiM

ACh), indicating that at 50 [tM DH3E the ACh response was completely inhibited.







68


220000






160000 ------------- -----------
...4(1 in I KCI
._

''"- DHIE aind- ,/






Well F12 0 012 AC H7 H8
1 6 0 0 0 0 ........................................................... .................................. ............................ ......................... ............ .. ....













Figure 5-4. Signal acquired for a4-2 nAChRs upon simultaneous application of DH-E
and ACh. During the first 17 seconds baseline fluorescence was measured for
each well. At 18 seconds well H8 (c) received 0.0005 pM DHjE + 5 pM
ACh, well F12 (0) received 0.05 [LM DH3E + 5 CtM ACh, well G12 (w)
received 50 [LM DH3E + 5 CtM ACh and well H7 (A) received only 20 mM
EPES/BSS. All wells received a final concentration of140 160 mM KC at 160
Time secondss
All of the -erythroidine alkaloids except for 3-Des-Met-E and Me-E were able


to fully inhibit ceSignal acquiresponse to 5 M AChRs upon simultaneous application owith

ratand ACh. During the first 17 se2 receptors at concentrations up to 20 dsM as measured by 3H-cytisine

displacement and it also did not inhibit the human (0) 2 ACh response at concentrations

up to 50 ACh, well F12 (o) received 0.05no measured binding to rat well at concentrations

recoup to 20 M but it did inhibit the human + 5 ACh and well H7 (A) received only 20 mMhighest

concentration tested (50 M). All wells received a finhibital concentratiothe n of 40 mM KC at 160
seconds










All response is 5.5-fold greater than that for 3--E as indicated by their ICand values of 0.067able

to fully inhibit cell respetively. THE, witAh all double 5-3)bonds in the A and B rings with

reduced, is the least potentrations up to 20 idines on human 2 with a measured by -cytisine
displacement and it also did not inhibit the human A4132 ACh response at concentrations

up to 50 |tM. 3-Des-Met-PE also had no measured binding to rat A4132 at concentrations

up to 20 |tM but it did inhibit the human A4132 ACh response by 50% at the highest

concentration tested (50 |tM). The potency of DH]E to inhibit the human A4132 ACh

response is 5.5-fold greater than that for ]3-E as indicated by their IC50 values of 0.067

|tM and 0.37 |tM respectively. TH]3E, with all double bonds in the A and B rings

reduced, is the least potent of the P-erythroidines on human oA42 with a measured IC50









of 2.5 [M. After DH3E, 3E-Diol had the second highest affinity (for both rat and human

a432) of the P-erythroidine compounds. 3E-Diol was also the second most potent of the

erythroidines on human a432 with an IC50 value of 0.065 [M.

Table 5-3. Inhibition of human a432 receptor ACh responses by natural product and
semi-synthetic P-erythroidines.
ICso (pM)/Percent Inhibition for
Compound Name or Abbreviation c
Human a402

Erythroidine compounds
P-E 0.37 + 0.055
DHPE 0.067 + 0.0035
THPE 2.5 0.20
3-Des-Met-pE 50% Inhibition at 50 [LM
Me-pE No inhibition up to 50 [M
PE-Diol 0.065 + 0.012
The compounds and ACh (5 [M) were administered simultaneously. Each value
represents the mean SEM of > four separate wells.

To determine the importance of the C-ring for binding to rat ca42 a

homoerythrinan compound from another plant genus, Phelline, was studied for its ability

to displace 3H-cytisine. This compound, named O-methylisophellibiline, is similar in

structure to DHpE except that it has a seven membered C-ring, which increases the size

of the molecule and alters the 3-dimensional position of the lactone carbonyl oxygen

(Figure 5-5). Enlarging the C-ring resulted in a decreased affinity for rat ca42. There

was over 50% displacement of 3H-cytisine from rat a4p2.















MeO

Figure 5-5. Structure of a Phelline alkaloid, O-methylisophellibiline. This alkaloid is
similar in structure to DH3E except for its seven-membered C-ring.

Aromatic Alkaloids

Erythraline (ELA), an aromatic alkaloid, lacks the D-ring lactone moiety of the

erythroidines and instead has a benzenoid ring (Table 5-4). ELA has an additional

(1,3-dioxolane) ring fused to the D-ring. The measured affinity (Ki value = 0.056 [LM) of

ELA is greater than that of DHPE (Ki value = 0.14 [LM) for rat a432. ELA does have an

affinity, although low, for the rat a7 receptor (SR = 46).

The aromatic alkaloids, erysovine and erysodine (ERV and ERD), are isomers

(Table 5-4). ERV contains a hydroxy group at position 15 and a methoxy group at

position 16 on its D-ring. In ERD the hydroxy and methoxy attached to the D-ring are

reversed. The reversal of those two substituents has a large influence upon the affinity

for rat a432. ERV has a high affinity for rat a432 (12 nM), which is 18 times greater

than the affinity ofDHPE for rat a432. ERD, with the hydroxy and methoxy groups

reversed, had a much lower affinity than ERV, 23-fold less, for rat a432 and 8.9-fold less

for human a432. ERV also exhibited an increased affinity for rat a7, with a selectivity

ratio of 50.8. ERD had no measurable interaction with rat a7 at up to 20 [LM.









Table 5-4. Structures, nomenclature and binding results for the aromatic alkaloids
erysovine, erysodine and erythraline.

Ki (AM)
Rat Human
Compound Name 125I-a-Btx 3H-Cytisine 3H-Cytisine
(a7) (a4p2) (a4p2)
MeO
Erysovine ,N
NP HO
0.61 0.13 0.012 0.0032 0.027 0.0032
ERV* MeO'

HO
Erysodine
Np MeO
>20 0.28 0.087 0.24 0.074
ERD e-
MeO' `


Erythraline
NP <
ELA* o 2.6 1.4 0.056 0.0035 0.081 0.018

MeO"''

Parameters were the same as in Table 5-1. Abbreviations for the analogs are indicated by
an asterisk.

ERT has a methoxy group at both position 15 and 16 on the D-ring (Table 5-5).

The presence of the two methoxy groups resulted in an affinity of 0.33 [LM for rat ca42,

which was similar to that of ERD for rat ca42. It did however have a minimum of

26.6-fold greater affinity for rat a7 as compared to ERD. Gluco-ERD is ERD with a

glucose molecule attached. Gluco-ERD is a natural product and like other phenolic

D-ring Erythrina compounds is attached to large sugar molecules until they are liberated

by application of an acid. Even with this large substituent Gluco-ERD had an affinity (Ki

value = 0.032 LiM) 4.4-fold greater than that of DHPE for rat ca42. It had no measurable

affinity for rat a7 at concentrations up to 20 LiM (SR = a minimum of 625).









Table 5-5. Structures, nomenclature and binding results for the aromatic alkaloids
erysotrine, and glucoerysodine.

Ki (9M)
Rat Human
Compound Name 25I-a-Btx (a7) 3H-Cytisine 3H-Cytisine

(a4p2) (a4p2)

MeO
Erysotrine Me
NP MeO
ERT* 0.75 + 0.20 0.33 + 0.070 0.86 0.22
MeO'"

Glucoerysodine NP
Gluco-ERD*
OHCH2
HO- \ O,^
HO OH N >20 0.032 + 0.0085 2.2 + 0.74
MeO

MeO'
Parameters were the same as in Table 5-1. Abbreviations for the analogs are indicated by
an asterisk. The diamond indicates a significant difference between the Ki values for rat
and human with 3H-cytisine.

Interestingly, the only alkaloid that had a significantly different Ki value (P< 0.043)

between rat (Ki value = 0.032 [M) and human (Ki value = 2.2 [M) a4p2 nAChR was

Gluco-ERD, which is a natural product. The other aromatic alkaloids had similar rat and

human a4p2 affinities.









Table 5-6. Inhibition of human a432 receptor ACh responses by natural product
aromatic Erythrina alkaloids.

Compound Name or Abbreviation ICso (rM) for Human a432
Aromatic compounds
ERV 0.054 0.00049
ERD 0.092 + 0.0035
ELA 0.13 0.0049
ERT 1.6 + 0.52
Gluco-ERD 0.43 + 0.079
The compounds and ACh (5 [M) were administered simultaneously. Each value
represents the mean SEM of > four separate wells.

Several of the aromatic compounds were as potent as DH3E in inhibiting human

a4p2 response to ACh. ERV was the most potent of all the alkaloids, it had an IC50

value of 0.054 [M for human a4p2 (Table 5-6). As indicated earlier, ERV also had the

highest affinity for both the rat and human a4p2-receptor. Although ERD had a 8.9-fold

decrease in affinity (for human a4p2) as compared to ERV, it was the second most

potent aromatic compound with an IC50 of 0.092 [M on human a4p2. ELA was the third

most potent of the aromatic compounds (on human a4p2) with and IC5o value of 0.13

[M for human a4p2. ERT was the least potent (IC50 = 1.6 [M) of the aromatic alkaloids

on human a4p2. The final aromatic compound, Gluco-ERD was the second to last

potent with an IC5o value of 0.43 [M.














CHAPTER 6
DISCUSSION

Nicotine and dihydro-P-erythroidine (DH3E) have both been used extensively as

tools for studying nAChRs. The reason being, that nicotine binds to and activates various

subtypes of nAChRs with its highest affinity and potency for a432 nAChRs, while

DHPE is one of the only antagonists with a relative selectivity for 32-containing

receptors. Although their functional effects for the receptor differ, both are competing

for the acetylcholine (ACh) binding site. The role of the a432 receptor in nicotine

addiction as well as its involvement in several neurological dysfunctions warrants further

investigation of the interactions of these compounds with 32-subunit containing

receptors.

There are various methods that have been employed to study the interaction of the

a432 nAChR with specific compounds in order to elucidate properties of the binding

site. The a432 receptor has undergone mutagenesis and chimeric studies in order to

determine residues important in allowing for activation or inhibition. Another approach

to understanding ligand and receptor interaction is to study the ligand directly. Through

alteration of the ligand's structure the consequential effects upon binding and function

tell a story about requirements of a molecule for interacting with ca42 (structure activity

relationship approach). The goal of this dissertation was to determine structure activity

relationships to understand what aspects of their structures allow for nicotine and DH3E

to bind and either activate or inhibit the a432 receptor. Determination of ligand









structural properties important for receptor interaction will provide a basis for further

manipulation of these molecules to develop higher affinity selective probes for in vitro

and in vivo studies of the a432 nAChR as well as possible drugs to treat nicotine

addiction.

Substituents at Four Different Positions on Nicotine Decrease Affinity for the a432
nAChR and Confer Partial Agonist and Antagonist Properties

This structure activity relationship study of nicotine analogs built on previously

published data of several substituted nicotine analogs as well as explored several novel

nicotine compounds. The previous studies (Damaj et al. 1996; Dukat et al. 1996;

Glassco et al. 1994; Kim et al. 1996; Lin et al. 1994) however only examined the ability

of the nicotine analogs to bind to the a432 nAChR as measured with either 3H-cytisine or

3H-nicotine. The studies conducted in this dissertation further explore the ability of

nicotine analogs to bind to the other high affinity neuronal nAChR, c7, for the first time.

We also investigate the effects of these compounds on the human a432 receptor

expressed in a mammalian cell line.

Substitutions were made at the 5'-, 1'-, and 3'-pyrrolidine positions as well as at

the 5-pyridyl position on nicotine. The substituents at the 5-pyridyl position, either a

phenyl or pyridyl ring, were large. They decreased the affinity of these compounds for

rat and human a432 receptors as compared to that of (S)-nicotine. The presence of the

tertiary nitrogen in the 5-pyridyl ring did not increase the affinity of this compound for

either the rat or human a432 receptor as compared to the nitrogen lacking phenyl ring. It

may have been expected that the tertiary nitrogen would provide an additional group for

possible hydrogen bonding or other additional electrostatic interactions with the binding

site and thus increase the affinity, but this was not the case. The Ki values for the









interactions of these two compounds with the rat a7 receptor were greater than 20 [tM.

This was an important finding because the selectivity ratios (Ki a7/ Ki a432) for both

compounds were greater than that of (S)-nicotine. Thus, the large substituents added at

the 5-position resulted in an enhanced selectivity for the high affinity receptor (as seen

for the rat a7 and a432 receptors). The 5-pyridyl substituted analogs were the only

nicotine analogs with significantly different Ki values for rat and human a432 receptors

(P = 0.0326 for 5-phe-nic and P = 0.0334 for 5-pyr-nic).

The compound DMXBA displays a variation in potency between human and

animal (rat) forms of a nicotinic receptor, specifically a7 (Meyer et al. 1998; Papke and

Porter-Papke, 2002). Stokes et al. (2004) determined by mutational analysis that this

variation in potency between human and rat was due to differences in residues within the

C loop (serl84 in human to asnl84 in rat; argl86 in human to lysl86 in rat) and the F

loop (gly167 in human to serl67 in rat) with some influence from the E loop. Although

there are two residue differences between the rat and human a432 receptors, only one of

them is likely to impact binding. The a4 subunit is the primary, or principal subunit, thus

loops A, B and C from this subunit interact with loops D, E and F from the 32, or

complimentary, subunit. The only residue within the binding loops that might be

anticipated to differently influence ligand interaction between rat and human a432 is a

glutamic acid in the human receptor in place of an aspartic acid in the rat. This residue

difference seems small; there is an additional methyl group on glutamic acid, but both

residues are polar and charged. There is also the possibility that supporting residues

outside of the six binding loops may be influencing (restricting) the ability of the

5-pyridyl analogs to enter the binding site of the human a432 receptor as compared to









rat. Another possible explanation of the difference between rat and human Ki values is

that in the CNS there exists a heterogenous population of a432 receptors. It has been

shown that a5-subunits are localized with a432 receptors in the chick brain (Conroy and

Berg, 1998) as well as in the cortex of rat (Mao et al. 2005). Therefore, if 3H-cytisine is

labeling both populations of receptors it may explain the differential interaction of several

of the nicotine analogs (as well as Erythrina alkaloids) between rat brain a432 and

human a432 receptors expressed in the tsA201 cell line.

Both 5-position substituents prevented the ligands from producing any activation of

the human a432 receptor. However, these compounds displayed antagonistic properties

with similar potencies to each other as measured using a membrane potential fluorescent

dye. Dukat et al. (2002) also observed the influence of smaller substituents at the

5-pyridyl position on the functional properties of rat a432 receptors and determined

using electrophysiological recordings from Xenopus oocytes that modifications resulted

in partial agonist and antagonist properties. Also, Carroll et al. (2001) found that a

phenyl group added at the 3'-position on epibatidine produced similar effects

(antagonism).

The method we used to study receptor activation or inhibition by these nicotine

analogs involves the measurement of changes in membrane potential using a fluorescent

dye. The dye fluoresces upon membrane depolarization resultant from receptor

activation. Fitch et al. (2003) published data on the effects of nicotinic receptors with

membrane potential and calcium dye and measurements from various cell lines

expressing nAChRs. They measured an EC50 for nicotine of 0.86 [LM on the human

a432 nAChR expressing cell line they utilized (K-177 cells which are a HEK-293









derived cell line). The ECso values measured in their experiments for agonists were 2 to

3-fold less than those reported in the literature for radiolabeled rubidium efflux assays

whereas the IC5o values for antagonists were higher (5 to 20-fold or greater) for ca334

expressing cells. They note that these estimates may possibly be affected by spare

receptors, only a sub-maximal occupation of receptors may be required in order to

depolarize the membrane resulting in an apparent increase in potency. The cells used for

this study are tsA201 cells, a human embryonic kidney cell subclone (HEK-293) obtained

from Jon Lindstrom (Kuryatov et al. 2005). Since HEK-293 cells endogenously express

Ml muscarinic acetylcholine receptors (Mundell and Benovic, 2000), 1 mM atropine (a

muscarinic antagonist) was used in the Flexstation assays. Nelson et al. (2003)

determined that a population of the cells had nicotine and ACh potencies similar to

oocytes expressing human a432 receptors. They also determined that the partial agonist

cytisine had an efficacy too low to correctly measure potency. Overall, Nelson et al.

(2003) concluded that the receptors expressed in the tsA201 cells exist in two

stoichiometries: the majority exist with three a4-and two 32-subunits, while the minority

population possesses two a4-subunits and three 32-subunits.

The substituents at the 5'-pyrrolidine position were methyl groups in either the

trans or cis configuration. Kim et al. (1996) had determined that the addition of the

methyl at the cis-position resulted in a 35-fold decrease in affinity for rat a432 as

compared to that of the methyl group in the trans configuration. The results in this

dissertation indicated at least a 90-fold difference in affinity between the trans and cis

conformations for both the rat and human a432 with the 5'-cis-met-nic having no

interaction with the a432 (human and rat) receptor at concentrations up to 20 LtM.









5'-Trans-met-nic had a decreased selectivity ratio of 14 as compared to the selectivity

ratio of nicotine (SR = 83). 5'-Cis-met-nic also had no interaction with the a7 receptor at

concentrations up to 20 [LM. The functional measurements of these two analogs

corroborate the binding results for human a432 in indicating preferential interaction of

the trans analog with the receptor and the relative lack of interaction between the receptor

and the cis analog. Trans-5'-met-nic produced 50% inhibition (at 50 [LM) of the human

a432 ACh response.

The l'-N-pyrrolidine analogs provided information on the influence of substituent

size on binding and function. Increasing the size of substituents on the nitrogen of the

pyrrolidine ring resulted in a decreased affinity for the rat a432 receptor. 1'-N-Ethyl-nor

had a measured affinity (Ki = 0.14 [LM) comparable to (S)-nornicotine (Ki = 0.12 [LM) for

rat a4p2. It also had a selectivity ratio of 18, which is less than that of (S)-nicotine (SR =

83). There was an inverse relationship between the size of the substituent on the

pyrrolidine nitrogen and affinity. As the size of the substituent increased from an ethyl to

propyl and cyclopropyl, the affinity for a432 decreased. Neither l'-N-propyl-nor or

l'-N-cyclopropyl-met-nor had measurable binding to rat or human a432 or rat a7

receptors at concentrations up to 20 [LM. Glassco et al. (1994) also measured this trend

of decreasing affinity for rat a432 with increasing 1'-N-substituent size. They found a

20-fold decrease in affinity (as compared to (S)-nicotine) when the size of the substituent

at the 1'-N-position was increased from a methyl to an ethyl, a 369-fold decrease (as

compared to (S)-nicotine) when substituting a propyl-group and greater than 7,000-fold

decrease when substituting a cyclopropyl-group. Ferretti et al. (2003) determined that a









methyl group was optimal for binding to rat a432 such that nornicotine or the addition of

a methyl or benzyl group to the pyrrolidine nitrogen resulted in reduced affinity.

Interestingly, although the l'-N-pyrrolidine substituted analogs displayed

decreased affinity for the rat a432 receptor as compared to (S)-nicotine, they were found

(in separate experiments) to both activate and inhibit the human a432 receptor. Analogs

containing the ethyl or propyl substituent produced about a 40% activation of the receptor

as compared to the ACh maximum response for human a432. However, it is possible

that a higher concentration would produce greater activation. The cyclopropyl-methyl

substituent produced about a 75% activation of the receptor as compared to ACh. Upon

simultaneous application with ACh, each of the three of the analogs produced an

inhibition ranging from 35-80% (1'-N-ethyl-nor = 35%; 1'-N-propyl-nor = 45%;

l'-N-ethyl-nor = 80%). Channel block at high concentrations of these analogs might

explain the resultant inhibition, but it does not account for the ability of these compounds

to activate the receptor. Also, the greater percent inhibition of human a432 as compared

to percent activation by the 1'-N-cyclopropyl-nor indicates the involvement of spare

receptors. The possible involvement of spare receptors would increase the apparent

potency of these compounds resulting in a weak partial agonist appearing more potent

and efficacious than if its effects were measured under voltage-clamp conditions. Based

upon their sub-maximal activation at high concentrations as well as the predicted

influence of spare receptors, these nicotine analogs are most likely functioning as partial

agonists.

The results from the final group of nicotine compounds, the 3'-pyrrolidine

substituted analogs, paralleled those of the l'-N-pyrrolidine substituted analogs. With









increasing substituent size there was a decrease in affinity for both the rat a432 and rat

a7 receptors. Adding a small substituent (methyl) at the 3'-position resulted in an

affinity for the rat a432 receptor that was 40-fold less than that of (S)-nicotine for the

receptor. This analog also interacted with rat a7 with a Ki value of 4.2 [tM (SR for

3'-met-nic = 11 for rat a432 and 6.7 for human a432). The only other 3'-substitued

analog that had measurable binding interaction with rat a432 was DMXBN, which had a

Ki value of 2.8 pM. The other two 3'-position analogs, like DMXBN, had large benzene

ring-containing substituents. The structures of these two compounds (DMXBN and

DMXBM) resemble that of DMXBA except that the benzyl or benzylidene moieties are

attached to nornicotine and myosmine, respectively, instead of anabaseine. Whereas

DMXBA has an affinity of 0.13 [tM for rat a7 and 0.25 [tM for rat a432 (Kem et al.

2004a), neither DMXBN nor DMXBM had measurable Ki values for rat a7. The low

activity of DMXBM was probably due to its small degree of ionization at physiological

pH. Myosmine has a pKa of 5.5, which is well below the pKa (8.05) of nicotine (pKa of

nornicotine is 9.12) (Fujita et al. 1971); Glennon and Dukat, 2000). DMXBM is

predicted to have a pKa no higher than 6.0 (Kem, personal communication), so it would

be largely unionized at physiological pH (7.4).

The functional properties of the 3'-substituted analogs upon human a432 measured

with membrane potential fluorescent dye varied with the compound. Three of the

3'-pyrrolidine substituted analogs produced activation of human a432 receptors;

DMXBN produced 80% activation, DMXBM produced 20% activation and 3'-met-nic

produced 40% activation. The only compound that produced an inhibition of the human

a4P2 ACh response was 3'-met-nic (20% inhibition).









Overall, my results for the nicotine analogs indicate that substituents at any of the

four positions examined in this dissertation result in a decreased affinity for the human

and rat a432 nAChR as compared to (S)-nicotine. However, the selectivity ratios for the

5-pyridyl substituted analogs were greater than that of (S)-nicotine. Therefore,

substituents at this position confer a greater selectivity for rat a432 and merit further

investigation for possible use as smoking cessation drug. The substituted nicotine

analogs in this study also greatly influence the function of these compounds by

converting those that interact with the receptor into partial agonists and antagonists.

Rowland et al. (2003) also found that 5'-trans-met-nic produced a dose-dependent

inhibition of nicotine self-administration in rats.

Previous studies have addressed the influence of pKa for both the pyridyl and

pyrrolidine ring nitrogens. Their results indicate that differences in the basicity of these

nitrogens could not alone account for variation in affinities of nicotine analogs for the

a4P2 receptor (Dukat et al. 1998; Copeland et al. 1991). To further understand the

interaction of nicotine with the ACh binding site, Celie et al. (2004) crystallized nicotine

bound to the AChBP. Their results indicate that nicotine is primarily in contact with the

principal side of the binding site (loops A, B, and C). Also, both nitrogens form

hydrogen bonds with residues within these loops. Specifically, the pyrrolidine nitrogen

forms a hydrogen bond with tryptophan 143 within the B-loop and the pyridyl nitrogen

forms its hydrogen bond with both methionine 114 and leucine 102 within the E-loop.

Their results also indicate that the primary differences in the binding site of the AChBP

versus the a432 receptor are three amino acids. The three residue differences in a432

may provide for better contact with nicotine. The nicotine substitutions reported in this









dissertation may be preventing the proper orientation of the analogs within the binding

site for establishing the hydrogen bonds necessary for the tight binding. The 5-pyridyl

substituted nicotine analogs likely fit the binding site because the large phenyl or

pyridyl-ring does not interfere with the primary interactions between the analog and the

receptor. This may explain the ability of these analogs to retain an affinity for rat and

human a432.

Separated Nicotine Analog Enantiomers Display a Difference in Affinity for a432
Unlike Nicotine

Interestingly we found that unlike previous studies, there was no significant

difference between (S)- and (R)-nicotine in binding to a4p2 (Copeland et al. 1991;

Zhang and Nordberg, 1993). We measured the affinity for human a4p2 whereas the

previous publications were performed using rat brain. It is possible that in a

heterogenous population of receptors, as in rat brain, these previous studies were not

measuring 3H-nicotine displacement from only a4p2 receptors but that their results were

influenced by other subtypes of nAChRs. Our results for the enantiomers of nornicotine

agree with those published by Copeland et al. (1991) in that the binding affinities for

each enantiomer were very similar; their results were done with rat brain tissue whereas

our results were measured for the human a4p2-subtype. The higher affinity for the

enantiomers of nicotine over those of nornicotine is most likely not due to the differences

in pKa of each molecule. Copeland et al. (1991) discuss that the difference in the

basicity (pKa) of the nomicotine pyrrolidine nitrogen relative to that of nicotine does not

produce a difference significant enough to result in the differing affinities of the two

compounds. Nicotine is however more lipophilic than nomicotine and the intimate









interaction of nicotine's methyl group may enhance the cation-7T interaction with the

binding site and account for nicotine's higher affinity.

The results of the nomicotine enantiomers on the activation of human a432

receptors expressed in tsA201 cells indicated that they are less efficacious than the

nicotine enantiomers. The EC5o values for the nornicotine enantiomers indicated that

(S)-nornicotine is more potent than its (R)-enantiomer. This contradicts previously

published data acquired from dopamine release assays on the nucleus accumbens

indicating that (R)-nornicotine is more potent than its enantiomer (Green et al. 2001).

The results of the separated nicotine analogs, l'-N-ethyl-nor and 5-phe-nic are in

better agreement with the published literature (Copeland et al. 1991; Zhang and

Nordberg, 1993) on nicotine for the increased affinity of the (S)-enantiomer (presumably

peaks 1 for both analogs). It is important to note however, that both nicotine analogs

were studied for their affinities to rat brain membranes like the previously reported

findings for nicotine and nornicotine. Our results for the separated enantiomers do

indicate that the selectivity of the (S)-enantiomer (presumed peak 1) of 5-phe-nic for rat

a432 over c7 would be even greater than that of racemic 5-phe-nic (SR = 303).

Substituents on the D-ring of the Erythrina Alkaloids Allow for High Affinity
Binding to the a432 nAChR and Inhibition of the a4p2 Acetylcholine Response

In the literature that has been published on DH3E, it is often referred to as a

competitive antagonist selective for the a4p2 receptor (Williams and Robinson, 1984;

Anderson and Arneric, 1994; Harvey and Luetje, 1996; Harvey et al. 1996). The

characterization of DH3E along with several other natural product and semi-synthetic

Erythrina alkaloids as conducted in these studies have provided a structure activity

relationship between the a4p2 receptor and these competitive antagonists. Only one









other Erythrina alkaloid has been studied for its effects upon nicotinic receptors. Decker

et al. (1995) published that the natural product erysodine has a seven-fold higher affinity

for rat a432 than DH3E. Although our results vary from Decker et al. (1995) in that we

found DHPE to have a higher affinity (as compared to erysodine) for rat a432, it was

determined that several structural trends exist among the alkaloids, which result in an

increased affinity for the rat and human a432 receptors.

The Erythrina alkaloids were divided into two groups containing different D-rings:

the lactone-bearing erythroidines and the aromatic D-ring compounds. The importance

of the double bonds between the A and B rings of the erythroidine compounds was

determined from the binding data. By partial reduction, the two double bonds in the A

and B rings of 3-E are converted to the one double bond of DHPE; there was about an

eight-fold increase in affinity for the rat a432 receptor. Reduction of the remaining

double bond between the A and B ring results in the structure of TH3E. This compound

had an affinity for the rat a432 receptor that was less than that of 3-E. These results

indicate that the optimal number of bonds between the A and B ring is one. Neither 3-E,

nor DHPE nor TH3E had any binding interaction with rat a7 receptors at concentrations

up to 50 |JM with DHPE having the highest selectivity ratio (357) of these three

P-erythroidine alkaloids.

The possible influence of three double bonds among the A and B rings also was

studied using the 3-Des-Met-pE compound. Consistent with the idea that a single double

bond among these two rings is optimal for binding, the three double bonds in

3-Des-Met-pE (along with the lack of the 3-methoxy) abolished measurable binding to

the rat a432 receptor at concentrations up to 20 [LM. This also caused a movement of the









C and D rings with respect to the A and B rings. 3-Des-Met-pE also had no measurable

binding to the rat a7 receptors at concentrations up to 20 [LM.

Me-pE, unlike the other P-erythroidine compounds, has a charged quaternary

ammonium group. Me-pE had no interaction with rat a432 or a7 at concentrations up to

20 [LM. The addition of the methyl group may be preventing the nitrogen from forming

the necessary hydrogen bonds required for binding. The remaining 3-erythroidine

compound, 3E-Diol, allowed the importance of the D-ring in binding to be investigated.

Interestingly, the opening of the D-ring still allowed for an interaction with rat a432 that

is comparable to that of DHPE. The opening of the D-ring may allow for additional

hydrogen bonding and possibly increase the flexibility of the molecule, thus resulting in a

greater affinity than that of 3-E. Like the other erythroidine compounds 3E-Diol also had

no interaction with rat a7 at concentrations up to 50 [LM and its selectivity ratio (161)

was less than that of DHPE. Alteration in the size of the C-ring (increasing from a 6 to

7-membered ring), as determined with O-methylisophellibiline, resulted in a decreased

affinity for rat ca42. This indicates the importance of the smaller C-ring size for binding

to rat ca42.

All of the aromatic compounds were obtained as natural products. Their D-rings

lack the lactone moiety of the erythroidine compounds, and are replaced by a substituted

phenyl ring. Several of these alkaloids had measured affinities greater than that of

DHpE. ELA and Gluco-ERD were similar in that they both had large substituents on

their D-ring. With the increase in affinity for rat ca42, there was also an increased

affinity for the rat a7 receptor. Gluco-ERD, which has a large sugar molecule attached to









the D-ring, had an affinity greater than that of ELA or DH3E for rat a432. It did not

however have any interaction with the rat a7 receptor at concentrations up to 20 [tM.

The importance of a hydroxyl group at position 15 was determined from the binding

results of ERV, ERD and EST. ERV had the highest affinity for the rat a432 receptor of

all the Erythrina compounds studied. ERD is identical in structure to ERV except that

the hydroxy and methoxy groups are reversed. This is important because there is a large

decrease in affinity, 23 times less, of ERD for rat a432 as compared to ERV. The four

fused rings of these alkaloids create rigidity in the 3-dimensional structures. It is possible

that when the methoxy group is attached at position 15 there may be repulsion between

the methyl of the methoxy group and a side chain of the receptor. Also, it is possible that

the 15-hydroxy group is a H-bond donor (methoxy group is not a H-bond donor) that

enhances overall binding.

The results from the rat membrane binding studies indicate that one double bond

between the A and B ring is optimal for nAChR binding. Various manipulations and

substituents to the D-ring still allow for high affinity binding to a432. Finally, the

aromatic compounds on average have a higher affinity for both the a432 and a7

nAChRs.

All of the Erythrina alkaloids that had a measurable binding affinity for the

a4P2-receptor functioned as antagonists according to measurements performed with the

membrane potential fluorescent dye. ERV, which had the highest affinity of the

Erythrina alkaloids for rat and human a432, also had the highest potency for inhibiting

the human a432 ACh response. Although there was a significant difference (P value =

0.0186) in binding (for human a432) between ERV and ERD as measured by 3H-cytisine









displacement, there was less than a two-fold difference in inhibitory potency (for human

ca42) between the two alkaloids.

There exist several published reports on the administration of several Erythrina

alkaloids in vivo. In 1955, Megirian et al. found that 3-E, DH3E, and 3-Des-Met-pE

produced ganglionic and neuromuscular blocking action on mice, rats and cats. Several

groups determined the effects of DHPE in the CNS. Corrigall et al. (1994) administered

DHPE directly to the ventral tegmental area of rats and found that DH3E attenuated

self-administration (intravenous) of nicotine. Systemic administration of DHPE can

antagonize the locomotor activating effect of nicotine in rats (Stolerman et al. 1997).

When injected intraventricularly it was determined that DH3E disrupted spatial memory

and inhibited nicotine's stimulatory effects (Curzon et al. 1996). As an herbal medicine,

Erythrina leaves and bark (which contain alkaloids) have been found to produce

anxiolytic effects (Onusic et al. 2003).

The discovery of the acetylcholine binding protein (AChBP) has allowed for the

crystallization of ligands bound within the ACh binding sites. Until recently only

agonists were crystallized in the binding site. In 2005, two groups crystallized

antagonists, an a-conotoxin (PnIA) and an a-neurotoxin (a-cobratoxin), within the

binding sites (Celie et al. 2005; Bourne et al. 2005). Both studies found that the C-loop

was in a conformation distinctly different (projects away from the adjacent subunit) from

that of the HEPES or nicotine bound AChBP. Both groups suggest that these antagonists

are binding to the resting state as opposed to the desensitized or open state of the

receptor.