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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2008-02-29.

Permanent Link: http://ufdc.ufl.edu/UFE0021471/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2008-02-29.
Physical Description: Book
Language: english
Creator: Macdougall, Kelly N
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Kelly N Macdougall.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kem, William R.
Electronic Access: INACCESSIBLE UNTIL 2008-02-29

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021471:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021471/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2008-02-29.
Physical Description: Book
Language: english
Creator: Macdougall, Kelly N
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicine -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Kelly N Macdougall.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kem, William R.
Electronic Access: INACCESSIBLE UNTIL 2008-02-29

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021471:00001


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1 EFFECTS OF 5-HYDROXYINDOLE ON TH E AFFINITY OF AGONISTS FOR THE ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR By KELLY N. MACDOUGALL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 Kelly N. MacDougall

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3 TABLE OF CONTENTS page LIST OF TABLES................................................................................................................. ..........4 LIST OF FIGURES................................................................................................................ .........5 ABSTRACT....................................................................................................................... ..............6 CHAPTER 1 INTRODUCTION................................................................................................................... .7 2 MATERIALS AND METHODS...........................................................................................11 Chemicals...................................................................................................................... .........11 Rat Brain Membranes............................................................................................................ .11 Cell Culture................................................................................................................... ..........11 Radioligand Binding Experiments..........................................................................................12 Binding Assay Data Analysis.................................................................................................13 3 RESULTS........................................................................................................................ .......14 IC50 Data...................................................................................................................... ..........14 5-HI IC50 Effects.............................................................................................................. .....14 Hill Slope Data................................................................................................................ .......15 5-HI Hill Slope Effects........................................................................................................ ...15 Effects of Other Allosteric Modulators on DMXBA Binding................................................15 Effects of Other Indoles Similar to 5-HI................................................................................16 Effect of 5-HI on [3H]-Epibatidine Binding...........................................................................16 4 DISCUSSION..................................................................................................................... ....34 LIST OF REFERENCES............................................................................................................. ..38 BIOGRAPHICAL SKETCH.........................................................................................................42

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4 LIST OF TABLES Table page 3-1 IC50 values for anabaseine compounds without 5-HI, with 5-HI and IC50 ratios............17 3-2 IC50 values for tertiary amines without 5-HI, with 5-HI and their IC50 ratios................17 3-3 IC50 values for quaternary ammonium a gonists without 5-HI, with 5-HI and their IC50 ratios.................................................................................................................... ......18 3-4 Reported efficacies for the compounds used with 5-HI.....................................................19 3-6 Hill slope values for tertiary amine co mpounds without 5-HI, with 5-HI and their Hill slope ratios.............................................................................................................. ....23 3-7 Hill slope values for quaternary ammoni um compounds without 5-HI, with 5-HI and their Hill slop e ratios........................................................................................................ ..24

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5 LIST OF FIGURES Figure page 1-1 Examples of chemical structures from each of the three groups looked at along with 5-HT and 5-HI.................................................................................................................. ..10 3-1 Graph of anabaseine compound efficacies vs. anabaseine compound IC50 ratios............20 3-3 Graph of tertiary amine compound effi cacies vs. tertiary amine IC50 ratios....................21 3-2 Graph of quaternary ammonium compound efficacies vs. quaternary ammonium IC50 ratios.................................................................................................................... ......22 3-4 Graph of anabaseine compound efficacies vs. anabaseine compound Hill slope ratios....25 3-5 Graph of tertiary amine compound efficaci es vs. tertiary amine compound Hill slope ratios......................................................................................................................... ..........26 3-6 Graph of quaternary ammonium compound efficacies vs. quaternary ammonium compound Hill slope ratios................................................................................................27 3-7 Graphs of competition binding assa ys performed with DMXBA and PAMs...................29 3-8 Graphs of competition binding assays performed with DMXBA and other indoles.........31 3-9 Graph of competition binding assay of DMXBA with 5-HI vs. [3H]-epibatidine in SH-EP1 cells expressin human 7 nAChRs......................................................................32 3-10 Graph of competition binding assay between 5-HI vs. [3H]-epibatidine in SH-EP1 cells expressing human 7 nAChRs..................................................................................32 3-11 Graph of competition binding assay between 5-HI vs. [125I] -bungarotoxin in rat brain membranes................................................................................................................33 4-1 Chemical structures of choline and succinylcholine..........................................................37

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6 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF 5-HYDROXYINDOLE ON TH E AFFINITY OF AGONISTS FOR THE ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR By Kelly N. MacDougall August 2007 Chair: William R. Kem Major: Medical Sciences The two most abundant neuronal nicotinic acetylcholine receptor (nAChR) subtypes are the 7 and the 4 2. The 7 subtype is characterized by high affinity binding to -bungarotoxin and rapid desensitization. DMXBA (G TS-21) is a partial agonist for the 7 receptor that has been shown to enhance cognition. Positive allo steric modulators (PAMs) function by enhancing agonist activity without causing desensitization. 5-hydroxyindole (5-H I) is a positive allosteric modulator (PAM) of the 7 nAChR. I examined the effects of 5-HI on 7 receptor binding by: anabaseine-type agonists, tertiary amines, and quaternary ammonium agonists. Using radioligand competition binding assays we have shown that 5-HI enhances 7 receptor affinities for all three types of agonists. It was also found that 5-HI redu ced the Hill slope for binding of all of the agonists excep t choline and succinylcholine. The anabaseine agonists and quaternary ammonium agonists (omitting choline) s how a correlation between efficacy and IC50 ratios. Only tertiary amines displayed an apparent in crease in Hill slope ratio with efficacy. Other known PAMs did not show DMXBA binding enhancemen ts like 5-HI. Other indoles similar to 5-HI also did not change the IC50 or Hill slope values of DMXBA. We determined that 5-HI is not competing for the binding sites of the agonist epibatidine or the antagonist -bungarotoxin on the 7 receptor.

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7 CHAPTER 1 INTRODUCTION Nicotinic acetylcholine recepto rs (nAChRs) are members of a superfamily of ligand-gated ion channels. Other members of this family include GABAA, GABAC, glycine, and serotonin (5HT3) receptors (Romanelli et al., 2007). Cu rrently, 17 nAChR subunits have been cloned including five muscle-type subunits ( 1, 1, and ) and 12 neuronal subunits ( 210 and 24). Unlike the muscle-type sub units, the neuronal subunits ca n exist in a wide array of combinations to form many different subtypes of receptors each with unique properties (Jensen et al., 2004). Neuronal nAChRs are distributed throughout th e central nervous system (CNS) and the peripheral nervous system where they participate in cholinergic synaptic transmission and also modulate the activity of non-chol inergic synapses (Gotti and Clementi, 2004). The two most abundant brain receptor subtypes are the heteromeric 4 2 and the homomeric 7 nAChRs, which respectively bind nico tine and the snake toxin -bungarotoxin ( -BTX) with high affinity. These subtypes can be found on cortical and hip pocampal neurons and have been of interest lately because of their proposed involvement in neuronal pathologies such as Alzheimers disease (AD). AD is the most common form of de mentia in the elderly, affecting nearly 10% of the population over age 65. It is characterized by plaques of -amyloid peptides and neurofibrillary tangles in the br ain. Decreased cholinergic transm ission may manifest itself in the form of memory loss and cognitive impairme nts (Hogg et al., 2003). Brains of patients with AD show a loss of nAChRs in regi ons that correlate with the loss of choliner gic transmission. In the cerebral cortex, while leve ls of receptors containing the 4 subunit are greatly (~50%) reduced, 7 subunit receptor levels show much smaller decreases (Albuquerque et al., 2001). The 7 receptor is also highly perm eable to calcium, an activator of many signal transduction

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8 pathways in the cell. The cytopr otective properties seen by activating 7 nAChRs may be caused at least partly by its e ffects on calcium signaling (Dajas -Bailador et al., 2000). This makes the homomeric 7 receptor a potential target for de veloping drugs intended to increase cholinergic transmission and prot ect neuronal signaling pathways in neurodegenerative diseases (Kem, 2000). GTS-21 (DMXBA) is a selective partial agonist for the 7 receptor that has been shown to enhance cognition and neuroprotec tion in animal studies and in clinical trials (Kem et al., 2004; Kem, 2006). Kitagawa et al. (2003) reported a phase I clinical tria l on healthy young male volunteers that indicated improveme nts in attention. In 2006 Olincy et al. reported another phase I clinical trial on GTS-21. The drug was given to patients who suffered from schizophrenia. They found that GTS-21 had a positive effect on cognition. Treating nAChRs with nicotinic agonists for therapeutic purposes may have some limitations. First, even nAChR subtype specific dr ugs might have unwanted side effects in other parts of the body where the nAChR is present (C lementi et al., 2000). Another problem is nAChR desensitization by agonists. This poses a particular issue for synaptic transmission mediated by 7 nAChRs because of their extremely rapi d desensitization (Uteshev et al., 2002). An alternative therapeutic appr oach that would avoid the dese nsitizing effects of exogenous agonists would be the use of positive allosteric m odulators (PAMs). For instance, diazepam is a PAM for certain GABAA receptors. PAMs bind to their receptor target and enhance the probability that endogenous agonists will bind to and open the channel, without opening the channel when acting alone (Hogg et al., 2005). PA Ms could aid in the development of drugs for disorders such as AD because they reduce the problem of desensitization and other unwanted side effects associated with exogenous agonists.

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9 A few compounds such as iver mectin (Krause et al., 1998), galantamine (Samochocki et al., 2003), PNU-120596 (Hurst et al., 2005), and 5-hydroxyindole (5-HI) (Zwart et al., 2002) have been found to be PAMs of 7 nAChRs. 5-HI, the aromatic moiety of 5-HT, was first shown to slow desensitization of 5-HT3 receptors (Kooyman et al., 1993). In 2002, Zwart et al. showed that 5-HI also increased the potency and efficacy of ACh on 7 receptors as recorded electrophysiologically with Xenopus oocytes. 5-HI produced a maxi mal effect at concentrations around 2mM. Interestingly, in contrast wi th findings of Kooyman et al. for 5-HT3 receptors, 5HI did not affect the desensitiz ation kinetics of ACh currents on 7 nAChRs (Zwart et al., 2002). Grantham et al. (2004) found that 5-HI enhanced the affinity for both sele ctive and non-selective agonists for the 7 receptor, but not antagonists. They al so reported that 5-HI does not compete directly for the ACh binding site with competitive antagonists on the 7 receptor. We were curious to see if the degree of bi nding affinity enhancement by 5-HI on various 7 agonists correlated with their repo rted efficacies. In that case, 5-HI could be a useful tool in predicting whether new compounds are partial or full agonists at the 7 receptor by performing radioligand binding assays in the presence and abse nce of 5-HI. We chose to look at agonists of varying efficacies from three groups of agonists based on their chemical structures. The groups consisted of benzylidene-anabaseines, other te rtiary amines and quate rnary ammonium agonists (Figure 1-1). We also tested so me of the other previously menti oned PAMs to determine if they produced similar effects to 5-HI on DMXBA bindi ng. Finally, we investigated whether some indoles with structures similar to 5-HI can also produce a similar enhancement of receptor affinity for DMXBA.

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10 N N N H O H N+ O O N H N H H O H N Anabaseine 5-Hydroxyindole Acetylcholine 5-HT Tropane Figure 1-1. Examples of chemical structures fr om each of the three groups looked at along with 5-HT and 5-HI. Anabaseine is an example of an anabaseine compound. Acetylcholine is an example of a quate rnary ammonium compound. Tropane is an example of a tertiary amine compound. 5-HT is an endogenous agonist for the 5HT3 receptor. 5-HI is a metabolite of 5-HT that acts as a PAM at 7 nAChRs.

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11 CHAPTER 2 MATERIALS AND METHODS Chemicals Dr. Ferenc Soti synthesized all benzylidene anabaseine compounds in the Kem lab. NMR was used to confirm the compound structure. Al l other chemicals were obtained from either Sigma Chemical Co. (St. Louis, MO), Tocris Bioscience (Ellisville, MO), or from Fisher Scientific (Fair Lawn, New Jersey). Rat Brain Membranes Whole Sprague-Dawley rat brains were receive d frozen and unstripped from males only from Pel-Freez Biologicals (Rogers, AR). They were prepared according to Marks and Collins (1982). The amount of protein was calculated using the BCA protein a ssay reagent kit from Pierce (Rockford, IL). The br ain suspension was centrifuged at 11,000 rpm for 10 minutes. The pellet was collected and homogenized in binding saline (120 mM NaCl, 50 mM Tris-buffer, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2; pH=7.4) containing 2 mg/m l of bovine serum albumin (Sigma, St. Louis, MO). The homogenized br ain was added to test tubes containing 200 l of 2 mg/ml bovine serum albumin disso lved in binding saline, 50 l of compound, and 50 l of radioligand. For experiments with acetylcholine, the brain membrane suspension was incubated for 30 minutes at room temperature in a 200 M concentration of DFP (Sigma, St. Louis, MO) to inhibit the enzyme acetylcholine este rase. Binding experiments used 200 g of homogenized rat brain protein in a final volume of 500 l. Cell Culture Cells from the SH-EP1 human epithelial cell line transfected to express the human 7 nAChR were obtained from Dr. Ronald J. Lukas (Barrow Neurological Institute, Phoenix, AZ). Cells were grown in Dulbeccos modified Eagle s medium from Irvine Scientific (Santa Ana,

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12 CA). Each 500 ml bottle of medium was supplemented w ith 25 ml fetal bovine serum (MediaTech Inc., Herdon, VA), 50 ml heat inactiv ated horse serum (Gibco, Carlsbad, CA), 5 ml sodium pyruvate (100 mM, 11.0 mg/ml) (Irvine Scie ntific, Santa Ana, CA), 10 ml L-glutamine (200 mM, 29.2 mg/ml) (Irvine Scie ntific, Santa Ana, CA), 5 ml penicillin-streptomycin (10,000 units/ml) (Irvine Scientific, Santa Ana, CA), and 100 l of 10 mg/ml amphotericin B (Sigma, St. Louis, MO). Hygromycin B (Calbiochem, La Jolla, CA) was added to the medium at a final concentration of 0.4 mg/ml. The cells were kept in a humidified environment with 5% CO2 at 37 C. After reaching confluence, cells were co llected in binding saline for binding experiments and prepared in the same manner as the rat brain. 70 g of membrane protein was used for binding experiments in a final volume of 500 l. Radioligand Binding Experiments [125I] -bungarotoxin (150 Ci/mmole) and [3H]epibatidine (47 Ci/mmole) were obtained from PerkinElmer Life and Analytical Sciences (Billerica, MA) and GE Healthcare Bio-sciences Corp. (Piscataway, NJ). The final concentration of the radioligands in the experiment was 1 nM. 1 mM nicotine was used to cal culate non-specific binding. In [125I] -bungarotoxin experiments the membranes were incubated for 2.5 hours at 37 C, while in [3H]epibatidine binding experiments the membranes were incubated for 3 hours at room temperature. After incubation the samples were vacuum filtered on a Brandel cell harvester (Gaithersburg, MD) using Whatman GF/C glass filters that were presoaked for 45 minutes in 0.5% polyethylenimine. The samples were filtered three times with 3 ml of ice-cold binding saline with the same composition used for preparing the rat brain membranes. The filters containing [3H]epibatidine were placed in 8 ml of 30% Scintisafe scintillation fluid (F isher). Radioactivity was counted for 5 minutes using a Beckman 5500B biogamma counter for the [125I] -bungarotoxin, or a Beckman LS-6500

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13 liquid scintillation counter for the [3H]epibatidine. Each concentration of drug used for competition binding assays was performed in quadruplicate. Binding Assay Data Analysis Binding assay data were analyzed using Gra phPad Prism software (San Diego, CA). The counts per minute were calculated based on the fi ve minute counts to calculate radioactivity. Saturation assays were analyzed using a one site binding curv e with the equation Y=Bmax*X/(Kd+X). The software then calculated the Bmax for the tissue and Kd for the radioligand. Scatchard plots were derived from the saturation assays using the software in order to better visualize the data. Data was normaliz ed by setting 100% equal to the specific binding value which was calculated by subt racting non-specific bi nding from total bi nding. The data was also normalized by setting zero equal to zero perc ent. When a curve had an obvious plateau at the top that did not equal 100%, the data was constrained by nor malizing 100% to the average of the values of the points in the plateau. When a curve had an obvious plateau at the bottom that did not equal zero percent, the data was constr ained by normalizing zero percent equal to the average of the values of the points in the pl ateau. Competition binding assays were analyzed using a sigmoidal dose response curve with a va riable slope using the equation Y=Bottom+(TopBottom)/(1+10^((LogIC50-X)*Hill slope. Hill slope and IC50 values were given by the software and obtained from the previous equation. Ki values were calculated using the Cheng-Prusoff equation (Ki=IC50/(1+(Ligand)/Kd)). The Kd for [125I] -bungarotoxin binding to rat brain membranes was experimentally determined to be 0.32 nM. The Kd value for [3H]epibatidine in SH-EP1 cells was experimentally determined to be 10 nM. Statistical values were calculated using GraphPad Prism software to pe rform unpaired, two-tailed T-tests.

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14 CHAPTER 3 RESULTS The effects of 5-HI on three chemically distinct groups of agonists for the 7 nAChRs were investigated. Competition binding assays performed with rat brain membranes were used to determine Hill slope and IC50 values for the agonists alone a nd in the presence of 5-HI at a concentration (2 mM) shown in pilot expe riments to produce maximal effects. IC50 Data The 5-HI effect on the IC50 was examined for each of the three agonist groups. The IC50 values for the anabaseine agonists were cons istently decreased by factors ranging from 2.43 1.71 to 4.64 0.670 (Table 3-1). The IC50 ratios for the tertiary amine compounds range from 3.43 0.626 to 6.11 0.359 (Table 3-2). The IC50 values of the quaternary ammonium compounds were decreased as well by factor s ranging from 1.64 0.178 to 4.96 0.0379 (Table 3-3). Choline and succinylcholin e show ratios that are lower then the other ratios in the quaternary ammonium compound group. 5-HI IC50 Effects To test our hypothesis that th e efficacy of the agonist mi ght be predicted from binding enhancement by 5-HI, we examined whether efficacies and IC50 changes were related for each of the three compound groups. Table 34 lists the reported efficacies of the compounds we tested. Figure 3-1 shows a graph of the efficacies compared to the IC50 ratios for the anabaseine compounds. There is an apparent relati onship between the efficacies and the IC50 ratios for these agonists. It appears that the IC50 ratios increase as the efficacy increases. The tertiary amine data (Figure 3-2) does not show a relationship between IC50 ratio and efficacy. Next, the efficacies of the five quaternary ammoni um compounds were compared to their IC50 ratios. It was found that there was no apparent relationship between the two paramete rs unless the choline

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15 point was omitted (Figure 3-3). The rationale for eliminating this compound from consideration in this figure will be discussed later. Hill Slope Data The data for the anabaseine compounds is f ound in Table 3-5. The average Hill slope of the anabaseine agonists was -2.94 (n=7). It was found that 5-HI consistently decreased the Hill slope of the anabaseine agonist s by a factor that varied from 1.41 0.188 (anabaseine) to 2.38 0.787 (4-TFMeOBA) (Table 3-5). The data for the tertiary amine agonists is summarized in Table 3-6. The mean Hill slope for the tertiary amine compounds was 2.30 (n=7). The average Hill slope for the quaternary ammonium agonist s was -2.98 (n=7). The Hill slopes of the quaternary ammonium agonists we re decreased to a lesser de gree by 5-HI, relative to the anabaseine compounds (Table 3-7). For both choline and succinylcholine there was not a significant effect of 5-HI on the Hill slopes. 5-HI Hill Slope Effects The efficacies of the anabaseine compounds were then compared to the degree of enhancement of the Hill slopes (Figure 3-4). The efficacies of the anabaseine compounds were not obviously related to the Hill slope change s caused by 5-HI. Next, the tertiary amine compounds were examined for a relationship betw een efficacy and Hill slope ratios. It seems that there may be a relation between these two parameters. It app ears that the Hill slope ratios increase as efficacy increases (Figure 3-5). The quaternary ammonium compounds did not show any apparent correlation between efficacy and cha nge in Hill slope, unless the point for choline is omitted (Figure 3-6). Effects of Other Allosteric Modulators on DMXBA Binding To determine if other PAMs affected agonist binding in the same way as 5-HI, DMXBA was used as a standard agonist to perform competition assays on three other known PAMs:

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16 ivermectin, galanthamine, and PNU-120596 (a Pfi zer compound). Figure 3-7 shows the results of the binding assays, which show no obvious changes in Hill slope or IC50 value for DMXBA in the presence of these three PAMs. Effects of Other Indoles Similar to 5-HI To determine if other indoles which had simila r core structures to 5-HI also behaved as PAMs, three other indoles were tested with DMXBA; 5-aminoindole, 5-methoxyindole, and indole-3-acetic acid. Figure 3-8 shows the resu lts for these three indoles, using DMXBA as the displacing ligand. The three indoles di d not influence the Hill slope or IC50 values for DMXBA. Effect of 5-HI on [3H]-Epibatidine Binding Epibatidine is a potent agonist wh ich has been found to bind to 7 receptors expressed in SH-EP1 cells (Peng et al., 2005). When 5-HI was added to DMXBA in the presence of radiolabled epibatidine, the binding curve was not shifted and the Hill slope did not change (Figure 3-9). This might be e xpected if 5-HI increased affi nity for DMXBA and epibatidine equally so that no change in their competition would be expected. Figur e 3-10 shows that 5-HI does not compete directly for the agonist bindin g site as epibatidine is not displaced with increasing concentrations of 5-HI. 5-HI also does not compete directly for the binding site of the antagonist -bungarotoxin as shown in figure 3-11.

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17 Table 3-1. IC50 values for anabaseine compounds without 5-HI, with 5-HI and IC50 ratios. IC50 values were determined using GraphPad Prism software. Each value is the mean SEM of three experiments. Concentrations were done in quadruplicate for each experiment. Table 3-2. IC50 values for tertiary amines without 5-HI, with 5-HI and their IC50 ratios. IC50 values were determined using GraphPad Prism software. Each value is the mean SEM of three experiments. Concentrations were done in quadruplicate for each experiment. Anabaseine Compounds (n=3) IC50 values ( M) without 5-HI IC50 values ( M) with 2 mM 5-HI IC50 Ratio (without/with) Anabaseine 1.09 0.0334 0.275 0.0248 3.96 0.241 DMXBA 0.496 0.0288 0.123 0.0204 4.03 0.625 2,4 DHBA 0.00226 0.000656 0.000487 0.000176 4.64 0.670 4-MeS-BA 1.20 0.0682 0.313 0.0448 3.83 0.404 4-TFMeOBA 10.4 1.57 3.13 0.343 3.32 0.373 OMPBA 3.21 0.257 1.32 0.820 2.43 1.71 4-OH-GTS-21 0.404 0.0270 0.0948 0.0105 4.26 0.362 Tertiary Amine Compounds (n=3) IC50 values ( M) without 5-HI IC50 values ( M) with 2 mM 5-HI IC50 Ratio (without/with) Tropane 49.9 18.4 8.17 2.64 6.11 0.359 Tropisetron 0.0666 0.00735 0.0143 0.00222 4.66 0.977 Tropinone 130 1.07 33.5 1.81 3.88 0.224 PNU-282987 0.0808 0.00521 0.0160 0.00308 5.05 1.25 AR-17779 0.748 0.0569 0.161 0.0142 4.65 0.154 Nicotine 2.52 0.372 0.734 0.152 3.43 0.626 Cytisine 3.50 0.433 0.750 0.0274 4.67 0.360

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18 Table 3-3. IC50 values for quaternary ammonium agonist s without 5-HI, with 5-HI and their IC50 ratios. IC50 values were determined using GraphPad Prism software. Each value is the mean SEM of three experiments. Concentrations were done in quadruplicate for each experiment. Quaternary Ammonium Compounds (n=3) IC50 values ( M) without 5-HI IC50 values ( M) with 2 mM 5-HI IC50 Ratio (without/with) ACh 13.2 0.724 2.66 0.151 4.96 0.0379 Choline 260 57.4 159 53.3 1.64 0.178 Succinylcholine 210 15.5 126 5.82 1.67 0.102 Carbachol 48.7 3.23 11.5 2.09 4.23 0.582 TMA 11.1 1.89 2.91 0.632 3.81 0.348 ETMA 13.3 2.04 3.54 0.790 3.76 0.852 MG-624 0.284 0.0267 0.0834 0.0158 3.41 0.328

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19 Table 3-4. Reported efficacies for the compounds used with 5-HI. Efficacies are given as determin ed electrophysiologically by in Xenopus oocytes for the given agonists in relation to acetylcholine. Compound Efficacy (ACh=100%) Receptor type Reference Benzylidene-anabaseines: Anabaseine 100% Rat (Kem et al., 2004) DMXBA 35% Rat (Stokes et al., 2004) 2,4 DHBA 65% Rat (Hunter et al., 1994) 4-MeS-BA 7.5% Rat (Papke et al., 2004 a) 4-TFMeOBA 5% Rat (Papke et al., 2004 a) OMPBA 0 Human Kem lab unpub. data 4-OH-GTS-21 40% Human (P apke et al., 2005 a) Tertiary Amines: Tropane 28% Human (Pa pke et al., 2005 b) Tropisetron 30% Human (P apke et al., 2005 b) Tropinone 64% Human (Pa pke et al., 2000 b) PNU-282987 77% Human (G ronlien et al., 2007) AR-17779 100% Human (Papket et al., 2005 a) Nicotine 60% Rat (Papke et al., 2007) Cytisine 90% Human (Papke et al., 2005 a) Quaternary Ammoniums: ACh 100% Human (Papke et al., 2005 a) Choline 100% Rat (Stokes et al., 2004) Succinylcholine 25% Rat (Placzek et al., 2004) Carbachol 60% Human Papke, personal comm. TMA N/A N/A N/A ETMA N/A N/A N/A MG-624 50% Human Kem lab unpub. data

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20 2.0 2.5 3.0 3.5 4.0 4.5 5.0 -0.5 0.0 0.5 1.0 1.5Anabaseine Compound IC50 Ratios Efficacy (ACh=1) Figure 3-1. Graph of anabaseine com pound efficacies vs. anabaseine compound IC50 ratios. The line was fitted using a linear regression analysis in GraphPad Prism software. The r2 value for the linear re gression fit is 0.4115.

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21 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 0.0 0.5 1.0 1.5Tertiary Amine IC50 RatiosEfficacy (ACh=1) Figure 3-2. Graph of tertiary amine compound efficacies vs. tertiary amine IC50 ratios. The line was fitted using a linear regression analys is in GraphPad Prism software. The r2 value for the linear regression fit is 0.8874.

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22 1 2 3 4 5 6 0.0 0.5 1.0 1.5Quaternary Ammonium Compound IC50 RatiosEfficacy (ACh=1) Figure 3-3. Graph of quaternary ammonium co mpound efficacies vs. quaternary ammonium IC50 ratios. The line was fitted using a linear regression analysis in GraphPad Prism software. The r2 value for linear regr ession fit is 0.09396.

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23 Table 3-5. Hill slope values for anabaseine com pounds without 5-HI, with 5-HI and their Hill slope ratios. Hill slope values were determined using GraphPad Prism software. Each value is the mean SEM of three experiments. Concentrations were done in quadruplicate for each experiment. Table 3-6. Hill slope values for tertiary amine co mpounds without 5-HI, with 5-HI and their Hill slope ratios. Hill slope values were determined using GraphPad Prism software. Each value is the mean SEM of three experiments. Concentrations we re done in quadruplicate for each experiment. Anabaseine Compounds (n=3) Hill Slope Without 5-HI Hill Slope With 2 mM 5-HI Hill Slope Ratio (without/with) Anabaseine -2.50 0.320 -1.77 0.0218 1.41 0.188 DMXBA -3.34 0.478 -1.59 0.103 2.10 0.294 2,4 DHBA -2.57 0.268 -1.59 0.296 1.62 0.137 4-MeS-BA -3.00 0.463 -1.74 0.217 1.72 0.249 4-TFMeOBA -3.62 1.09 -1.52 0.233 2.38 0.787 OMPBA -3.31 0.399 -2.10 0.243 1.58 0.379 4-OH-GTS-21 -2.23 0.0464 -1.52 0.200 1.47 0.214 Tropane -1.86 0.293 -1.62 0.0686 1.15 0.216 Tropisetron -1.70 0.0664 -1.60 0.0964 1.06 0.0384 Tropinone -2.20 0.2710 -1.70 0.0345 1.29 0.1690 PNU-282987 -2.27 0.121 -1.56 0.134 1.46 0.108 AR-17779 -2.86 0.728 -1.83 0.223 1.56 0.550 Nicotine -2.69 0.971 -1.77 0.222 1.52 0.353 Cytisine -2.55 0.898 -1.55 0.0954 1.65 0.131 Tertiary Amine Compounds (n=3) Hill Slope Without 5-HI Hill Slope With 2 mM 5-HI Hill Slope Ratio (without/with)

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24 Table 3-7. Hill slope values for quaternary amm onium compounds without 5-HI, with 5-HI and their Hill slope ratios. Hill slope values were determined using GraphPad Prism software. Each value is the mean SEM of three experiments. Concentrations were done in quadruplicat e for each experiment. ACh -2.32 0.174 -1.39 0.0473 1.67 0.176 Choline -4.96 0.868 -6.88 2.90 0.721 0.224 Succinylcholine -3.40 0.448 -3.66 0.231 0.929 0.183 Carbachol -2.66 0.327 -1.53 0.202 1.74 0.0910 TMA -3.06 0.663 -2.42 0.579 1.26 0.803 ETMA -1.98 0.746 -1.29 0.158 1.53 0.0991 MG-624 -2.49 0.139 -1.95 0.0464 1.28 0.0667 Quaternary Ammonium Compounds (n=3) Hill Slope without 5-HI Hill Slope with 2 mM 5-HI Hill Slope Ratio (without/with)

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25 1.25 1.50 1.75 2.00 2.25 2.50 0.0 0.5 1.0 1.5Anabaseine Compound Hill Slope RatiosEfficacy (ACh=1) Figure 3-4. Graph of anabaseine compound ef ficacies vs. anabaseine compound Hill slope ratios. The line was fitted using a linear regression analysis in GraphPad Prism software. The r2 value for linear re gression fit is 0.2701.

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26 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 0.0 0.5 1.0 1.5Tertiary Amine Hill Slope RatiosEfficacy (ACh=1) Figure 3-5. Graph of tertiary amine compound effi cacies vs. tertiary amin e compound Hill slope ratios. The line was fitted using a linear regression analysis in GraphPad Prism software. The r2 value for linear regr ession fit is 0.8087.

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27 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5Quaternary Ammonium Compound Hill Slope RatiosEfficacy (ACh=1) Figure 3-6. Graph of quaternary ammonium co mpound efficacies vs. quaternary ammonium compound Hill slope ratios. The line was fitte d using a linear regression analysis in GraphPad Prism software. The r2 value for linear regr ession fit is 0.01034.

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28 -9 -8 -7 -6 -5 -4 -50 0 50 100 150w/o 5-HI w/ 5-HI (2mM) A log [DMXBA], M% of Control -10 -9 -8 -7 -6 -5 -4 -50 0 50 100 150w/ galantamine (20 M) w/o galantamine B log [DMXBA], M% of Control

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29 -9 -8 -7 -6 -5 -50 0 50 100 150 w/ ivermectin (300 M) w/o Ivermectin C log [DMXBA], M% of Control -9 -8 -7 -6 -5 -50 0 50 100 150w/o PNU w/ PNU (300 M) log [DMXBA], M% of ControlD Figure 3-7. Graphs of competition binding assa ys performed with DMXBA in rat brain membranes using [125I]-bungarotoxin. A) DMXBA with 5-HI. B) DMXBA with galantamine. C) DMXBA with ivermectin. D) DMXBA with PNU-120596.

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30 -9 -8 -7 -6 -5 -4 -50 0 50 100 150w/o 5-HI w/ 5-HI (2mM) A log [DMXBA], M% of Control -9 -8 -7 -6 -5 -4 -50 0 50 100 150w/ 5-aminoindole (1mM) w/o 5-aminoindole B log [DMXBA], M% of Control

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31 -9 -8 -7 -6 -5 -4 -50 0 50 100 150w/ 5-Me w/o 5-Me (20 M) log [DMXBA], M% of ControlC -9 -8 -7 -6 -5 -50 0 50 100 150w/ 5-HI 3-acetic acid (2mM) w/o 5-HI 3-acetic acid log [DMXBA], M% of ControlD Figure 3-8. Graphs of competition binding assays performed in rat brain membranes using [125I] -bungarotoxin. A) DMXBA with 5-HI. B) DMXBA with 5-aminoindole. C) DMXBA with 5-methoxyindole. D) DMXBA with 5-hydr oxyindole acetic acid.

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32 -9 -8 -7 -6 -5 -4 -50 0 50 100 150w/o 5-HI w/ 5-HI (2mM) log [DMXBA], M% of Control Figure 3-9. Graph of competiti on binding assay of DMXBA vs. [3H]-epibatidine in SH-EP1 cells expressin human 7 nAChRs. -6 -5 -4 -3 -2 -1 0 0 50 100 150log [5-HI], M% of Control Figure 3-10. Graph of competition binding assay between 5-HI vs. [3H]-epibatidine in SH-EP1 cells expressing human 7 nAChRs.

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33 -4 -3 -2 -1 0 1 0 25 50 75 100 125 150 175log [5-HI], (M)% of Specific Binding Figure 3-11. Graph of competition binding assay between 5-HI vs. [125I] -bungarotoxin in rat brain membranes.

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34 CHAPTER 4 DISCUSSION We have shown that 5-HI enhances the appare nt affinity of a variety of agonists for the 7 nAChR. In addition, 5-HI reduces the Hill slopes for agonist displacement of [125I] bungarotoxin to the 7 nAChR. Although our data are from rat brain membranes we have also done preliminary experiments with SH -EP1 cells expressing the human 7 receptor that indicates that 5-HI works in the same manner on the human receptor. Our original hypothesis was that the IC50 enhancements would vary with efficacy. Ou r data has not been able to prove this hypothesis. We have found that performing competition binding a ssays with an agonist in the presence of 5-HI is not a reliable method to determine the efficacy of the agonist. Only the anabaseine type compounds, out of the three gr oups of agonists teste d, show that such a relationship between efficacy and IC50 enhancement may exist (Figure 3-1). The tertiary amine group shows no apparent correlation between IC50 ratios and efficacy (Figure 3-2). The efficacies are not known for all of the quaternar y ammonium agonists. Therefore, TMA and ETMA have been omitted from the graphs for the quaternary ammonium compounds. Figure 33 does not reveal an appare nt relation between the IC50 ratios and efficacy of the quaternary ammonium compounds, unless the point for choline is omitted. It should be considered that the reported efficacies used to determine relationships with IC50 and Hill slope ratios are a combination of rat and human receptor efficacies. When we discovered that 5-HI wa s also changing the Hill slope values for the agonists, we decided to look for a relationship between the change in Hill slope s and the efficacies. For the anabaseine agonists and the quate rnary ammonium agonists, we f ound that a relationship was not so obvious, unless the point for choline is omitted (F igures 3-4 and 3-6). The tertiary amines do show an apparent correlation betw een Hill slope ratios and efficacy (Figure 3-5). It may be that

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35 for the tertiary amines, the Hill slope is a more sensitive measurement of differences between the compounds within the group. However, we recognize that the Hill slope is a complex variable to measure and therefore our data does no t have a simplistic interpretation. Our results for the quaternary ammonium co mpounds show two compounds that do not fit the pattern of the rest of the compounds in th is category. Choline and succinylcholine both have Hill slope ratios that do not vary significantl y from 1.0. Apparently 5-HI does not produce a change in Hill slope for these two compounds, but it does still produce an enhancement of IC50 values. The Hill slope value for choline when 5-HI is present has a very large standard of error, so this experiment will need to be repeated in or der to ascertain that this data is consistent. Interestingly, the IC50 ratios of choline and su ccinylcholine also show di fferences from the other quaternary ammonium compounds. Their ratios are very small co mpared to not only the other quaternary ammonium compounds, but also compared to all of the agonists te sted. It is possible that the chemical structures (Figure 4-1) of c holine and succinylcholine allow them to compete with 5-HI for its binding site. Both molecule s have polar hydrogen bonding groups that might compete for a common binding site in the 5-HI bindi ng domain. It is possibl e that there are other quaternary ammonium agonists that we have not looked at that behave simila rily to choline and succinylcholine. Preliminary findings for OMPBA indicate th at this anabaseine compound may be an antagonist for the 7 receptor (Kem lab, unpublished results). If this is the case, then we would not suspect 5-HI to chan ge its Hill Slope and IC50 values. However, our data shows that 5-HI does change the Hill Slope and IC50 value for OMPBA. Further el ectrophysiological studies will need to be done on this compound to determine if it is indeed a competitive antagonist, a channel blocker or a desensitizing agent.

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36 The quaternary ammonium compound MG-624 was previously determined to be an antagonist for the chick 7 nAChR (Gotti et al., 2000). Since 5-HI enhanced both the Hill slope and IC50 of this compound we were prompted to te st its efficacy electrophysiologically. We found that MG-624 produced curren ts slightly greater then 50% of the ACh maximum current on human 7 receptors in Xenopus oocytes and therefore must be considered a partial agonist on this receptor (Kem lab, unpublished results). The site of 5-HI binding and its mechanisms of action to potentiate nicotinic transmission are still unknown. Electrophysiologi cal studies show that, unlike iv ermectin, 5-HI does not need to be pre-applied to the receptor in order to potentiate the response to agonists (Zwart et al., 2002). We have shown that 5-HI is not compe ting for the binding site of agonists such as epibatidine (Figure 3-7) or antagonists such as -bungarotoxin (Figure 3-8). 5-HI thus binds to an allosteric site on the receptor that apparently increases the probability of its activation when agonist is bound. Computer docking experiments could be helpfu l in locating the binding site for 5-HI. Another useful approach might be to us e the acetylcholine-binding protein isolated from molluscan glial cells th at binds to ACh (Talley et al., 2006) The ligand binding domain of the protein is similar to that of the 7 nAChR ligand binding domain. A crystal structure of 5-HI bound to the acetylcholine-binding protein might reveal where 5-HI binds on the 7 receptor. However, it is possible that 5-HI binds to site s on the receptor that are not present on the acetylcholine-binding protein. It is interesting that 5-HI, as well as most other nicotinic recept or PAMs, has only been shown to act on 7 nAChRs and not heteromeric nicotini c receptor subtypes. 5-HI also potentiates the actions of agonists on the homomeric 5-HT3 receptor, which is closely related in sequence to the 7 nAChR (Van Hooft et al., 1997; Papke et al., 2004). This suggests that 5-HI

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37 (and other 7-selective PAMs) may need to bind to more than the two or three binding sites offered by subunits in the heteromeric nAChRs to ex ert its PAM effect. Further binding and functional investigations of 5-HI with the 7 nAChR are needed to understand the mechanisms of action of this allosteric modulator. Figure 4-1. Chemical structures of choline and succinylcholine.

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38 APPENDIX A LIST OF REFERENCES Albuquerque EX, Santos MD, Alkondon M, Pereira EF R, and Maelicke A ( 2001) Modulation of nicotinic receptor activity in the central nervous system: A novel approach to the treatment of Alzheimer disease. Alzheimer Dis Assoc Disord 15: 19-25. Clementi F, Fornasari D, and Gotti C (2000) Neuronal nicotinic receptors, important new players in brain function. Eur J Pharmacol 393: 3-10. Dani JA and Bertrand D (2007) Nicotinic acetylc holine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol 47: 699-729. Dajas-Bailador FA, Lima PA, and Wonnacott S (2000) The alpha7 nicotinic acetylcholine receptor subtype mediates nicotine protection against NMDA excitotoxicity in primary hippocampal cultures through a Ca(2+) dependent mechanism. Neuropharmacology 39: 2799-2807. Gotti C, Carbonnelle E, Moretti M, Zwart R, and Clementi F (2000) Drugs selective for nicotinic receptor subtypes: a real possibility or a dream? Behav Brain Res 113: 183-192. Gotti C and Clementi F (2004) Neuronal nicotinic receptors: From structure to pathology. Prog Neurobiol 74: 363-396. Grantham C, Berben M, Ashton D, Lesage A, and Langlois X (2004) Indol e analogues increase 7 nicotinic receptor affinity for nicotinic agonists. Society For Neuroscience Abstracts 956.13. Gronlien JH, Haakerud M, Ween H, Thorin-Hag ene K, Briggs CA, Gopalakrishnan M, and Malysz J (2007) Distinct profiles of {a lpha}7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes. Mol Pharmacol (in press). Grutter T, de Carvalho LP, Dufresne V, Taly A, and Changeux JP (2006) Identification of two critical residues within the cys-loop sequen ce that determine fast-gating kinetics in a pentameric ligand-gated ion channel. J Mol Neurosci 30: 63-64. Hogg RC, Buisson B, and Bertrand D (2005) Alloster ic modulation of ligand-gated ion channels. Biochem Pharmacol 70: 1267-1276. Hogg RC, Raggenbass M, and Bertrand D (2003) Nicotinic acetylcholine receptors: From structure to brain function. Rev Physiol Biochem Pharmacol 147: 1-46. Hunter BE, de Fiebre CM, Papke RL, Kem WR, and Meyer EM (1994) A nove l nicotinic agonist facilitates induction of long-term potentiation in the rat hippocampus. Neurosci Lett 168: 130-134.

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39 Hurst RS, Hajs M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-Root KL, Berkenpas MT, Hoffmann WE, Piotrowski DW, Groppi VE, Allaman G, Ogier R, Bertrand S, Bertrand D, and Arneric SP (2005) A novel positiv e allosteric modulator of the 7 neuronal nicotinic acetylcholine receptor: In vitro and in vivo characterization. J Neurosci 25: 4396-4405. Jensen AA, Frlund B, Liljefors T, and Krogs gaard-Larsen P (2005) Neuronal nicotinic acetylcholine receptors: Structural revelations target identifications, and therapeutic inspirations. J Med Chem 48: 4705-4745. Kem WR (2000) The brain 7 nicotinic receptor may be an important therapeutic target for the treatment of Alzheimers disease: studies with DMXBA (GTS-21). Behav Brain Res 113: 169-181. Kem WR, Mahnir VM, Prokai L, Papke RL, Cao X, FeFrancois S, Wildeboer K, Prokai-Tatrai K, Porter-Papke J, and Soti F (2004) H ydroxy metabolites of the alzheimers drug candidate 3-[(2,4-dimethoxy)benzylidene]-ana baseine dihydrochloride (GTS-21): Their molecular properties, interactions with brain nicotinic receptors, a nd brain penetration. Mol Pharmacol 65: 56-67. Kem WR, Soti F, Wildeboer K, LeFrancois S, MacDougall K, Wei DQ, Chou KC, and Arias HR (2006) The nemertine toxin anabaseine and its derivative DMXBA (GTS-21): chemical and pharmacological properties. Mar Drugs 4: 255-273. Kitagawa H, Takenouchi T, Azuma R, Wesnes KA, Kramer WG, Clody DE, and Burnett AL (2003) Safety, pharmacokinetics, and effects on cognitive function of multiple doses of GTS-21 in healthy, male volunteers. Neuropsychopharmacology 3: 542-551. Kooyman AR, van Hooft JA, and Vijver berg HPM (1993) 5-Hydroxyindole slows desensitization of the 5-HT3 receptor-mediated ion current in N1E-115 neuroblastoma cells. Br J Pharmacol 108: 287-289. Krause RM, Buisson B, Bertrand S, Corringer PJ, Galzi JL, Changeux JP, and Bertrand D (1998) Ivermectin: A positive allosteric effector of the 7 neuronal nicotinic acetylcholine receptor. Mol Pharmacol 52: 283-294. Marks MJ and Collins AC (1982) Characterizatio n of nicotine binding in mouse brain and comparison with the binding of -bungarotoxin and quinuclidinyl benzilate. Mol Pharmacol 22: 554-564. Olincy A, Harris JG, Johnson LL, Pender V, Kongs S, Allensworth D, Ellis J, Zerbe GO, Leonard S, Stevens KE, Stevens JO, Martin L, Adler LE, Soti F, Kem WR, and Freedman R (2006) Proof-of-concept trial of an 7 nicotinic agonist in schizophrenia. Arch Gen Psychiatry 63: 630-638.

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40 Papke RL, Dwoskin LP, and Crooks PA (2007) Th e pharmacological activity of nicotine and nornicotine on nAChRs subtypes: relevance to nicotine dependence and drug discovery. J Neurochem 101: 160-167. Papke RL, McCormack TJ, Jack BA, Wang D, B ugaj-Gaweda B, Schiff HC, Buhr JD, Waber AJ, and Stokes C (2005 a) Rhesus monkey al pha7 nicotinic acetylcholine receptors: comparisons to human alpha7 receptors expressed in Xenopus oocytes. Eur J Pharmacol 524: 11-18. Papke RL, Meyer EM, Lavieri S, Bollampally SR, Papke TA, Horenstein NA, Itoh Y, and Porter Papke JK (2004 a) Effects at a distance in al pha 7 nAChR selective agonists: benzylidene substitutions that regulat e potency and efficacy. Neuropharmacology 46: 1023-1038. Papke RL, Porter Papke, JK, a nd Rose, GM (2004 b) Activity of 7-selective agonists at nicotinic and serotonin 5HT3 receptors expressed in Xenopus oocytes. Bioorg Med Chem Lett 14: 1849-1853. Papke RL, Schiff HC, Jack BA, and Horenstein NA (2005 b) Molecular dissection of tropisetron, an alpha7 nicotinic acetylcholine r eceptor-selective partial agonist. Neurosci Lett 378: 140-144. Peng JH, Fryer JD, Hurst RS, Schroeder KM, Geor ge AA, Morrissy S, Groppi VE, Leonard SS, and Lukas RJ (2005) High-affinity epibatidine binding of functional, human 7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells. J Pharmacol Exp Ther 313: 24-35. Placzek AN, Grassi F, Papke T, Meyer EM, a nd Papke RL (2004) A single point mutation confers properties of the muscle-type ni cotinic acetylcholine receptor to homomeric alpha7 receptors. Mol Pharmacol 66: 169-177. Romanelli MN, Gratteri P, Guandali ni L, Martini E, Bonaccini C, and Gualtieri F (2007) Central Nicotinic receptors: Structure, function, ligands, and therapeutic potential. ChemMedChem 2: 2-24. Samochocki M, Hffle A, Fehrenbacher A, Jostock R, Ludwig J, Christner C, Radina M, Zerlin M, Ullmer C, Pereira EFR, Lbbert H, Albuquerque EX, and Maelicke A (2003) Galantamine is an allosterically potentiati ng ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors. J Pharmacol Exp Ther 305: 1024-1036. Stokes C, Papke JK, Horenstein NA, Kem WR McCormack TJ, and Papke RL (2004) The structural basis for GTS-21 selectivity between human and rat nicotinic alpha7 receptors. Mol Pharmacol 66: 14-24.

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41 Talley TT, Yalda S, Ho KY, Tor Y, Soti FS, Kem WR, and Taylor P (2006) Spectroscopic analysis of benzylidene anabaseine comple xes with acetylcholine binding proteins as models for ligand-nicotinic receptor interactions. Biochemistry 45: 8894-8902. Uteshev VV, Meyer EM, and Papke RL (2002) Ac tivation and inhibition of native neuronal alpha-bungarotoxin-sensitive nicotinic ACh receptors. Brain Res 948: 33-46. Van Hooft JA, Van Der Harr E, and Vijverberg HPM (1997) Allosteric potentiation of the 5-HT3 receptor-mediated ion current in N1E-115 neuroblastoma cells by 5-hydroxyindole and analogues. Neuropharm 36: 649-653. Zwart R, De Filippi G, Broad LM, McPhie GI, Pearson KH, Baldwinson T, and Sher E (2002) 5Hydroxyindole potentiates human 7 nicotinic receptor-mediated responses and enhances acetylcholine-induced glutamate release in cerebellar slices. Neuropharmacology 43: 374384.

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42 BIOGRAPHICAL SKETCH Kelly Nicole MacDougall was born in 1981 in Duluth, Minnesota to Deborah Yvonne Wiesen and David Bruce Wiesen. She at tended The College of Saint Scholastica in Duluth, Minnesota where she completed her und ergraduate degree in biology. Upon graduation she entered the Inderdisciplinary Program in Biom edical Sciences at the University of Florida College of Medicine. She joined the laborator y of Dr. William R. Kem in the department of Pharmacology and Therapeutics to expand her knowledge of drug discovery and cognitive disorders. Kelly married Justin Allen MacDougall in 1999. Together they have two daughters, Madison and Brooklyn. After gradua tion Kelly plans to attend the Un iversity of Florida College of Pharmacy to pursue a Doctor of Pharmacy degree.