In Vitro Pharmacological Characterization of a Novel Allosteric Modulator of 7 Neuronal Acetylcholine Receptor, 4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1- yl)benzenesulfonamide (A-867744), Exhibiting Unique Pharmacological Profile

Targeting 7 neuronal acetylcholine receptors (nAChRs) with selective agonists and positive allosteric modulators (PAMs) is considered a therapeutic approach for managing cognitive deficits in schizophrenia and Alzheimer’s disease. In this study, we describe a novel type II 7 PAM, 4-(5-(4-chlorophenyl)-2-methyl3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), that exhibits a unique pharmacological profile. In oocytes expressing 7 nAChRs, A-867744 potentiated acetylcholine (ACh)-evoked currents, with an EC50 value of 1 M. At highest concentrations of A-867744 tested, ACh-evoked currents were essentially nondecaying. At lower concentrations, no evidence of a distinct secondary component was evident in contrast to 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), another type II 7 PAM. In the presence of A-867744, ACh concentration responses were potentiated by increases in potency, Hill slope, and maximal efficacy. When examined in rat hippocampus CA1 stratum radiatum interneurons or dentate gyrus granule cells, A-867744 (10 M) increased choline-evoked 7 currents and recovery from inhibition/desensitization, and enhanced spontaneous inhibitory postsynaptic current activity. A-867744, like other 7 PAMs tested [1-(5-chloro-2-hydroxyphenyl)-3-(2chloro-5-trifluoromethyl-phenyl)urea (NS1738), TQS, and 1-(5chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596)], did not displace the binding of [H]methyllycaconitine to rat cortex 7* nAChRs. However, unlike these PAMs, A-867744 displaced the binding of the agonist [H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2azoniabicyclo[2.2.1]heptane (A-585539) in rat cortex, with a Ki value of 23 nM. A-867744 neither increased agonist-evoked responses nor displaced the binding of [H]A-585539 in an 7/5-hydroxytryptamine3 ( 7/5-HT3) chimera, suggesting an interaction distinct from the 7 N terminus or M2-3 loop. In addition, A-867744 failed to potentiate responses mediated by 5-HT3 or 3 4 and 4 2 nAChRs. In summary, this study identifies a novel and selective 7 PAM showing activity at recombinant and native 7 nAChRs exhibiting a unique pharmacological interaction with the receptor. Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the pentameric superfamily of ligand-gated ion channels that includes 5-HT3, GABAA/C, and glycine receptors. These receptors are composed of either homomeric or hetThis work was supported by Abbott. All authors are employees of Abbott. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.151886. ABBREVIATIONS: nAChR, neuronal acetylcholine receptor; 5-HT, 5-hydroxytryptamine; PAM, positive allosteric modulator; SB-206553, 3,5-dihydro5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b ]di pyrrole-1(2H)-carboxamide; PNU-120596, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea; TQS, 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide; CCMI (XY4083), N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide; 5-HI, 5-hydroxyindole; A-867744, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1yl)-benzenesulfonamide; MLA, methyllycaconitine; PNU-282987, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride; A-585539, (1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane; NS6784, 4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-1,4-diaza-bicyclo[3.2.2]nonane; NS1738, 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea; TTX, tetrodotoxin; APV, 2-amino-5-phosphonovalerate; CNQX, 6-cyano-2,3-dihydroxy-7-nitroquinoxaline; FMP, fast membrane potential; HEK, human embryonic kidney; FLIPR, fluorometric imaging plate reader; LY-2087101, [2-(4-fluoro-phenylamino)-4-methyl-thiazol-5-yl]-thiopen-3-yl-methanone; ACSF, artificial cerebrospinal fluid; IPSC, inhibitory postsynaptic current; P, puff application; Hpot, holding potential. 0022-3565/09/3301-257–267$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 330, No. 1 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 151886/3487991 JPET 330:257–267, 2009 Printed in U.S.A. 257 at A PE T Jornals on M ay 9, 2017 jpet.asjournals.org D ow nladed from eromeric / subunit combinations. Currently, 12 neuronal nicotinic subunits have been identified ( 2– 10; 2– 4) (Paterson and Nordberg, 2000; Gotti et al., 2006). One subtype abundantly expressed in the central nervous system, including in regions involved in learning and memory (hippocampus and cerebral cortex), is the homomeric 7 subunit (Rubboli et al., 1994; Wevers et al., 1994). Homomeric 7 nAChRs, when expressed in heterologous expression systems, activate and desensitize rapidly, and they exhibit relatively higher calcium permeability compared with other nAChR combinations (Sands et al., 1993; Dajas-Bailador and Wonnacott, 2004). This increased Ca permeability is thought to stimulate downstream events, including extracellular signal-regulated kinase and cAMP response elementbinding protein pathways involved in learning and cognition (Sweatt, 2004; Bitner et al., 2007). In addition, activation of 7 nAChRs contributes to neuronal excitability (Frazier et al., 1998), modulates the release of excitatory and inhibitory neurotransmitters (Alkondon et al., 2000), exhibits neuroprotective effects in experimental in vitro models of cellular damage (Levin and Rezvani, 2002), and shows procognitive effects in in vivo animals models of learning and memory (Cincotta et al., 2008). Activation or enhancement of 7 nAChR function can occur via either direct agonist activation of the orthosteric site or via positive allosteric modulation. The recent focus on the latter mechanism has led to the identification of structurally diverse positive allosteric modulators (PAMs), including SB206553 (Dunlop et al., 2009), PNU-120596 (Hurst et al., 2005), TQS (Grønlien et al., 2007), CCMI (also referred as compound 6/Q or XY4083) (Ng et al., 2007), and NS1738 (Timmermann et al., 2007). In addition, genistein (Grønlien et al., 2007), galantamine (Samochocki et al., 2003), bovine serum albumin (Conroy et al., 2003), SLURP-1 (Chimienti et al., 2003), an acetylcholinesterase-derived peptide (Zbarsky et al., 2004), and (2-amino-5-keto)thiazole compounds (Broad et al., 2006) also function as PAMs. Based on biophysical properties at least two different profiles of 7 PAMs are distinguished: type I—exemplified by 5-HI and genistein— predominantly increasing peak current amplitude response, and type II—represented by PNU-120596 and TQS—affecting both peak current response and current decay profile (Bertrand and Gopalakrishnan, 2007; Grønlien et al., 2007). Both type I and II PAMs have been shown to exhibit efficacy in vivo behavioral models. For example, PNU-120596 reversed amphetamine-induced gating deficits in rats, and CCMI improved gating deficits in DBA/2 mice (Hurst et al., 2005; Ng et al., 2007). These observations show that 7 PAMs, belonging to both types, are effective in certain preclinical in vivo models. The differential spectrum of behavioral efficacy relevant to cognitive deficits in Alzheimer’s disease and schizophrenia remains to be fully elucidated. The mechanisms or sites by which PAMs interact with 7 nAChRs to enhance the receptor function are incompletely understood. Emerging structure-function studies using chimeric and single amino acid point mutation approaches have suggested distinct regions responsible for the PAM effects of NS1738 and PNU-120596, respectively, in the N terminus and transmembrane domains (Bertrand et al., 2008; Young et al., 2008). Given the structural diversity of 7 PAM chemotypes, it is likely that there are multiple allosteric sites on the receptor. Thus, the 7 PAM interaction with the nAChRs may be similar to PAM binding to GABAA receptors. At the GABAA receptors, three different allosteric sites have been proposed: 1) within the ion channel domain transmembrane 2, near the extracellular end; 2) in the proximity of the agonist binding sites; and 3) in the linker region stretching from the agonist site loop C to the top of the transmembrane 1 region (Olsen et al., 2004). In this study, we describe A-867744, as a novel type II PAM. We demonstrate that this compound selectively potentiates 7 but not 4 2, 3 4, or 5-HT3A receptors and that it displays unique pharmacology based on radioligand and functional studies distinguishing this compound from other known 7 PAMs. Materials and Methods Materials. Oocytes were obtained from adult female Xenopus laevis frogs (Blades Biological Ltd., Cowden, Edenbridge, Kent, UK) and male Sprague-Dawley rats (10–40 days old) from Charles River Breeding Laboratories (Portage, MI). Animals were cared for in accordance with the Institutional Animal Care Committee guidelines that meet the guidelines of the National Institutes of Health (Bethesda, MD). Acetylcholine, choline, methyllycaconitine (MLA), and nicotine were obtained from Sigma-Aldrich (St. Louis, MO or Oslo, Norway) or from Tocris Bioscience (Ellisville, MO or Bristol, UK). PNU-120596, TQS, PNU-282987, and [H]A-585539 were synthesized at Abbott (Abbott Park, IL). A-867744 was also synthesized at Abbott and dissolved in dimethyl sulfoxide as stock solution (10 or 30 mM) and diluted to appropriate test concentrations right before experiments to prevent the compound from precipitating out, especially at higher concentrations tested after extended preincubation. NS6784 was obtained from NeuroSearch (Ballerup, Denmark). [H]MLA, TTX, APV, CNQX, picrotoxin, 5-HT, and atropine were obtained from Tocris Bioscience or Sigma-Aldrich. All other chemicals and reagents were obtained from Sigma-Aldrich or Fisher Scientific (Essex, UK). Two-Electrode Voltage Clamp in X. laevis Oocytes. X. laevis oocytes were prepared for electrophysiological experiments as described previously (Grønlien et al., 2007; Briggs et al., 2008). In brief, three to four lobes from ovaries of female adult X. laevis frogs were removed, manually defolliculated, and treated with collagenase type 1A (2 mg/ml; Sigma-Aldrich) prepared in low-Ca Barth’s solution [90 mM NaCl, 1.0 mM KCl, 0.66 mM NaNO3, 2.4 mM NaHCO3, 10 mM HEPES, 2.5 mM sodium pyruvate, 0.82 mM MgCl2, and 0.5% (v/v) penicillin-streptomycin solution purchased from Sigma-Aldrich, pH 7.55] for 1.5 to 2 h at 18°C under constant agitation to obtain isolated oocytes. The oocytes were injected with 20 to 25 ng of human or rat 7 nAChR cRNA, or 1 to 5 ng of human 5-HT3A cRNA, kept at 18°C in a humidified incubator in modified Barth’s solution [90 mM NaCl, 1.0 mM KCl, 0.66 mM NaNO3, 2.4 mM NaHCO3, 10 mM HEPES, 2.5 mM sodium pyruvate, 0.74 mM CaCl2, 0.82 mM MgCl2, 0.5% (v/v) penicillin-streptomycin solution, pH 7.55] and used 2 to 7 days after injection. Responses were measured by two-electrode voltage-clamp technique using Parallel Oocyte Electrophysiology Test station (Trumbull et al., 2003). During recordings, the oocytes were bathed in Ba -OR2 solution (90 mM NaCl, 2.5 mM KCl, 2.5 mM BaCl2, 1.0 mM MgCl2, 5.0 mM HEPES, and 0.0005 mM atropine, pH 7.4) and held at 60 mV at room temperature ( 20°C). Agonists were applied to recording chambers at 6 ml/s as indicated. Modulator test compounds were applied for a minimum of 60 s before agonist application, allowing for sufficient preincubation unless otherwise stated. Agonist application after preincubation with modulator was always done in the presence of the test modulator. Calcium and FMP Imaging. Functional nAChR activities were assessed in HEK-293 cells expressing human 4 2 or 3 4 subunits and in IMR-32 cells by measuring intracellular calcium changes 258 Malysz et al. at A PE T Jornals on M ay 9, 2017 jpet.asjournals.org D ow nladed from using Fluo-4 acetoxymethyl ester or no-wash calcium dye (Applied Biosystems/MDS Analytical Technologies/Molecular Devices, Sunnyvale, CA or Invitrogen, Carlsbad, CA) and the fluorometric imaging plate reader (FLIPR) system (Applied Biosystems/MDS Analytical Technologies/Molecular Devices) as described previously (Grønlien et al., 2007; Briggs et al., 2008; Gopalakrishnan et al., 2008; Faghih et al., 2009). The responses in HEK-293 cells expressing 7/5-HT3 chimera (Bertrand et al., 2008) were determined using FMP dye (Molecular Devices). In brief, cells were plated at densities of 25,000 to 100,000 cells/well in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and appropriate antibiotic selection (HEK-293-derived cell lines) or minimum essential media supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate, 1% nonessential amino acids, and 1% antibiotic-antimycotic solution (IMR-32 cells) in 96-well clear-bottomed black walled plates precoated with poly-D-lysine and allowed to incubate for 24 to 48 h at 37°C in 5% CO2 in a humidified environment. After aspirating the media, cells were incubated for 45 to 60 min with Fluo-4 acetoxymethyl ester, no-wash calcium dye, or FMP indicator dye in the dark at room temperature. Calcium dyes were dissolved in Nmethyl-D-glucamine/Ringer’s buffer (140 mM N-methyl-D-glucamine, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM CaCl2, pH 7.4), whereas FMP dye was dissolved in Ca and Mg -free phosphate-buffered saline supplemented with 0.1 mM CaCl2, 0.1 mM MgCl2, and 10 mM HEPES. When required after dye loading, cells were gently washed with the same buffer, removing extracellular dye and leaving 100 l/well after the final wash. The protocol used in studies involving HEK-293-derived cell lines was as follows. Cells were placed in the FLIPR chamber where 50 l of 3 stock concentration of test modulators or buffer prepared in the loading buffer was applied in the first addition for 5 min. In the second addition also for 5 min, 50 l of 4 stock concentrations of agonist (nicotine or PNU-282987) or buffer was added. In IMR-32 cells, double addition protocol was also used. To determine the concentration responses of A-867744, various concentrations of A-867744 were given in the first addition followed by 1 M NS6784 in the second application. In another set of experiments described in Fig. 3 (reactivation by A-867744), NS6784 (1 M) was applied first, followed by MLA (100 or 300 nM) or blank, and last by A-867744 (10 M). All experiments were carried out at room temperature ( 20–22°C). Whole-Cell Patch-Clamp Recordings in Brain Slices. Hippocampal brain slices were prepared from male Sprague-Dawley rats, fully anesthetized with Ultane (sevoflurane) or with CO2 gas, sacrificed by decapitation. Brain was rapidly removed and placed into ice-cold either high-Mg artificial cerebral spinal fluid (ACSF: 130 mM NaCl, 2.8 mM KCl, 11.3 mM MgCl2, 2.5 mM CaCl2, 1.25 mM NaH2PO4, 10 mM dextrose, and 26 mM NaHCO3 gassed continuously with 95% O2, 5% CO2, pH 7.3–7.4 at ambient temperature) or regular Mg ACSF (1 mM MgCl2 used instead of 11.3 mM). Hippocampal brain slices (250–400 m in thickness, coronal or 30° parahorizontal) were prepared using standard procedures and cut at 1 to 3°C using a Vibratome slicer with temperature-controlled oxygenated ACSF bath. Slices were preincubated at 32°C for at least 1 h before use. For each experiment, one slice was selected, placed in a chamber perfused with ACSF at ambient temperature ( 22°C), and visualized using an E600FN microscope (Nikon, Tokyo, Japan) with infrared differential interference contrast optics. Whole-cell patchclamp recordings were obtained from either hippocampus CA1 interneurons (stratum radiatum) to access fast somatic 7 currents or from hilar dentate gyrus granule cells to measure spontaneous inhibitory postsynaptic currents (IPSCs). Borosilicate glass capillary pipettes were filled with internal solution containing 10 mM CsCl, 0.5 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, 0.3 mM Na-GTP, and 135 mM methanesulfonic acid, adjusted to pH 7.3 with CsOH. Whole-cell recording was established, and neuronal activity was validated using voltage steps to activate characteristic sodium currents. IPSCs, presumably mediated by GABAA transmission, were recorded at 0 mV so that inward cationic currents (e.g., glutamatergic -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid excitatory postsynaptic currents) would be minimized and anionic currents (GABAA IPSCs) would be outward. IPSCs occurring spontaneously during 5-min epochs were recorded using a MultiClamp 700A amplifier, Digidata 1322A converter, and pClamp 9 software (Axon Instruments, Foster City, CA). When recordings from CA1 interneurons were made, cells were held at 80 mV in the presence of TTX (0.5 M), APV (20–50 M), CNQX (20–50 M), picrotoxin (50 M), and atropine (0.5 M) added to the perfusing bath ACSF to minimize the contribution of neuronally mediated glutamatergic, GABAergic, and muscarinic activities. In the presence of the blockers, 7 agonist puffs—10 mM choline or 3 mM ACh in application pipette—were made using a Picospritzer II apparatus (General Valve, Fairfield, NJ) for 5to 50-ms duration and 10 to 30 psi pressure setting. Puff pipettes were positioned 5 to 20 m away from recording cells. The exact concentration of compound reaching the cells to evoke 7 currents cannot be precisely determined, but it would be expected to be considerably lower than in the puff pipette. In dentate gyrus experiments, compounds were applied by bath perfusion (1.8 ml/min flow rate; 600l chamber). Data were stored in a PC and analyzed using pClamp 9 or MiniAnalysis 6.0.3 software (Synaptsoft, Decatur, GA) as appropriate. Whole-Cell Patch-Clamp Recordings in HEK-293 Cells Expressing 7/5-HT3 Chimera. Standard whole-cell patch-clamp technique was used to record from HEK-293 cells stably expressing chimeric 7/5-HT3 receptors (i.e., chimera 2 in Bertrand et al., 2008). Cells were plated 48 to 96 h on round poly-D-lysine-precoated glass coverslips before recordings. Experiments were done using pClamp 8.0 or 9.0 (Applied Biosystems/MDS Analytical Technologies/Axon Instruments) installed on a Dell Pentium III computer connected to an Axon 200B amplifier (Applied Biosystems/MDS Analytical Technologies/Axon Instruments, Sunnyvale, CA). Changes in test solution conditions were obtained using a pulled theta tube controlled by Perfusion Fast-Step system (Warner Instruments, Hamden, CT). The pipette solution contained 120 mM KCl, 30 mM NaCl, 2 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.2 to 7.3. The bath solution contained 140 mM NaCl, 5 mM KCl, 0.1 mM CalCl2, 0.1 mM MgCl2,10 mM HEPES, and 0.5 M atropine. Cells were held at 80 mV unless specified otherwise. Radioligand Binding Experiments. Experiments were carried out as described previously (Anderson et al., 2008). Membrane homogenates were prepared from rat cortex and HEK-293 cells stably expressing chimera (i.e., chimera 2 in Bertrand et al., 2008). In ligand saturation experiments, [H]A-585539 and [H]MLA were used in concentrations ranging from 0.04 to 6 nM. In competition experiments, compounds were tested in increasing concentrations up to the highest of 30 M, for their abilities to displace [H]A-585539 or [H]MLA binding. Nonspecific binding was defined with 10 or 30 M MLA. Data Analysis. In two-electrode voltage-clamp studies, responses were quantified by measuring peak current amplitude. Peak current responses were expressed as percentage response to 100 M ACh ( 7 currents) or 2 to 3 M 5-HT (5-HT3A currents) in the presence of varying test modulator concentration responses, or to 1 mM ACh (without PAM) in agonist concentration responses ( 7 oocytes). In Ca imaging or FMP studies, raw fluorescence responses were corrected by subtracting fluorescence values from wells with buffer only added. Peak fluorescent responses were determined over the range of drug exposure using FLIPR software and expressed as a percentage of the reference peak response for 3 to 10 M nicotine ( 4 2 and 3 4) or 25 nM PNU-282987 (chimera studies) to determine the effects of A-867744. In FMP imaging studies, concentration-response experiments to PNU-292987 were normalized to the maximal control agonist response. Whole-cell patch-clamp responses determined in hippocampus brain slice were normalized to the choline-evoked responses obtained before the addition of A-867744. Radioligand binding Ki constants were calculated as described previously (Anderson et al., 2008). Concentration-response graphs were In Vitro Pharmacological Characterization of 7 PAM A-867744 259 at A PE T Jornals on M ay 9, 2017 jpet.asjournals.org D ow nladed from prepared using Prism (GraphPad Software Inc., San Diego, CA). Data are reported as means with 95% confidence intervals or S.E.M.), n is the number of independent determinations made, and p 0.05 was considered statistically significant.