Enhancement of α7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective α7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to α7 nAChRs [rat or human cortex, [3H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), Ki = 0.2–0.6 nM or [3H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-α7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman α3β4, chimeric (α6/α3)β4, or 5-HT3A receptors, and weak or negligible Ca2+ responses in human neuroblastoma IMR-32 cells (α3* function) and human α4β2 and α4β4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat α7 nAChR current responses in oocytes (EC50, 50–90 nM total charge, ∼80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked α7-like currents, which were inhibited by the α7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an α7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive α7 nAChR-mediated Ca2+ signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity α7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.
Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the pentameric superfamily of Cys-loop ligand gated ion channels. They are composed of either homomeric α or heteromeric α and β subunits assembled from a family of 12 distinct neuronal nicotinic subunits (α2–α10; β2–β4) (Dani and Bertrand, 2007; Albuquerque et al., 2009). The specific assembly of nAChR subunits determines the biophysical and pharmacological properties of recombinant and native receptors. For example, homomeric α7 nAChRs activate and desensitize rapidly in response to agonists, show relatively low potency to acetylcholine (ACh) and nicotine, and exhibit high calcium permeability compared with other nAChR subtypes (Fucile, 2004; Dani and Bertrand, 2007).
Activation of α7 nAChRs is thought to be procognitive, improving learning functions. Although the mechanisms linking α7 nAChRs to in vivo efficacy remain poorly understood, they may involve modulation of excitatory and/or inhibitory neurotransmitter pathways (Alkondon et al., 2000) and stimulation of downstream biochemical signals, including phosphorylated extracellular-regulated kinase (pERK) and phosphorylated cAMP response element binding protein (pCREB) in the cerebral cortex and hippocampus (Sweatt, 2004; Bitner et al., 2007). At the cellular level, α7 nAChRs contribute to neuronal excitability (Frazier et al., 1998) and exhibit neuroprotective effects in experimental in vitro models of cellular damage (Levin and Rezvani, 2002). These observations provide a working hypothesis supporting the utility of targeting the α7 nAChRs for treatment of neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia.
Much of the evidence for a role of α7 nAChRs in cognitive processing has been provided by studies using synthetic agonists. For example, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) enhanced cognitive performance in behavioral assays that involve working memory, short-term recognition memory, and long-term memory consolidation. A-582941 also normalized sensory auditory gating deficits modeling a schizophrenic phenotype (Bitner et al., 2007). Likewise, 1,4-diazabicyclo[3.2.2]nonane-4-carboxylic acid, 4-bromophenyl ester (SSR180711) enhanced episodic memory in the object recognition task, reversed dizocilpine (MK-801)-induced deficits in retention of episodic memory, and restored MK-801- or 1-(1-phenylcyclohexyl)piperidine (phencyclidine)-induced memory deficits in the water Morris or linear maze (Pichat et al., 2007). N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987) enhanced GABAergic synaptic activity and oscillatory activity in the hippocampus and reversed an amphetamine-induced sensory gating deficit (Hajós et al., 2005). Both PNU-282987 and SSR180711 also increased c-Fos in the prefrontal cortex and the shell of nucleus accumbens similar to the effect of antipsychotics (Hansen et al., 2007).
Several other novel α7 nAChR agonists—(2R)-spiro-[1-azabicyclo[2.2.2]octane-3,2(3H)-furo[2,3-b]pyridine]-tartrate (AZD-0328) (Sydserff et al., 2009), 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide (SEN-12333/WAY-317538) (Roncarati et al., 2009), (S)-(1-aza-bicyclo[2.2.2]oct-3-yl)-carbamic acid (S)-1-(2-fluoro-phenyl)-ethyl ester (JN-403) (Feuerbach et al., 2009), 2[2(4-bromophenyl)-2-oxoethyl]-1- methyl-pyridinium chloride (S24795) (Lopez-Hernandez et al., 2007), and N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]-1-benzofuran-2-carboxamide (TC-5619) (Hauser et al., 2009)—have been recently described. Although these compounds have provided considerable insight supporting the involvement of α7 nAChRs in cognitive and learning functions, the majority of them have inherent limitations. For example, A-582941 (Bitner et al., 2007), MEM3454 (Rezvani et al., 2009), and AZD-0328 (Sydserff et al., 2009) also exhibit relatively high affinity at 5-HT3 receptors, complicating interpretation of their pharmacological effects. Likewise, SEN-12333/WAY-317538 is a potent antagonist at H3 receptors. JN-403, S24795, and SSR180711 have relatively weak potency and/or low maximum efficacy as α7 agonists (Biton et al., 2007; Lopez-Hernandez et al., 2007; Feuerbach et al., 2009).
In this study, we describe the in vitro profile of a novel selective α7 agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). This compound has high affinity for the human and rat α7 nAChRs and submicromolar potency and nearly full efficacy at recombinant α7 nAChRs expressed in Xenopus oocytes. ABT-107 exhibits weak interaction with non-α7 nAChRs, 5-HT3, and H3 receptors. The effects of this compound are described herein at native α7 nAChRs, in an in vitro glutamate neurotoxicity model, and evaluated in the context of interaction with the selective α7 positive allosteric modulators (PAMs) N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596) and 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744) (Hurst et al., 2005; Malysz et al., 2009a). This study and the accompanying report by Bitner et al. (2010) show that selective targeting of α7 nAChRs has unique potential for modulating pathways of therapeutic relevance for both symptomatic alleviation and disease modification associated with central nervous system (CNS) disorders such as AD.
Materials and Methods
Adult female Xenopus laevis frogs were obtained from Blades Biological Ltd. (Cowden, Edenbridge, Kent, UK), and male Sprague-Dawley rats were from Charles River Breeding Laboratories (Portage, MI; 10–40 days old). Other animals used were obtained from commercial sources as indicated. Experimental procedures involving animals were conducted under protocols approved by the Institutional Animal Care and Use Committee and are consistent with American Association for Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. ACh, methyllycaconitine (MLA), and nicotine were obtained from Sigma-Aldrich (St. Louis, MO or Oslo, Norway) or Tocris Bioscience (Ellisville, MO or Bristol, UK). ABT-107, A-867744, PNU-120596, and [3H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539) were synthesized at Abbott (Abbott Park, IL). [3H]MLA was purchased from ARC (American Radiolabeled Chemicals, St. Louis, MO). Tetrodotoxin (TTX), (2R)-amino-5-phosphonovaleric acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), picrotoxin, and atropine were obtained from Tocris Bioscience or Sigma-Aldrich. All the other chemicals and reagents were obtained from Sigma-Aldrich or Thermo Fisher Scientific (Waltham, MA), or as indicated.
α7 nAChR binding in human and rat brain.
Both antagonist ([3H]MLA) and agonist ([3H]A-585539) radioligand assays were used to determine α7 nAChR affinity of ABT-107 as described previously (Anderson et al., 2008). Briefly, membrane-enriched fractions from rat brain minus cerebellum were purchased (ABS Inc., Wilmington, DE or Pel-Freez Biologicals, Rogers, AR) or prepared fresh from male Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA), 0.5 to 4 months old. Individual brains minus cerebellum were homogenized for 10 s in 0.32 M sucrose and 10 mM Tris-Cl, pH 7.4, using a Kinematica (Littau-Lucerne, Switzerland) Polytron homogenizer. The tissue suspensions were centrifuged at 1000g for 10 min at ∼4°C. The supernatants were centrifuged at 20,000g for 20 min (∼4°C). The resulting pellets were washed twice by centrifugation at 40,000g (∼4°C) for 15 min in 20 ml of H2O, and the final pellets were frozen at −80°C. Human cerebral cortex (purchased from ABS Inc.) membranes were prepared using the same protocol. Binding conditions for the α7 nAChR antagonist [3H]MLA were as described in detail elsewhere (Anderson et al., 2008). In competition experiments, membrane samples containing 100 to 200 μg of protein, ∼5 nM [3H]MLA (25 Ci/mmol; PerkinElmer Life and Analytical Sciences, Waltham, MA), and 0.1% bovine serum albumin (BSA) in Balanced Salt Solution (BSS)-Tris buffer, pH 7.4, were incubated in duplicate with various concentrations of ABT-107 in a final volume of 500 μl for 60 min at 22°C. Nonspecific binding was determined in the presence of 10 to 30 μM MLA. Bound radioactivity was isolated by vacuum filtration onto glass fiber filter plates presoaked with 2% BSA (Millipore Corporation, Billerica, MA) using a 96-well filtration apparatus (PerkinElmer Life and Analytical Sciences Waltham, MA) and washed with 2 ml of ice-cold BSS. Forty microliters of scintillant were added to each well, and radioactivity was determined using scintillation counting in a TopCount (model NXT; PerkinElmer Life and Analytical Sciences). Binding conditions for the α7 nAChR agonist [3H]A-585539 (∼0.4 nM, 62 Ci/mmol) were essentially the same except that the incubation was carried out at 4°C for ∼75 min without BSA and the filter plates were presoaked with 0.3% polyethyleneimine (Sigma-Aldrich). IC50 values were determined by regression analysis using Microsoft (Redmond, WA) Excel or Assay Explorer (Symyx, Sunnyvale, CA). Ki values were calculated from the IC50 values using the Cheng-Prusoff equation using Kd values of 0.063 and 1.26 nM for [3H]A-585539 and [3H]MLA, respectively (Anderson et al., 2008).
α4β2* nAChR binding in rat brain.
Tissue was the same as that used for the rat brain α7 nAChR binding assays, and binding conditions were similar. [3H]Cytisine binding to the α4β2* subtype in rat brain membranes was determined using the method described previously (Anderson et al., 2008). Membrane-enriched fractions were slowly thawed at 4°C, washed, and resuspended in 30 volumes of BSS-Tris buffer (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 50 mM Tris-Cl, pH 7.4, 4°C). Samples containing 100 to 200 μg of protein, ∼0.75 nM [3H]cytisine (30 Ci/mmol; PerkinElmer Life and Analytical Sciences), and various concentrations of ABT-107 were incubated in a final volume of 500 μl for 75 min at ∼4°C. Nonspecific binding was determined in the presence of 10 μM (−)-nicotine. Bound radioactivity was collected and measured as described for the [3H]A-585539 radioligand binding assay. Ki values were calculated from the IC50 values using the Cheng-Prusoff equation using a Kd value of 0.077 nM.
α1β1γδ muscle receptor binding in TE671.
TE671 human medulloblastoma cells were obtained from American Type Culture Collection (ATCC; Manassas, VA) and grown at 37°C in polystyrene culture flasks with Dulbecco's modified Eagle's medium containing 10% (v/v) horse serum and 5% (v/v) fetal calf serum under a humidified atmosphere of 5% CO2 in air. At confluence, TE671 cells were rinsed with phosphate-buffered saline, and intact cells were harvested mechanically and frozen as a cell suspension at −80°C. Before use, the frozen cell suspension was slowly thawed and centrifuged at 2°C for 10 min (27,000g). After two washes of the pellet in 20 mM HEPES buffer containing 118 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, and 2.5 mM CaCl2, pH 7.5, the final pellet was resuspended in 20 mM HEPES buffer containing 0.01% BSA to 4 × 106 cells/ml and used for binding assays as described by Lukas (1986). Aliquots of 500 μl of suspension were added to 25 μl of test solution and 25 μl of [3H]α-bungarotoxin (∼1 nM final concentration, 60 Ci/mmol; GE Healthcare, Little Chalfont, Buckinghamshire, UK), mixed, and incubated for 2 h at 37°C. Nonspecific binding was determined with d-tubocurarine (100 μM). After incubation, the samples were poured directly onto Whatman (GE Healthcare, Piscataway, NJ) GF/C glass fiber filters (presoaked in 0.1% polyethyleneimine for at least 30 min) under suction and immediately washed twice with 5 ml of ice-cold buffer. Bound radioactivity on the filters was counted. A Kd value of 0.81 nM for [3H]α-bungarotoxin was used for calculation of Ki values using the Cheng-Prusoff equation.
Methods followed the protocols described elsewhere (Jørgensen et al., 2008). Dopamine transporter (DAT)-containing synaptosomes were prepared from striatum dissected out from Wistar rats (150–200 g; Taconic, Ry, Denmark). Tissue was homogenized for 5 to 10 s in 100 volumes of ice-cold 0.32 M sucrose containing 1 mM pargyline using a motor-driven Teflon (DuPont, Wilmington, DE) pestle in a glass homogenizing vessel. The homogenate was centrifuged at 1000g for 10 min. The resulting supernatant was centrifuged at 27,000g for 50 min at 4°C. The supernatant was discarded, and the crude synaptosomal pellet was resuspended (2000 ml/g of original tissue) in oxygenated Krebs-Ringer incubation buffer (NaCl, 122 mM; EDTA, 0.16 mM; KCl, 4.8 mM; Na2HPO4, 12.7 mM; NaH2PO4, 3 mM; MgSO4, 1.2; CaCl2, 0.97 mM; glucose, 10 mM; ascorbic acid, 1 mM; adjusted to pH 7.2). The preparation of norepinephrine transporter (NET)- and serotonin transporter (SERT)-expressing synaptosomes was performed as described for DAT-expressing synaptosomes using hippocampi and cerebral cortices, respectively, diluted to 500 ml/g of original tissue. Aliquots of 900 μl of tissue suspension were added to 50 μl of test solution and 50 μl of [3H]dopamine, [3H]norepinephrine, or [3H]5-hydroxytryptamine, respectively (all 5 nM, final concentration), mixed, and incubated at 37°C. Nonspecific uptake was determined in the presence of 10 μM benztropine, 1 μM desipramine, or 1 μM citalopram for DAT-, NET-, and SERT-expressing synaptosomes, respectively. Incubation (30 min for 5-hydroxytryptamine, 90 min for norepinephrine, and 25 min for dopamine) was terminated by filtration of the samples over UniFilter GF/C glass fiber filters (PerkinElmer Life and Analytical Sciences) using a Tomtec (Hamden, CT) cell harvester (Harvester 96), and the filters were washed with approximately 5 ml of ice-cold 0.9% (w/v) NaCl solution. The amount of radioactivity on the filters was determined by conventional liquid scintillation counting using a TopCount liquid scintillation counter (model NXT; PerkinElmer Life and Analytical Sciences).
Electrophysiological Recordings Using Oocytes.
The preparation of X. laevis oocytes, injection of cRNA, maintenance of the oocytes, and experimental procedures and analyses generally followed standard procedures described previously (Grønlien et al., 2007; Briggs et al., 2009; Malysz et al., 2009b). The oocytes were injected with human or rat α7 nAChR cRNA, human or ferret α3 plus β4 cRNA, human chimeric (α6/α3)—containing α61–207–α3208–446 sequence as described by Kuryatov et al. (2000)—plus human β4 cRNA or human 5-HT3A cRNA within 24 h of their preparation and were used 2 to 7 days after injection. Experiments were conducted in OR2 solution (90 mM NaCl, 2.5 mM KCl, 2.5 mM BaCl2, 1 mM MgCl2, and 5 mM Na-HEPES, pH 7.4) with 0.5 μM atropine added to block muscarinic receptors. Compounds were applied, and responses were measured under two-electrode voltage clamp (−60 mV cell potential) in the parallel oocyte electrophysiology test station apparatus (Malysz et al., 2009b). Responses were normalized to control effects of ACh (either 1 or 10 mM ACh as specified for agonist activity or 0.1 mM ACh for assessment of antagonist/inhibitor profile) for nAChR-mediated currents and to 5-HT (10 μM in agonist and 2–3 μM in antagonist/inhibitor experiments) for determination of human 5-HT3A receptor activity. The normalized values were used to determine concentration-response curve parameters by nonlinear curve fitting of the Hill equation using GraphPad Software Inc. (San Diego, CA) Prism software or a similar algorithm incorporated in the parallel oocyte electrophysiology test station software (Malysz et al., 2009b).
Electrophysiological Recordings in Human Embryonic Kidney 293 Cells Expressing Human α3β4 nAChRs.
Human embryonic kidney (HEK)-293 cells expressing human α3β4 nAChRs were plated on poly-d-lysine-coated glass coverslips in growth media for at least 24 h. Before experiments, coverslips were transferred to a perfusion chamber, and cells were visualized with an inverted microscope. The cells were superfused with extracellular buffer containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM Na-HEPES, pH 7.4. Micropipets were made from borosilicate glass and filled with intracellular buffer containing 120 mM K-gluconate, 6 mM KCl, 5 mM NaCl, 2 mM MgCl2, 0.5 mM EGTA, 2 mM ATP, 0.2 mM GTP, and 10 mM K-HEPES, pH 7.4. Pipet resistances were 2 to 5 MΩ, and series resistance was compensated by 80%. Voltage-clamp recordings (−60 mV) were made using an EPC-9 amplifier (HEKA, Lambrecht/Pfalz, Germany) operated through a Macintosh G4 computer (Apple Computer, Cupertino, CA). Data were sampled at 20 kHz and low pass-filtered at 6.7 kHz. ABT-107 and ACh were applied for 1 s with at least 30-s washout between agonist applications to allow for nAChR recovery from desensitization. Compounds were delivered using a two-barreled micropipet fashioned from theta-type glass capillary tubing with a tip opening of 50 to 100 μm. Solutions were applied through the two halves of the theta-tube via Teflon (DuPont) tubing. The tip of the theta-tube was positioned in the immediate vicinity of the voltage-clamped cell under visual guidance. The lateral position of the theta-tube was controlled via a piezoceramic device (Burleigh Instruments, Fishers, NY), allowing the environment of the cell to be rapidly switched between the buffers flowing from the two barrels (e.g., with or without agonist).
Fluorescence Imaging Plate Reader Calcium Imaging Assay.
Experiments were carried out according to methods described recently (Grønlien et al., 2007; Briggs et al., 2009). HEK-293 cell lines stably expressing human α4β2 or α4β4 nAChRs were established and maintained using standard procedures. IMR-32 cells were obtained from ATCC and maintained using the procedures provided by the vendor. Agonist-evoked Ca2+ increases were measured using Fluo-4/acetoxymethyl to detect intracellular Ca2+ in conjunction with a fluorescence imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA; Danaher, Washington, DC) equipped with an argon laser and a charge-coupled device camera (Molecular Devices; Danaher). Black-walled 96-well plates were used to reduce light scattering. The cell permeant acetoxymethyl ester form of Fluo-4 was prepared to a concentration of 1 mM. The dye was then diluted to a final concentration of 2 μM in growth medium and placed with the cells for 60 to 90 min at room temperature. The unincorporated dye was removed from the cells by washing with assay buffer containing 140 mM N-methyl-d-glucamine, 5 mM KCl, 1 mM MgCl2, 10 mM CaCl2, and 10 mM HEPES, pH 7.4. After addition of various concentrations of ABT-107, the Ca2+ signals were measured in FLIPR. The percentage of maximal fluorescence intensity relative to that induced by 100 μM (−)-nicotine, applied as reference control to each plate, was used to determine the concentration-response parameters for ABT-107 by nonlinear curve fitting. Inhibitory effects were determined by first exposing the cells to various concentrations of ABT-107 followed by a second addition of 100 μM (−)-nicotine. The degree of inhibition was determined by comparison of the nicotine-evoked response after previous test compound addition.
In additional experiments, the α7 nAChR response in IMR-32 cells and cortical cultures was assessed by using a combination of a selective α7 nAChR PAM, A-867744, or PNU-120596 and the α7 agonist ABT-107 according to methods published previously (Malysz et al., 2009a; Ween et al., 2010). The responses were normalized to wells receiving coapplication of ABT-107 (0.1 μM) and either 10 μM A-867744 or PNU-120596.
Hippocampal Brain Slice Electrophysiology.
Whole cell patch-clamp recordings from brain slices were carried out as described recently (Malysz et al., 2009a). Briefly, hippocampus brain slices were prepared from 15- to 22-day-old male Sprague-Dawley rats (Charles River Laboratories, Inc.). Rats were fully anesthetized with Ultane (sevoflurane; Abbott, Abbott Park, IL), sacrificed by decapitation, and the brain was rapidly removed into ice-cold regular or high-Mg2+ artificial cerebral spinal fluid (ACSF; 130 mM NaCl, 2.8 mM KCl, 11.3 mM or 1 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). Hippocampus brain slices (250–350 μm thick, coronal or 30° parahorizontal) were prepared using standard procedures and cut at 1 to 3°C using a vibratome 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 a Nikon (Melville, NY) E600FN microscope with infrared differential interference contrast optics. Patch-clamp recordings were obtained using borosilicate glass capillary pipets 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 monitored at −70 mV, and neuronal activity was validated using voltage steps to activate characteristic sodium currents. Inhibitory postsynaptic currents (IPSCs), presumably mediated by GABAA transmission, were recorded at 0 mV so that cationic currents (such as glutamatergic α-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 (Axon Instruments, Sunnyvale, CA), Digidata 1322A converter (Molecular Devices; Danaher), and pClamp 9 software (Molecular Devices). Compounds were applied by bath perfusion (1.8 ml/min flow rate, 600-μl chamber). Data were stored in a personal computer and analyzed using pClamp 9 or MiniAnalysis 6.0.3 (Synaptsoft, Fort Lee, NJ) software to detect IPSC activity. Fast somatic currents were recorded from hippocampus CA1 stratum radiatum interneurons by ABT-107 application using a Picospritzer II (General Valve, Fairfield, NJ; pressure pulses, 5–100 ms and 20–30 psi) in the presence of the blockers (0.5 μM atropine, 10–30 μM CNQX, 10–50 μM APV, 50 μM picrotoxin, and 0.5 μM TTX) at the holding potential of −80 mV.
pERK Kinase Signaling Assay.
PC-12 cells were obtained from ATCC and maintained in Ham's F-12 medium, 2.5% fetal bovine serum (Invitrogen, Carlsbad, CA), and 15% horse serum (Sigma-Aldrich) on poly-d-lysine-coated 96-well plates using procedures specified by ATCC. Twenty-four hours before treatment, the medium was replaced with unsupplemented Ham's F-12 medium to reduce basal phosphorylation. All the treatments were conducted with compounds diluted in Hank's balanced salt solution (HBSS) at 37°C. Cells were preincubated for 30 min in 50 μl of HBSS without or with test inhibitors, followed by addition of another 50 μl of HBSS and further 10-min preincubation, followed by addition of another 50 μl of HBSS containing test compounds, as indicated, for a final 7-min incubation. The incubation was terminated by placing the plate on ice and removing the incubation medium by aspiration. The cells were then treated on ice for 15 min with 5 μl of Insect Cell Lysis Buffer (BD Pharmingen, San Jose, CA) plus a mixture containing 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, and 1% protease inhibitor mixture (Sigma-Aldrich) plus 100 units/ml benzonase (Novagen, Madison, WI) and then stored at −80°C. Because of potential edge effects, two half-plates in a 6 × 8 array format, omitting outer wells, were run on one 96-lane gel.
Glutamate Cytotoxicity Assay in Cortical Cell Cultures.
Primary rat neonatal cortical neurons were isolated from newborn rat pups and cultured as described elsewhere (Manelli and Puttfarcken, 1995). Primary cortical cultures (7–9 days in culture) were incubated with ABT-107 or MK-801 for 2 h before a 15-min exposure to 300 μM glutamate/10 μM glycine. After exposure, the cells were washed twice and placed overnight in low glucose (5 mM) containing Dulbecco's modified Eagle's medium, with or without ABT-107 or MK-801. Cell viability was determined by measuring ATP levels (CellTiter-Glo Luminescent Cell Viability Assay; Promega, Madison, WI) 24 h after exposure to glutamate/glycine. Values were normalized to control wells, which were not exposed to glutamate/glycine insult.
In two electrode voltage-clamp studies, responses were quantified by measuring peak current amplitude or total charge transfer (integral) and normalized as described. In Ca2+ imaging experiments, 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 control responses (defined separately for each condition). Radioligand binding Ki constants were calculated using the Cheng-Prusoff equation as described previously (Anderson et al., 2008). Concentration-response graphs were prepared using GraphPad Prism (GraphPad Software Inc.). Data are reported as means with 95% confidence intervals (CI) or S.E.M. and the number of independent determinations made (n). Statistical significance was considered with p < 0.05 (Student's t test).
Radioligand Binding Profile of ABT-107.
Cerebral cortical homogenates express α7 and α4β2* nAChRs that selectively bind radioligands [3H]A-585539 and [3H]MLA (for α7 nAChRs) and [3H]cytisine (for α4β2* nAChRs). Affinities of ABT-107 for these receptors were examined in displacement experiments. As shown in Fig. 1b and summarized in Table 1, ABT-107 showed comparable Ki values of ∼0.2 to 0.6 nM for human and rat α7 nAChRs when examined by [3H]A-585539 displacement and a Ki value of 7 nM for antagonist [3H]MLA displacement. At other nAChRs examined, ABT-107 showed at least ∼200-fold selectivity (Fig. 1b; Table 1). Ki values at α4β2* in rat brain and α1β1γδ (TE671 cells) nAChRs were 4 and 1.8 μM, respectively.
To further characterize the binding interaction of ABT-107 with the α7 nAChR, [3H]A-585539 competition displacement experiments were carried out in the presence and absence of the α7 PAM PNU-120596 in rat cortical homogenates. Similar competition experiments were also done with MLA. PNU-120596 alone did not displace [3H]A-585539 binding up to the highest concentration tested of 10 μM (Malysz et al., 2009a). As shown in Fig. 1c, the Ki constants in the absence and presence of PNU-120596 (10 μM) were, respectively, 1.0 nM (0.8–1.4 nM, 95% CI, n = 3–5) and 1.3 nM (1.0–1.8 nM, n = 3–5, p > 0.05) for ABT-107, and 2.5 nM (1.7–3.6 nM) and 3.8 nM (2.6–5.6 nM, n = 3–5, p > 0.05) for MLA. Hence, there were no significant effects of PNU-120596 on ABT-107 or MLA binding to the α7 nAChRs revealed by the displacement of [3H]A-585539. The effect of ABT-107 was further examined in a radioligand binding panel (Cerep, Seattle, WA) consisting of 81 receptors, ion channels, and transporters. ABT-107 showed only modest interaction with human 5-HT2A [80% displacement at 10 μM and Ki of 3.9 μM, [3H]ketanserin displacement, recombinant receptor expression in Chinese hamster ovary (CHO) cells] and rat sigma [89% displacement at 10 μM, [3H]1,3-di-o-tolylguanidine (DTG), rat cerebral cortex] receptors (Cerep). At human muscarinic receptors [M2, [3H]N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5, 6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4]benzodiazepine-11-carboxamide (AF-DX384), recombinant expression in CHO cells; M3, [3H]1,1-dimethyl-4-diphenylacetoxypiperidinium (4-DAMP), recombinant expression in CHO cells; M4,[3H]4-DAMP, recombinant expression in CHO cells; M5,[3H]4-DAMP, recombinant expression in CHO cells], the maximum displacement was less than 55% with the exception of M3 (67%). At human 5-HT3 receptors [[3H]1-methyl-N-[(3-endo)-9-methyl-9-azabicyclo[3.3.1]non3-yl]-1H-indazole-3-carboxamide hydrochloride (BRL43694), recombinant expression in HEK-293 cells], the displacement was negligible (4% at 10 μM). ABT-107 bound only weakly to human H3 receptors [Ki = 1.1 μM and 79% displacement, [3H]N-methyl-1H-imidazole-4-ethanamine (NAMH), recombinant expression in C6 cells, data not shown].
Functional Selectivity of ABT-107.
Consistent with the weak or negligible interaction with other nAChRs, ABT-107 failed to evoke currents in Xenopus oocytes expressing human or ferret α3β4 or human chimeric (α6/α3)β4 subunits up to the highest concentration tested of 316 μM (Table 2) under conditions where the positive control, ACh, evoked robust currents. The ACh EC50 values were 273 μM (227–327 μM, 95% CI, n = 2) and 71 μM (59–86 μM, n = 2) at α3β4 and (α6/α3)β4 receptors, respectively. Likewise, ABT-107 failed to directly activate human α4β2 receptors expressed in HEK-293 cells and native α3* nAChRs in IMR-32 cells (Fig. 1d; Table 3). ABT-107 was weak or negligible evoking Ca2+ responses in HEK-293 cells expressing human α4β4 nAChRs with a maximum efficacy of ∼10% (EC50, ∼3 μM). As an antagonist/inhibitor, ABT-107 caused some effect at heteromeric nAChRs but generally with low potency (IC50 values in the range of approximately 1 to 20 μM; see Tables 4 and 5). At 5-HT3 receptors, ABT-107 was not an agonist and only a very weak antagonist (20% inhibition at 100 μM; see Table 5), confirming the result observed in the radioligand 5-HT3 displacement assay. In neurotransmitter uptake experiments, ABT-107 did not inhibit the uptake of [3H]norepinephrine up to 10 μM but did have moderate effects on [3H]dopamine and [3H]5-HT uptake with IC50 values of approximately 7 and 0.4 μM, respectively (Table 6).
Activation of Recombinant α7 Currents by ABT-107.
Concentration-dependent effects of ABT-107 were examined at human and rat α7 nAChRs expressed in Xenopus oocytes. As shown in Fig. 2, a and b, and Table 2, ABT-107 activated both receptors with comparable EC50 values of ∼50 to 90 nM (total charge) and ∼300 nM (peak amplitude analysis) and a maximum efficacy of ∼80% (both types of analysis) relative to ACh. In additional experiments, the desensitizing/inhibition property of ABT-107 was examined using a double addition protocol where oocytes were preincubated with increasing concentrations of ABT-107 before stimulation with ACh. As shown in Fig. 2c, ABT-107 pretreatment attenuated the responses to ACh with an IC50 value of ∼10 nM (peak amplitude response analysis), close to its binding Ki at the α7 nAChRs. Responses to ABT-107 were also examined in the presence of the recently described α7 PAM A-867744. As shown in Fig. 2c, A-867744 enhanced the responses to ACh (peak amplitude analysis) as reflected by increases in potency (from 232 to 9.7 nM), the Hill slope (1.2 to 3), and maximum efficacy (79 to 98%). These experiments, interestingly, illustrate that the IC50 value for inhibition of agonist evoked responses and the EC50 value in the presence of the PAM were essentially identical (∼10 nM).
Activation of Native α7 nAChRs by ABT-107.
Rat hippocampus stratum radiatum CA1 interneurons express somatic α7 nAChRs that can be activated by pressure application of an α7 agonist such as ACh or choline-evoking characteristic α7 currents. This approach was used to evaluate the ability of ABT-107 (300 nM in the application micropipet) to activate native α7 nAChRs. As shown in Fig. 3a, pressure application of ABT-107 evoked currents with a profile characteristic of α7 currents displaying very fast onset and rapid current decay. In 9 of 12 tested cells, such α7-like currents exhibited mean current amplitude of −332.5 pA (−172 to −494 pA, 95% CI) and current density of −7.6 pA/pF (−4.3 to −10.9 pA/pF). In four cells, addition of the α7 antagonist MLA (10 nM) to the superfusing ACSF completely blocked the ABT-107-evoked response within 5 to 10 min of exposure. Reversal of this inhibition was only partial after prolonged exposure to MLA-free ACSF (Fig. 3a).
Whole cell patch-clamp experiments were also carried out in dentate gyrus granule cells measuring spontaneous IPSC (GABAergic) synaptic activity. The application of an α7 agonist alone did not evoke significant changes in the activity profile (data not shown). However, in the presence of the α7 PAM A-867744 (10 μM), which by itself had little or no effect on IPSC activity (Malysz et al., 2009a), 30 and 300 nM ABT-107 increased the IPSC activity (measured by the number of events in a 5-min recording) by 49 ± 16% (n = 4, p < 0.05, Student's t test) and 192 ± 34% (n = 3, p < 0.05, Student's t test), respectively. A representative recording from this series of experiments is shown in Fig. 3b.
Other cellular preparations expressing native α7 nAChRs are rat cortical cultures and human IMR-32 neuroblastoma cells. We have studied the responses to ABT-107 in these cell types by intracellular Ca2+ imaging. As depicted in Fig. 4d, the addition of ABT-107 alone did not evoke detectable Ca2+ signals under the imaging conditions of this study. In contrast, when the responses were examined in the presence of A-867744 or PNU-120596 (added as pretreatment before ABT-107), robust concentration-dependent increases in Ca2+ signals were obtained in both cell types (Fig. 4). The EC50 values for ABT-107 were ∼0.7 and 13 nM in the presence of PNU-120596 and A-867744, respectively (Fig. 4; Table 3), illustrating dependence on the PAM chemotype used. Figure 4, b and c, also illustrates representative fluorescence responses and mean concentration-responses for ABT-107 in the presence of PNU-120596 with and without pretreatment with MLA. As shown, the antagonist shifted the ABT-107-evoked responses to the right by ∼15-fold (EC50 value shifting from 0.7 nM to 10 nM), consistent with a competitive interaction between MLA and ABT-107. The somewhat reduced maximum efficacy to ABT-107 in the presence of the antagonist was most likely caused by the experimental condition being carried out at nonequilibrium.
Effect of ABT-107 on Extracellular Signal-Regulated Kinase Phosphorylation in Differentiated PC-12 Cells.
Nerve growth factor (NGF)-differentiated PC-12 cells offer a unique opportunity for the study of the α7 nAChR function. In this study, we have used this assay to investigate the effect of ABT-107 on one such biochemical marker, phosphorylation of extracellular signal-regulated kinase [ratio of pERK/total extracellular-regulated kinase (tERK)], a signaling event of relevance for biochemical processing underlying cognition (Bitner et al., 2007). As shown in Fig. 5, a and b, analysis of Western immunoblots revealed the ability of ABT-107, especially in the presence of the α7 PAM A-867744, to increase the level of pERK/tERK ratio in a concentration-dependent manner. In the absence of this PAM, a modest increase of ∼1.3-fold (Fig. 5a, inset) was noted for ABT-107, whereas in its presence, the value increased to ∼14-fold. The effects to ABT-107 and another selective α7 agonist, PNU-282987, in the presence of A-867744 were attenuated by MLA, confirming their dependence on α7 nAChR activation (Fig. 5b).
Effect of ABT-107 against Glutamate-Induced Toxicity in Cortical Cultures.
To further investigate the in vitro properties of ABT-107, the effects of this compound were examined in a cellular model of neurotoxicity triggered by exposure of cortical cultures to glutamate/glycine. As shown in Fig. 6, this neurotoxic insult caused ∼30% reduction in the cellular ATP content assayed under the conditions of this study. Pretreatment with ABT-107 completely reversed the effect of glutamate/glycine coapplication, similar to the effect of the NMDA antagonist MK-801. The ability of ABT-107 to mitigate this excitotoxicity suggests its potential for neuroprotection.
In this study, we describe the in vitro pharmacological properties of ABT-107, a novel α7 nAChR agonist showing high affinity and at least 100-fold selectivity versus other nicotinic subtypes and receptors examined. Functionally, ABT-107 potently activated α7 nAChRs with nearly full efficacy compared with the neurotransmitter ACh and exhibited very weak or negligible agonist activity at related α3*, α4β2, chimeric (α6/α3)β4, α4β4, and 5-HT3 receptors. ABT-107 displayed comparable efficacy at human and rat α7 nAChRs and was also active at native rat α7 nAChRs. Responses to ABT-107 were inhibited by the α7 antagonist MLA and potentiated by the selective α7 PAM A-867744, confirming dependence on the α7 nAChR activation. When coapplied with A-867744 or PNU-120596, ABT-107 evoked robust increases in intracellular Ca2+ and extracellular signal-regulated kinase phosphorylation in human neuroblastoma IMR-32 cells, rat cortical cultures, or rat PC-12 cells. ABT-107 also exhibited activity consistent with neuroprotection in cortical cultures as measured by the glutamate excitotoxicity model.
α7 nAChRs are considered a viable target for the development of therapeutics aimed at CNS disorders such as AD and schizophrenia (reviewed in Steinlein and Bertrand, 2008; Taly et al., 2009). This focus has led to the discovery of a number of distinct α7 agonists, including the spirofused quinuclidines (5S)-spiro[1,3-oxazolidine-5,8′-1-azabicyclo[2.2.2]octane]-2-one (AR-R17779) (Mullen et al., 2000) and AZD-0328 (Sydserff et al., 2009), quinuclidine amides including PNU-282987 (Bodnar et al., 2005), N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide (PHA-543613) (Wishka et al., 2006), TC-5619 (Hauser et al., 2009), and the related carbamate JN-403 (Feuerbach et al., 2009), SSR180711 (Biton et al., 2007; Pichat et al., 2007), methyl pyridinium S24795 (Lopez-Hernandez et al., 2007), morpholinyl-pentanamide SEN-12333/WAY-317538 (Haydar et al., 2009; Roncarati et al., 2009), octahydro-pyrrolopyrrole A-582941 (Bitner et al., 2007; Tietje et al., 2008), and other yet undisclosed molecules (Taly et al., 2009). The availability of these compounds has advanced the understanding of the physiological roles of α7 nAChRs. As such, α7 agonists have shown procognitive effects in learning and memory cognition models and in improving sensory gating deficits. In addition, neuroprotective effects of α7 agonists were observed in the in vitro cellular toxicity models involving differentiated PC-12 cells, cortical neuron cultures, and hippocampus neuron cultures (Dani and Bertrand, 2007; Albuquerque et al., 2009). Consistent with this, ABT-107 attenuated glutamate-induced insult in cortical cultures (Fig. 6).
Despite the availability of various α7 nAChR agonist tools, uncertainty still remains linking various behavioral and/or biochemical effects with the α7 nAChR agonist profile. To fully characterize the potential relationships, α7 agonists with varying activation efficacies (full, partial, or zero efficacy desensitizing) (Briggs et al., 2009; Malysz et al., 2009b) are needed. Thus far, most of the available α7 agonists, such as SSR180711, AZD-0328, S24795, SEN-12333, and JN-403, show partial agonist activity (i.e., maximum efficacy <75% versus ACh) and/or relatively weak potency. Additional α7 agonists displaying high affinity and potency, full agonist profile, and favorable pharmacokinetics and absorption, distribution, metabolism, and excretion properties are needed. In this study, we describe ABT-107 as a compound fulfilling these in vitro pharmacological criteria. We show that ABT-107 exhibits high affinity and selectivity for the α7 nAChRs. Functionally, ABT-107 activated recombinant human and rat α7 nAChRs with high potency (EC50 values of ∼50–90 nM based on charge transfer analysis and ∼300 nM, peak amplitude analysis) and nearly full agonist profile compared with the neurotransmitter ACh. Although it may be speculated that the level of agonist efficacy and/or potency plays a role in driving certain physiological and cognitive functions, studies with the very weak partial agonist 1,4-diazabicyclo[3.2.2]nonan-4-yl(5-(3-(trifluoromethyl)phenyl)furan-2-yl)methanone (NS6740) (Briggs et al., 2009), the partial A-582941 (Bitner et al., 2007), and the nearly full agonist ABT-107 show that in vivo efficacy can be realized by ligands belonging to these profiles. More efficacious agonists, nonetheless, may be superior in at least some cognition domains as shown by the passive avoidance model (Briggs et al., 2009).
In terms of activation profile, ABT-107 is similar to the recently described full agonist TC-5619 (EC50, ∼30 nM, total charge analysis) (Hauser et al., 2009). TC-5619 bound to α7 nAChRs with a Ki value of ∼1 nM, with at least 2000-fold selectivity versus α4β2* nAChRs. It did not evoke Rb+ flux mediated by the muscle type or α3* (PC-12)-containing nAChRs, indicating lack of agonist activity. Because antagonist profiles, activities at native nAChRs, and interaction with α7 PAMs (as shown in this study for ABT-107) were not described, the effects of TC-5619 remain to be fully characterized. In vivo behavioral studies with TC-5619 revealed efficacy, particularly in models for cognition (novel object recognition in rats) and for schizophrenia, including positive (prepulse inhibition in transgenic th(tk−)/th(tk−) mice additive with antipsychotic clozapine) and negative aspects (social investigation in transgenic th(tk−)/th(tk−) mice and apomorphine-induced prepulse inhibition deficit in rats). Consistent with the potential utility of α7 agonists in improving deficits in schizophrenia and cognitive dysfunction, ABT-107 improved sensory gating in DBA/2 mice, a genetic mouse strain expressing auditory gating deficits (H. M. Robb, K. E. Stevens, K. L. Kohlhaas, R. S. Bitner, J Ji, W. H. Bunnelle, L. E. Rueter, M. Gopalakrishnan, and R. J. Radek, unpublished observation), and enhanced cognitive performance in monkey delayed matching to sample, short-term rat social recognition, and long-term (24 h) mouse two-trial inhibitory avoidance (Bitner et al., 2010). Furthermore, in vivo studies with ABT-107 showed increases in rat cortical and hippocampal phosphorylation of the inhibitory residue (Ser9) of glycogen synthase kinase 3β, a primary tau kinase associated with AD pathology, and reduced spinal tau hyperphosphorylation after continuous (2-week) infusion of ABT-107 in tau/amyloid precursor protein transgenic AD mice (Bitner et al., 2010, companion article).
The α7 Ki constants for ABT-107 depended on the radioligand used. Displacement of [3H]MLA and [3H]A-585539 by ABT-107 yielded Ki values of 7 nM and 0.4 to 0.6 nM, respectively. Similar differences in Ki values were noted for other α7 ligands, including PNU-282987, A-582941, (±)-AR-R17779, varenicline, and A-585539, when comparing [3H]MLA and [3H]A-585539 displacement data (Anderson et al., 2008). The reason for these differences remains unclear and requires further studies. However, a potential explanation may involve experimental conditions with [3H]A-585539 binding carried out at 4°C and [3H]MLA binding at ∼22°C. In fact, the Kd value for [3H]A-585539 shifted from ∼0.060 nM at 4°C to 0.2 nM at 22°C and 1 nM at 37°C (Anderson et al., 2008). Alternatively, ABT-107 may show higher affinity for the agonist [3H]A-585539 over the antagonist [3H]MLA as a result of preferential displacement of an agonist radioligand.
As an inhibitor, ABT-107 was also selective for the α7 nAChR with an IC50 value of ∼10 nM, whereas IC50 values at other receptors examined were >0.3 μM (Tables 4 and 5). The inhibitory profile of ABT-107 at the α7 nAChR was probably caused by desensitization. Because the desensitized state of the α7 nAChR exhibits a high affinity receptor state and this is the receptor form that is thought to be predominantly expressed during radioligand binding equilibrium conditions, good agreement for α7 ligands is expected for electrophysiologically determined IC50 values and their affinity Ki constants. Consistent with this view, we noted the IC50 value of ∼10 nM and Ki value in the range of 0.4 to 8 nM for ABT-107 in this study, similar to data previously reported for other α7 nAChR ligands (Briggs and McKenna, 1998; Anderson et al., 2008).
To further characterize the interaction of ABT-107 with the α7 nAChR in vitro, the effects of this agonist were examined in the presence of selective α7 PAMs, A-867744 and PNU-120596 (Hurst et al., 2005; Malysz et al., 2009a). Preaddition of the PAM A-867744 enhanced ABT-107-evoked α7 currents by increasing potency, Hill slope, and maximum efficacy (Fig. 2c). It is noteworthy that the EC50 value of ∼10 nM for ABT-107 in the presence of the α7 PAM was essentially the same as its IC50 value for inhibition of ACh-evoked α7 responses and also approximated its α7 Ki value. The enhancement of responses to ABT-107 by the α7 PAM suggests an increase in agonist-window current (overlap between activation and inactivation curves) (see Grønlien et al., 2007) and greater efficacy in pharmacological studies. In support, we showed that PAM coapplication with ABT-107 was able to robustly increase GABAergic IPSC activity in rat dentate gyrus, Ca2+ transient generation in IMR-32 neuroblastoma cells, and pERK/tERK ratio in PC-12 cells. The effects of ABT-107 in the presence of PAM were similar to those reported previously for choline and A-867744-evoking IPSC activity in dentate gyrus (Malysz et al., 2009a), for a number of other α7 agonists in IMR-32 cells examined in the presence of a type II α7 PAM by Ca2+ imaging (Ween et al., 2010; S. Gopalakrishnan, B. M. Philip, J. H. Gronlien, J. Malysz, D. J. Anderson, M. Gopalakrishnan, U. Warrior, and D. J. Burns, unpublished observations), and in PC-12 cells for pERK/tERK studied by the In-Cell Western method (El Kouhen et al., 2009). These studies collectively link ion flux through the α7 nAChRs to increases in intracellular Ca2+ and pERK/tERK levels, supporting use of ABT-107 as a pharmacological tool compound.
Taken together, this study shows ABT-107 to be a high affinity and selective α7 nAChR ligand exhibiting favorable agonist potency and efficacy. The data provided substantiate ABT-107 as a suitable tool compound for characterizing the roles of α7 nAChRs and further defining their relevance in physiology and in CNS disorders such as AD and schizophrenia.
This study was supported by funding from Abbott and NeuroSearch.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- nicotinic acetylcholine receptor
- phosphorylated extracellular-regulated kinase
- phosphorylated cAMP response element binding protein
- Alzheimer's disease
- 1,4-diazabicyclo[3.2.2]nonane-4-carboxylic acid, 4-bromophenyl ester
- N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride
- positive allosteric modulator
- N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride
- central nervous system
- bovine serum albumin
- American Type Culture Collection
- human embryonic kidney
- fluorescence imaging plate reader
- artificial cerebral spinal fluid
- inhibitory postsynaptic current
- Hank's balanced salt solution
- confidence interval
- Chinese hamster ovary
- N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5, 6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4]benzodiazepine-11-carboxamide
- 1-methyl-N-[(3-endo)-9-methyl-9-azabicyclo[3.3.1]non3-yl]-1H-indazole-3-carboxamide hydrochloride
- total extracellular-regulated kinase
- 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide
- (S)-(1-aza-bicyclo[2.2.2]oct-3-yl)-carbamic acid (S)-1-(2-fluoro-phenyl)-ethyl ester
- 2[2(4-bromophenyl)-2-oxoethyl]-1- methyl-pyridinium chloride
- (2R)-amino-5-phosphonovaleric acid
- balanced salt solution
- dopamine transporter
- norepinephrine transporter
- serotonin transporter
- nerve growth factor
- 1,4-diazabicyclo [3.2.2]nonan-4-yl(5-(3-(trifluoromethyl)phenyl)furan-2-yl)methanone.
- Received February 11, 2010.
- Accepted May 17, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics