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1.  Dissociation between duration of action in the forced swim test and nicotinic acetylcholine receptor occupancy with sazetidine, varenicline, and 5-I-A85380 
Psychopharmacology  2011;217(2):199-210.
Nicotinic acetylcholine receptor (nAChR) agonists, partial agonists and antagonists have antidepressant-like effects in rodent models and reduce symptoms of depression in humans.
The aim of this study was to determine if the β2* partial agonist sazetidine-A (sazetidine) showed an antidepressant-like effect in the forced swim test that was mediated by β2* nAChRs activation or desensitization.
Sazetidine, the less selective β2* partial agonist varenicline and the full β2* agonist 5-I-A8350, exhibited acute antidepressant-like effects in the forced swim test. The role of β2* nAChRs was confirmed by results showing 1) reversal of sazetidine’s antidepressant-like effects in the forced swim test by nAChR antagonists mecamylamine and dihydro-β-erythroidine (DHβE); 2) no effect of sazetidine in mice lacking the β2 subunit of the nAChR; and 3) a high correspondence between behaviorally active doses of sazetidine and β2* receptor occupancy. β2* receptor occupancy following acute sazetidine, varenicline, and 5-I-A8350 extended beyond the duration of action in the forced swim test. The long lasting receptor occupancy of sazetidine did not diminish behavioral efficacy in the forced swim test following repeated dosing.
These results demonstrate that activation of β2* nAChRs mediate sazetidine’s antidepressant-like actions and suggest that ligands that activate β2* nAChRs would be promising targets for the development of a new class of antidepressant.
PMCID: PMC3266849  PMID: 21487659
nicotinic receptor; antidepressant; sazetidine-A; AMOP-H-OH; varenicline; 5-I-A85380; receptor occupancy; forced swim
2.  Chemistry and Pharmacology of Nicotinic Ligands Based on 6-[5-(Azetidin-2-ylmethoxy) pyridin-3-yl]hex-5-yn-1-ol (AMOP-H-OH) for Possible Use in Depression 
ChemMedChem  2009;4(8):1279-1291.
AMOP-H-OH (6-[5-(azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol) and some of its sulfur-bearing analogs were tested for their actions in vitro at human α4β2-, α4β4-, α3β4*- and α1*-nicotinic acetylcholine receptors (nAChRs). AMOP-H-OH also was assessed in a model of antidepressant efficacy. AMOP-H-OH and some of its analogs have high potency and selectivity for α4β2-nAChRs over other nAChR subtypes. Effects are manifest as partial agonism, perhaps reflecting selectivity for high sensitivity (α4)3(β2)2-nAChRs. More prolonged exposure to AMOP-H-OH and its analogs produces inhibition of subsequent responses to acute challenges with nicotinic full agonists, again selectively for α4β2-nAChRs over other nAChR subtypes. The inhibition is mediated either via antagonism or desensitization of nAChR function, but the degree of inhibition of α4β2-nAChRs is limited by the drugs’ activities as partial agonists. Certain aspects of the in vitro pharmacology suggest that AMOP-H-OH and some of its analogs have a set of binding sites on α4β2-nAChRs that are distinct from those for full agonists. The in vitro pharmacological profile suggests that peripheral side effects of AMOP-H-OH or its analogs would be minimal and that their behavioral effects would be dominated by central nAChR actions. AMOP-H-OH also has profound and high potency antidepressant-like effects in the forced swim test. The net action of prolonged exposure to AMOP-H-OH or its analogs, as for nicotine, seems to be a selective decrease in α4β2-nAChR function. Inactivation of nAChRs may be a common neurochemical endpoint for nicotine dependence, its treatment, and some of its manifestations, including relief from depression.
PMCID: PMC2955514  PMID: 19569163
nicotine; nAChRs; depression; α4β2; Sazetidine-A; AMOP-H-OH
3.  Chemistry and Behavioral Studies Identify Chiral Cyclopropanes as Selective α4β2-Nicotinic Acetylcholine Receptor Partial Agonists Exhibiting an Antidepressant Profile 
Journal of Medicinal Chemistry  2012;55(2):717-724.
Despite their discovery in the early 20th century and intensive study over the last twenty years, nicotinic acetylcholine receptors (nAChRs) are still far from being well understood. Only a few chemical entities targeting nAChRs are currently undergoing clinical trials, and even fewer have reached the marketplace. In our efforts to discover novel and truly selective nAChR ligands, we designed and synthesized a series of chiral cyclopropane-containing α4β2-specific ligands that display low nanomolar binding affinities and excellent subtype selectivity, while acting as partial agonists at α4β2-nAChRs. Their favorable antidepressant-like properties were demonstrated in the classical mouse forced swim test. Preliminary ADMET studies and broad screening towards other common neurotransmitter receptors were also carried out to further evaluate their safety profile and eliminate their potential off-target activity. These highly potent cyclopropane ligands possess superior subtype selectivity compared to other α4β2-nAChR agonists reported to date, including the marketed drug varenicline, and therefore may fully satisfy the crucial prerequisite for avoiding adverse side effects. These novel chemical entities could potentially be advanced to the clinic as new drug candidates for treating depression.
PMCID: PMC3292870  PMID: 22171543
4.  Nicotinic Receptor Antagonists as Treatments for Nicotine Abuse 
Despite the proven efficacy of current pharmacotherapies for tobacco dependence, relapse rates continue to be high, indicating that novel medications are needed. Currently, several smoking cessation agents are available, including varenicline (Chantix®), bupropion (Zyban®), and cytisine (Tabex®). Varenicline and cytisine are partial agonists at the α4β2* nicotinic acetylcholine receptor (nAChR). Bupropion is an antidepressant but is also an antagonist at α3β2* ganglionic nAChRs. The rewarding effects of nicotine are mediated, in part, by nicotine-evoked dopamine (DA) release leading to sensitization, which is associated with repeated nicotine administration and nicotine addiction. Receptor antagonists that selectivity target central nAChR subtypes mediating nicotine-evoked DA release should have efficacy as tobacco use cessation agents with the therapeutic advantage of a limited side-effect profile. While α-conotoxin MII (α-CtxMII)-insensitive nAChRs (e.g., α4β2*) contribute to nicotine-evoked DA release, these nAChRs are widely distributed in the brain, and inhibition of these receptors may lead to nonselective and untoward effects. In contrast, α-CtxMII-sensitive nAChRs mediating nicotine-evoked DA release offer an advantage as targets for smoking cessation, due to their more restricted localization primarily to dopaminergic neurons. Small drug-like molecules that are selective antagonists at α-CtxMII-sensitive nAChR subtypes that contain α6 and β2 subunits have now been identified. Early research identified a variety of quaternary ammonium analogs that were potent and selective antagonists at nAChRs mediating nicotine-evoked DA release. More recent data have shown that novel, non-quaternary bis-1,2,5,6-tetrahydropyridine analogs potently inhibit (IC50<1 nM) nicotine-evoked DA release in vitro by acting as antagonists at α-CtxMII-sensitive nAChR subtypes; these compounds also decrease NIC self-administration in rats.
PMCID: PMC4110698  PMID: 24484986
5.  The potent and selective α4β2*/α6*-nicotinic acetylcholine receptor partial agonist 2-[5-[5-((S)Azetidin-2-ylmethoxy)-3-pyridinyl]-3-isoxazolyl]ethanol demonstrates antidepressive-like behavior in animal models and a favorable ADME-tox profile 
Preclinical and clinical studies demonstrated that the inhibition of cholinergic supersensitivity through nicotinic antagonists and partial agonists can be used successfully to treat depressed patients, especially those who are poor responders to selective serotonin reuptake inhibitors (SSRIs). In our effort to develop novel antidepressant drugs, LF-3-88 was identified as a potent nicotinic acetylcholine receptor (nAChR) partial agonist with subnanomolar to nanomolar affinities for β2-containing nAChRs (α2β2, α3β2, α4β2, and α4β2*) and superior selectivity away from α3β4 − (Ki > 104 nmol/L) and α7-nAChRs (Ki > 104 nmol/L) as well as 51 other central nervous system (CNS)-related neurotransmitter receptors and transporters. Functional activities at different nAChR subtypes were characterized utilizing 86Rb+ ion efflux assays, two-electrode voltage-clamp (TEVC) recording in oocytes, and whole-cell current recording measurements. In mouse models, administration of LF-3-88 resulted in antidepressive-like behavioral signatures 15 min post injection in the SmartCube® test (5 and 10 mg/kg, i.p.; about 45-min session), decreased immobility in the forced swim test (1–3 mg/kg, i.p.; 1–10 mg/kg, p.o.; 30 min pretreatment, 6-min trial), and decreased latency to approach food in the novelty-suppressed feeding test after 29 days chronic administration once daily (5 mg/kg but not 10 mg/kg, p.o.; 15-min trial). In addition, LF-3-88 exhibited a favorable profile in pharmacokinetic/ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) assays. This compound was also shown to cause no mortality in wild-type Balb/CJ mice when tested at 300 mg/kg. These results further support the potential of potent and selective nicotinic partial agonists for use in the treatment of depression.
PMCID: PMC4184702  PMID: 25505580
Antidepressive-like behavior; nicotinic acetylcholine receptor; partial agonist; selectivity
6.  α4β2 nicotinic acetylcholine receptor partial agonists with low intrinsic efficacy have antidepressant-like properties 
Behavioural pharmacology  2011;22(4):291-299.
Previous studies have suggested that treatment with antagonists or partial agonists of nicotinic acetylcholine receptors containing the β2 subunit (β2* nAChRs) results in antidepressant-like effects. In the current study we tested 3 novel compounds with different affinity and functional efficacy at α4β2* nAChRs, which were synthesized as part of nAChR discovery projects at Pfizer in the tail suspension, forced swim and novelty-suppressed feeding tests of antidepressant efficacy. All compounds tested reduced immobility in the forced swim test and one of the compounds also reduced immobility in the tail suspension test. All the compounds appeared to affect food intake on their own, with 2 compounds reducing feeding significantly in the home cage, precluding a clear interpretation of the results in the novelty-suppressed feeding test. None of the compounds altered locomotor activity at the doses and time points used here. Therefore, a subset of these compounds has pharmacological and behavioral properties that demonstrate the potential of nicotinic compounds as a treatment of mood disorders. Further development of nicotinic-based antidepressants should focus on increasing nAChR subtype selectivity to obtain consistent antidepressant properties with an acceptable side effect profile.
PMCID: PMC3227135  PMID: 21566524
Nicotinic acetylcholine receptors; partial agonists; depression; forced swim test; tail suspension test; novelty-suppressed feeding; mice
7.  Positive allosteric modulators as an approach to nicotinic acetylcholine receptor- targeted therapeutics: advantages and limitations 
Biochemical pharmacology  2011;82(8):915-930.
Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.
PMCID: PMC3162128  PMID: 21575610
Alzheimer’s disease; schizophrenia; drug development; electrophysiology; modeling
8.  Identification of Novel α4β2-Nicotinic Acetylcholine Receptor (nAChR) Agonists Based on an Isoxazole Ether Scaffold that Demonstrate Antidepressant-like Activity 
Journal of Medicinal Chemistry  2012;55(2):812-823.
There is considerable evidence to support the hypothesis that the blockade of nAChR is responsible for the antidepressant action of nicotinic ligands. The nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine, has been shown to be an effective add-on in patients that do not respond to selective serotonin reuptake inhibitors. This suggests that nAChR ligands may address an unmet clinical need by providing relief from depressive symptoms in refractory patients. In this study, a new series of nAChR ligands based on an isoxazole-ether scaffold have been designed and synthesized for binding and functional assays. Preliminary structure-activity relationship (SAR) efforts identified a lead compound 43, which possesses potent antidepressant-like activity (1 mg/kg, IP; 5 mg/kg, PO) in the classical mouse forced swim test. Early stage absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) studies also suggested favorable drug-like properties, and broad screening towards other common neurotransmitter receptors indicated that compound 43 is highly selective for nAChRs over the other 45 neurotransmitter receptors and transporters tested.
PMCID: PMC3272775  PMID: 22148173
9.  Acetylcholine receptors in the retinas of the α7 nicotinic acetylcholine receptor knockout mouse 
Molecular Vision  2014;20:1328-1356.
The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer’s disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.
To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.
In the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3–α5 and α9-containing nAChR subunits and m1–m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.
Rather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.
PMCID: PMC4169779  PMID: 25352741
10.  Selectivity of ABT-089 for α4β2* and α6β2* nicotinic acetylcholine receptors in brain 
Biochemical pharmacology  2009;78(7):795-802.
Numerous pharmaceutical efforts have targeted neuronal nicotinic receptors (nAChRs) for amelioration of cognitive deficits. While α4β2 and α7 are the more prominent nAChR in brain, other heteromeric nAChR can have important impact on agonist pharmacology. ABT-089 is a pioneer nAChR agonist found to enhance cognitive function with an exceptionally low incidence of adverse effects. To further investigate the mechanism of action of ABT-089, we evaluated its function in mouse brain preparations in which we have characterized the subunit composition of native nAChR. Among α4β2*- nAChR, ABT-089 had partial agonist activity (7-23% of nicotine) and high selectivity for α4α5β2 nAChR as evidenced by loss of activity in thalamus of α5-/- mice. ABT-089 stimulated [3H]-dopamine release (57%) exceeded the activity at α4β2* nAChR, that could be explained by the activity at α6β2* nAChR The concentration-response relationship for ABT-089 stimulation of α6β2* nAChR was biphasic. EC50 and efficacy values for ABT-089, respectively, were 28 μM and 98% at the less sensitive α6β2* nAChR and 0.11 μM and 36% at the more sensitive subtype (the most sensitive target for ABT-089 identified to date). ABT-089 had essentially no agonist or antagonist activity at concentrations ≤ 300 μM at α3β4-nAChR measured by [3H]-acetylcholine release from interpeduncular nucleus. Thus, ABT-089 is a β2* nAChR ligand with demonstrable agonist activity at α4β2* and α6β2* receptors. As one form of α6β2* nAChR is sensitive to sub-μM concentrations, we propose that that this receptor in particular may contribute to the enhanced cognitive performance following low doses of ABT-089.
PMCID: PMC2772152  PMID: 19481067
nicotinic acetylcholine receptor; dopamine; thalamus; striatum; cortex; desensitization
11.  Chemistry and Pharmacological Characterization of Novel Nitrogen Analogs of AMOP-H-OH (Sazetidine-A; 6-[5-(Azetidin-2-ylmethoxy)pyridin-3-yl]hex-5-yn-1-ol) as α4β2-Nicotinic Acetylcholine Receptor-Selective Partial Agonists 
Journal of medicinal chemistry  2010;53(19):6973-6985.
In order to advance therapeutic applications of nicotinic ligands, continuing research efforts are being directed toward the identification and characterization of novel nicotinic acetylcholine receptor (nAChR) ligands that are both potent and subtype selective. Herein we report the synthesis and pharmacological evaluation of members of a new series of 3-alkoxy-5-aminopyridine derivatives that display good selectivity for the α4β2-nAChR subtype based on ligand binding and functional evaluations. The most potent ligand in this series, compound 64, showed high radioligand binding affinity and selectivity for rat α4β2-nAChR with a Ki value of 1.2 nM and 4700-fold selectivity for α4β2-over α3β4-nAChR, and ~100-fold selectivity for functional, high-sensitivity, human α4β2-nAChR over α3β4*-nAChR. In the mouse forced swim test, compound 64 exhibited antidepressant-like effects. Structure-activity relationship (SAR) analyses suggest that the introduction of additional substituents to the amino group present on the pyridine ring of the N-demethylated analogue of compound 17 can provide potent α4β2-nAChR-selective ligands for possible use in treatment of neurological and psychiatric disorders including depression.
PMCID: PMC2957884  PMID: 20822184
12.  Blockade of Neuronal α7-nAChR by α-Conotoxin ImI Explained by Computational Scanning and Energy Calculations 
PLoS Computational Biology  2011;7(3):e1002011.
α-Conotoxins potently inhibit isoforms of nicotinic acetylcholine receptors (nAChRs), which are essential for neuronal and neuromuscular transmission. They are also used as neurochemical tools to study nAChR physiology and are being evaluated as drug leads to treat various neuronal disorders. A number of experimental studies have been performed to investigate the structure-activity relationships of conotoxin/nAChR complexes. However, the structural determinants of their binding interactions are still ambiguous in the absence of experimental structures of conotoxin-receptor complexes. In this study, the binding modes of α-conotoxin ImI to the α7-nAChR, currently the best-studied system experimentally, were investigated using comparative modeling and molecular dynamics simulations. The structures of more than 30 single point mutants of either the conotoxin or the receptor were modeled and analyzed. The models were used to explain qualitatively the change of affinities measured experimentally, including some nAChR positions located outside the binding site. Mutational energies were calculated using different methods that combine a conformational refinement procedure (minimization with a distance dependent dielectric constant or explicit water, or molecular dynamics using five restraint strategies) and a binding energy function (MM-GB/SA or MM-PB/SA). The protocol using explicit water energy minimization and MM-GB/SA gave the best correlations with experimental binding affinities, with an R2 value of 0.74. The van der Waals and non-polar desolvation components were found to be the main driving force for binding of the conotoxin to the nAChR. The electrostatic component was responsible for the selectivity of the various ImI mutants. Overall, this study provides novel insights into the binding mechanism of α-conotoxins to nAChRs and the methodological developments reported here open avenues for computational scanning studies of a rapidly expanding range of wild-type and chemically modified α-conotoxins.
Author Summary
Conotoxins are peptide toxins extracted from the venom of carnivorous marine cone snails. Members of the α-conotoxin subfamily potently block nicotinic acetylcholine receptors (nAChRs), which are involved in signal transmission between two neurons or between neurons and muscle fibers. nAChRs are important pharmacological targets due to their involvement in the transmission of pain stimuli and also in numerous neurone diseases and disorders. Their potency and specificity have led to the development of α-conotoxins as drug leads, and also to their use in the investigation of the role of nAChRs in various physiological processes. The most studied conotoxin/nAChR system, ImI/α7, was modeled in this study, and several computational methods were tested for their ability to explain the perturbations observed experimentally after introducing single point mutations into either ImI or the α7 receptor. The aim of this study was to establish a theoretical basis to rapidly identify new α-conotoxin mutants that might have improved specificity and affinity for a given receptor subtype. Furthermore, hundreds of thousands of conotoxins are predicted to exist, and computational methods are needed to help streamline the discovery of their molecular targets.
PMCID: PMC3048385  PMID: 21390272
13.  Chemistry and Pharmacological Studies of 3-Alkoxy-2,5-Disubstituted-Pyridinyl Compounds as Novel Selective α4β2 Nicotinic Acetylcholine Receptor Ligands That Reduces Alcohol Intake in Rats 
Journal of medicinal chemistry  2013;56(7):10.1021/jm4000374.
Neuronal acetylcholine receptors mediate the addictive effects of nicotine and may also be involved in alcohol addiction. Varenicline, an approved smoking cessation medication, showed clear efficacy in reducing alcohol consumption in heavy-drinking smokers. More recently, sazetidine-A, which selectively desensitizes α4β2 nicotinic receptors, was shown to significantly reduce alcohol intake in a rat model. To develop novel therapeutics for treating alcohol use disorder, we designed and synthesized novel sazetidine-A analogs containing a methyl group at the 2-position of the pyridine ring. In vitro pharmacological studies revealed that some of the novel compounds showed similar overall pharmacological property profiles with that of sazetidine-A, but exhibited reduced agonist activity across all nicotinic receptor subtypes tested. In animal studies, compound (S)-9 significantly reduced alcohol uptake in rats. More importantly, preliminary results from studies in a ferret model indicate that these novel nAChR ligands have an improved adverse side-effect profile in comparison with that of varenicline.
PMCID: PMC3809750  PMID: 23540678
nicotinic acetylcholine receptors; sazetidine-A; varenicline; desensitization; addiction; alcohol use disorders
14.  Varenicline Blocks β2*-nAChR–Mediated Response and Activates β4*-nAChR–Mediated Responses in Mice In Vivo 
Nicotine & Tobacco Research  2012;14(6):711-719.
The smoking cessation aid, varenicline, has higher affinity for the alpha4beta2-subtype of the nicotinic acetylcholine receptor (α4β2*-nAChR) than for other subtypes of nAChRs by in vitro assays. The mechanism of action of acute varenicline was studied in vivo to determine (a) subtype activation associated with physiological effects and (b) dose relationship as an antagonist of nicotine.
Acute doses of saline, nicotine, and varenicline were given to mice, and locomotor depression and hypothermia were measured. Subunit null mutant mice as well as selective antagonists were used to study mode of action of varenicline as an agonist. Varenicline as an antagonist of nicotine was also investigated.
Varenicline evokes locomotor depression and hypothermia at higher doses than necessary for nicotine. Null mutation of the α7- or β2-nAChR subunit did not decrease the effectiveness of varenicline; however, null mutation of the β4 subunit significantly decreased the magnitude of the varenicline effect. Effects of the highest dose studied were blocked by mecamylamine (general nAChR antagonist) and partially antagonized by hexamethonium (largely peripheral nAChR antagonist). No significant block was seen with ondansetron antagonist of 5-hydroxytryptamine 3 receptor. Using a dose of nicotine selective for β2*-nAChR subtype effects with these tests, dose-dependent antagonism by varenicline was seen. Effective inhibitory doses were determined and appear to be in a range consistent with binding affinity or desensitization of β2*-nAChRs.
Varenicline acts as a functional antagonist of β2*-nAChRs, blocking certain effects of nicotine. At higher doses, varenicline is an agonist of β4*-nAChRs producing physiological changes in mice.
PMCID: PMC3356295  PMID: 22241831
15.  The Subtypes of Nicotinic Acetylcholine Receptors on Dopaminergic Terminals of Mouse Striatum 
Biochemical pharmacology  2007;74(8):1235-1246.
This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [125I]-α-bungarotoxin and [3H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and α-conotoxin MII (α-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the α6 subunit (α4α6β2β3, α6β2β3, α6β2) and bind α-CtxMII with high affinity. One of these three subtypes (α4α6β2β3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for α-CtxMII (α4β2, α4α5β2) are somewhat more numerous than the α6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.
PMCID: PMC2735219  PMID: 17825262
16.  Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system 
Acta Pharmacologica Sinica  2009;30(6):673-680.
Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors (nAChRs), they are grouped into either heteromeric (comprising both α and β subunits) or homomeric (comprising only α subunits) receptors. The nAChRs are known to be differentially permeable to calcium ions, with the α7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the α-bungarotoxin-sensitive α7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals: (1) direct calcium influx through the nAChRs, (2) indirect calcium influx through voltage-dependent calcium channels (VDCCs) which are activated by the nAChR-mediated depolarization, and (3) calcium-induced calcium release (CICR) (triggered by the first two sources) from the endoplasmic reticulum (ER) through the ryanodine receptors and inositol (1,4,5)-triphosphate receptors (IP3Rs). Downstream signaling events mediated by nAChR-mediated calcium responses can be grouped into instantaneous effects (such as neurotransmitter release, which can occur in milliseconds after nAChR activation), short-term effects (such as the recovery of nAChR desensitization through cellular signaling cascades), and long-term effects (such as neuroprotection via gene expression). In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understanding the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.
PMCID: PMC4002362  PMID: 19448647
nicotinic acetylcholine receptors; calcium; cellular signaling; nervous system
17.  Nicotinic receptor modulation to treat alcohol and drug dependence 
Alcohol and drug dependence are serious public health problems worldwide. The prevalence of alcohol and drug dependence in the United States and other parts of the world is significant. Given the limitations in the efficacy of current pharmacotherapies to treat these disorders, research in developing alternative pharmacotherapies continues. Preclinical and clinical evidence thus far has indicated that brain nicotinic acetylcholine receptors (nAChRs) are important pharmacological targets for the development of medications to treat alcohol and drug dependence. The nAChRs are a super family of ligand gated ion channels, and are expressed throughout the brain with twelve neuronal nAChR subunits (α2–α10 and β2–β4) identified. Here, we review preclinical and clinical evidence involving a number of nAChR ligands that target different nAChR subtypes in alcohol and nicotine addiction. The important ligands include cytisine, lobeline, mecamylamine, varenicline, sazetidine A and others that target α4β2* nAChR subtypes as small molecule modulators of the brain nicotinic cholinergic system are also discussed. Taken together, both preclinical and clinical data exist that support nAChR–based ligands as promising therapeutic agents for the treatment of alcohol and drug dependence.
PMCID: PMC4295535  PMID: 25642160
nicotinic receptor; alcohol dependence; nicotine addiction; drug addiction; CNS disorders; drug development; animal models
18.  Structural Characterization of Binding Mode of Smoking Cessation Drugs to Nicotinic Acetylcholine Receptors through Study of Ligand Complexes with Acetylcholine-binding Protein* 
The Journal of Biological Chemistry  2012;287(28):23283-23293.
Background: Cytisine and varenicline are smoking cessation drugs binding to nicotinic receptors (nAChRs).
Results: We studied crystal structures of cytisine and varenicline with AChBP and analyzed binding of α4β2-like or α7-like AChBP mutants to cytisine.
Conclusion: Ligand selectivity relies on residues beyond the binding site primary shell.
Significance: These structures will contribute to designing novel compounds targeting specific nAChR subtypes.
Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4β2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.
PMCID: PMC3390607  PMID: 22553201
Crystal Structure; Cys-loop Receptors; Ion Channels; Ligand-binding Protein; Nicotinic Acetylcholine Receptors; Acetylcholine-binding Protein; α4β2-selective Ligands; Cytisine; Varenicline
19.  Muscarinic and Nicotinic Acetylcholine Receptor Agonists and Allosteric Modulators for the Treatment of Schizophrenia 
Neuropsychopharmacology  2011;37(1):16-42.
Muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs) are emerging as important targets for the development of novel treatments for the symptoms associated with schizophrenia. Preclinical and early proof-of-concept clinical studies have provided strong evidence that activators of specific mAChR (M1 and M4) and nAChR (α7 and α2β4) subtypes are effective in animal models of antipsychotic-like activity and/or cognitive enhancement, and in the treatment of positive and cognitive symptoms in patients with schizophrenia. While early attempts to develop selective mAChR and nAChR agonists provided important preliminary findings, these compounds have ultimately failed in clinical development due to a lack of true subtype selectivity and subsequent dose-limiting adverse effects. In recent years, there have been major advances in the discovery of highly selective activators for the different mAChR and nAChR subtypes with suitable properties for optimization as potential candidates for clinical trials. One novel strategy has been to identify ligands that activate a specific receptor subtype through actions at sites that are distinct from the highly conserved ACh-binding site, termed allosteric sites. These allosteric activators, both allosteric agonists and positive allosteric modulators, of mAChR and nAChR subtypes demonstrate unique mechanisms of action and high selectivity in vivo, and may provide innovative treatment strategies for schizophrenia.
PMCID: PMC3238081  PMID: 21956443
acetylcholine; schizophrenia and antipsychotics; drug discovery and drug development; schizophrenia
20.  A Genome-Wide RNAi Screen in Caenorhabditis elegans Identifies the Nicotinic Acetylcholine Receptor Subunit ACR-7 as an Antipsychotic Drug Target 
PLoS Genetics  2013;9(2):e1003313.
We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest (scla genes) in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD) targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.
Author Summary
Clozapine is the most effective medication for treatment-refractory schizophrenia but produces toxic side effects such as agranulocytosis, metabolic syndrome, and developmental defects after exposure early in life. However, clozapine's molecular mechanisms of action remain poorly understood. In past studies, we showed that pharmacogenomic experiments in C. elegans identify novel signaling pathways through which clozapine exerts its biological effects. Here, we report the first genetic suppressor screen for antipsychotic (APD) drug targets in an animal and identify 40 suppressors of clozapine-induced larval arrest, including the α-like nicotinic acetylcholine receptor (nAChR) acr-7. We validate our RNAi result by showing that an acr-7 knockout suppresses clozapine-induced larval arrest and inhibition of pharyngeal pumping. Expression of a full-length translational acr-7::GFP (Green Fluorescent Protein) construct in the acr-7 mutant rescues suppression of these phenotypes. Clozapine-induced phenotypes are phenocopied by nAChR agonists and blocked by nAChR antagonists. The results suggest that clozapine induces these phenotypes through activation of the ACR-7 receptor. Recent studies have underscored the potential importance of nAChRs in the pathophysiology of schizophrenia. A clearer understanding of APD mechanisms would facilitate the design of improved drugs and may inform our understanding, not only of drug mechanisms, but also of disease pathogenesis.
PMCID: PMC3585123  PMID: 23468647
21.  Structural differences determine the relative selectivity of nicotinic compounds for native α4β2*-, α6β2*-, α3β4*- and α7-nicotine acetylcholine receptors 
Neuropharmacology  2010;58(7):1054-1066.
Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, α4β2* and α6β2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The α4β2*-nAChRs are widely distributed, while α6β2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the meso-accumbens pathway thought to be essential for reward and addiction. α4β2*-nAChR also modulate GABA release in these areas.
Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing hα7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, α4β2*, α6β2*, α7 and α3β4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at α4β2* and α6β2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for α6β2*-nAChR.
PMCID: PMC2849849  PMID: 20114055
TC2429; TC2403; TC1698; TC2242; TC6951; varenicline
22.  Bupropion Binds to Two Sites in the Torpedo Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analog [125I]-SADU-3-72 
Biochemistry  2012;51(12):2425-2435.
Bupropion, a clinically-used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analog, (±)-2-(N-tert-butylamino)-3′-[125I]-iodo-4′-azidopropiophenone (SADU-3-72). Based upon inhibition of [125I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC50 = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with three-fold higher affinity in the desensitized (IC50 = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [125I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [125I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu265, βLeu257) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP, but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr213 in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket.
PMCID: PMC3315157  PMID: 22394379
23.  Cytisine, a partial agonist of high affinity nicotinic acetylcholine receptors, has antidepressant-like properties in male C57BL/6J mice 
Neuropharmacology  2007;52(5):1256-1262.
The nicotine in tobacco is thought to modulate neuronal systems regulating mood. Moreover, it appears possible that blockade rather than activation of β2-containing (β2*) nicotinic acetylcholine receptors (nAChRs) may lead to antidepressant-like effects. We used cytisine, a partial agonist of α4/β2* nAChRs and a full agonist at α3/β4* nAChRs, in several tests of antidepressant efficacy. Further, we used c-fos expression to identify potential neurobiological correlates of the antidepressant-like effects of cytisine. Cytisine had antidepressant-like effects in several animal models of antidepressant efficacy. In addition, immunohistochemical analyses indicated that cytisine could reduce c-fos immunoreactivity in the basolateral amygdala by ~ 50%. These data show that cytisine acts like classical antidepressants in rodent models of antidepressant efficacy. In addition, cytisine’s ability to block α4/β2* nAChRs may be responsible for its antidepressant-like properties, and these may be mediated through a reduction of neuronal activity in the basolateral amygdala. These studies also suggest that both antagonists and partial agonists of α4/β2* nAChRs would be interesting targets for the development of novel antidepressant drugs.
PMCID: PMC1959230  PMID: 17320916
Nicotinic acetylcholine receptors; depression; nicotinic partial agonist; C57BL/6J male mice; c-fos
24.  Activation of α4* nAChRs is necessary and sufficient for varenicline-induced reduction of alcohol consumption 
Recently, the smoking cessation therapeutic varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist, has been shown to reduce alcohol consumption. However, the mechanism and nAChR subtype(s) involved are unknown. Here we demonstrate that varenicline and alcohol exposure, either alone or in combination, selectively activates dopaminergic (DAergic) neurons within the posterior, but not the anterior, ventral tegmental area (VTA). To gain insight into which nAChR subtypes may be involved in the response to alcohol, we analyzed nAChR subunit gene expression in posterior VTA DAergic neurons. Ethanol-activated DAergic neurons expressed higher levels of α4, α6, and β3 subunit genes compared to non-activated neurons. To examine the role of nicotinic receptors containing the α4 subunit (α4* nAChRs) in varenicline-induced reduction of alcohol consumption, we examined the effect of the drug in two complementary mouse models, a knockout line that does not express the α4 subunit (α4 KO) and another line that expresses α4* nAChRs hypersensitive to agonist (Leu9′Ala). While varenicline (0.1 – 0.3 mg/kg, i.p.) reduced 2 % and 20 % alcohol consumption in wildtype (WT) mice, the drug did not significantly reduce consumption in α4 KO animals. Conversely, low doses of varenicline (0.0125 – 0.05 mg/kg, i.p.) that had little effect in WT mice dramatically reduced ethanol intake in Leu9′fAla mice. Infusion of varenicline into the posterior, but not the anterior VTA was sufficient to reduce alcohol consumption. Together, our data indicate that activation of α4* nAChRs is necessary and sufficient for varenicline reduction of alcohol consumption.
PMCID: PMC2941435  PMID: 20668200
alcoholism; ethanol; varenicline; nicotinic acetylcholine receptors; dopamine; mice
25.  alpha4beta2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief 
Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addition, mental illness and movement control in humans. We developed a unique model system to examine the role of alpha4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the alpha4 subunit from dopaminergic neurons in mice. The loss alpha4 mRNA and alpha4beta2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of alpha4beta2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. Alpha4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. Alpha4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze and elimination of alpha4-beta2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of alpha4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression, however nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine behaviors.
PMCID: PMC3539812  PMID: 21795541

Results 1-25 (651630)