Clark A. Briggs

Distinct Profiles of α7 nAChR Positive Allosteric Modulation Revealed by Structurally Diverse Chemotypes

Selective modulation of α7 nicotinic acetylcholine receptors (nAChRs) is thought to regulate processes impaired in schizophrenia, Alzheimers disease, and other dementias. One approach to target α7 nAChRs is by positive allosteric modulation. Structurally diverse compounds, including PNU-120596, 4-naphthalene-1-yl-3a,4,5,9b-tetrahydro-3-H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), and 5-hydroxyindole (5-HI) have been identified as positive allosteric modulators (PAMs), but their receptor interactions and pharmacological profiles remain to be fully elucidated. In this study, we investigated interactions of these compounds at human α7 nAChRs, expressed in Xenopus laevis oocytes, along with genistein, a tyrosine kinase inhibitor. Genistein was found to function as a PAM. Two types of PAM profiles were observed. 5-HI and genistein predominantly affected the apparent peak current (type I) whereas PNU-120596 and TQS increased the apparent peak current and evoked a distinct weakly decaying current (type II). Concentration-responses to agonists [ACh, 3-[(3E)-3-[(2,4-dimethoxyphenyl)methylidene]-5,6-dihydro-4H-pyridin-2-yl]pyridine dihydrochloride (GTS-21), and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)] were potentiated by both types, although type II PAMs had greater effects. When applied after α7 nAChRs were desensitized, type II, but not type I, PAMs could reactivate α7 currents. Both types of PAMs also increased the ACh-evoked α7 window currents, with type II PAMs generally showing larger potentiation. None of the PAMs tested increased nicotine-evoked Ca2+ transients in human embryonic kidney 293 cells expressing human α4β2 or α3β4 nAChRs, although some inhibition was noted for 5-HI, genistein, and TQS. In summary, our studies reveal two distinct α7 PAM profiles, which could offer unique opportunities for modulating α7 nAChRs in vivo and in the development of novel therapeutics for central nervous system indications.

Broad-Spectrum Efficacy across Cognitive Domains by α7 Nicotinic Acetylcholine Receptor Agonism Correlates with Activation of ERK1/2 and CREB Phosphorylation Pathways

The α7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel α7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (K i = 10.8 nm) and human (K i = 16.7 nm) α7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the α7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that α7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of α7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked α7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that α7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimers disease and schizophrenia.

An Allosteric Modulator of the α7 Nicotinic Acetylcholine Receptor Possessing Cognition-Enhancing Properties in Vivo

Augmentation of nicotinic α7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimers disease and schizophrenia. In the present report, a novel positive allosteric modulator of the α7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through α7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of α7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (–)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (–)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that α7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

Functional characterization of the novel neuronal nicotinic acetylcholine receptor ligand GTS-21 in vitro and in vivo

(2.4)-Dimethoxybenzylidene anabaseine dihydrochloride (GTS-21), a compound that interacts with rat neuronal nicotinic acetylcholine receptors (nAChRs), was evaluated using human recombinant nAChRs in vitro and various pharmacokinetic and behavioral models in rodents, dogs and monkeys. GTS-21 bound to human alpha 4 beta 2 nAChR (K1-20 nM) 100-fold more potently than to human alpha 7 nAChR, and was 18- and 2-fold less potent than (-)-nicotine at human alpha 4 beta 2 and alpha 7 nAChR, respectively. Functionally. GTS-21 stimulated [5H]dopamine release from rat striatal slices with an EC50 of 10 +/- 2 microM (250-fold less potent and 70% as efficacious as (-)-nicotine), an effect blocked by the nAChR antagonist dihydro-beta-erythroidine. However, GTS-21 did not stimulate human alpha 4 beta 2 nor human ganglionic nAChRs significantly. In vivo, GTS-21 had no adverse effect on dog blood pressure (< or = 2.5 micromol/kg i.v. bolus infusion), in marked contrast with (-)-nicotine, GTS-21 (-62 micromol/kg.s.e.) also did not cross-discriminate significantly with (-)-nicotine in rats and did not reduce temperature or locomotion in mice. Neither was it active in the elevated plus maze anxiety model (0.19-6.2 micromol/kg.IP) in normal mice. However, GTS-21 did improve learning performance of monkeys in the delayed matching-to-sample task (32-130 nmol/kg.i.m.).

A-85380 [3-(2(S)-azetidinylmethoxy) pyridine]: In Vitro pharmacological properties of a novel, high affinity α4β2 nicotinic acetylcholine receptor ligand

Abstract The in vitro pharmacological properties of a novel cholinergic channel ligand, A-85380 [3-(2( S )-azetidinylmethoxy) pyridine], were examined using tissue preparations that express different putative nAChR subtypes. In radioligand binding studies, A-85380 is shown to be a potent and selective ligand for the human α4β2 nAChR subtype ( K i = 0.05 ± 0.01 nM) relative to the human α7 ( K i = 148 ± 13 nM) and the muscle α1β1dg subtype expressed in Torpedo electroplax ( K i = 314 ± 12 nM). The R -enantiomer of A-85380, A-159470, displays little enantioselectivity towards the α4β2 and α1β1δγ subtypes but does display 12-fold enantioselectivity towards the α7 subtype ( K i = 1275 ± 199 nM). (+)- and (−)-Epibatidine display similar potencies at the human human α4β2 ( K i = 0.04 ± 0.02 nM and 0.07 ± 0.02 nM, respectively), human α7 ( K i = 16 ± 2 nM and 22 ± 3 nM, respectively) and muscle α1β1δg ( K i = 2.5 ± 0.9 nM and 5.7 ± 1.0 nM, respectively) nAChRs. Functionally, A-85380 is a potent activator of cation efflux through the human α4β2 (EC 50 = 0.7 ± 0.1 μ M) and ganglionic (EC 50 = 0.8 ± 0.09 μ M) subtypes, effects that are attenuated by pretreatment with mecamylamine (10 μM). Further, A-85380 can activate (EC 50 = 8.9 ± 1.9 μ M) currents through channels formed by injection of the human α7 subunit into Xenopus oocytes, effects that are attenuated by pretreatment with the α7 nAChR antagonist, methyllycaconitine (10 nM). In all cases, A-85380 is more potent than (−)-nicotine but less potent than (±)-epibatidine. In neurotransmitter release studies, A-85380 stimulates the release of dopamine with an EC 50 value of 0.003 ± 0.001 μ M which is equipotent to (±)-epibatidine, and 20-fold more potent that (−)-nicotine (EC 50 = 0.04 ± 0.009 μ M). Thus, A-85380 displays a profile of robust activation of a number of nAChR subtypes with substantially less affinity for [ 125 I]α-BgT sites than [ 3 H](-)-cytisine sites, suggesting that it may serve as a more selective pharmacologic probe for the αx4β2 subtype relative to the α7 and α1β1δg nAChRs than (±)-epibatidine.

Activation and inhibition of the human α7 nicotinic acetylcholine receptor by agonists

To better understand the effects of weak as well as strong agonists at the human alpha7 nicotinic acetylcholine receptor (human alpha7 nAChR), the abilities of several classic nAChR agonists to both activate and inhibit (desensitize) the human alpha7 nAChR expressed in Xenopus oocytes were quantified and compared. Activation was measured during 0.2-20 s agonist application, as required to elicit a peak response. Inhibition was measured as the reduction in the agonist response to 200 microM ACh in the presence of inhibitor during a 5-20 min incubation. Acetylcholine (ACh), (-)-nicotine, (+)-nicotine, and 1,1-dimethyl-4-phenylpiperazinium (DMPP) were 62- to 130-fold more potent as inhibitors than as activators, with excellent correlation between the IC50 and EC50 values (r2 = 0.924). Agonist concentrations that elicited only 0.6-1.2% nAChR activation were sufficient to inhibit the response to ACh by 50%. Thus, even a very weak agonist could appear to be a potent and effective inhibitor through receptor desensitization. (-)-Lobeline, in contrast, acted as an antagonist at the human alpha7 nAChR, eliciting no detectable agonist-like response at concentrations up to 1 mM, but inhibiting the response to ACh with an IC50 value of 8.5 microM. (-)-Cotinine and the novel ligand ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine] acted as weak agonists at the human alpha7 nAChR (1 and 1.5% response at 1 mM, respectively) and inhibited the response to ACh with IC50) values of 175 and 48 microM, respectively. These effects could be explained by receptor desensitization, at least in part.

Preclinical Characterization of A‐582941: A Novel α7 Neuronal Nicotinic Receptor Agonist with Broad Spectrum Cognition‐Enhancing Properties

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the α7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine α7 nAChR agonist, A‐582941. A‐582941 was found to exhibit high‐affinity binding and partial agonism at α7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A‐582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A‐582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short‐term recognition memory, memory consolidation, and sensory gating deficit. A‐582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that α7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

Activation of the 5‐HT1c receptor expressed in Xenopus oocytes by the benzazepines SCH 23390 and SKF 38393

1 A cloned 5‐HT1C receptor expressed in Xenopus laevis oocytes was used to characterize the action of four dopamine D1‐selective benzazepines at the 5‐HT1C receptor. Additionally, the apparent binding of the D1‐selective benzazepines to 5‐HT1C receptors was measured in the choroid plexus of the pig. 2 In voltage‐clamped oocytes expressing the cloned 5‐HT1C receptor, 5‐hydroxytryptamine (5‐HT) elicited a characteristic inward current response with an EC50 of 13 nm. SCH 23390 acted as a stereoselective agonist (or partial agonist) with an EC50 of about 550 nm. SKF 38393 (1 μm–1 mm), SKF 77434 (100 μm), and SKF 82958 (100 μm) also acted as agonists (or partial agonists) at the cloned 5‐HT1C receptor. SKF 38393 was not stereoselective at the 5‐HT1C receptor. 3 The response to SCH 23390 activated slowly and, although the response contained many oscillations characteristic of the activation of the phosphatidylinositol signal transduction system, SCH 23390 rarely elicited the rapid spike‐like response seen routinely in response to 5‐HT. However, the responses to SKF 38393, SKF 77434, and SKF 82958 were identical in appearance to the response to 5‐HT, except that the responses to the benzazepines were smaller. These comparisons were made by applying both a benzazepine and 5‐HT to each individual oocyte expressing the cloned 5‐HT1C receptor. 4 Consistent with the responses measured in oocytes, SCH 23390 bound stereoselectively to 5‐HT1C receptors in the choroid plexus of the pig (Ki = 6.3 nm), and SKF 38393 bound non‐stereoselectively with lower affinity (Ki = 2.0–2.2 μm). 5 It is concluded that while these benzazepines demonstrate selectivity for the dopamine D1 receptor, they also can act as agonists or partial agonists at the 5‐HT1C receptor in situ and as expressed in Xenopus oocytes. The oocyte expression system is useful for studies of the functional pharmacology of these 5‐HT1C receptors. Information about the pharmacological actions and variations in stereoselectivity among dopamine and 5‐HT receptors should be of interest in modelling the interactions of ligands with these G‐protein coupled receptors, and in the testing of such models through receptor mutagenesis.;

A68930: a potent agonist selective for the dopamine D1 receptor

A68930, (1R,3S)-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman HCl, is a potent (EC50 = 2.5 nM), partial (intrinsic activity = 66% of dopamine) agonist in the fish retina dopamine-sensitive adenylate cyclase model of the D1 dopamine receptor. In the rat caudate-putamen model of the D1 dopamine receptor, A68930 is a potent (EC50 = 2.1 nM) full agonist. In contrast, A68930 is a much weaker (EC50 = 3920 nM) full agonist in a biochemical model of the dopamine D2 receptor. The orientation of the 3-phenyl substituent in the molecule is critical for the affinity and selectivity of the molecule towards the dopamine D1 receptor. A68930 also displays weak alpha 2-agonist activity but the molecule is virtually inactive at the alpha 1- and beta-adrenoceptors. When tested in rats bearing a unilateral 6-OHDA lesion of the nigro-neostriatal neurons, A68930 elicits prolonged (greater than 20 h) contralateral turning that is antagonized by dopamine D1 receptor selective doses of SCH 23390 but not by D2 receptor selective doses of haloperidol. In this lesioned rat model, A68930 increases 2-deoxyglucose accumulation in the lesioned substantia nigra, pars reticulata. When tested in normal rats, A68930 elicits hyperactivity and, at higher doses, produces a forelimb clonus.

Effect of MK-801 at the human α7 nicotinic acetylcholine receptor

Abstract Responses of the human α7 nicotinic acetylcholine receptor (α7 nAChR) expressed in Xenopus laevis oocytes were quantified in the presence of barium (10 mM) to prevent secondary activation of Ca 2+ -dependent Cl − currents and atropine (2 μM) to block endogenous muscarinic receptors. Acetylcholine (ACh) elicited responses with EC 50 values of 177 ± 32 μM to 272 ± 26 μM in different experiments. Responses to ACh (200 μM) were blocked by the nAChR antagonists α-bungarotoxin (IC 50 = 0.54 ± 0.04 nM), methyllycaconitine (IC 50 = 0.64 ± 0.08 nM) and mecamylamine (IC 50 = 1.8 ± 02 μM). Additionally, MK-801, a non-competitive blocker of N-methyl- d -aspartate (NMDA) sensitive glutamate receptor channels, inhibited the human α7 nAChR. This effect was not stereoselective; the IC 50 for (+)-MK-801 was 15 ± 3 μM while that for (−)-MK-801 was 14 ± 3 μM. The inhibition by MK-801, in contrast to methyllycaconitine, was dependent upon cell potential, consistent with a mechanism involving channel blockade. The inhibition by MK-801 reversed slowly (time constant approximately 20 min) compared to that by methyllycaconitine (100% recovery within 10 min). However, MK-801 did not appear to be trapped in the channel because the recovery from inhibition showed little dependence upon stimulation rate or cell potential. Thus, MK-801 acted as a non-stereoselective α7 nAChR inhibitor that was only about 8-fold less potent than the nAChR antagonist mecamylamine and probably acted through channel blockade.