Allosteric Modulator Of Α7 nAChRs Research Articles

The α7 nAChR allosteric modulator PNU-120596 amends neuroinflammatory and motor consequences of parkinsonism in rats: Role of JAK2/NF-κB/GSk3β/ TNF-α pathway

Parkinson’s disease (PD) is the second most common neurodegenerative disorder and a leading cause of disability. The current gold standard for PD treatment, L-Dopa, has limited clinical efficacy and multiple side effects. Evidence suggests that activation of α7 nicotinic acetylcholine receptors (α7nAChRs) abrogates neuronal and inflammatory insults. Here we tested whether PNU-120596 (PNU), a type II positive allosteric modulator of α7 nAChR, has a critical role in regulating motor dysfunction and neuroinflammation correlated with the associated PD dysfunction. Neuroprotective mechanisms were investigated through neurobehavioral, molecular, histopathological, and immunohistochemical studies. PNU reversed motor incoordination and hypokinesia induced via the intrastriatal injection of 6-hydroxydopamine and manifested by lower falling latency in the rotarod test, short ambulation time and low rearing incidence in open field test. Tyrosine hydroxylase immunostaining showed a significant restoration of dopaminergic neurons following PNU treatment, in addition to histopathological restoration in nigrostriatal tissues. PNU halted striatal neuroinflammation manifested as a suppressed expression of JAK2/NF-κB/GSk3β accompanied by a parallel decline in the protein expression of TNF-α in nigrostriatal tissue denoting the modulator anti-inflammatory capacity. Moreover, the protective effects of PNU were partially reversed by the α7 nAChR antagonist, methyllycaconitine, indicating the role of α7 nAChR modulation in the mechanism of action of PNU. This is the first study to reveal the positive effects of PNU-120596 on motor derangements of PD via JAK2/NF-κB/GSk3β/ TNF-α neuroinflammatory pathways, which could offer a potential therapeutic strategy for PD.

JWX-A0108, a positive allosteric modulator of α7 nAChR, attenuates cognitive deficits in APP/PS1 mice by suppressing NF-κB-mediated inflammation

Neuroinflammation plays an early and prominent role in the pathology of Alzheimer’s disease (AD). Studies have shown that cholinergic lesion is a contributor for the pathophysiology of AD. The α7 nicotinic acetylcholine receptors (nAChRs), a subtype of nAChRs, are abundantly expressed in the brain regions related to cognition and memory, such as hippocampus and frontal cortex. The α7 nAChR is rapidly activated and desensitized by agonists. JWX-A0108 is a type I positive allosteric modulator (PAM) of α7 nAChR, which mainly enhances agonist-evoked peak currents. Here, we used the Morris Water Maze to evaluate the effect of JWX-A0108 on cognition and memory functions in APP/PS1 mice, and the mechanism related to anti-inflammatory effect. The results showed that JWX-A0108 could improve the learning and memory function of APP/PS1 transgenic mice in Morris water maze, decrease the expression of IL-1β, TNF-α, IL-6 in the brain and lower the phosphorylation level of IκBα (Ser32/36) and NF-κB p65 (Ser536), decrease the expression of Iba1, the microglia activation marker. Nissl staining showed that the CA3 and DG regions of hippocampus were damaged in APP/PS1 mice, which was improved by JWX-A0108. All of these effects of JWX-A0108 were reversed by MLA (α7 nAChR specific blocker). Taken together, the results reveal that JWX-A0108 improved the learning and memory function of APP/PS1 mice by enhancing the anti-inflammatory effect of the endogenous choline system through α7 nAChR, inhibited the activation of the NF-κB signaling pathway by inhibiting IκB phosphorylation, and ultimately inhibited inflammatory responses.

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR

Many synthetic compounds to which we attribute specific activities are produced as racemic mixtures of stereoisomers, and it may be that all the desired activity comes from a single enantiomer. We have previously shown this to be the case with the α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM) 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) and the α7 ago-PAM 4BP-TQS. Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (2,3,5,6TMP-TQS), previously published as a "silent allosteric modulator" and an antagonist of α7 allosteric activation, shares the same scaffold with three chiral centers as the aforementioned compounds. We isolated the enantiomers of 2,3,5,6TMP-TQS and determined that the (-) isomer was a significantly better antagonist than the (+) isomer of the allosteric activation of both wild-type α7 and the nonorthosterically activatible C190A α7 mutant by the ago-PAM GAT107 (the active isomer of 4BP-TQS). In contrast, (+)2,3,5,6TMP-TQS proved to be an α7 PAM. (-)2,3,5,6TMP-TQS was shown to antagonize the allosteric activation of α7 by the structurally unrelated ago-PAM B-973B as well as the allosteric activation of the TQS-sensitive α4β2L15'M mutant. In silico docking of 2,3,5,6TMP-TQS in the putative allosteric activation binding site suggested a specific interaction of the (-) enantiomer with α7T106, and allosteric activation of α7T106 mutants was not inhibited by (-)2,3,5,6TMP-TQS, confirming the importance of this interaction and supporting the model of the allosteric binding site. Comparisons and contrasts between 2,3,5,6TMP-TQS isomers and active and inactive enantiomers of other TQS-related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of PAM activity. SIGNIFICANCE STATEMENT: Many synthetic ligands are in use as racemic preparations. We show that one enantiomer of the TQS analog Cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-te-trahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, originally reported to lack activity when used as a racemic preparation, is an α7 nicotinic acetylcholine receptor positive allosteric modulator (PAM). The other enantiomer is not a PAM, but it is an effective allosteric antagonist. In silico studies and structural comparisons identify essential elements of both the allosteric ligands and receptor binding sites important for these allosteric activities.

Chemical conversion of nicotinamide into type I positive allosteric modulator of α7 nAChRs.

Structural modifications of nicotinamide, a form of vitamin B3, gave rise to a series of compounds (8aa-8ce) that exhibit activities as type I positive allosteric modulators (PAMs) of human α7 nAChR expressed in Xenopus oocytes in two-electrode voltage clamp assay. The compound 8ai was a potent and efficacious PAM with an EC50 = 3.34 ± 1.13 μM and the maximum activation effect of α7 current over 1474 ± 246% in the presence of acetylcholine (100 μM). It is highly specific to α7 nAChR over other subtypes of nAChR and 5-HT3A receptors. The structure-activity relationship analysis identified a key skeleton of nicotinamide nucleus critical for biological activity. Taken together, the 8ai as a type I PAM of α7 nAChR may be beneficial for improvement of cognitive deficit.

Discovery of BNC375, a Potent, Selective, and Orally Available Type I Positive Allosteric Modulator of α7 nAChRs.

Positive allosteric modulators (PAMs) of α7 nAChRs can have different properties with respect to their effects on channel kinetics. Type I PAMs amplify peak channel response to acetylcholine but do not appear to influence channel desensitization kinetics, whereas Type II PAMs both increase channel response and delay receptor desensitization. Both Type I and Type II PAMs are reported in literature, but there are limited reports describing their structure-kinetic profile relationships. Here, we report a novel class of compounds with either Type I or Type II behavior that can be tuned by the relative stereochemistry around the central cyclopropyl ring: for example, (R,R)-13 (BNC375) and its analogues with RR stereochemistry around the central cyclopropyl ring are Type I PAMs, whereas compounds in the same series with SS stereochemistry (e.g., (S,S)-13) are Type II PAMs as measured using patch-clamp electrophysiology. Further fine control over the kinetics has been achieved by changing the substitutions on the aniline ring: generally the substitution of aniline with strong electron withdrawing groups reduces the Type II character of these compounds. Our structure-activity optimization efforts have led to the discovery of BNC375, a small molecule with good CNS-drug like properties and clinical candidate potential.

Pharmacological characterization of JWX-A0108 as a novel type I positive allosteric modulator of α7 nAChR that can reverse acoustic gating deficits in a mouse prepulse inhibition model.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca2+-permeable homopentameric ion channel implicated in cognition and neuropsychiatric disorders. Pharmacological enhancement of α7 nAChR function has been suggested for improvement of cognitive deficits. In the present study, we characterized a thiazolyl heterocyclic derivative, 6-(2-chloro-6-methylphenyl)-2-((3-fluoro-4-methylphenyl)amino)thiazolo[4,5-d]pyrimidin-7(6H)-one (JWX-A0108), as a novel type I α7 nAChR positive allosteric modulator (PAM), and evaluated its ability to reverse auditory gating and spatial working memory deficits in mice. In Xenopus oocytes expressing human nAChR channels, application of JWX-A0108 selectively enhanced α7 nAChR-mediated inward current in the presence of the agonist ACh (EC50 value = 4.35 ± 0.12 µM). In hippocampal slices, co-application of ACh and JWX-A0108 (10 µM for each) markedly increased both the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded in pyramidal neurons, but JWX-A0108 did not affect GABA-induced current in oocytes expressing human GABAA receptor α1β3γ2 and α5β3γ2 subtypes. In mice with MK-801-induced deficits in auditory gating, administration of JWX-A0108 (1, 3, and 10 mg/kg, i.p.) dose-dependently attenuates MK-801-induced auditory gating deficits in five prepulse intensities (72, 76, 80, 84, and 88 dB). Furthermore, administration of JWX-A0108 (0.03, 0.1, or 0.3 mg/kg, i.p.) significantly reversed MK-801-induced impaired spatial working memory in mice. Our results demonstrate that JWX-A0108 is a novel type I PAM of α7 nAChR, which may be beneficial for improvement of cognitive deficits commonly found in neuropsychiatric disorders such as schizophrenia and Alzheimer's disease.

Pharmacological Properties and Evaluations of a Novel Positive Allosteric Modulator of α7 nAChR for Attenuation of Schizophrenia-Like Behavior in Mice

The alpha-7 neuronal nicotinic acetylcholine receptor (α7 nAChR), consisting of homomeric α7 subunits, is a ligand-gated Ca2+-permeable ion channel critical for cognition, sensory processing, attention, and working memory. Dysfunctional α7 nAChR is implicated in cognition and neuropsychiatric disorders. Therefore, enhancement of α7 nAChR function is considered to be therapeutic strategy aimed at ameliorating cognitive deficits of neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia. We recently synthesized a novel series of 6-(2-Chloro-6-methylphenyl)-2-((3-fluoro-4-methylphenyl)amino)thiazolo[4,5-d]pyrimidin-7(6H)-ones as positive allosteric modulators (PAM) of α7 nAChR. In this study we evaluated the effect of a representative compound JWX-A0108 on activation of α7 and auditory gating or cognitive deficits in mice using a combination of assays including two-electrode voltage clamp (TEVC) recordings of Xenopus oocytes expressing human α7 nAChR, brain slice recordings, pre-pulse inhibition (PPI) for startle response and Y-maze spontaneous alternation test. Compound JWX-A0108 exhibits as type I PAM that specifically activates α7 current in the presence of 100 µM ACh in dose-dependent manner with EC50 at 5.7 ± 0.7 µM and 6-fold increase of the current. Application of JWX-A0108 (1-10 mg/kg, i.p.) dose-dependently reversed the auditory gating deficit induced by NMDA antagonist MK-801 in prepulse inhibition assay. In Y-maze, JWX-A0108 (0.03-0.3 mg/kg, i.p.) significantly increased the time spent in novel arm from 45% to 63% in mice treated with MK-801. We also evaluated the effect of JWX-A0108 on locomotor activity, and fount that JWX-A0108 (0.03 mg/kg, i.p.) significantly reduced MK-801-induced hyperactivity. The effects of JWX-A0108 on selectivity, pERK signaling and synaptic transmissions are currently being investigated.

Boosting Endogenous Resistance of Brain to Ischemia.

Most survivors of ischemic stroke remain physically disabled and require prolonged rehabilitation. However, some stroke victims achieve a full neurological recovery suggesting that the human brain can defend itself against ischemic injury, but the protective mechanisms are unknown. This study used selective pharmacological agents and a rat model of cerebral ischemic stroke to detect endogenous brain protective mechanisms that require activation of α7 nicotinic acetylcholine receptors (nAChRs). This endogenous protection was found to be (1) limited to less severe injuries; (2) significantly augmented by intranasal administration of a positive allosteric modulator of α7 nAChRs, significantly reducing brain injury and neurological deficits after more severe ischemic injuries; and (3) reduced by inhibition of calcium/calmodulin-dependent kinase-II. The physiological role of α7 nAChRs remains largely unknown. The therapeutic activation of α7 nAChRs after cerebral ischemia may serve as an important physiological responsibility of these ubiquitous receptors and holds a significant translational potential.

A Type-II positive allosteric modulator of α7 nAChRs significantly reduces brain injury and improves neurological function after focal cerebral ischemia in rats

A Type-II positive allosteric modulator of α7 nAChRs significantly reduces brain injury and improves neurological function after focal cerebral ischemia in rats

A Type-II Positive Allosteric Modulator of α7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats

In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.

Modulation of dopamine release by α7-type nicotinic acetylcholine receptors

Disentangling receptor kinetics and network dynamics is a prerequisite for understanding and predicting the systemic effects of pharmacological compounds. These effects may result from a complex balance between activation and desensitization of various receptor subtypes that can be located on excitatory or inhibitory neurons or their afferent fibres. We therefore combined receptor and network modelling [1] to explain the suppressive effect that TC-7020 [2,3], a partial agonist of α7-type nicotinic acetylcholine receptors (nAChRs), was observed to exert on the release of dopamine (DA) in the nucleus accumbens of anesthetized rats. As nAChRs are ion channels permeating Na+ and Ca2+, and as i.v. injection of nicotine itself enhanced DA release, partial agonists would rather be expected to enhance DA release as well. Using the model, we evaluated several potential effects of TC-7020 on DA-ergic and GABA-ergic neurons in the ventral tegmental area (VTA) and its glutamatergic afferents from neocortex. Potential effects of TC-7020 included on the one hand a reduced nAChR channel conductance on DA neurons through competitive or non-competitive inhibition, or through receptor desensitization. On the other hand, enhancing the nAChR conductance on GABA-neurons (through direct activation of nAChRs, or through priming of their response to background ACh [4]) could have produced, through inhibition of DA neurons, a similar suppressive effect on DA release. The model showed that rapid desensitization of the α7 nAChRs on DA neurons explained both the sign and time-course of the voltammetric DA response, provided that a background cholinergic tone was present that contributed, by activating α7 nAChRs without desensitizing them, to the baseline level of DA release. Another surprising finding of the voltammetric DA recordings was that PNU-120596, a positive allosteric modulator of α7 nAChRs, prevented and abolished the effect of TC-7020 on DA release, instead of amplifying it. The model proposes two possible but contrasting mechanisms: a well-established relieve of α7 nAChRs from the weak state of desensitization into which they were brought by TC-7020, or, in contrast, the induction of a strong desensitization in α7 nAChRs due to channel over-excitation by background ACh under the amplifying effect of PNU-120596 [5]. These results show that tonic levels of neurotransmitters and neuromodulators like acetylcholine and dopamine may profoundly affect the dynamics, and hence the systemic effect, of pharmacological compounds. As these background levels may be altered in in-vitro preparations, circuit modelling may become an indispensible tool in drug development.

Nicotinic acetylcholine receptors (nAchRs) in supraoptic nucleus (SON) neurons: Potential role in regulation of food intake

Nicotine induces anorexia. nAchRs are expressed in SON of the hypothalamus, where oxytocin (OT) and vasopressin (VP) are synthesized. Nicotine stimulates release of VP as well as OT, an anorexic agent. Deficits in OT are associated with human obesity. Thus, nicotine may reduce food intake, in part, via modulation of OT neurons in SON. In our current studies, live‐cell calcium imaging was used to determine if acetylcholine (Ach) or nicotine altered intracellular calcium ([Ca++]i)signaling in SON neurons. Hypothalamic explants that include SON were loaded with the calcium sensitive dye, Fura‐2AM. Changes in [Ca++]i were monitored by changes in the 340/380 nm emission. A short application of Ach (5 mM) to the bath caused an increase in [Ca++]i in a subset of SON neurons, in the presence or absence of PNU‐120596 (10 μM), a positive allosteric modulator of α7 nAchRs. Ca++ response to Ach was reduced but still existed in the presence of atropine (10 μM) to block muscarinic AchRs. Nicotine (20–100 μM) also induced [Ca++]i increase in a portion of SON neurons. These findings are consistent with activation of nAchRs in SON neurons. Future studies will further characterize the specific subtypes of nAchRs in SON and identify the peptidergic phenotype of responsive neurons. Supported by AHA 0735329N.

In Vitro neuroprotective effects of ABT-779, a positive allosteric modulator of α7 nAChRs

In Vitro neuroprotective effects of ABT-779, a positive allosteric modulator of α7 nAChRs

In vitro pharmacological characterization of ABT-779, a novel positive allosteric modulator of α7 nAChRs

In vitro pharmacological characterization of ABT-779, a novel positive allosteric modulator of α7 nAChRs

Activation of Functional α7-Containing nAChRs in Hippocampal CA1 Pyramidal Neurons by Physiological Levels of Choline in the Presence of PNU-120596

BackgroundThe level of expression of functional α7-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal CA1 pyramidal neurons is believed to be very low compared to hippocampal CA1 interneurons, and for many years this expression was largely overlooked. However, high densities of expression of functional α7-containing nAChRs in CA1 pyramidal neurons may not be necessary for triggering important cellular and network functions, especially if activation of α7-containing nAChRs occurs in the presence of positive allosteric modulators such as PNU-120596.Methodology/Principal FindingsAn approach previously developed for α7-containing nAChRs expressed in tuberomammillary neurons was applied to investigate functional CA1 pyramidal α7-containing nAChRs using rat coronal hippocampal slices and patch-clamp electrophysiology. The majority (∼71%) of tested CA1 pyramidal neurons expressed low densities of functional α7-containing nAChRs as evidenced by small whole-cell responses to choline, a selective endogenous agonist of α7 nAChRs. These responses were potentiated by PNU-120596, a novel positive allosteric modulator of α7 nAChRs. The density of functional α7-containing nAChRs expressed in CA1 pyramidal neurons (and thus, the normalized net effect of activation, i.e., response net charge per unit of membrane capacitance per unit of time) was estimated to be ∼5% of the density observed in CA1 interneurons. The results of this study demonstrate that despite low levels of expression of functional pyramidal α7-containing nAChRs, physiological levels of choline (∼10 µM) are sufficient to activate these receptors and transiently depolarize and even excite CA1 pyramidal neurons in the presence of PNU-120596. The observed effects are possible because in the presence of 10 µM choline and 1–5 µM PNU-120596, a single opening of an individual pyramidal α7-containing nAChR ion channel appears to transiently depolarize (∼4 mV) the entire pyramidal neuron and occasionally trigger action potentials.Conclusions1) The majority of hippocampal CA1 pyramidal neurons express functional α7-containing nAChRs. In the absence of PNU-120596, a positive allosteric modulator of α7 nAChRs, a lack of responsiveness of some hippocampal CA1 pyramidal neurons to focal application of 0.5–1 mM choline does not imply a lack of expression of functional α7-containing nAChRs in these neurons. Rather, it may indicate a lack of detection of α7-containing nAChR-mediated currents by patch-clamp electrophysiology. 2) PNU-120596 can serve as a powerful tool for detection and enhancement of responsiveness of low densities of functional α7-containing nAChRs such as those present in hippocampal CA1 pyramidal neurons. 3) In the presence of PNU-120596, physiological concentrations of choline activate functional CA1 pyramidal α7-containing nAChRs and produce step-like currents that cause repetitive step-like depolarizations, occasionally triggering bursts of action potentials in CA1 pyramidal neurons. Therefore, the results of this study suggest that in the presence of PNU-120596 and possibly other positive allosteric modulators, endogenous choline may persistently activate CA1 pyramidal α7-containing nAChRs, enhance the excitability of CA1 pyramidal neurons and thus act as a potent therapeutic agent with potential neuroprotective and cognition-enhancing properties.