Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M₄ muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M₄ mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist–receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [³⁵S]GTPγS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3β phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M₄ mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [³H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M₄ mAChR knockout mice, validating the M₄ mAChR as a key target of action of this novel allosteric ligand.     

alpha7 nicotinic acetylcholine receptor agonist properties of tilorone and related tricyclic analogues

Background and purpose:

The α7 nicotinic acetylcholine receptor (nAChR) has attracted considerable interest as a target for cognitive enhancement in schizophrenia and Alzheimer''s Disease. However, most recently described α7 agonists are derived from the quinuclidine structural class. Alternatively, the present study identifies tilorone as a novel α7-selective agonist and characterizes analogues developed from this lead.

Experimental approach:

Activity and selectivity were determined from rat brain α7 and α4β2 nAChR binding, recombinant nAChR activation, and native α7 nAChR mediated stimulation of ERK1/2 phosphorylation in PC12 cells.

Key results:

Tilorone bound α7 nAChR (IC₅₀ 110 nM) with high selectivity relative to α4β2 (IC₅₀ 70 000 nM), activated human α7 nAChR with an EC₅₀ value of 2.5 μM and maximal response of 67% relative to acetylcholine, and showed little agonist effect at human α3β4 or α4β2 nAChRs. However, the rat α7 nAChR maximal response was only 34%. Lead optimization led to 2-(5-methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-xanthen-9-one (A-844606) with improved binding (α7 IC₅₀ 11 nM, α4β2 IC₅₀>30 000 nM) and activity at both human and rat α7 nAChR (EC₅₀s 1.4 and 2.2 μM and apparent efficacies 61 and 63%, respectively). These compounds also activated native α7 nAChR, stimulating ERK1/2 phosphorylation in PC12 cells.

Conclusions and implications:

Tilorone, known as an interferon inducer, is a selective α7 nAChR agonist, suggesting utility of the fluorenone pharmacophore for the development of α7 nAChR selective agonists. Whether α7 stimulation mediates interferon induction, or whether interferon induction may influence the potential anti-inflammatory properties of α7 nAChR agonists remains to be elucidated.     

Selective ��4��2 Nicotinic Acetylcholine Receptor Agonists Target Epigenetic Mechanisms in Cortical GABAergic Neurons

Nicotine improves cognitive performance and attention in both experimental animals and in human subjects, including patients affected by neuropsychiatric disorders. However, the specific molecular mechanisms underlying nicotine-induced behavioral changes remain unclear. We have recently shown in mice that repeated injections of nicotine, which achieve plasma concentrations comparable to those reported in high cigarette smokers, result in an epigenetically induced increase of glutamic acid decarboxylase 67 (GAD₆₇) expression. Here we explored the impact of synthetic α₄β₂ and α₇ nAChR agonists on GABAergic epigenetic parameters. Varenicline (VAR), a high-affinity partial agonist at α₄β₂ and a lower affinity full agonist at α₇ neuronal nAChR, injected in doses of 1–5 mg/kg/s.c. twice daily for 5 days, elicited a 30–40% decrease of cortical DNA methyltransferase (DNMT)1 mRNA and an increased expression of GAD₆₇ mRNA and protein. This upregulation of GAD₆₇ was abolished by the nAChR antagonist mecamylamine. Furthermore, the level of MeCP₂ binding to GAD₆₇ promoters was significantly reduced following VAR administration. This effect was abolished when VAR was administered with mecamylamine. Similar effects on cortical DNMT1 and GAD₆₇ expression were obtained after administration of A–85380, an agonist that binds to α₄β₂ but has negligible affinity for α₃β₄ or α₇ subtypes containing nAChR. In contrast, PNU–282987, an agonist of the homomeric α₇ nAChR, failed to decrease cortical DNMT1 mRNA or to induce GAD₆₇ expression. The present study suggests that the α₄β₂ nAChR agonists may be better suited to control the epigenetic alterations of GABAergic neurons in schizophrenia than the α₇ nAChR agonists.     

Calcium signalling mediated through ��7 and non-��7 nAChR stimulation is differentially regulated in bovine chromaffin cells to induce catecholamine release

BACKGROUND AND PURPOSE

Ca²⁺ signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the α7 nAChR, in bovine chromaffin cells are still matters of debate.

EXPERIMENTAL APPROACH

We have used chromaffin cell cultures loaded with Fluo-4 or transfected with aequorins directed to the cytosol or mitochondria, several nAChR agonists (nicotine, 5-iodo-A-85380, PNU282987 and choline), and the α7 nAChR allosteric modulator PNU120596.

KEY RESULTS

Minimal [Ca²⁺]_c transients, induced by low concentrations of selective α7 nAChR agonists and nicotine, were markedly increased by the α7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the α7 nAChR antagonist α-bungarotoxin (α7-modulated-response). Conversely, high concentrations of the α7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca²⁺]_c transients, that were blocked by mecamylamine but were unaffected by α-bungarotoxin (non-α7 response). [Ca²⁺]_c increases mediated by α7 nAChR were related to Ca²⁺ entry through non-L-type Ca²⁺ channels, whereas non-α7 nAChR-mediated signals were related to L-type Ca²⁺ channels; Ca²⁺-induced Ca²⁺-release contributed to both responses. Mitochondrial involvement in the control of [Ca²⁺]_c transients, mediated by either receptor, was minimal. Catecholamine release coupled to α7 nAChRs was more efficient in terms of catecholamine released/[Ca²⁺]_c.

CONCLUSIONS AND IMPLICATIONS

[Ca²⁺]_c and catecholamine release mediated by α7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the α7 nAChR response was lost and the non-α7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of α7 nAChRs.     

Impact of species variability and 'probe-dependence' on the detection and in vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor

BACKGROUND AND PURPOSE

We recently characterized LY2033298 as a novel allosteric modulator and agonist at M₄ muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M₄ mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions.

EXPERIMENTAL APPROACH

LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M₄ mAChRs, using assays of agonist-induced ERK1/2 or GSK-3α phosphorylation, [³⁵S]-GTPγS binding, or effects on equilibrium binding of [³H]-NMS and ACh. The in vivo actions of LY2033298 were investigated in a mouse model of amphetamine-induced locomotor activity. The function of LY2033298 was examined in combination with ACh, oxotremorine or xanomeline.

KEY RESULTS

LY2033298 had similar affinities for the human and mouse M₄ mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M₄ mAChR. At the mouse M₄ mAChR, LY2033298 showed higher co-operativity with oxotremorine than with ACh or xanomeline. The different degrees of co-operativity between LY2033298 and each agonist at the mouse relative to the human M₄ mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M₄ mAChR, and also highlight how the interaction between LY2033298 and different orthosteric ligands is subject to ‘probe dependence’. This has implications for the validation of allosteric modulator actions in vivo.     

Alpha7 Nicotinic Acetylcholine Receptors Modulate Motivation to Self-Administer Nicotine: Implications for Smoking and Schizophrenia

Individuals diagnosed with schizophrenia have an exceptionally high risk for tobacco dependence. Postmortem studies show that these individuals have significant reductions in α7 nicotinic acetylcholine receptors (nAChRs) in several brain areas. Decreased α7-mediated function might not only be linked to schizophrenia but also to increased tobacco consumption. The purpose of this study was to determine whether pharmacological blockade of α7 nAChRs would increase motivation of rats to intravenously self-administer nicotine (NIC) during a progressive ratio schedule of reinforcement (PR). Before PR, rats received local infusions of 0, 10, or 20 pmol of a selective α7 nAChR antagonist, α-conotoxin ArIB [V11L,V16D] (ArIB) into the nucleus accumbens (NAc) shell or the anterior cingulate cortex, brain areas that contribute to motivation for drug reward. We additionally sought to determine whether local infusion of 0, 10, or 40 nmol of a selective α7 nAChR agonist, PNU 282987, into these brain areas would decrease motivation for NIC use. Infusion of ArIB into the NAc shell and anterior cingulate cortex resulted in a significant increase in active lever pressing, breakpoints, and NIC intake, suggesting that a decrease in α7 nAChR function increases motivation to work for NIC. In contrast, PNU 282987 infusion resulted in reductions in these measures when administered into the NAc shell, but had no effect after administration into the anterior cingulate cortex. These data identify reduction of α7 nAChR function as a potential mechanism for elevated tobacco use in schizophrenia and also identify activation of α7 nAChRs as a potential strategy for tobacco cessation therapy.     

Antipsychotic Drug-Like Effects of the Selective M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0152100

Accumulating evidence suggests that selective M₄ muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M₄ activators were unsuccessful because of the lack of M₄ mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M₄ positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M₄ on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M₄-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M₄ modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M₄ PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.     

Muscarinic receptor pharmacology and circuitry for the modulation of cognition

The muscarinic cholinergic system constitutes an important part of the neuronal circuitry that modulates normal cognition. Muscarinic receptor antagonists are well known to produce or exacerbate impairments in attention, learning, and memory. Conversely, both direct-acting muscarinic receptor agonists and indirect-acting muscarinic cholinergic agonists, such as acetylcholinesterase inhibitors, have shown cognition-enhancing properties, including improvements in normal cognitive function, reversal of cognitive deficits induced by muscarinic receptor antagonists, and attenuation of cognitive deficits in psychiatric and neurological disorders, such as Alzheimer's disease and schizophrenia. However, until recently, the lack of small molecule ligands that antagonize or activate specific muscarinic acetylcholine receptor (mAChR) subtypes with high selectivity has been a major obstacle in defining the relative contributions of individual mAChRs to different aspects of cognitive function and for the development of novel therapeutic agents. These limitations may be potentially overcome by the recent discovery of novel mAChR subtype-selective compounds, notably allosteric agonists and positive allosteric modulators, which exhibit greater selectivity for individual mAChR subtypes than previous mAChR orthosteric agonists. In preclinical studies, these novel ligands have shown promising efficacy in several models for the enhancement of cognition. In this chapter, we will review the muscarinic cholinergic circuitry and pharmacology of mAChR agonists and antagonists relevant to the modulation of different aspects of cognition in animals and clinical populations.     

AC-260584, an orally bioavailable M1 muscarinic receptor allosteric agonist, improves cognitive performance in an animal model

The recent discovery of allosteric potentiators and agonists of the muscarinic M₁ receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC₅₀ 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M₁ receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M₁ receptor over the M₂, M₃, M₄ and M₅ muscarinic receptor subtypes. Using GTPγS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M₁ receptor activation since it was not observed in M₁ knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M₁ receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M₁ selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease.     

Muscarinic acetylcholine receptors for psychotic disorders: bench-side to clinic

Modern interest in muscarinic acetylcholine receptor (mAChR) activators for schizophrenia began in the 1990s when xanomeline, an M₁/M₄-preferring mAChR agonist developed for cognitive symptoms of Alzheimer's disease (AD), had unexpected antipsychotic activity. However, strategies to address tolerability concerns associated with activation of peripheral mAChRs were not available at that time. The discovery of specific targeted ligands and combination treatments to reduce peripheral mAChR engagement have advanced the potential of mAChR activators as effective treatments for psychotic disorders. This review provides perspectives on the background of the identification of mAChRs as potential antipsychotics, advances in the preclinical understanding of mAChRs as targets, and the current state of mAChR activators under active clinical development for schizophrenia.     

Partial Agonists of the ��3��4* Neuronal Nicotinic Acetylcholine Receptor Reduce Ethanol Consumption and Seeking in Rats

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3–CHRNA5–CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2^* nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.     

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [³⁵S]GTPγS binding, using the anti-Gα_q antibody sc-393. Increased specific [³⁵S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gα_q, selectively coupled with the M₁ mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M₁ mAChR agonists in the present study were generally lower than previously reported in M₁-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [³⁵S]GTPγS binding to Gα_q was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M₁-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the G_q-coupled M₁ mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.     

��4��2 Nicotinic acetylcholine receptors, willing if able

Li and Steinbach apply nonstationary noise analysis to the whole-cell current responses of low sensitivity α4β2 nAChR stably-expressed in HEK cells. These receptors represent one of the most important nAChR subtypes in brain, and also one of the most difficult to study in native tissues. They found the activating properties of the full agonists ACh and nicotine to be similar with regard to P_open and single channel conductance, whereas the weak α4β2 partial agonist cytisine caused channels to open with low probability but increased single channel conductance. When optimally stimulated by either of the full agonists, approximately 80% of the available receptors opened at the peak of the response. However, comparisons of whole-cell current to estimates of total cell surface receptors, indicated that only about 7% of the total receptor population can be activated. These observations provide important and intriguing new pieces of the brain nicotine receptor puzzle.     

The selectivity of ��-adrenoceptor agonists at human ��1-, ��2- and ��3-adrenoceptors

Background and purpose:

There are two important properties of receptor–ligand interactions: affinity (the ability of the ligand to bind to the receptor) and efficacy (the ability of the receptor–ligand complex to induce a response). Ligands are classified as agonists or antagonists depending on whether or not they have efficacy. In theory, it is possible to develop selective agonists based on selective affinity, selective intrinsic efficacy or both. This study examined the affinity and intrinsic efficacy of 31 β-adrenoceptor agonists at the three human β-adrenoceptors to determine whether the current agonists are subtype selective because of affinity or intrinsic efficacy.

Experimental approach:

Stable clonal CHO-K1 cell lines, transfected with either the human β₁, β₂ or β₃-adrenoceptor, were used, and whole-cell [³H]-CGP 12177 radioligand binding and [³H]-cAMP accumulation were measured.

Key results:

Several agonists were found to be highly subtype selective because of selective affinity (e.g. salmeterol and formoterol, for the β₂-adrenoceptor over the β₁ or β₃), while others (e.g. isoprenaline) had little affinity–selectivity. However, the intrinsic efficacy of salmeterol, formoterol and isoprenaline was similar across all three receptor subtypes. Other ligands (e.g. denopamine for β₁; clenbuterol, AZ 40140d, salbutamol for β₂) were found to have subtype-selective intrinsic efficacy. Several ligands appeared to activate two agonist conformations of the β₁- and β₃-adrenoceptors.

Conclusions and implications:

There are agonists with subtype selectivity based upon both selective affinity and selective intrinsic efficacy. Therefore, there is scope to develop better selective agonists based upon both selective affinity and selective intrinsic efficacy.This article is commented on by Kenakin, pp. 1045–1047 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00764.x     

Discovery of Novel N-Substituted Oxindoles as Selective M1 and M4 Muscarinic Acetylcholine Receptors Partial Agonists

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Pharmacology of α7 nicotinic acetylcholine receptor mediated extracellular signal-regulated kinase signalling in PC12 cells

Background and purpose:

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate cell survival and memory processing. The involvement of specific nAChR subtypes in downstream signalling events has been ill defined thus far, because of a lack of subtype-selective ligands. In this study, we investigated activation and modulation of α7 nAChR-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) in PC12 cells, using selective agonists and positive allosteric modulators.

Experimental approach:

We used undifferentiated PC12 cells endogenously expressing α7 nAChR for both biochemical and functional studies. ERK phosphorylation changes were measured by using a novel In-Cell Western procedure. α7 nAChR-mediated Ca²⁺ signalling was determined by using the fluorometric imaging plate reader assay.

Key results:

Robust induction of ERK phosphorylation followed exposure of PC12 cells to the selective agonist PNU-282987 in the presence of the α7 nAChR modulator PNU-120596. ERK phosphorylation was transient and was attenuated by the selective antagonist methyllycaconitine. Consistent with allosteric modulation of α7 nAChRs, PNU-120596 enhanced both the agonist potency and efficacy in activating ERK. Moreover, α7 nAChR agonists could be quantitatively differentiated based on their potency in activating ERK signalling. The rank order of potencies correlated fairly well with the corresponding binding K_i values of these α7 nAChR agonists.

Conclusions and implications:

The present work extends previous observations demonstrating the involvement of α7 nAChRs in ERK1/2 phosphorylation in PC12 cells. The In-Cell Western procedure allowed a detailed investigation of α7 nAChR function and downstream ERK signalling in response to agonist and allosteric modulators.     

Role of channel activation in cognitive enhancement mediated by α7 nicotinic acetylcholine receptors

Background and purpose:

Several agonists of the α7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid α7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved.

Experimental approach:

Two structurally related α7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm.

Key results:

NS6784 activated human and rat α7 nAChR with EC₅₀s of 0.72 and 0.88 µM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at α7 nAChR (<2% in oocytes, ≤8% in GH4C1 cells), although its agonist-like properties were revealed by adding a positive allosteric modulator of α7 nAChRs or using the slowly desensitizing α7V274T receptor. In mouse inhibitory avoidance (IA) memory retention, NS6784 enhanced performance as did the 60% partial agonist A-582941. In contrast, NS6740 did not enhance performance, but blocked effects of A-582941.

Conclusions and implications:

Collectively, these findings suggest that a degree of α7 nAChR agonist efficacy is required for behavioural effects in the IA paradigm, and that such behavioural efficacy is not due to α7 nAChR desensitization. Also, a partial agonist of very low efficacy for this receptor could be used as an inhibitor, in the absence of α7 nAChR antagonists with favourable CNS penetration.     

Enhancement of Attentional Performance by Selective Stimulation of ��4��2* nAChRs: Underlying Cholinergic Mechanisms

Impairments in attention are a major component of the cognitive symptoms of neuropsychiatric and neurodegenerative disorders. Using an operant sustained attention task (SAT), including a distractor condition (dSAT), we assessed the putative pro-attentional effects of the selective α4β2^* nicotinic acetylcholine receptor (nAChR) agonist S 38232 in comparison with the non-selective agonist nicotine. Neither drug benefited SAT performance. However, in interaction with the increased task demands implemented by distractor presentation, the selective agonist, but not nicotine, enhanced the detection of signals during the post-distractor recovery period. This effect is consistent with the hypothesis that second-long increases in cholinergic activity (‘transients'') mediate the detection of cues and that nAChR agonists augment such transients. Electrochemical recordings of prefrontal cholinergic transients evoked by S 38232 and nicotine indicated that the α4β2^* nAChR agonist evoked cholinergic transients that were characterized by a faster rise time and more rapid decay than those evoked by nicotine. Blockade of the α7 nAChR ‘sharpens'' nicotine-evoked transients; therefore, we determined the effects of co-administration of nicotine and the α7 nAChR antagonist methyllycaconitine on dSAT performance. Compared with vehicle and nicotine alone, this combined treatment significantly enhanced the detection of signals. These results indicate that compared with nicotine, α4β2^* nAChR agonists significantly enhance attentional performance and that the dSAT represents a useful behavioral screening tool. The combined behavioral and electrochemical evidence supports the hypothesis that nAChR agonist-evoked cholinergic transients, which are characterized by rapid rise time and fast decay, predict robust drug-induced enhancement of attentional performance.     

Selective Muscarinic Receptor Agonists and Antagonists

Abstract: Muscarinic receptors are composed of a family of four subtypes each of which can be distinguished pharmacologically and structurally. The physiological role of each subtype in the central and peripheral nervous systems remains to be clarified, due, in part, to a lack of agonists and antagonists with adequate subtype selectivity. Nonetheless, several agonists with functional selectivity for M₁ receptors are now in advanced clinical evaluation for Alzheimer's disease, while selective M₁/M₃ antagonists may prove useful in the treatment of disorders of smooth muscle function. These novel compounds thus provide an advance over earlier therapeutics with which the clinical efficacy was compromised by the side effect profile. This review attempts to assess novel, selective agonists and antagonists, both in terms of their use in defining muscarinic receptor subtypes and their potential clinical utility.