Therapeutic Targeting of α7 Nicotinic Acetylcholine Receptors.

The α7-type nicotinic acetylcholine receptor is one of the most unique and interesting of all the members of the cys-loop superfamily of ligand-gated ion channels. Since it was first identified initially as a binding site for α-bungarotoxin in mammalian brain and later as a functional homomeric receptor with relatively high calcium permeability, it has been pursued as a potential therapeutic target for numerous indications, from Alzheimer disease to asthma. In this review, we discuss the history and state of the art for targeting α7 receptors, beginning with subtype-selective agonists and the basic pharmacophore for the selective activation of α7 receptors. A key feature of α7 receptors is their rapid desensitization by standard "orthosteric" agonist, and we discuss insights into the conformational landscape of α7 receptors that has been gained by the development of ligands binding to allosteric sites. Some of these sites are targeted by positive allosteric modulators that have a wide range of effects on the activation profile of the receptors. Other sites are targeted by direct allosteric agonist or antagonists. We include a perspective on the potential importance of α7 receptors for metabotropic as well as ionotropic signaling. We outline the challenges that exist for future development of drugs to target this important receptor and approaches that may be considered to address those challenges. SIGNIFICANCE STATEMENT: The α7-type nicotinic acetylcholine receptor (nAChR) is acknowledged as a potentially important therapeutic target with functional properties associated with both ionotropic and metabotropic signaling. The functional properties of α7 nAChR can be regulated in diverse ways with the variety of orthosteric and allosteric ligands described in this review.

Insights Into the Differential Desensitization of α4ß2 Nicotinic Acetylcholine Receptor Isoforms Obtained With Positive Allosteric Modulation of Mutant Receptors.

The development of highly efficacious positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChR) has proven useful in defining the ligand dependence of the conformational dynamics of α7 receptors. No such effective modulators are known to exist for the α4ß2 nAChR of the brain, limiting our ability to understand the importance of desensitization for the activity profile of specific ligands. In this study, we used mutant ß2 subunits that allowed the use of the α7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) to probe the desensitizing effects of nicotinic ligands on the two forms of α4ß2 receptors; high sensitivity (HS) (two α4 and three ß2 subunits) and low sensitivity (LS) (three α4 and two ß2 subunits). A total of 28 different ligands of 8 different categories, based on activity and selectivity, were tested for their ability to induce TQS-sensitive desensitization of HS and LS α4ß2 receptors. Results confirm that HS α4ß2 receptor responses are strongly limited by desensitization, by at least an order of magnitude more so than the responses of LS receptors. The activation of α4ß2 receptors by the smoking-cessation drugs cytisine and varenicline is strongly limited by desensitization, as is the activation of LS receptors by the HS-selective agonists 6-[5-[(2S)-2-Azetidinylmethoxy]-3-pyridinyl]-5-hexyn-1-ol dihydrochloride and 4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine difumarate. The evaluation of drugs previously identified as α7-selective agonists revealed varying patterns of α4ß2 cross-desensitization that were predictive of the effects of these drugs on the activation of wild-type α4ß2 receptors by acetylcholine, supporting the utility of TQS-sensitive receptors for the development of focused therapeutics. SIGNIFICANCE STATEMENT: To varying degrees, ligands regulate the balance of active and desensitized states of the two forms of the primary nAChR subtypes in brain. Using mutant beta subunits, an allosteric modulator can reverse ligand-induced desensitization, revealing the differential desensitization of the receptors by specific ligands. This study shows that drugs believed to be selective for therapeutic targets may cross-desensitize other targets and that, within a class of drugs, improved specificity can be achieved by using agents that reduce such cross-desensitization.

Silent agonists for α7 nicotinic acetylcholine receptors.

We discuss models for the activation and desensitization of α7 nicotinic acetylcholine receptors (nAChRs) and the effects of efficacious type II positive allosteric modulators (PAMs) that destabilize α7 desensitized states. Type II PAMs such as PNU-120596 can be used to distinguish inactive compounds from silent agonists, compounds that produce little or no channel activation but stabilize the non-conducting conformations associated with desensitization. We discuss the effects of α7 nAChRs in cells of the immune system and their roles in modulating inflammation and pain through what has come to be known as the cholinergic anti-inflammatory system (CAS). Cells controlling CAS do not generate ion channel currents but rather respond to α7 drugs by modulating intracellular signaling pathways analogous to the effects of metabotropic receptors. Metabotropic signaling by α7 receptors appears to be mediated by receptors in nonconducting conformations and can be accomplished by silent agonists. We discuss electrophysiological structure-activity relationships for α7 silent agonists and their use in cell-based and in vivo assays for CAS regulation. We discuss the strongly desensitizing partial agonist GTS-21 and its effectiveness in modulation of CAS. We also review the properties of the silent agonist NS6740, which is remarkably effective at maintaining α7 receptors in PAM-sensitive desensitized states. Most silent agonists bind to sites overlapping those for orthosteric agonists, but some appear to bind to allosteric sites. Finally, we discuss α9* nAChRs and their potential role in CAS, and ligands that will be useful in defining and distinguishing the specific roles of α7 and α9 in CAS.

Differential Activation and Desensitization States Promoted by Noncanonical α7 Nicotinic Acetylcholine Receptor Agonists.

A series of dipicolyl amine pyrimidines (DPPs) were previously identified as potential α7 agonists by means of a calcium influx assay in the presence of the positive allosteric modulator (PAM) 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596). The compounds lack the quaternary or strongly basic nitrogens of typical nicotinic agonists. Although differing in structure from typical nicotinic agonists, based on crystallographic data with the acetylcholine binding protein, they appeared to engage the site shared by such typical orthosteric agonists. Using oocytes expressing human α7 receptors, we found that the DPPs were efficacious activators of the receptor, with currents showing rapid desensitization characteristic of α7 receptors. However, we note that the rate of recovery from this desensitization depends strongly on structural features within the DPP family. Although the activation of receptors by DPP was blocked by the competitive antagonist methyllycaconitine (MLA), MLA had no effect on the DPP-induced desensitization, suggesting multiple modes of DPP binding. As expected, the desensitized conformational states could be reactivated by PAMs. Mutants made insensitive to acetylcholine by the C190A mutation in the agonist binding site were weakly activated by DPPs. The observation that activation of C190A mutants by the DPP compounds was resistant to the allosteric antagonist (-)cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide supports the hypothesis that the activity of these noncanonical agonists in the orthosteric binding sites was not entirely dependent on the classic epitopes controlling activation by typical agonists and that perhaps they may access alternative modes for promoting the conformational changes associated with activation and desensitization. SIGNIFICANCE STATEMENT: This study reports a family of nicotinic acetylcholine receptor agonists that break the rules about what the structure of a nicotinic acetylcholine receptor agonist should be. It shows that the activity of these noncanonical agonists in the orthosteric binding sites is not dependent on the classical epitopes controlling activation by typical agonists and that through different binding poses, they promote unique conformational changes associated with receptor activation and desensitization.

Betel quid: New insights into an ancient addiction.

The use of areca nuts (areca) in the form of betel quids constitutes the fourth most common addiction in the world, associated with high risk for oral disease and cancer. Areca is a complex natural product, making it difficult to identify specific components associated with the addictive and carcinogenic properties. It is commonly believed that the muscarinic agonist arecoline is at the core of the addiction. However, muscarinic receptor activation is not generally believed to support drug-taking behaviour. Subjective accounts of areca use include descriptions of both sedative and stimulatory effects, consistent with the presence of multiple psychoactive agents. We have previously reported partial agonism of α4-containing nicotinic acetylcholine receptors by arecoline and subsequent inhibition of those receptors by whole areca broth. In the present study, we report the inhibition of nicotinic acetylcholine receptors and other types of neurotransmitter receptors with compounds of high molecular weight in areca and the ability of low molecular weight areca extract to activate GABA and glutamate receptors. We confirm the presence of a high concentration of GABA and glutamate in areca. Additionally, data also indicate the presence of a dopamine and serotonin transporter blocking activity in areca that could account for the reported stimulant and antidepressant activity. Our data suggest that toxic elements of high molecular weight may contribute to the oral health liability of betel quid use, while two distinct low molecular weight components may provide elements of reward, and the nicotinic activity of arecoline contributes to the physical dependence of addiction.

Sulfonium Ligands of the α7 nAChR.

The α7 nicotinic acetylcholine receptor (nAChR) is an important target given its role in cognitive function as well as in the cholinergic anti-inflammatory pathway, where ligands that are effective at stabilizing desensitized states of the receptor are of particular interest. The typical structural element associated with a good desensitizer is the ammonium pharmacophore, but recent work has identified that a trivalent sulfur, in the positively charged sulfonium form, can substitute for the nitrogen in the ammonium pharmacophore. However, the breadth and scope of employing the sulfonium group is largely unexplored. In this work, we have surveyed a disparate group of sulfonium compounds for their functional activity with α7 as well as other nAChR subtypes. Amongst them, we found that there is a wide range of ability to induce α7 desensitization, with 4-hydroxyphenyldimethylsulfonium and suplatast sulfonium salts being the most desensitizing. The smallest sulfonium compound, trimethylsulfonium, was a partial agonist for α7 and other neuronal nAChR. Molecular docking into the α7 receptor extracellular domain revealed preferred poses in the orthosteric binding site for all but one compound, with typical cation-pi interactions as seen with traditional ammonium compounds. A number of the compounds tested may serve as useful platforms for further development of α7 desensitizing ability and for receptor subtype selectivity.

Allosterically Potentiated α7 Nicotinic Acetylcholine Receptors: Reduced Calcium Permeability and Current-Independent Control of Intracellular Calcium.

The currents of α7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of α7 receptors differ from normal α7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of α7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer's solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the allosteric agonist-PAMs (ago-PAMs) (3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (GAT107) and 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propenamide (B-973B) showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively, compared with currents activated by ACh alone, indicative of reduced calcium permeability. Currents potentiated by 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide were also linear and showed no calcium-dependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically activated α7 nicotinic ACh receptor may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptor can increase channel activation by two or more orders of magnitude, raising the concern that, due to the relatively high calcium permeability of α7 receptors activated by acetylcholine alone, such efficacious PAMs may have cytotoxic side effects. We show that PAMs alter the ion conduction pathway and, in general, reduce the calcium permeability of the channels. This supports the hypothesis that α7 effects on intracellular calcium may be independent of channel-mediated calcium influx.

Nicotinic Acetylcholine Receptor Accessory Subunits Determine the Activity Profile of Epibatidine Derivatives.

Epibatidine is a potent analgetic agent with very high affinity for brain nicotinic acetylcholine receptors (nAChR). We determined the activity profiles of three epibatidine derivatives, RTI-36, RTI-76, and RTI-102, which have affinity for brain nAChR equivalent to that of epibatidine but reduced analgetic activity. RNAs coding for nAChR monomeric subunits and/or concatamers were injected into Xenopus oocytes to obtain receptors of defined subunit composition and stoichiometry. The epibatidine analogs produced protracted activation of high sensitivity (HS) α4- and α2-containing receptors with the stoichiometry of 2alpha:3beta subunits but not low sensitivity (LS) receptors with the reverse ratio of alpha and beta subunits. Although not strongly activated by the epibatidine analogs, LS α4- and α2-containing receptors were potently desensitized by the epibatidine analogs. In general, the responses of α4(2)ß2(2)α5 and ß3α4ß2α6ß2 receptors were similar to those of the HS α4ß2 receptors. RTI-36, the analog closest in structure to epibatidine, was the most efficacious of the three compounds, also effectively activating α7 and α3ß4 receptors, albeit with lower potency and less desensitizing effect. Although not the most efficacious agonist, RTI-76 was the most potent desensitizer of α4- and α2-containing receptors. RTI-102, a strong partial agonist for HS α4ß2 receptors, was effectively an antagonist for LS α4ß2 receptors. Our results highlight the importance of subunit stoichiometry and the presence or absence of specific accessory subunits for determining the activity of these drugs on brain nAChR, affecting the interpretation of in vivo studies since in most cases these structural details are not known. SIGNIFICANCE STATEMENT: Epibatidine and related compounds are potent ligands for the high-affinity nicotine receptors of the brain, which are therapeutic targets and mediators of nicotine addiction. Far from being a homogeneous population, these receptors are diverse in subunit composition and vary in subunit stoichiometry. We show the importance of these structural details for drug activity profiles, which present a challenge for the interpretation of in vivo experiments since conventional methods, such as in situ hybridization and immunohistochemistry, cannot illuminate these details.

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 α4ß2L15'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.

A silent agonist of α7 nicotinic acetylcholine receptors modulates inflammation ex vivo and attenuates EAE.

Nicotinic acetylcholine receptors (nAChRs) are best known to function as ligand-gated ion channels in the nervous system. However, recent evidence suggests that nicotine modulates inflammation by desensitizing non-neuronal nAChRs, rather than by inducing channel opening. Silent agonists are molecules that selectively induce the desensitized state of nAChRs while producing little or no channel opening. A silent agonist of α7 nAChRs has recently been shown to reduce inflammation in an animal model of inflammatory pain. The objective of this study was to determine whether a silent agonist of α7 nAChRs can also effectively modulate inflammation and disease manifestation in an animal model of multiple sclerosis. We first evaluated the effects of various nAChR ligands and of an α7 nAChR-selective silent agonist, 1-ethyl-4-(3-(bromo)phenyl)piperazine (m-bromo PEP), on the modulation of mouse bone marrow-derived monocyte/macrophage (BMDM) numbers, phenotype and cytokine production. The non-competitive antagonist mecamylamine and the silent agonist m-bromo PEP reduced pro-inflammatory BMDM numbers by affecting their viability and proliferation. Both molecules also significantly reduced cytokine production by mouse BMDMs and significantly ameliorated disease in experimental autoimmune encephalomyelitis. Finally, m-bromo PEP also reduced chronic inflammatory pain in mice. Taken together, our results further support the hypothesis that nAChRs may modulate inflammation via receptor desensitization rather than channel opening. α7 nAChR-selective silent agonists may thus be a novel source of anti-inflammatory compounds that could be used for the treatment of inflammatory disorders.

Betel Nut (areca) and Smokeless Tobacco Use in Myanmar.

Background: Betel nut (areca) is the world's fourth most commonly used addictive substance. Arecoline, a muscarinic agonist in areca, is also a partial agonist for the addiction-related high-affinity brain nicotine receptors. In many countries, smokeless tobacco is commonly mixed with areca. Objective: We sought to evaluate the knowledge of self-harm, and addiction associated betel quid use in an unban population. Methods: We conducted a survey study of 200 betel quid users in Yangon, Myanmar, and a survey of betel quid vendors to determine the relative amounts of areca and tobacco in the available quids. Results: The data determined that a large majority of the survey subjects (84%) used tobacco with their areca. Users had a general awareness that betel chewing was "a bad habit" (85%) and 80% were aware of the cancer risks. Understanding areca addiction remains a challenge since, aside from the strong muscarinic activity of arecoline stimulating salivation, overt neurologic effects are difficult for even the users to identify. Fifty eight percent of the respondents indicated that chewing betel quid had effects like drinking coffee, and 55.5% indicated that it had effects like drinking alcohol. Data obtained from the quid vendors indicated that 75% added tobacco in equal amounts to areca. Conclusion: The concomitant use of nicotine and areca indicates that betel quid addiction includes a significant component of nicotine dependence. However, the additional activities of areca, including the muscarinic effects of arecoline, indicate that potential cessation therapies should optimally address other factors as well.

Macroscopic and Microscopic Activation of α7 Nicotinic Acetylcholine Receptors by the Structurally Unrelated Allosteric Agonist-Positive Allosteric Modulators (ago-PAMs) B-973B and GAT107.

B-973 is an efficacious type II positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptors that, like 4BP-TQS and its active isomer GAT107, can produce direct allosteric activation in addition to potentiation of orthosteric agonist activity, which identifies it as an allosteric activating (ago)-PAM. We compared the properties of B-973B, the active enantiomer of B-973, with those of GAT107 regarding the separation of allosteric potentiation and activation. Both ago-PAMs can strongly activate mutants of α7 that are insensitive to standard orthosteric agonists like acetylcholine. Likewise, the activity of both ago-PAMs is largely eliminated by the M254L mutation in the putative transmembrane PAM-binding site. Allosteric activation by B-973B appeared more protracted than that produced by GAT107, and B-973B responses were relatively insensitive to the noncompetitive antagonist mecamylamine compared with GAT107 responses. Similar differences are also seen in the single-channel currents. The two agents generate unique profiles of full-conductance and subconductance states, with B-973B producing protracted bursts, even in the presence of mecamylamine. Modeling and docking studies suggest that the molecular basis for these effects depends on specific interactions in both the extracellular and transmembrane domains of the receptor.

Allosteric Agonism of α7 Nicotinic Acetylcholine Receptors: Receptor Modulation Outside the Orthosteric Site.

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. Typically, channel activation follows the binding of agonists to the orthosteric binding sites of the receptor. α7 nAChRs have a very low probability of channel activation, which can be reversed by the binding of α7 selective positive allosteric modulators (PAMs) to putative sites within the transmembrane domains. Although typical PAMs, like PNU-120596, require coapplication of an orthosteric agonist to produce large channel activations, some, like GAT107 and B-973B [(S)-3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide], are characterized as allosteric activating PAMs, which also bind to an allosteric activation (AA) site in the extracellular domain and activate the α7 ion channel by themselves. We had previously characterized N,N-diethyl-N'-phenylpiperazine analogs with various functions. In this work, we docked members of this family to a homology model of the α7 receptor extracellular domain. The compound 1,1-diethyl-4(naphthalene-2-yl)piperazin-1-ium (2NDEP) a weak partial agonist, showed particularly favorable docking and binding energies at the putative AA site of the receptor. We hypothesized that 2NDEP could couple with PAMs through the AA site. This hypothesis was tested with the α7 mutant C190A, which is not activated by orthosteric agonists but is effectively activated by GAT107. The results showed that 2NDEP acts as an allosteric agonist of α7C190A when coapplied with the PAM PNU-120596. Also, the allosteric activity was nearly abolished upon coapplication with the AA site-selective antagonist 2,3,5,6MP-TQS (cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), consistent with AA site involvement. Overall, our findings show a novel mode of agonism through an allosteric site in the extracellular domain of α7 nAChR.

Heteromeric Neuronal Nicotinic Acetylcholine Receptors with Mutant ß Subunits Acquire Sensitivity to α7-Selective Positive Allosteric Modulators.

Homomeric α7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by α7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to α7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing α4 and ß2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the α7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the ß2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the α4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant ß subunit, regardless of the position of the mutant ß subunit within the pentameric complex. Similar results were obtained when ß2L15'M was coexpressed with α2 or α3 and when the L15'M mutation was placed in ß4 and coexpressed with α2, α3, or α4. Functional receptors were not observed when ß1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the α4ß2L15'M receptor compared with α7 and the availability of high-resolution α4ß2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of α7 subunits but are insensitive to PAMs, which greatly increase the open probability of α7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in α7. Specifically, the mutation of the TM2 15' leucine to methionine in α subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal ß subunits allows heteromeric receptors to respond to select α7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

Cracking the Betel Nut: Cholinergic Activity of Areca Alkaloids and Related Compounds.

INTRODUCTION: The use of betel quid is the most understudied major addiction in the world. The neuropsychological activity of betel quid has been attributed to alkaloids of Areca catechu. With the goal of developing novel addiction treatments, we evaluate the muscarinic and nicotinic activity of the four major Areca alkaloids: arecoline, arecaidine, guvacoline, and guvacine and four structurally related compounds. METHODS: Acetylcholine receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. RESULTS: Both arecoline- and guvacoline-activated muscarinic acetylcholine receptors (mAChR), while only arecoline produced significant activation of nicotinic AChR (nAChR). We characterized four additional arecoline-related compounds, seeking an analog that would retain selective activity for a α4* nAChR, with diminished effects on mAChR and not be a desensitizer of α7 nAChR. We show that this profile is largely met by isoarecolone. Three additional arecoline analogs were characterized. While the quaternary dimethyl analog had a broad range of activities, including activation of mAChR and muscle-type nAChR, the methyl analog only activated a range of α4* nAChR, albeit with low potency. The ethyl analog had no detectable cholinergic activity. CONCLUSIONS: Evidence indicates that α4* nAChR are at the root of nicotine addiction, and this may also be the case for betel addiction. Our characterization of isoarecolone and 1-(4-methylpiperazin-1-yl) ethanone as truly selective α4*nAChR selective partial agonists with low muscarinic activity may point toward a promising new direction for the development of drugs to treat both nicotine and betel addiction. IMPLICATIONS: Nearly 600 million people use Areca nut, often with tobacco. Two of the Areca alkaloids are muscarinic acetylcholine receptor agonists, and one, arecoline, is a partial agonist for the α4* nicotinic acetylcholine receptors (nAChR) associated with tobacco addiction. The profile of arecoline activity suggested its potential to be used as a scaffold for developing new tobacco cessation drugs if analogs can be identified that retain the same nicotinic receptor selectivity without muscarinic activity. We report that isoarecolone is a selective partial agonist for α4* nAChR with minimal muscarinic activity and 1-(4-methylpiperazin-1-yl) ethanone has similar nAChR selectivity and no detectable muscarinic action.

The Antinociceptive and Anti-Inflammatory Properties of the α7 nAChR Weak Partial Agonist p-CF3 N,N-diethyl-N'-phenylpiperazine.

Chronic pain and inflammatory diseases can be regulated by complex mechanisms involving α7 nicotinic acetylcholine receptors (nAChRs), making this subtype a promising drug target for anti-inflammatory therapies. Recent evidence suggests that suchtreatment of inflammatory pain may rely on metabotropic-like rather than ionotropic activation of the α7 receptor subtype in non-neuronal cells. We previously identified para-trifluoromethyl (p-CF3) N,N-diethyl-N'-phenylpiperazinium (diEPP) iodide to be among the compounds classified as silent agonists, which are very weak α7 partial agonists that are able to induce positive allosteric modulator (PAM)-sensitive desensitization. Such drugs have been shown to selectively promote α7 ionotropic-independent functions. Therefore, we here further investigated the electrophysiological profile of p-CF3 diEPP and its in vivo antinociceptive activity using Xenopus oocytes expressing α7, α4ß2, or α3ß4 nAChRs. The evoked currents confirmed p-CF3 diEPP to be α7-selective with a maximal agonism 5% that of acetylcholine (ACh). Coapplication of p-CF3 diEPP with the type II PAM 4-naphthalene-1-yl-3a,4,5,9b-tetrahydro-3-H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS) produced desensitization that could be converted to PAM-potentiated currents, which at a negative holding potential were up to 13-fold greater than ACh controls. Voltage-dependence experiments indicated that channel block may limit both control ACh and TQS-potentiated responses. Although no p-CF3 diEPP agonist activity was detected for the heteromeric nAChRs, it was a noncompetitive antagonist of these receptors. The compound displayed remarkable antihyperalgesic and antiedema effects in in vivo assays. The antinociceptive activity was dose and time dependent. The anti-inflammatory components were sensitive to the α7-selective antagonist methyllycaconitine, which supports the idea that these effects are mediated by the α7 nAChR.

Critical Molecular Determinants of α7 Nicotinic Acetylcholine Receptor Allosteric Activation: SEPARATION OF DIRECT ALLOSTERIC ACTIVATION AND POSITIVE ALLOSTERIC MODULATION.

The α7 nicotinic acetylcholine receptors (nAChRs) are uniquely sensitive to selective positive allosteric modulators (PAMs), which increase the efficiency of channel activation to a level greater than that of other nAChRs. Although PAMs must work in concert with "orthosteric" agonists, compounds such as GAT107 ((3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) have the combined properties of agonists and PAMs (ago-PAM) and produce very effective channel activation (direct allosteric activation (DAA)) by operating at two distinct sites in the absence of added agonist. One site is likely to be the same transmembrane site where PAMs like PNU-120596 function. We show that the other site, required for direct activation, is likely to be solvent-accessible at the extracellular domain vestibule. We identify key attributes of molecules in this family that are able to act at the DAA site through variation at the aryl ring substituent of the tetrahydroquinoline ring system and with two different classes of competitive antagonists of DAA. Analyses of molecular features of effective allosteric agonists allow us to propose a binding model for the DAA site, featuring a largely non-polar pocket accessed from the extracellular vestibule with an important role for Asp-101. This hypothesis is supported with data from site-directed mutants. Future refinement of the model and the characterization of specific GAT107 analogs will allow us to define critical structural elements that can be mapped onto the receptor surface for an improved understanding of this novel way to target α7 nAChR therapeutically.

Design, synthesis, and biological activity of 5'-phenyl-1,2,5,6-tetrahydro-3,3'-bipyridine analogues as potential antagonists of nicotinic acetylcholine receptors.

Starting from a known non-specific agonist (1) of nicotinic acetylcholine receptors (nAChRs), rationally guided structural-based design resulted in the discovery of a small series of 5'-phenyl-1,2,5,6-tetrahydro-3,3'-bipyridines (3a-3e) incorporating a phenyl ring off the pyridine core of 1. The compounds were synthesized via successive Suzuki couplings on a suitably functionalized pyridine starting monomer 4 to append phenyl and pyridyl substituents off the 3- and 5-positions, respectively, and then subsequent modifications were made on the flanking pyridyl ring to provide target compounds. Compound 3a is a novel antagonist, which is highly selective for α3ß4 nAChR (Ki=123nM) over the α4ß2 and α7 receptors.

Dissection of N,N-diethyl-N'-phenylpiperazines as α7 nicotinic receptor silent agonists.

The α7 nicotinic acetylcholine receptor (nAChR) is a target for control of inflammation-related phenomena via compounds that are able to selectively induce desensitized states of the receptor. Compounds that selectively desensitize, without facilitating significant channel activation, are termed 'silent agonists' because they can be discriminated from antagonists by the currents evoked with co-application with type II positive allosteric modulators (PAMs). One example is N,N-diethyl-N'-phenyl-piperazine (diEPP) (J. Pharm. Exp. Ther.2014, 350, 665). We used Ullmann-type aryl amination to synthesize a panel of 27 compounds related to diEPP by substitutions at the aryl ring and in the linkage between the piperazine and phenyl rings. Two-electrode voltage clamping of the human α7 nAChR expressed in Xenopus oocytes revealed that it was possible to tune the behavior of compounds to show enhanced desensitization without corresponding partial agonist activity such that trifluoromethyl and carboxamide aryl substituents showed 33 to 46-fold larger PAM-dependent net-charge responses, indicating selective partitioning of the ligand receptor complexes into the desensitized state.

The activity of GAT107, an allosteric activator and positive modulator of α7 nicotinic acetylcholine receptors (nAChR), is regulated by aromatic amino acids that span the subunit interface.

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.