Explorations of Agonist Selectivity for the α9* nAChR with Novel Substituted Carbamoyl/Amido/Heteroaryl Dialkylpiperazinium Salts and Their Therapeutic Implications in Pain and Inflammation.

There is an urgent need for nonopioid treatments for chronic and neuropathic pain to provide effective alternatives amid the escalating opioid crisis. This study introduces novel compounds targeting the α9 nicotinic acetylcholine receptor (nAChR) subunit, which is crucial for pain regulation, inflammation, and inner ear functions. Specifically, it identifies novel substituted carbamoyl/amido/heteroaryl dialkylpiperazinium iodides as potent agonists selective for human α9 and α9α10 over α7 nAChRs, particularly compounds 3f, 3h, and 3j. Compound 3h (GAT2711) demonstrated a 230 nM potency as a full agonist at α9 nAChRs, being 340-fold selective over α7. Compound 3c was 10-fold selective for α9α10 over α9 nAChR. Compounds 2, 3f, and 3h inhibited ATP-induced interleukin-1ß release in THP-1 cells. The analgesic activity of 3h was fully retained in α7 knockout mice, suggesting that analgesic effects were potentially mediated through α9* nAChRs. Our findings provide a blueprint for developing α9*-specific therapeutics for pain.

Functions and pharmacology of α2ß2 nicotinic acetylcholine receptors; in and out of the shadow of α4ß2 nicotinic acetylcholine receptors.

Although α2 was the first neuronal nicotinic acetylcholine receptor (nAChR) receptor subunit to be cloned, due to its low level of expression in rodent brain, its study has largely been neglected. This study provides a comparison of the α2 and α4 structures and their functional similarities, especially in regard to the existence of low and high sensitivity forms based on subunit stoichiometry. We show that the pharmacological profiles of the low and high sensitivity forms of α2ß2 and α4ß2 receptors are very similar in their responses to nicotine, with high sensitivity receptors showing protracted responses. Sazetidine A, an agonist that is selective for the high sensitivity α4 receptors also selectively activates high sensitivity α2 receptors. Likewise, α2 receptors have similar responses as α4 receptors to the positive allosteric modulators (PAMs) desformylflustrabromine (dFBr) and NS9283. We show that the partial agonists for α4ß2 receptors, cytisine and varenicline are also partial agonists for α2ß2 receptors. Studies have shown that levels of α2 expression may be much higher in the brains of primates than those of rodents, suggesting a potential importance for human therapeutics. High-affinity nAChR have been studied in humans with PET ligands such as flubatine. We show that flubatine has similar activity with α2ß2 and α4ß2 receptors so that α2 receptors will also be detected in PET studies that have previously presumed to selectively detect α4ß2 receptors. Therefore, α2 receptors need more consideration in the development of therapeutics to manage nicotine addiction and declining cholinergic function in age and disease.

The many enigmas of nicotine.

This review discusses the diverse effects of nicotine on the various nicotinic acetylcholine receptors of the central and peripheral nervous system and how those effects may promote the usage and addiction to tobacco products.

Investigating the role of nicotinic acetylcholine receptors in menthol's effects in mice.

The use of menthol in tobacco products has been linked to an increased likelihood of developing nicotine dependence. The widespread use of menthol can be attributed to its unique sensory characteristics; however, emerging evidence suggests that menthol also alters sensitivity to nicotine through modulation of nicotinic acetylcholine receptors (nAChRs). Nicotinic subunits, such as ß2 and α5, are of interest due to their implications in nicotine reward, reinforcement, intake regulation, and aversion. This study, therefore, examined the in vivo relevance of ß2 and α5 nicotinic subunits on the pharmacological and behavioral effects of menthol. Data suggests that the α5 nicotinic subunit modulates menthol intake in mice. Overall, deletion or a reduction in function of the α5 subunit lessened aversion to menthol. α5 KO mice and mice possessing the humanized α5 SNP, a variant that confers a nicotine dependence phenotype in humans, demonstrated increased menthol intake compared to their WT counterparts and in a sex-related fashion for α5 SNP mice. We further reported that the modulatory effects of the α5 subunit do not extend to other aversive tastants like quinine, suggesting that deficits in α5* nAChR signaling may not abolish general sensitivity to the aversive effects of other noxious chemicals. Further probing into the role of α5 in other pharmacological properties of menthol revealed that the α5 subunit does not modulate the antinociceptive properties of menthol in mice and suggests that the in vivo differences observed are likely not due to the direct effects of menthol on α5-containing nAChRs in vitro.

New Alpha9 nAChR Ligands Based on a 5-(Quinuclidin-3-ylmethyl)-1,2,4-oxadiazole Scaffold.

Several lines of evidence have indicated that nicotinic acetylcholine receptors (nAChR) that contain α9 subunits, probably in combination with α10 subunits, may be valuable targets for the management of pain associated with inflammatory diseases through a cholinergic anti-inflammatory system (CAS), which has also been associated with α7 nAChR. Both α7- and α9-containing neuronal nAChR can be pharmacologically distinguished from the high-affinity nicotinic receptors of the brain by their sensitivity to α-bungarotoxin, but in other ways, they have quite distinct pharmacological profiles. The early association of α7 with CAS led to the development of numerous new ligands, variously characterized as α7 agonists, partial agonists, or silent agonists that desensitized α7 receptors without activation. Subsequent reinvestigation of one such family of α7 ligands based on an N,N-diethyl-N'-phenylpiperazine scaffold led to the identification of potent agonists and antagonists for α9. In this paper, we characterize the α9/α10 activity of a series of compounds based on a 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole (QMO) scaffold and identify two new potent ligands of α9, QMO-28, an agonist, and QMO-17, an antagonist. We separated the stereoisomers of these compounds to identify the most potent agonist and discovered that only the 3R isomer of QMO-17 was an α9 antagonist, permitting an in silico model of α9 antagonism to be developed. The α9 activity of these compounds was confirmed to be potentially useful for CAS management of inflammatory pain in cell-based assays of cytokine release.

Pharmacological profiles and anti-inflammatory activity of pCN-diEPP and mCN-diEPP, new alpha9alpha10 nicotinic receptor ligands.

Pain due to inflammation can be reduced by targeting the noncanonical nicotinic receptors (NCNR) in cells of the immune system that regulate the synthesis and release of pro- and anti-inflammatory cytokines. Although NCNR do not generate ion channel currents, the pharmacology of ion-channel forms of the receptors can predict drugs which may be effective regulators of the cholinergic anti-inflammatory system (CAS). Agonists of α7 type receptors have been definitively associated with CAS. Receptors containing α9 and α10 subunits have also been implicated. We have recently characterized two small molecules, pCN-diEPP and mCN-diEPP, as selective α9α10 agonists and antagonists, respectively. We used these drugs, along with nicotine, an α7 agonist and α9α10 antagonist, to probe the mixed populations of receptors that are formed when α7, α9, and α10 are all expressed together in Xenopus oocytes. We also evaluated the effects of the CN-diEPP compounds on regulating the ATP-induced release of interleukin-1ß from monocytic THP-1 cells, which express NCNR. The compounds successfully identified separate populations of receptors when all three subunits were co-expressed, including a potential population of homomeric α10 receptors. The α9α10 agonist pCN-diEPP was the more effective regulator of interleukin-1ß release in THP-1 cells. pCN-diEPP was also fully effective in a mouse model of inflammatory pain, while mCN-diEPP had only partial effects, requiring a higher dosage. The analgetic effects of pCN-diEPP and mCN-diEPP were retained in α7 knockout mice. Taken together, our results suggest that drugs that selectively activate α9α10 receptors may useful to reduce inflammatory pain through the CAS.

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.

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.

Pharmacological characterization of 5-iodo-A-85380, a ß2-selective nicotinic receptor agonist, in mice.

BACKGROUND: Because of their implications in several pathological conditions, α4ß2* nicotinic acetylcholine receptors (nAChRs) are potential targets for the treatment of nicotine dependence, pain, and many psychiatric and neurodegenerative diseases. However, they exist in various subtypes, and finding selective tools to investigate them has proved challenging. The nicotinic receptor agonist, 5-iodo-A-85380 (5IA), has helped in delineating the function of ß2-containing subtypes in vitro; however, much is still unknown about its behavioral effects. Furthermore, its effectiveness on α6-containing subtypes is limited. AIMS: To investigate the effects of 5IA on nociception (formalin, hot-plate, and tail-flick tests), locomotion, hypothermia, and conditioned reward after acute and repeated administration, and to examine the potential role of ß2 and α6 nAChR subunits in these effects. Lastly, its selectivity for expressed low sensitivity (LS) and high sensitivity (HS) α4ß2 receptors is investigated. RESULTS: 5IA dose-dependently induced hypothermia, locomotion suppression, conditioned place preference, and antinociception (only in the formalin test but not in the hot-plate or tail-flick tests). Furthermore, these effects were mediated by ß2 but not α6 nicotinic subunits. Finally, we show that 5-iodo-A-85380 potently activates both stoichiometries of α4ß2 nAChRs with differential efficacies, being a full agonist on HS α4(2)ß2(3) nAChRs, and a partial agonist on LS α4(3)ß2(2) nAChRs and α6-containing subtypes as well.

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.

Coffee and cigarettes: Modulation of high and low sensitivity α4ß2 nicotinic acetylcholine receptors by n-MP, a biomarker of coffee consumption.

Smokers report particular appreciation for coffee with their first cigarettes of the day. We investigated with voltage-clamp experiments, effects of aqueous extracts (coffees) of unroasted and roasted coffee beans on the activity of human brain nicotinic acetylcholine receptor (nAChR) subtypes expressed in Xenopus oocytes, looking at complex brews, low molecular weight (LMW) fractions, and specific compounds present in coffee. When co-applied with PNU-120596, a positive allosteric modulator (PAM), the coffees stimulated currents from cells expressing α7 nAChR that were larger than ACh controls. The PAM-dependent responses to green bean coffee were three-fold greater than those to dark roasted coffee, consistent with α7 receptor activation by choline, a component of coffee that is partially degraded in the roasting process. Coffees were tested on both high sensitivity (HS) and low sensitivity (LS) forms of α4ß2 nAChR, which are associated with nicotine addiction. To varying degrees, these receptors were both activated and inhibited by the coffees and LMW extracts. We also examined the activity of nine small molecules present in coffee. Only two compounds, 1-methylpyridinium and 1-1-dimethylpiperidium, produced during the process of roasting coffee beans, showed significant effects on nAChR. The compounds were competitive antagonists of the HS α4ß2 receptors, but were PAMs for LS α4ß2 receptors. HS receptors in smokers are likely to progressively desensitize through a day of smoking but may be hypersensitive in the mornings when brain nicotine levels are low. A smoker's first cup of coffee may therefore balance the effects of the day's first cigarette in the brain.

Comparison of the Anti-inflammatory Properties of Two Nicotinic Acetylcholine Receptor Ligands, Phosphocholine and pCF3-diEPP.

Activation of nicotinic acetylcholine receptors (nAChRs) expressed by innate immune cells can attenuate pro-inflammatory responses. Silent nAChR agonists, which down-modulate inflammation but have little or no ionotropic activity, are of outstanding clinical interest for the prevention and therapy of numerous inflammatory diseases. Here, we compare two silent nAChR agonists, phosphocholine, which is known to interact with nAChR subunits α7, α9, and α10, and pCF3-N,N-diethyl-N'-phenyl-piperazine (pCF3-diEPP), a previously identified α7 nAChR silent agonist, regarding their anti-inflammatory properties and their effects on ionotropic nAChR functions. The lipopolysaccharide (LPS)-induced release of interleukin (IL)-6 by primary murine macrophages was inhibited by pCF3-diEPP, while phosphocholine was ineffective presumably because of instability. In human whole blood cultures pCF3-diEPP inhibited the LPS-induced secretion of IL-6, TNF-α and IL-1ß. The ATP-mediated release of IL-1ß by LPS-primed human peripheral blood mononuclear leukocytes, monocytic THP-1 cells and THP-1-derived M1-like macrophages was reduced by both phosphocholine and femtomolar concentrations of pCF3-diEPP. These effects were sensitive to mecamylamine and to conopeptides RgIA4 and [V11L; V16D]ArIB, suggesting the involvement of nAChR subunits α7, α9 and/or α10. In two-electrode voltage-clamp measurements pCF3-diEPP functioned as a partial agonist and a strong desensitizer of classical human α9 and α9α10 nAChRs. Interestingly, pCF3-diEPP was more effective as an ionotropic agonist at these nAChRs than at α7 nAChR. In conclusion, phosphocholine and pCF3-diEPP are potent agonists at unconventional nAChRs expressed by monocytic and macrophage-like cells. pCF3-diEPP inhibits the LPS-induced release of pro-inflammatory cytokines, while phosphocholine is ineffective. However, both agonists signal via nAChR subunits α7, α9 and/or α10 to efficiently down-modulate the ATP-induced release of IL-1ß. Compared to phosphocholine, pCF3-diEPP is expected to have better pharmacological properties. Thus, low concentrations of pCF3-diEPP may be a therapeutic option for the treatment of inflammatory diseases including trauma-induced sterile inflammation.

Selective Agonists and Antagonists of α9 Versus α7 Nicotinic Acetylcholine Receptors.

Nicotinic acetylcholine receptors containing α9 subunits are essential for the auditory function and have been implicated, along with α7-containing nicotinic receptors, as potential targets for the treatment of inflammatory and neuropathic pain. The study of α9-containing receptors has been hampered by the lack of selective agonists. The only α9-selective antagonists previously identified are peptide conotoxins. Curiously, the activity of α7 and α9 receptors as modulators of inflammatory pain appears to not rely strictly on ion channel activation, which led to the identification of α7 "silent agonists" and phosphocholine as an "unconventional agonist" for α9 containing receptors. The parallel testing of the α7 silent agonist p-CF3-diEPP and phosphocholine led to the discovery that p-CF3-diEPP was an α9 agonist. In this report, we compared the activity of α7 and α9 with a family of structurally related compounds, most of which were previously shown to be α7 partial or silent agonists. We identify several potent α9-selective agonists as well as numerous potent and selective α9 antagonists and describe the structural basis for these activities. Several of these compounds have previously been shown to be effective in animal models of inflammatory pain, an activity that was assumed to be due to α7 silent agonism but may, in fact, be due to α9 activity. The α9-selective conotoxin antagonists have also been shown to reduce pain in similar models. Our identification of these new α9 agonists and antagonists may prove to be invaluable for defining an optimal approach for treating pain, allowing for reduced use of opioid drugs.

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.

The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site.

α-conotoxins are 13-19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. α-conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets α7 nAChR. Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site.

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.

Stable desensitization of α7 nicotinic acetylcholine receptors by NS6740 requires interaction with S36 in the orthosteric agonist binding site.

NS6740 is an α7 nicotinic acetylcholine receptor-selective partial agonist with low efficacy for channel activation, capable of promoting the stable conversion of the receptors to nonconducting (desensitized) states that can be reactivated with the application of positive allosteric modulators (PAMs). In spite of its low efficacy for channel activation, NS6740 is an effective activator of the cholinergic anti-inflammatory pathway. We observed that the concentration-response relationships for channel activation, both when applied alone and when co-applied with the PAM PNU-120596 are inverted-U shaped with inhibitory/desensitizing activities dominant at high concentrations. We evaluated the potential importance of recently identified binding sites for allosteric activators and tested the hypotheses that the stable desensitization produced by NS6740 may be due to binding to these sites. Our experiments were guided by molecular modeling of NS6740 binding to both the allosteric and orthosteric activation sites on the receptor. Our results indicate that with α7C190A mutants, which have compromised orthosteric activation sites, NS6740 may work at the allosteric activation sites to promote transient PAM-dependent currents but not the stable desensitization seen with wild-type α7 receptors. Modeling NS6740 in the orthosteric binding sites identified S36 as an important residue for NS6740 binding and predicted that an S36V mutation would limit NS6740 activity. The efficacy of NS6740 for α7S36V receptors was reduced to zero, and applications of the compound to α7S36V receptors failed to induce the desensitization observed with wild-type receptors. The results indicate that the unique properties of NS6740 are due primarily to binding at the sites for orthosteric agonists.

Effects of α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulator on BDNF, NKCC1 and KCC2 Expression in the Hippocampus following Lipopolysaccharide-Induced Allodynia and Hyperalgesia in a Mouse Model of Inflammatory Pain.

BACKGROUND & OBJECTIVES: Hyperalgesia and allodynia are frequent symptoms of inflammatory pain. Neuronal excitability induced by the Brain-Derived Neurotrophic Factor (BDNF)-tyrosine receptor kinase B (TrkB) cascade has a role in the modulation of inflammatory pain. The effects of 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an α7 nicotinic Acetylcholine Receptor Positive Allosteric Modulator (nAChR PAM), on hippocampal BDNF, cation-chloride cotransporters, NKCC1 and KCC2, expression in inflammatory pain are not known. The objective of the study was to determine the effects of TQS on BDNF, NKCC1, and KCC2 expression in the hippocampus following lipopolysaccharide (LPS)-induced allodynia and hyperalgesia in a mouse model of inflammatory pain. METHODS: Mice were treated with TQS followed by LPS (1 mg/kg, ip) administration. The effects of TQS on mRNA and BDNF in the hippocampus were examined using qRT-PCR and Western blot, respectively. Immunoreactivity of BDNF, NKCC1, and KCC2 in the hippocampus was measured after LPS administration using immunofluorescence assay. Allodynia and hyperalgesia were determined using von Frey filaments and hot plate, respectively. RESULTS: The LPS (1 mg/kg) upregulates mRNA of BDNF and downregulates mRNA of KCC2 in the hippocampus and pretreatment of TQS (4 mg/kg) reversed the effects induced by LPS. In addition, the TQS decreased LPS-induced upregulation of BDNF and p-NKCC1 immunoreactivity in the dentate gyrus and CA1 region of the hippocampus. BDNF receptor (TrkB) antagonist, ANA12 (0.50 mg/kg), and NKCC1 inhibitor bumetanide (30 mg/kg) reduced LPS-induced allodynia and hyperalgesia. Blockade of TrkB with ANA12 (0.25 mg/kg) enhanced the effects of TQS (1 mg/kg) against LPS-induced allodynia and hyperalgesia. Similarly, bumetanide (10 mg/kg) enhanced the effects of TQS (1 mg/kg) against allodynia and hyperalgesia. CONCLUSION: These results suggest that antinociceptive effects of α7 nAChR PAM are associated with downregulation of hippocampal BDNF and p-NKCC1 and upregulation of KCC2 in a mouse model of inflammatory pain.