A silent agonist of ?7 nicotinic acetylcholine receptors modulates inflammation ex vivo and attenuates EAE.
ABSTRACT: 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 ?7 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 ?7 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 ?7 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. ?7 nAChR-selective silent agonists may thus be a novel source of anti-inflammatory compounds that could be used for the treatment of inflammatory disorders.
Project description:Nicotinic acetylcholine receptor (nAChR) ligands that lack agonist activity but enhance activation in the presence of an agonist are called positive allosteric modulators (PAMs). nAChR PAMs have therapeutic potential for the treatment of nicotine addiction and several neuropsychiatric disorders. PAMs need to be selectively targeted toward certain nAChR subtypes to tap this potential. We previously discovered a novel PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which selectively potentiates the opening of ?4?2*, ?2?2*, ?2?4*, and (?4?4)2?4 nAChRs and reactivates some of these subtypes when desensitized (* indicates the presence of other subunits). We located the Br-PBTC-binding site through mutagenesis and docking in ?4. The amino acids Glu-282 and Phe-286 near the extracellular domain on the third transmembrane helix were found to be crucial for Br-PBTC's PAM effect. E282Q abolishes Br-PBTC potentiation. Using (?4E282Q?2)2?5 nAChRs, we discovered that the trifluoromethylated derivatives of Br-PBTC can potentiate channel opening of ?5-containing nAChRs. Mutating Tyr-430 in the ?5 M4 domain changed ?5-selectivity among Br-PBTC derivatives. There are two kinds of ?4 subunits in ?4?2 nAChRs. Primary ?4 forms an agonist-binding site with another ?2 subunit. Accessory ?4 forms an agonist-binding site with another ?4 subunit. The pharmacological effect of Br-PBTC depends both on its own and agonists' occupancy of primary and accessory ?4 subunits. Br-PBTC reactivates desensitized (?4?2)2?4 nAChRs. Its full efficacy requires intact Br-PBTC sites in at least one accessory and one primary ?4 subunit. PAM potency increases with higher occupancy of the agonist sites. Br-PBTC and its derivatives should prove useful as ? subunit-selective nAChR PAMs.
Project description:The ?7 nicotinic acetylcholine receptor (nAChR) silent agonists, able to induce receptor desensitization and promote the ?7 metabotropic function, are emerging as new promising therapeutic anti-inflammatory agents. Herein, we report the structure-activity relationship investigation of the archetypal silent agonist NS6740 (1,4-diazabicyclo[3.2.2]nonan-4-yl(5-(3-(trifluoromethyl)-phenyl)-furan-2-yl)methanone) (1) to elucidate the ligand-receptor interactions responsible for the ?7 silent activation. In this study, NS6740 fragments 11-16 and analogs 17-32 were designed, synthesized, and assayed on human ?7 nAChRs expressed in Xenopus laevis oocytes with two-electrode voltage clamping experiments. All together the structural portions of NS6740 were critical to engender its peculiar activity profile. The diazabicyclic nucleus was essential but not sufficient for inducing ?7 silent activation. The central hydrogen-bond acceptor core and the aromatic moiety were crucial for promoting prolonged ?7 receptor binding and sustained desensitization. Compounds 13 and 17 were efficacious partial agonists. Compounds 12, 21, 23-26, and 30 strongly desensitized ?7 nAChR and therefore may be of interest for additional investigation of inflammation responses. We gained key structural information useful for further silent agonist development.
Project description:Recently, ?7 nicotinic acetylcholine receptors (nAChRs), primarily activated by binding of orthosteric agonists, represent a target for anti-inflammatory and analgesic drug development. These receptors may also be modulated by positive allosteric modulators (PAMs), ago-allosteric ligands (ago-PAMs), and ?7-silent agonists. Activation of ?7 nAChRs has been reported to increase the brain levels of endogenous ligands for nuclear peroxisome proliferator-activated receptors type-? (PPAR-?), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in a Ca2+-dependent manner. Here, we investigated potential crosstalk between ?7 nAChR and PPAR-?, using the formalin test, a mouse model of tonic pain. Using pharmacological and genetic approaches, we found that PNU282987, a full ?7 agonist, attenuated formalin-induced nociceptive behavior in ?7-dependent manner. Interestingly, the selective PPAR-? antagonist GW6471 blocked the antinociceptive effects of PNU282987, but did not alter the antinociceptive responses evoked by the ?7 nAChR PAM PNU120596, ago-PAM GAT107, and silent agonist NS6740. Moreover, GW6471 administered systemically or spinally, but not via the intraplantar surface of the formalin-injected paw blocked PNU282987-induced antinociception. Conversely, exogenous administration of the naturally occurring PPAR-? agonist PEA potentiated the antinociceptive effects of PNU282987. In contrast, the cannabinoid CB1 antagonist rimonabant and the CB2 antagonist SR144528 failed to reverse the antinociceptive effects of PNU282987. These findings suggest that PPAR-? plays a key role in a putative antinociceptive ?7 nicotinic signaling pathway.
Project description:The existence of a cholinergic anti-inflammatory pathway negatively modulating the inflammatory and immune responses in various clinical conditions and experimental models has long been postulated. In particular, the protective involvement of the vagus nerve and of nicotinic Ach receptors (nAChRs) has been proposed in intestinal inflammation and repeatedly investigated in DSS- and TNBS-induced colitis. However, the role of ?7 nAChRs stimulation is still controversial and the potential contribution of ?4?2 nAChRs has never been explored in this experimental condition. Our aims were therefore to pharmacologically investigate the role played by both ?7 and ?4?2 nAChRs in the modulation of the local and systemic inflammatory responses activated in TNBS-induced colitis in mice and to assess the involvement of the spleen in nicotinic responses. To this end, TNBS-exposed mice were sub-acutely treated with various subcutaneous doses of highly selective agonists (AR-R17779 and TC-2403) and antagonists (methyllycaconitine and dihydro-?-erythroidine) of ?7 and ?4?2 nAChRs, respectively, or with sulfasalazine 50 mg/kg per os and clinical and inflammatory responses were evaluated by means of biochemical, histological and flow cytometry assays. ?4?2 ligands evoked weak and contradictory effects, while ?7 nAChR agonist AR-R17779 emerged as the most beneficial treatment, able to attenuate several local markers of colitis severity and to revert the rise in splenic T-cells and in colonic inflammatory cytokines levels induced by haptenization. After splenectomy, AR-R17779 lost its protective effects, demonstrating for the first time that, in TNBS-model of experimental colitis, the anti-inflammatory effect of exogenous ?7 nAChR stimulation is strictly spleen-dependent. Our findings showed that the selective ?7 nAChRs agonist AR-R17779 exerted beneficial effects in a model of intestinal inflammation characterized by activation of the adaptive immune system and that the spleen is essential to mediate this cholinergic protection.
Project description:We introduce the term 'silent agonists' to describe ligands that can place the ?7 nicotinic acetylcholine receptor (nAChR) into a desensitized state with little or no apparent activation of the ion channel, forming a complex that can subsequently generate currents when treated with an allosteric modulator. KC-1 (5'-phenylanabaseine) was synthesized and identified as a new silent agonist for the ?7 nAChR; it binds to the receptor but does not activate ?7 nAChR channel opening when applied alone, and its agonism is revealed by co-application with the type II positive allosteric modulator PNU-120596 in the Xenopus oocyte system. The concise synthesis was accomplished in three steps with the C-C bonds formed via Pd-catalyzed mono-arylation and organolithium coupling with N-Boc piperidinone. Comparative structural analyses indicate that a positive charge, an H-bond acceptor, and an aryl ring in a proper arrangement are needed to constitute one class of silent agonist for the ?7 nAChR. Because silent agonists may act on signaling pathways not involving ion channel opening, this class of ?7 nAChR ligands may constitute a new alternative for the development of ?7 nAChR therapeutics.
Project description:?7 Nicotinic acetylcholine receptors (nAChRs) reportedly reduce inflammation by blocking effects of the important pro-inflammatory transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NF?B). The ?7 nAChR partial agonist GTS-21 reduces secretion of pro-inflammatory cytokines including interleukin-6 (IL6) and tumor-necrosis factor (TNF) in models of endotoxemia and sepsis, and its anti-inflammatory effects are widely ascribed to ?7 nAChR activation. However, mechanistic details of ?7 nAChR involvement in GTS-21 effects on inflammatory pathways remain unclear. Here, we investigate how GTS-21 acts in two cell systems including the non-immune rat pituitary cell line GH4C1 expressing an NF?B-driven reporter gene and cytokine secretion by ex vivo cultures of primary mouse macrophages activated by lipopolysaccharide (LPS). GTS-21 does not change TNF-stimulated NF?B signaling in GH4C1 cells expressing rat ?7 nAChRs, suggesting that GTS-21 requires additional unidentified factors besides ?7 nAChR expression to allow anti-inflammatory effects in these cells. In contrast, GTS-21 dose-dependently suppresses LPS-induced IL6 and TNF secretion in primary mouse macrophages endogenously expressing ?7 nAChRs. GTS-21 also blocks TNF-induced phosphorylation of NF?B inhibitor alpha (I?B?), an important intermediary in NF?B signaling. However, ?7 antagonists methyllycaconitine and ?-bungarotoxin only partially reverse GTS-21 blockade of IL6 and TNF secretion. Further, GTS-21 significantly inhibited LPS-induced IL6 and TNF secretion in macrophages isolated from knockout mice lacking ?7 nAChRs. These data indicate that even though a discrete component of the anti-inflammatory effects of GTS-21 requires expression of ?7 nAChRs in macrophages, GTS-21 also has anti-inflammatory effects independent of these receptors depending on the cellular context.
Project description:Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of ?2* and ?4* nAChRs but is without effect on ?3* or ?6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of ?4 subunits, which is also a PAM site for steroid hormone estrogens such as 17?-estradiol. Br-PBTC is much more potent than estrogens. Like 17?-estradiol, the non-steroid Br-PBTC only requires one ?4 subunit to potentiate nAChR function, and its potentiation is stronger with more ?4 subunits. This feature enables Br-BPTC to potentiate activation of (?4?2)(?6?2)?3 but not (?6?2)2?3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different ?6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two ?4 subunits but not those with only one. Three ?4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.
Project description:Partial agonists of the α4β2 nicotinic acetylcholine receptor (nAChR), such as varenicline, are therapeutically used in smoking cessation treatment. These drugs derive their therapeutic effect from fundamental molecular actions, which are to desensitize α4β2 nAChRs and induce channel opening with higher affinity, but lower efficacy than a full agonist at equal receptor occupancy. Here, we report X-ray crystal structures of a unique acetylcholine binding protein (AChBP) from the annelid Capitella teleta, Ct-AChBP, in complex with varenicline or lobeline, which are both partial agonists. These structures highlight the architecture for molecular recognition of these ligands, indicating the contact residues that potentially mediate their molecular actions in α4β2 nAChRs. We then used structure-guided mutagenesis and electrophysiological recordings to pinpoint crucial interactions of varenicline with residues on the complementary face of the binding site in α4β2 nAChRs. We observe that residues in loops D and E are molecular determinants of desensitization and channel opening with limited efficacy by the partial agonist varenicline. Together, this study analyzes molecular recognition of smoking cessation drugs by nAChRs in a structural context.
Project description:Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([(3)H]phencyclidine; IC50 = 4 μM) than in the resting state ([(3)H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([(3)H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [(3)H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [(3)H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [(3)H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity.
Project description:<h4>Background and purpose</h4>Ca(2+) signalling and exocytosis mediated by nicotinic receptor (nAChR) subtypes, especially the ?7 nAChR, in bovine chromaffin cells are still matters of debate.<h4>Experimental approach</h4>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 ?7 nAChR allosteric modulator PNU120596.<h4>Key results</h4>Minimal [Ca(2+) ](c) transients, induced by low concentrations of selective ?7 nAChR agonists and nicotine, were markedly increased by the ?7 nAChR allosteric modulator PNU120596. These potentiated responses were completely blocked by the ?7 nAChR antagonist ?-bungarotoxin (?7-modulated-response). Conversely, high concentrations of the ?7 nAChR agonists, nicotine or 5-iodo-A-85380 induced larger [Ca(2+) ](c) transients, that were blocked by mecamylamine but were unaffected by ?-bungarotoxin (non-?7 response). [Ca(2+) ](c) increases mediated by ?7 nAChR were related to Ca(2+) entry through non-L-type Ca(2+) channels, whereas non-?7 nAChR-mediated signals were related to L-type Ca(2+) channels; Ca(2+) -induced Ca(2+) -release contributed to both responses. Mitochondrial involvement in the control of [Ca(2+) ](c) transients, mediated by either receptor, was minimal. Catecholamine release coupled to ?7 nAChRs was more efficient in terms of catecholamine released/[Ca(2+) ](c) .<h4>Conclusions and implications</h4>[Ca(2+) ](c) and catecholamine release mediated by ?7 nAChRs required an allosteric modulator and low doses of the agonist. At higher agonist concentrations, the ?7 nAChR response was lost and the non-?7 nAChRs were activated. Catecholamine release might therefore be regulated by different nAChR subtypes, depending on agonist concentrations and the presence of allosteric modulators of ?7 nAChRs.