RESUMEN
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.
Asunto(s)
Analgésicos , Inflamación , Piperazinas , Receptores Nicotínicos , Animales , Humanos , Masculino , Ratones , Analgésicos/farmacología , Analgésicos/química , Analgésicos/síntesis química , Analgésicos/uso terapéutico , Inflamación/tratamiento farmacológico , Ratones Noqueados , Agonistas Nicotínicos/farmacología , Agonistas Nicotínicos/química , Agonistas Nicotínicos/uso terapéutico , Agonistas Nicotínicos/síntesis química , Dolor/tratamiento farmacológico , Piperazinas/farmacología , Piperazinas/química , Piperazinas/síntesis química , Piperazinas/uso terapéutico , Receptores Nicotínicos/metabolismo , Sales (Química)/química , Sales (Química)/farmacología , Relación Estructura-Actividad , Yoduros/químicaRESUMEN
The dopamine D1 receptor (D1R), is a class A G protein coupled-receptor (GPCR) which has been a promising drug target for psychiatric and neurological disorders such as Parkinson's disease (PD). Previous studies have suggested that therapeutic effects can be realized by targeting the ß-arrestin signaling pathway of dopamine receptors, while overactivation of the G protein-dependent pathways leads to side effects, such as dyskinesias. Therefore, it is highly desirable to develop a D1R ligand that selectively regulates the ß-arrestin pathway. Currently, most D1R agonists are signaling-balanced and stimulate both G protein and ß-arrestin pathways, with a few reports of G protein biased ligands. However, identification and characterization of ß-arrestin biased D1R agonists has been a challenge thus far. In this study, we implemented Gaussian accelerated molecular dynamics (GaMD) simulations to provide valuable computational insights into the possible underlying molecular mechanism of the different signaling properties of two catechol and two non-catechol D1R agonists that are either G protein biased or signaling-balanced. Dynamic network analysis further identified critical residues in the allosteric signaling network of D1R for each ligand at different conformational or binding states. Some of these residues are crucial for G protein or arrestin signals of GPCRs based on previous studies. Finally, we provided a molecular design strategy which can be utilized by medicinal chemists to develop potential ß-arrestin biased D1R ligands. The proposed hypotheses are experimentally testable and can guide the development of safer and more effective medications for a variety of CNS disorders.
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Proteínas de Unión al GTP , Transducción de Señal , beta-Arrestinas/metabolismo , Ligandos , Proteínas de Unión al GTP/metabolismo , Agonistas de Dopamina/química , Agonistas de Dopamina/farmacología , Receptores de Dopamina D1/metabolismoRESUMEN
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.
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Conducta Adictiva , Receptores Nicotínicos , Areca , Arecolina/farmacología , Ácido gamma-AminobutíricoRESUMEN
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.
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Receptores Nicotínicos , Productos de Tabaco , Animales , Biomarcadores , Humanos , Nicotina/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through D1- (D1R and D5R) and D2-class (D2R, D3R, and D4R) receptors and activates both G protein- and ß-arrestin-dependent signaling pathways. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinson's disease or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling toward the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity relationship (SAR) studies have embarked on discovering ß-arrestin-biased dopamine agonists, focused on D2 partial agonists, noncatechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R ß-arrestin-biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold to identify chemical motifs responsible for ß-arrestin-biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein-biased, but none of them were exclusively arrestin-biased. Additionally, various small-fragment molecular probes displayed weak bias toward the ß-arrestin pathway. Continued in-depth SFSR (structure-functional selectivity relationship) studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate the discovery of potent and efficacious arrestin-biased dopamine receptor ligands.
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Agonistas de Dopamina , Dopamina , Animales , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Proteínas de Unión al GTP/metabolismo , Ligandos , Quinolonas , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/metabolismo , Tiofenos , beta-Arrestinas/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Conotoxinas , Neuralgia , Receptores Nicotínicos , Animales , Conotoxinas/farmacología , Neuralgia/tratamiento farmacológico , Antagonistas Nicotínicos/farmacología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
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.
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Receptor Nicotínico de Acetilcolina alfa 7 , Animales , Sitios de Unión , Humanos , Ligandos , Simulación del Acoplamiento MolecularRESUMEN
Azasugars are monosaccharide analogs in which the ring oxygen is replaced with a nitrogen atom. These well-known glycosidase inhibitors are of interest as therapeutics, yet several aspects of azasugars remain unknown including their distribution, structural diversity, and chemical ecology. The hallmark signature of bacterial azasugar biosynthesis is a three gene cluster (3GC) coding for aminotransferase, phosphatase, and dehydrogenase enzymes. Using the bioinformatics platform Enzyme Similarity Tool (EST), we identified hundreds of putative three gene clusters coding for azasugar production in microbial species. In the course of this work, we also report a consensus sequence for the aminotransferase involved in azasugar biosynthesis as being: SGNXFRXXXFPNXXXXXXXLXVPXPYCXRC. Most clusters are found in Bacillus and Streptomyces species which typically inhabit soil and the rhizosphere, but some clusters are found with diverse species representation such as Photorhabdus and Xenorhabdus which are symbiotic with entomopathogenic nematodes; the human skin commensal Cutibacterium acnes, and the marine Bacillus rugosus SPB7, a symbiont to the sea sponge Spongia officinalis. This pan-taxonomic survey of the azasugar 3GC signature may lead to the identification of new azasugar producers, facilitate studies of their natural functions, and lead to new potential therapeutics.
RESUMEN
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.
Asunto(s)
Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Regulación Alostérica , Sitio Alostérico , Animales , Sitios de Unión , Humanos , Ligandos , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
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.
Asunto(s)
Compuestos de Azabiciclo/farmacología , Furanos/farmacología , Agonistas Nicotínicos/farmacología , Serina/química , Serina/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Acetilcolina/agonistas , Acetilcolina/antagonistas & inhibidores , Regulación Alostérica , Animales , Compuestos de Azabiciclo/agonistas , Sitios de Unión , Agonismo Parcial de Drogas , Furanos/agonistas , Isoxazoles/farmacología , Simulación del Acoplamiento Molecular , Compuestos de Fenilurea/farmacología , Dominios Proteicos , Xenopus laevis/genética , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
The α7 nicotinic acetylcholine receptor is a homopentameric ion channel from the Cys-loop receptor superfamily targeted for psychiatric indications and inflammatory pain. Molecular dynamics studies of the receptor have focused on residue mobility and global conformational changes to address receptor function. However, a comparative analysis of α7 with its homologs that cannot trigger channel opening has not been made so far. To identify the residues involved in α7 activation, we ran triplicate 500-ns molecular dynamics simulations with an α7 extracellular domain homology model and two acetylcholine-binding protein homologs. We tested the effect of ligand binding and amino acid sequence on the structure and dynamics of the three proteins. We found that mobile regions identified based on root mean-square deviation and root mean-square fluctuation values are not always consistent among the individual α7 extracellular domain simulations. Comparison of the replica-average properties of the three proteins based on dynamic cross-correlation maps showed that ligand binding affects the coupling between the C-loop and the Cys-loop, vestibular loop, and ß1-ß2 loops. In addition, the main-immunogenic-region-like domain of α7 went through correlated motions with multiple domains of the receptor. These correlated motions were absent or diminished in α7 homologs, suggesting a unique role in α7 activation.
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Simulación de Dinámica Molecular , Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Secuencia de Aminoácidos , Sitios de Unión , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
The α7 nicotinic acetylcholine receptor (nAChR) silent agonists, able to induce receptor desensitization and promote the α7 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 α7 silent activation. In this study, NS6740 fragments 11-16 and analogs 17-32 were designed, synthesized, and assayed on human α7 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 α7 silent activation. The central hydrogen-bond acceptor core and the aromatic moiety were crucial for promoting prolonged α7 receptor binding and sustained desensitization. Compounds 13 and 17 were efficacious partial agonists. Compounds 12, 21, 23-26, and 30 strongly desensitized α7 nAChR and therefore may be of interest for additional investigation of inflammation responses. We gained key structural information useful for further silent agonist development.
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Compuestos de Azabiciclo/farmacología , Diseño de Fármacos , Fenómenos Electrofisiológicos/efectos de los fármacos , Furanos/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Animales , Compuestos de Azabiciclo/química , Técnicas de Química Sintética , Furanos/química , Humanos , Enlace de Hidrógeno , Ligandos , Relación Estructura-Actividad , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
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.
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Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Xenopus laevis/genética , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Sitios de Unión , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Mutación , Estereoisomerismo , Sulfonamidas/química , Xenopus laevis/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Manganese exposure produces Parkinson's-like neurologic symptoms, suggesting a selective dysregulation of dopamine transmission. It is unknown, however, how manganese accumulates in dopaminergic brain regions or how it regulates the activity of dopamine neurons. Our in vivo studies in male C57BLJ mice suggest that manganese accumulates in dopamine neurons of the VTA and substantia nigra via nifedipine-sensitive Ca2+ channels. Manganese produces a Ca2+ channel-mediated current, which increases neurotransmitter release and rhythmic firing activity of dopamine neurons. These increases are prevented by blockade of Ca2+ channels and depend on downstream recruitment of Ca2+-activated potassium channels to the plasma membrane. These findings demonstrate the mechanism of manganese-induced dysfunction of dopamine neurons, and reveal a potential therapeutic target to attenuate manganese-induced impairment of dopamine transmission.SIGNIFICANCE STATEMENT Manganese is a trace element critical to many physiological processes. Overexposure to manganese is an environmental risk factor for neurologic disorders, such as a Parkinson's disease-like syndrome known as manganism. We found that manganese concentration-dependently increased the excitability of dopamine neurons, decreased the amplitude of action potentials, and narrowed action potential width. Blockade of Ca2+ channels prevented these effects as well as manganese accumulation in the mouse midbrain in vivo Our data provide a potential mechanism for manganese regulation of dopaminergic neurons.
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Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Manganeso/metabolismo , Manganeso/toxicidad , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de ÓrganosRESUMEN
The α7 nicotinic acetylcholine receptor is a homopentameric ion-channel of the Cys-loop superfamily characterized by its low probability of opening, high calcium permeability, and rapid desensitization. The α7 receptor has been targeted for the treatment of the cognitive symptoms of schizophrenia, depression, and Alzheimer's disease, but it is also involved in inflammatory modulation as a part of the cholinergic anti-inflammatory pathway. Despite its functional importance, in silico studies of the α7 receptor cannot produce a general model explaining the structural features of receptor activation, nor predict the mode of action for various ligand classes. Two particular problems in modeling the α7 nAChR are the absence of a high-resolution structure and the presence of five potentially nonequivalent orthosteric ligand binding sites. There is wide variability regarding the templates used for homology modeling, types of ligands investigated, simulation methods, and simulation times. However, a systematic survey focusing on the methodological similarities and differences in modeling α7 has not been done. In this work, we make a critical analysis of the modeling literature of α7 nAChR by comparing the findings of computational studies with each other and with experimental studies under the main topics of structural studies, ligand binding studies, and comparisons with other nAChR. In light of our findings, we also summarize current problems in the field and make suggestions for future studies concerning modeling of the α7 receptor.
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Simulación por Computador , Descubrimiento de Drogas , Modelos Moleculares , Transducción de Señal , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Humanos , Receptor Nicotínico de Acetilcolina alfa 7/químicaRESUMEN
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.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Receptores Nicotínicos , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Ratones , Agonistas Nicotínicos/farmacología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Azasugars, "nitrogen in the ring" analogues of monosaccharides, are known to be distributed in select plant, fungal. and bacterial species. We identify Chitinophaga pinensis DSM 2588 as the first bacterial source of the plant pyrrolidine azasugar 1,4-dideoxy-1,4-aminoarabinitol (DAB-1). Comparative sequence analyses identified C. pinensis as a putative azasugar producer, via observation of a three-gene cluster coding for putative aminotransferase, alcohol dehydrogenase, and sugar phosphatase enzymes, similar to the previously reported azasugar biosynthetic signature identified in Bacillus amyloliquefaciens FZB42. Multistep fractionation of C. pinensis culture media guided by a maltase inhibition assay yielded a component with a mass consistent with the structure of DAB-1. Heterologous expression of the three-gene cluster in E. coli, a non-azasugar producer, led to the isolation of nectrisine, a biosynthetic precursor to DAB-1, which displayed potent slow tight binding inhibition of maltase. Reduction of nectrisine with NaBH4 removed the slow tight binding inhibition kinetics, and MS analysis provided evidence for the production of a compound matching that of the isolated DAB-1 from C. pinensis. 1H NMR analysis of the nectrisine produced in E. coli after NaBD4 reduction produced a spectrum consistent with DAB-1 deuterated at C-1, primarily at the pro-S position. These results support the idea that the azasugar three-gene cluster represents a general biosynthetic path leading to several different compounds, which may prove useful for the identification of other azasugar-producing organisms.
Asunto(s)
Compuestos Aza/metabolismo , Bacteroidetes/metabolismo , Genes Bacterianos , Familia de Multigenes , Pirrolidinas/metabolismo , Azúcares/metabolismo , Proteínas Bacterianas/metabolismo , Espectroscopía de Protones por Resonancia Magnética , Pirrolidinas/químicaRESUMEN
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.
Asunto(s)
Mutación , Neuronas/metabolismo , Multimerización de Proteína , Subunidades de Proteína/genética , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Regulación Alostérica/genética , Secuencia de Aminoácidos , Animales , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
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.