RESUMEN
This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and ß2 subunits (α7ß2-nAChR subtype). Basal forebrain cholinergic neurons express α7ß2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-ß associated with early Alzheimer's disease. Additional work indicates that α7ß2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7ß2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7ß2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7ß2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7ß2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7ß2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7ß2-nAChR and detailed investigations of their physiological roles.
Asunto(s)
Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Colinérgicos , Sitios de Unión , Neuronas GABAérgicas/metabolismo , Antagonistas Nicotínicos/farmacologíaRESUMEN
Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.
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Conotoxinas , Receptores Nicotínicos , Conotoxinas/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Antagonistas Nicotínicos/química , Mutación , Simulación de Dinámica MolecularRESUMEN
BACKGROUND: Clinical and preclinical research have demonstrated that short-term exposure to nicotine during the initial experimentation stage can lead to early manifestation of withdrawal-like signs, indicating the state of "acute dependence". As drug withdrawal is a major factor driving the progression toward regular drug intake, characterizing and understanding the features of early nicotine withdrawal may be important for the prevention and treatment of drug addiction. In this study, we corroborate the previous studies by showing that withdrawal-like signs can be precipitated after short-term nicotine exposure in mice, providing a potential animal model of acute dependence on nicotine. RESULTS: To model nicotine exposure from light tobacco use during the initial experimentation stage, mice were treated with 0.5 mg/kg (-)-nicotine ditartrate once daily for 3 days. On the following day, the behavioral tests were conducted after implementing spontaneous or mecamylamine-precipitated withdrawal. In the open field test, precipitated nicotine withdrawal reduced locomotor activity and time spent in the center zone. In the elevated plus maze test, the mecamylamine challenge increased the time spent in the closed arm and reduced the number of entries irrespective of nicotine experience. In the examination of the somatic aspect, precipitated nicotine withdrawal enhanced the number of somatic signs. Finally, nicotine withdrawal did not affect cognitive functioning or social behavior in the passive avoidance, spatial object recognition, or social interaction test. CONCLUSIONS: Collectively, our data demonstrate that early nicotine withdrawal-like signs could be precipitated by the nicotinic antagonist mecamylamine in mice, and that early withdrawal from nicotine primarily causes physical symptoms.
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Nicotina , Síndrome de Abstinencia a Sustancias , Ratones , Animales , Nicotina/efectos adversos , Mecamilamina/farmacología , Mecamilamina/uso terapéutico , Síndrome de Abstinencia a Sustancias/tratamiento farmacológico , Síndrome de Abstinencia a Sustancias/etiología , Síndrome de Abstinencia a Sustancias/psicología , Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/uso terapéutico , AutoestimulaciónRESUMEN
Nicotinic acetylcholine receptors (nAChRs) regulate pain pathways with various outcomes depending on receptor subtypes, neuron types, and locations. But it remains unknown whether α4ß2 nAChRs abundantly expressed in the substantia nigra pars reticulata (SNr) have potential to mitigate hyperalgesia in pain states. We observed that injection of nAChR antagonists into the SNr reduced pain thresholds in naïve mice, whereas injection of nAChR agonists into the SNr relieved hyperalgesia in mice, subjected to capsaicin injection into the lower hind leg, spinal nerve injury, chronic constriction injury, or chronic nicotine exposure. The analgesic effects of nAChR agonists were mimicked by optogenetic stimulation of cholinergic inputs from the pedunculopontine nucleus (PPN) to the SNr, but attenuated upon downregulation of α4 nAChRs on SNr GABAergic neurons and injection of dihydro-ß-erythroidine into the SNr. Chronic nicotine-induced hyperalgesia depended on α4 nAChRs in SNr GABAergic neurons and was associated with the reduction of ACh release in the SNr. Either activation of α4 nAChRs in the SNr or optogenetic stimulation of the PPN-SNr cholinergic projection mitigated chronic nicotine-induced hyperalgesia. Interestingly, mechanical stimulation-induced ACh release was significantly attenuated in mice subjected to either capsaicin injection into the lower hind leg or SNI. These results suggest that α4 nAChRs on GABAergic neurons mediate a cholinergic analgesic circuit in the SNr, and these receptors may be effective therapeutic targets to relieve hyperalgesia in acute and chronic pain, and chronic nicotine exposure.
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Neuronas GABAérgicas , Hiperalgesia , Ratones Endogámicos C57BL , Receptores Nicotínicos , Animales , Receptores Nicotínicos/metabolismo , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/fisiología , Masculino , Hiperalgesia/metabolismo , Hiperalgesia/tratamiento farmacológico , Ratones , Porción Reticular de la Sustancia Negra/metabolismo , Porción Reticular de la Sustancia Negra/efectos de los fármacos , Nicotina/farmacología , Analgésicos/farmacología , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Capsaicina/farmacología , Acetilcolina/metabolismo , Optogenética , Umbral del Dolor/efectos de los fármacosRESUMEN
Macrocyclic imine phycotoxins are an emerging class of chemical compounds associated with harmful algal blooms and shellfish toxicity. Earlier binding and electrophysiology experiments on nAChR subtypes and their soluble AChBP surrogates evidenced common trends for substantial antagonism, binding affinities, and receptor-subtype selectivity. Earlier, complementary crystal structures of AChBP complexes showed that common determinants within the binding nest at each subunit interface confer high-affinity toxin binding, while distinctive determinants from the flexible loop C, and either capping the nest or extending toward peripheral subsites, dictate broad versus narrow receptor subtype selectivity. From these data, small spiroimine enantiomers mimicking the functional core motif of phycotoxins were chemically synthesized and characterized. Voltage-clamp analyses involving three nAChR subtypes revealed preserved antagonism for both enantiomers, despite lower subtype specificity and binding affinities associated with faster reversibility compared with their macrocyclic relatives. Binding and structural analyses involving two AChBPs pointed to modest affinities and positional variability of the spiroimines, along with a range of AChBP loop-C conformations denoting a prevalence of antagonistic properties. These data highlight the major contribution of the spiroimine core to binding within the nAChR nest and confirm the need for an extended interaction network as established by the macrocyclic toxins to define high affinities and marked subtype specificity. This study identifies a minimal set of functional pharmacophores and binding determinants as templates for designing new antagonists targeting disease-associated nAChR subtypes.
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Iminas , Toxinas Marinas , Antagonistas Nicotínicos , Receptores Nicotínicos , Toxinas Marinas/química , Toxinas Marinas/farmacología , Toxinas Marinas/toxicidad , Iminas/química , Iminas/farmacología , Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/química , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Animales , Compuestos Macrocíclicos/farmacología , Compuestos Macrocíclicos/química , Relación Estructura-ActividadRESUMEN
α-Conotoxins, as selective nAChR antagonists, can be valuable tools for targeted drug delivery and fluorescent labeling, while conotoxin-drug or conotoxin-fluorescent conjugates through the disulfide bond are rarely reported. Herein, we demonstrate the [2,4] disulfide bond of α-conotoxin as a feasible new chemical modification site. In this study, analogs of the α-conotoxin LsIA cysteine[2,4] were synthesized by stapling with five linkers, and their inhibitory activities against human α7 and rat α3ß2 nAChRs were maintained. To further apply this method in targeted delivery, the alkynylbenzyl bromide linker was synthesized and conjugated with Coumarin 120 (AMC) and Camptothecin (CPT) by copper-catalyzed click chemistry, and then stapled between cysteine[2,4] of the LsIA to construct a fluorescent probe and two peptide-drug conjugates. The maximum emission wavelength of the LsIA fluorescent probe was 402.2 nm, which was essentially unchanged compared with AMC. The cytotoxic activity of the LsIA peptide-drug conjugates on human A549 was maintained in vitro. The results demonstrate that the stapling of cysteine[2,4] with alkynylbenzyl bromide is a simple and feasible strategy for the exploitation and utilization of the α-conotoxin LsIA.
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Conotoxinas , Cisteína , Humanos , Conotoxinas/química , Conotoxinas/farmacología , Cisteína/química , Animales , Disulfuros/química , Células A549 , Sistemas de Liberación de Medicamentos , Ratas , Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/química , Colorantes Fluorescentes/química , Receptores Nicotínicos/metabolismo , Cumarinas/química , Cumarinas/farmacología , Química ClicRESUMEN
α7 nicotinic acetylcholine receptors (nAChRs) are mainly distributed in the central nervous system (CNS), including the hippocampus, striatum, and cortex of the brain. The α7 nAChR has high Ca2+ permeability and can be quickly activated and desensitized, and is closely related to Alzheimer's disease (AD), epilepsy, schizophrenia, lung cancer, Parkinson's disease (PD), inflammation, and other diseases. α-conotoxins from marine cone snail venom are typically short, disulfide-rich neuropeptides targeting nAChRs and can distinguish various subtypes, providing vital pharmacological tools for the functional research of nAChRs. [Q1G, ΔR14]LvΙB is a rat α7 nAChRs selective antagonist, modified from α-conotoxin LvΙB. In this study, we utilized three types of fluorescein after N-Hydroxy succinimide (NHS) activation treatment: 6-TAMRA-SE, Cy3 NHS, and BODIPY-FL NHS, labeling the N-Terminal of [Q1G, ΔR14]LvΙB under weak alkaline conditions, obtaining three fluorescent analogs: LvIB-R, LvIB-C, and LvIB-B, respectively. The potency of [Q1G, ΔR14]LvΙB fluorescent analogs was evaluated at rat α7 nAChRs expressed in Xenopus laevis oocytes. Using a two-electrode voltage clamp (TEVC), the half-maximal inhibitory concentration (IC50) values of LvIB-R, LvIB-C, and LvIB-B were 643.3 nM, 298.0 nM, and 186.9 nM, respectively. The stability of cerebrospinal fluid analysis showed that after incubation for 12 h, the retention rates of the three fluorescent analogs were 52.2%, 22.1%, and 0%, respectively. [Q1G, ΔR14]LvΙB fluorescent analogs were applied to explore the distribution of α7 nAChRs in the hippocampus and striatum of rat brain tissue and it was found that Cy3- and BODIPY FL-labeled [Q1G, ΔR14]LvΙB exhibited better imaging characteristics than 6-TAMARA-. It was also found that α7 nAChRs are widely distributed in the cerebral cortex and cerebellar lobules. Taking into account potency, imaging, and stability, [Q1G, ΔR14]LvΙB -BODIPY FL is an ideal pharmacological tool to investigate the tissue distribution and function of α7 nAChRs. Our findings not only provide a foundation for the development of conotoxins as visual pharmacological probes, but also demonstrate the distribution of α7 nAChRs in the rat brain.
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Encéfalo , Conotoxinas , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7 , Animales , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Conotoxinas/farmacología , Conotoxinas/química , Ratas , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Antagonistas Nicotínicos/farmacología , Colorantes Fluorescentes , Ratas Sprague-Dawley , Masculino , FemeninoRESUMEN
Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting painful neuropathy that occurs commonly during cancer management, which often leads to the discontinuation of medication. Previous studies suggest that the α9α10 nicotinic acetylcholine receptor (nAChR)-specific antagonist αO-conotoxin GeXIVA[1,2] is effective in CIPN models; however, the related mechanisms remain unclear. Here, we analyzed the preventive effect of GeXIVA[1,2] on neuropathic pain in the long-term oxaliplatin injection-induced CIPN model. At the end of treatment, lumbar (L4-L6) spinal cord was extracted, and RNA sequencing and bioinformatic analysis were performed to investigate the potential genes and pathways related to CIPN and GeXIVA[1,2]. GeXIVA[1,2] inhibited the development of mechanical allodynia induced by chronic oxaliplatin treatment. Repeated injections of GeXIVA[1,2] for 3 weeks had no effect on the mice's normal pain threshold or locomotor activity and anxiety-like behavior, as evaluated in the open field test (OFT) and elevated plus maze (EPM). Our RNA sequencing results identified 209 differentially expressed genes (DEGs) in the CIPN model, and simultaneously injecting GeXIVA[1,2] with oxaliplatin altered 53 of the identified DEGs. These reverted genes were significantly enriched in immune-related pathways represented by the cytokine-cytokine receptor interaction pathway. Our findings suggest that GeXIVA[1,2] could be a potential therapeutic compound for chronic oxaliplatin-induced CIPN management.
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Antineoplásicos , Conotoxinas , Neuralgia , Ratones , Animales , Oxaliplatino/efectos adversos , Conotoxinas/farmacología , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Neuralgia/genética , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/genética , Modelos Animales de Enfermedad , Antagonistas Nicotínicos/farmacología , Expresión Génica , Antineoplásicos/efectos adversosRESUMEN
Nicotine binds to nicotinic acetylcholine receptors (nAChRs) that are overexpressed in different cancer cells, promoting tumor growth and resistance to chemotherapy. In this study, we aimed to investigate the potential of APS7-2 and APS8-2, synthetic analogs of a marine sponge toxin, to inhibit nicotine-mediated effects on A549 human lung cancer cells. Our electrophysiological measurements confirmed that APS7-2 and APS8-2 act as α7 nAChR antagonists. APS8-2 showed no cytotoxicity in A549 cells, while APS7-2 showed concentration-dependent cytotoxicity in A549 cells. The different cytotoxic responses of APS7-2 and APS8-2 emphasize the importance of the chemical structure in determining their cytotoxicity on cancer cells. Nicotine-mediated effects include increased cell viability and proliferation, elevated intracellular calcium levels, and reduced cisplatin-induced cytotoxicity and reactive oxygen species production (ROS) in A549 cells. These effects of nicotine were effectively attenuated by APS8-2, whereas APS7-2 was less effective. Our results suggest that APS8-2 is a promising new therapeutic agent in the chemotherapy of lung cancer.
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Antineoplásicos , Supervivencia Celular , Neoplasias Pulmonares , Nicotina , Especies Reactivas de Oxígeno , Receptor Nicotínico de Acetilcolina alfa 7 , Humanos , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Células A549 , Nicotina/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Animales , Antagonistas Nicotínicos/farmacología , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Calcio/metabolismo , Poríferos/químicaRESUMEN
The α9- and α7-containing nicotinic acetylcholine receptors (nAChRs) mediate numerous physiological and pathological processes by complex mechanisms that are currently the subject of intensive study and debate. In this regard, selective ligands serve as invaluable investigative tools and, in many cases, potential therapeutics for the treatment of various CNS disfunctions and diseases, neuropathic pain, inflammation, and cancer. However, the present scenario differs significantly between the two aforementioned nicotinic subtypes. Over the past few decades, a large number of selective α7-nAChR ligands, including full, partial and silent agonists, antagonists, and allosteric modulators, have been described and reviewed. Conversely, reports on selective α9-containing nAChR ligands are relatively scarce, also due to a more recent characterization of this receptor subtype, and hardly any focusing on small molecules. In this review, we focus on the latter, providing a comprehensive overview, while providing only an update over the last five years for α7-nAChR ligands.
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Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Ligandos , Nicotina , Antagonistas NicotínicosRESUMEN
The study of nicotinic acetylcholine receptors (nAChRs) has significantly progressed in the last decade, due to a) the improved techniques available for structural studies; b) the identification of ligands interacting at orthosteric and allosteric recognition sites on the nAChR proteins, able to tune channel conformational states; c) the better functional characterization of receptor subtypes/subunits and their therapeutic potential; d) the availability of novel pharmacological agents able to activate or block nicotinic-mediated cholinergic responses with subtype or stoichiometry selectivity. The copious literature on nAChRs is related to the pharmacological profile of new, promising subtype selective derivatives as well as the encouraging preclinical and early clinical evaluation of known ligands. However, recently approved therapeutic derivatives are still missing, and examples of ligands discontinued in advanced CNS clinical trials include drug candidates acting at both neuronal homomeric and heteromeric receptors. In this review, we have selected heteromeric nAChRs as the target and comment on literature reports of the past five years dealing with the discovery of new small molecule ligands or the advanced pharmacological/preclinical investigation of more promising compounds. The results obtained with bifunctional nicotinic ligands and a light-activated ligand as well as the applications of promising radiopharmaceuticals for heteromeric subtypes are also discussed.
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Receptores Nicotínicos , Receptores Nicotínicos/metabolismo , Ligandos , Regulación Alostérica , Neuronas/metabolismo , Transmisión Sináptica , Nicotina , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacologíaRESUMEN
The pentameric nicotinic acetylcholine receptors (nAChRs) are typically classed as muscle- or neuronal-type, however, the latter has also been reported in non-neuronal cells. Given their broad distribution, nAChRs mediate numerous physiological and pathological processes including synaptic transmission, presynaptic modulation of transmitter release, neuropathic pain, inflammation, and cancer. There are 17 different nAChR subunits and combinations of these subunits produce subtypes with diverse pharmacological properties. The expression and role of some nAChR subtypes have been extensively deciphered with the aid of knock-out models. Many nAChR subtypes expressed in heterologous systems are selectively targeted by the disulfide-rich α-conotoxins. α-Conotoxins are small peptides isolated from the venom of cone snails, and a number of them have potential pharmaceutical value.
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Conotoxinas , Receptores Nicotínicos , Conotoxinas/farmacología , Conotoxinas/química , Conotoxinas/metabolismo , Receptores Nicotínicos/metabolismo , Péptidos/farmacología , Membrana Celular/metabolismo , Neuronas/metabolismo , Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/uso terapéuticoRESUMEN
The use of alcohol causes significant morbidity and mortality across the globe. Alcohol use disorder (AUD) is defined by the excessive use of this drug despite a negative impact on the individual's life. While there are currently medications available to treat AUD, they have limited efficacy and several side effects. As such, it is essential to continue to look for novel therapeutics. One target for novel therapeutics is nicotinic acetylcholine receptors (nAChRs). Here we systematically review the literature on the involvement of nAChRs in alcohol consumption. Data from both genetic and pharmacology studies provide evidence that nAChRs modulate alcohol intake. Interestingly, pharmacological modulation of all nAChR subtypes examined can decrease alcohol consumption. The reviewed literature demonstrates that nAChRs should continue to be investigated as novel therapeutics for AUD.
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Consumo de Bebidas Alcohólicas , Receptores Nicotínicos , Humanos , Consumo de Bebidas Alcohólicas/efectos adversos , Alcoholismo/tratamiento farmacológico , Etanol , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/genéticaRESUMEN
α4/6-conotoxin TxID, which was identified from Conus textile, simultaneously blocks rat (r) α3ß4 and rα6/α3ß4 nicotinic acetylcholine receptors (nAChRs) with IC50 values of 3.6 nM and 33.9 nM, respectively. In order to identify the effects of loop2 size on the potency of TxID, alanine (Ala) insertion and truncation mutants were designed and synthesized in this study. An electrophysiological assay was used to evaluate the activity of TxID and its loop2-modified mutants. The results showed that the inhibition of 4/7-subfamily mutants [+9A]TxID, [+10A]TxID, [+14A]TxID, and all the 4/5-subfamily mutants against rα3ß4 and rα6/α3ß4 nAChRs decreased. Overall, ala-insertion or truncation of the 9th, 10th, and 11th amino acid results in a loss of inhibition and the truncation of loop2 has more obvious impacts on its functions. Our findings have strengthened the understanding of α-conotoxin, provided guidance for further modifications, and offered a perspective for future studies on the molecular mechanism of the interaction between α-conotoxins and nAChRs.
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Conotoxinas , Caracol Conus , Receptores Nicotínicos , Ratas , Animales , Conotoxinas/química , Caracol Conus/química , Receptores Nicotínicos/metabolismo , Alanina , Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/químicaRESUMEN
α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 ß2ß3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.
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Conotoxinas , Caracol Conus , Receptores Nicotínicos , Animales , Ratas , Conotoxinas/farmacología , Conotoxinas/química , Caracol Conus/química , Caracol Conus/fisiología , Antagonistas Nicotínicos/farmacología , Caracoles , PolinesiaRESUMEN
Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.
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Conotoxinas , Caracol Conus , Receptores Nicotínicos , Ratones , Humanos , Animales , Conotoxinas/farmacología , Caracol Conus/metabolismo , Ponzoñas , Receptores Nicotínicos/metabolismo , Péptidos/metabolismo , Antagonistas Nicotínicos/farmacologíaRESUMEN
Conotoxins are a class of disulfide-rich peptides found in the venom of cone snails, which have attracted considerable attention in recent years due to their potent activity on ion channels and potential for therapeutics. Among them, α-conotoxin RgIA, a 13-residue peptide, has shown great promise as a potent inhibitor of α9α10 nAChRs for pain management. In this study, we investigated the effect of substituting the naturally occurring L-type arginine at position 11 of the RgIA sequence with its D-type amino acid. Our results indicate that this substitution abrogated the ability of RgIA to block α9α10 nAChRs, but instead endowed the peptide with the ability to block α7 nAChR activity. Structural analyses revealed that this substitution induced significant alteration of the secondary structure of RgIA[11r], which consequently affected its activity. Our findings underscore the potential of D-type amino acid substitution as a promising strategy for designing novel conotoxin-based ligands targeting different types of nAChRs.
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Conotoxinas , Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Receptores Nicotínicos/metabolismo , Conotoxinas/química , Péptidos/farmacología , Péptidos/metabolismo , Arginina/farmacología , Antagonistas Nicotínicos/químicaRESUMEN
Tobacco use is a persistent public health issue. It kills up to half its users and is the cause of nearly 90% of all lung cancers. The main psychoactive component of tobacco is nicotine, primarily responsible for its abuse-related effects. Accordingly, most pharmacotherapies for smoking cessation target nicotinic acetylcholine receptors (nAChRs), nicotine's major site of action in the brain. The goal of the current review is twofold: first, to provide a brief overview of the most commonly used behavioral procedures for evaluating smoking cessation pharmacotherapies and an introduction to pharmacokinetic and pharmacodynamic properties of nicotine important for consideration in the development of new pharmacotherapies; and second, to discuss current and potential future pharmacological interventions aimed at decreasing tobacco use. Attention will focus on the potential for allosteric modulators of nAChRs to offer an improvement over currently approved pharmacotherapies. Additionally, given increasing public concern for the potential health consequences of using electronic nicotine delivery systems, which allow users to inhale aerosolized solutions as an alternative to smoking tobacco, an effort will be made throughout this review to address the implications of this relatively new form of nicotine delivery, specifically as it relates to smoking cessation. SIGNIFICANCE STATEMENT: Despite decades of research that have vastly improved our understanding of nicotine and its effects on the body, only a handful of pharmacotherapies have been successfully developed for use in smoking cessation. Thus, investigation of alternative pharmacological strategies for treating tobacco use disorder remains active; allosteric modulators of nicotinic acetylcholine receptors represent one class of compounds currently under development for this purpose.
Asunto(s)
Antagonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/uso terapéutico , Tabaquismo/tratamiento farmacológico , Animales , Ensayos Clínicos Fase III como Asunto , Humanos , Nicotina/farmacocinética , Receptores Nicotínicos/metabolismo , Cese del Hábito de Fumar/métodos , Tabaquismo/metabolismoRESUMEN
The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.
Asunto(s)
Vías Eferentes/fisiología , Sistema de la Línea Lateral/fisiología , Sistema Nervioso Parasimpático/fisiología , Sinapsis/fisiología , Animales , Bungarotoxinas/farmacología , Señalización del Calcio/efectos de los fármacos , Regulación de la Expresión Génica , Células Ciliadas Auditivas/fisiología , Antagonistas Nicotínicos/farmacología , Oocitos , Estimulación Física , Receptores Nicotínicos/efectos de los fármacos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Estricnina/farmacología , Xenopus , Pez CebraRESUMEN
Previously, bupropion (BUP), a norepinephrine (NE)/dopamine (DA) transporter blocker and nicotinic acetylcholine receptors (nAChRs) antagonist, was found to intensify methamphetamine (METH) craving behaviours in mice. Intense craving causes relapse in drug dependence. This study characterized local field potential (LFP) patterns in the brain regions associated with METH-conditioned place preference (CPP) enhanced by BUP. Male Swiss albino ICR mice were implanted with LFP electrodes to the ventral tegmental area (VTA), medial prefrontal cortex (mPFC) and the nucleus accumbens core (NAcc). Animals received sessions to learn the association between injection effects (1 mg/kg METH and normal saline) with contextual environments (METH- and saline-paired compartments) during the conditioning phase. A total of 20 mg/kg BUP was given to animals before LFP, and behaviour recording in the CPP apparatus during the post-conditioning phase. The results showed that increased CPP scores and % number of entries to the METH-paired zone, as well as changes in VTA, mPFC and NAcc spectral powers and coherence among these areas, were associated with METH-CPP. Treatment with BUP increased VTA delta and gamma I, decreased mPFC alpha, increased NAcc gamma I and decreased gamma II powers. Coherence analyses revealed that BUP decreased gamma II VTA-mPFC and increased beta and gamma I VTA-NAcc connectivity. Altogether, BUP produced additional effects to that of METH-CPP alone. These findings demonstrated changes in neural circuit activities associated with METH-CPP intensified by BUP. Moreover, modulation of NE/DA systems and/or nAChRs actions in the VTA-cortico-accumbens loop might underlie METH craving and dependence.