RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng flowers, which are the buds of the traditional Chinese medicinal herb Sanqi, are widely used in China for their cough-ameliorating properties, with demonstrated therapeutic effects in the treatment of both acute and chronic coughs. However, both the antitussive mechanism and active compound basis of P. notoginseng flowers remain poorly understood. AIM OF THE STUDY: We investigated the antitussive effects of P. notoginseng flowers, identified the bioactive constituents responsible for alleviating cough symptoms, and elucidated the underlying pharmacological mechanisms. MATERIALS AND METHODS: We analyzed the major chemical constituents of aqueous extracts of P. notoginseng flowers using liquid chromatography-mass spectrometry and quantitatively analyzed the key component, 20S-ginsenoside Rh2, using high-performance liquid chromatography. Using a cough reflex model in healthy mice and an ovalbumin-induced, highly sensitive guinea pig cough model, we verified the suppressive effects of P. notoginseng flowers and their saponin constituents on coughing. Furthermore, we explored the mechanisms of action of the key ion channels, NaV1.7 and TRPV1, using whole-cell patch-clamp techniques and molecular docking. Finally, the therapeutic mechanisms of P. notoginseng flowers on pathological cough were revealed using hematoxylin and eosin staining, immunohistochemistry, and western blotting. RESULTS: The active components of P. notoginseng flowers were primarily protopanaxadiol-type saponins, among which 20S-ginsenoside Rh2 had the highest content (51.46 mg/g). In the mouse model, P. notoginseng flowers exhibited antitussive effects comparable to those of pentoxyverine citrate. Although its main saponin component, 20S-ginsenoside Rh2, showed slightly weaker effects, it still demonstrated concentration-dependent inhibition of channel activity. The whole-cell patch-clamp technique and virtual molecular docking showed that Rh2 might exert its effects by directly binding to the NaV1.7 and TRPV1 channels. In the guinea pig model, P. notoginseng flowers and their saponin components not only reduced cough frequency and prolonged the latency period before cough onset, but also significantly inhibited tracheal and pulmonary inflammation and the overexpression of TRPV1. CONCLUSIONS: 20S-Ginsenoside Rh2, the major bioactive saponin in P. notoginseng flowers, exhibits potent antitussive effects. The potential mechanism of action of 20S-Ginsenoside Rh2 in the treatment of cough may involve inhibiting NaV1.7 and TRPV1 channel currents through direct binding to core protein active sites and downregulating TRPV1 expression.
Asunto(s)
Antitusígenos , Tos , Regulación hacia Abajo , Flores , Ginsenósidos , Canal de Sodio Activado por Voltaje NAV1.7 , Panax notoginseng , Canales Catiónicos TRPV , Animales , Canales Catiónicos TRPV/metabolismo , Cobayas , Flores/química , Tos/tratamiento farmacológico , Ginsenósidos/farmacología , Antitusígenos/farmacología , Masculino , Ratones , Panax notoginseng/química , Regulación hacia Abajo/efectos de los fármacos , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Células HEK293 , Simulación del Acoplamiento Molecular , Cricetulus , Modelos Animales de Enfermedad , Células CHO , Saponinas/farmacología , OvalbúminaRESUMEN
Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to NaV1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.
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Inteligencia Artificial , Descubrimiento de Drogas , Simulación del Acoplamiento Molecular , Descubrimiento de Drogas/métodos , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/química , Unión Proteica , Cristalografía por Rayos X , Ligandos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Evaluación Preclínica de Medicamentos/métodosRESUMEN
The relationship between transcription and protein expression is complex. We identified polysome-associated RNA transcripts in the somata and central terminals of mouse sensory neurons in control, painful (plus nerve growth factor), and pain-free conditions (Nav1.7-null mice). The majority (98%) of translated transcripts are shared between male and female mice in both the somata and terminals. Some transcripts are highly enriched in the somata or terminals. Changes in the translatome in painful and pain-free conditions include novel and known regulators of pain pathways. Antisense knockdown of selected somatic and terminal polysome-associated transcripts that correlate with pain states diminished pain behavior. Terminal-enriched transcripts included those encoding synaptic proteins (e.g., synaptotagmin), non-coding RNAs, transcription factors (e.g., Znf431), proteins associated with transsynaptic trafficking (HoxC9), GABA-generating enzymes (Gad1 and Gad2), and neuropeptides (Penk). Thus, central terminal translation may well be a significant regulatory locus for peripheral input from sensory neurons.
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Dolor , Células Receptoras Sensoriales , Animales , Células Receptoras Sensoriales/metabolismo , Ratones , Masculino , Femenino , Dolor/metabolismo , Biosíntesis de Proteínas , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Glutamato Descarboxilasa/metabolismo , Glutamato Descarboxilasa/genética , Polirribosomas/metabolismo , Ratones Endogámicos C57BL , Ganglios Espinales/metabolismoRESUMEN
Cholesterol is a major component of plasma membranes and plays a significant role in actively regulating the functioning of several membrane proteins in humans. In this study, we focus on the role of cholesterol depletion on the voltage-gated sodium channel Nav1.7, which is primarily expressed in the peripheral sensory neurons and linked to various chronic inherited pain syndromes. Coarse-grained molecular dynamics simulations revealed key dynamic changes of Nav1.7 upon membrane cholesterol depletion: A loss of rigidity in the structural motifs linked to activation and fast-inactivation is observed, suggesting an easier transition of the channel between different gating states. In-vitro whole-cell patch clamp experiments on HEK293t cells expressing Nav1.7 validated these predictions at the functional level: Hyperpolarizing shifts in the voltage-dependence of activation and fast-inactivation were observed along with an acceleration of the time to peak and onset kinetics of fast inactivation. These results underline the critical role of membrane composition, and of cholesterol in particular, in influencing Nav1.7 gating characteristics. Furthermore, our results also point to cholesterol-driven changes of the geometry of drug-binding regions, hinting to a key role of the membrane environment in the regulation of drug effects.
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Membrana Celular , Colesterol , Simulación de Dinámica Molecular , Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/química , Canal de Sodio Activado por Voltaje NAV1.7/genética , Humanos , Colesterol/metabolismo , Colesterol/química , Membrana Celular/metabolismo , Membrana Celular/química , Células HEK293 , Activación del Canal IónicoRESUMEN
Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.
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Analgésicos , Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.7 , Neuronas , Tetrodotoxina , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/citología , Neuronas/efectos de los fármacos , Ratones , Tetrodotoxina/farmacología , Analgésicos/farmacología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/farmacología , Masculino , Crotalus , Potenciales de la Membrana/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , PéptidosRESUMEN
There has been significant progress in our understanding of the molecular basis by which nociceptors transduce and transmit noxious (tissue damaging) stimuli. This is dependent on ion channels, many of which are selectively expressed in nociceptors. Mutations in such proteins have recently been linked to inherited pain disorders in humans. An exemplar is the voltage-gated sodium channel (VGSC) NaV1.7. Loss of function mutations in NaV1.7 result in congenital inability to experience pain while gain-of-function mutations can cause a number of distinct neuropathic pain disorders, including erythromelalgia, paroxysmal extreme pain disorder, and small-fiber neuropathy. Furthermore, variants in the VGSCs 1.8 and 1.9 have also been linked to human pain disorders. There is a correlation between the impact of mutations on the biophysical properties of the ion channel and the severity of the clinical phenotype. Pain channelopathies are not restricted to VGSCs: a mutation in the ligand-gated ion channel TRPA1, (which responds to environmental irritants) causes a familial episodic pain disorder. Ion channel variants have also been linked to more common neuropathic pain disorders such as painful diabetic neuropathy. Not only do these ion channels present targets for novel analgesics, but stratification based on genotype may improve treatment selection of existing analgesics.
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Canalopatías , Humanos , Canalopatías/genética , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.7/genética , Dolor/genética , Neuralgia/genéticaRESUMEN
Familial episodic pain syndrome (FEPS) is an early childhood onset disorder of severe episodic limb pain caused mainly by pathogenic variants of SCN11A, SCN10A, and SCN9A, which encode three voltage-gated sodium channels (VGSCs) expressed as key determinants of nociceptor excitability in primary sensory neurons. There may still be many undiagnosed patients with FEPS. A better understanding of the associated pathogenesis, epidemiology, and clinical characteristics is needed to provide appropriate diagnosis and care. For this study, nationwide recruitment of Japanese patients was conducted using provisional clinical diagnostic criteria, followed by genetic testing for SCN11A, SCN10A, and SCN9A. In the cohort of 212 recruited patients, genetic testing revealed that 64 patients (30.2%) harbored pathogenic or likely pathogenic variants of these genes, consisting of 42 (19.8%), 14 (6.60%), and 8 (3.77%) patients with variants of SCN11A, SCN10A, and SCN9A, respectively. Meanwhile, the proportions of patients meeting the tentative clinical criteria were 89.1%, 52.0%, and 54.5% among patients with pathogenic or likely pathogenic variants of each of the three genes, suggesting the validity of these clinical criteria, especially for patients with SCN11A variants. These clinical diagnostic criteria of FEPS will accelerate the recruitment of patients with underlying pathogenic variants who are unexpectedly prevalent in Japan.
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Pruebas Genéticas , Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.9/genética , Japón/epidemiología , Canal de Sodio Activado por Voltaje NAV1.8/genética , Masculino , Femenino , Pruebas Genéticas/métodos , Adulto , Adolescente , Niño , Predisposición Genética a la Enfermedad , Adulto Joven , Preescolar , Mutación , Dolor , Recto/anomalíasRESUMEN
The dorsal root ganglion (DRG) is the primary neuron responsible for transmitting peripheral pain signals to the central nervous system and plays a crucial role in pain transduction. Modulation of DRG excitability is considered a viable approach for pain management. Neuronal excitability is intricately linked to the ion channels on the neurons. The small and medium-sized DRG neurons are chiefly engaged in pain conduction and have high levels of TTX-S sodium channels, with Nav1.7 accounting for approximately 80% of the current. Voltage-gated sodium channel (VGSC or Nav) blockers are vital targets for the management of central nervous system diseases, particularly chronic pain. VGSCs play a key role in controlling cellular excitability. Clinical research has shown that Nav1.7 plays a crucial role in pain sensation, and there is strong genetic evidence linking Nav1.7 and its encoding gene SCN9A gene to painful disorders in humans. Many studies have shown that Nav1.7 plays an important role in pain management. The role of Nav1.7 in pain signaling pathways makes it an attractive target for the potential development of new pain drugs. Meanwhile, understanding the architecture of Nav1.7 may help to develop the next generation of painkillers. This review provides updates on the recently reported molecular inhibitors targeting the Nav1.7 pathway, summarizes their structure-activity relationships (SARs), and discusses their therapeutic effects on painful diseases. Pharmaceutical chemists are working to improve the therapeutic index of Nav1.7 inhibitors, achieve better analgesic effects, and reduce side effects. We hope that this review will contribute to the development of novel Nav1.7 inhibitors as potential drugs.
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Canal de Sodio Activado por Voltaje NAV1.7 , Bloqueadores del Canal de Sodio Activado por Voltaje , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/uso terapéutico , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Dolor en Cáncer/tratamiento farmacológico , Dolor en Cáncer/metabolismo , Analgésicos/química , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Relación Estructura-Actividad , Manejo del Dolor/métodos , Estructura Molecular , Neoplasias/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/farmacología , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/uso terapéuticoRESUMEN
(1) Background: Irritable bowel syndrome (IBS) is a common disease in the gastrointestinal (GI) tract. Atractylodes macrocephala Koidz (AMK) is known as one of the traditional medicines that shows a good efficacy in the GI tract. (2) Methods: We investigated the effect of AMK in a network pharmacology and zymosan-induced IBS animal model. In addition, we performed electrophysiological experiments to confirm the regulatory mechanisms related to IBS. (3) Results: Various characteristics of AMK were investigated using TCMSP data and various analysis systems. AMK restored the macroscopic changes and weight to normal. Colonic mucosa and inflammatory factors were reduced. These effects were similar to those of amitriptyline and sulfasalazine. In addition, transient receptor potential (TRP) V1, voltage-gated Na+ (NaV) 1.5, and NaV1.7 channels were inhibited. (4) Conclusion: These results suggest that AMK may be a promising therapeutic candidate for IBS management through the regulation of ion channels.
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Atractylodes , Modelos Animales de Enfermedad , Síndrome del Colon Irritable , Canales Catiónicos TRPV , Zimosan , Animales , Síndrome del Colon Irritable/tratamiento farmacológico , Síndrome del Colon Irritable/inducido químicamente , Canales Catiónicos TRPV/metabolismo , Ratones , Atractylodes/química , Masculino , Extractos Vegetales/farmacología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacosRESUMEN
This study reports that targeting intrinsically disordered regions of the voltage-gated sodium channel 1.7 (NaV1.7) protein facilitates discovery of sodium channel inhibitory peptide aptamers (NaViPA) for adeno-associated virus-mediated (AAV-mediated), sensory neuron-specific analgesia. A multipronged inhibition of INa1.7, INa1.6, INa1.3, and INa1.1 - but not INa1.5 and INa1.8 - was found for a prototype and named NaViPA1, which was derived from the NaV1.7 intracellular loop 1, and is conserved among the TTXs NaV subtypes. NaViPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs INa but not TTXr INa. DRG injection of AAV6-encoded NaViPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current clamp of the PSNs showed that NaViPA1 expression normalized PSN excitability in TNI rats, suggesting that NaViPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. IHC revealed efficient NaViPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaViPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaViPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs NaVs, has potential as a peripheral nerve-restricted analgesic therapeutic.
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Dependovirus , Canal de Sodio Activado por Voltaje NAV1.7 , Células Receptoras Sensoriales , Animales , Ratas , Dependovirus/genética , Células Receptoras Sensoriales/metabolismo , Masculino , Humanos , Femenino , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Ganglios Espinales/metabolismo , Ratas Sprague-Dawley , Neuralgia/metabolismo , Neuralgia/genética , Neuralgia/tratamiento farmacológico , AnalgesiaRESUMEN
Voltage-gated sodium channel subtypes, Nav1.7, Nav1.8, and Nav1.9 are predominantly expressed in peripheral sensory neurons. Recent genetic studies have revealed that they are involved in pathological pain processing and that the blockade of Nav1.7, Nav1.8, or Nav1.9 will become a promising pharmacotherapy especially for neuropathic pain. A growing number of drug discovery programs have targeted either of the subtypes to obtain a selective inhibitor which can provide pain relief without affecting the cardiovascular and central nervous systems, though none of them has been approved yet. Here we describe the in vitro characteristics of ANP-230, a novel sodium channel blocker under clinical development. Surprisingly, ANP-230 was shown to block three pain-related subtypes, human Nav1.7, Nav1.8, and Nav1.9 with similar potency, but had only low inhibitory activity to human cardiac Nav1.5 channel and rat central Nav channels. The voltage clamp experiments using different step pulse protocols revealed that ANP-230 had a "tonic block" mode of action without state- and use-dependency. In addition, ANP-230 caused a depolarizing shift of the activation curve and decelerated gating kinetics in human Nav1.7-stably expressing cells. The depolarizing shift of activation curve was commonly observed in human Nav1.8-stably expressing cells as well as rat dorsal root ganglion neurons. These data suggested a quite unique mechanism of Nav channel inhibition by ANP-230. Finally, ANP-230 reduced excitability of rat dorsal root ganglion neurons in a concentration dependent manner. Collectively, these promising results indicate that ANP-230 could be a potent drug for neuropathic pain.
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Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.8 , Canal de Sodio Activado por Voltaje NAV1.9 , Bloqueadores de los Canales de Sodio , Humanos , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8/genética , Animales , Ratas , Canal de Sodio Activado por Voltaje NAV1.9/metabolismo , Canal de Sodio Activado por Voltaje NAV1.9/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Bloqueadores de los Canales de Sodio/farmacología , Células HEK293 , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/citologíaRESUMEN
BACKGROUND AND PURPOSE: The voltage-gated sodium channel isoform NaV1.7 is a high-interest target for the development of non-opioid analgesics due to its preferential expression in pain-sensing neurons. NaV1.7 is also expressed in autonomic neurons, yet its contribution to involuntary visceral reflexes has received limited attention. The small molecule inhibitor ST-2560 was advanced into pain behaviour and cardiovascular models to understand the pharmacodynamic effects of selective inhibition of NaV1.7. EXPERIMENTAL APPROACH: Potency of ST-2560 at NaV1.7 and off-target ion channels was evaluated by whole-cell patch-clamp electrophysiology. Effects on nocifensive reflexes were assessed in non-human primate (NHP) behavioural models, employing the chemical capsaicin and mechanical stimuli. Cardiovascular parameters were monitored continuously in freely-moving, telemetered NHPs following administration of vehicle and ST-2560. KEY RESULTS: ST-2560 is a potent inhibitor (IC50 = 39 nM) of NaV1.7 in primates with ≥1000-fold selectivity over other isoforms of the human NaV1.x family. Following systemic administration, ST-2560 (0.1-0.3 mg·kg-1, s.c.) suppressed noxious mechanical- and chemical-evoked reflexes at free plasma concentrations threefold to fivefold above NaV1.7 IC50. ST-2560 (0.1-1.0 mg·kg-1, s.c.) also produced changes in haemodynamic parameters, most notably a 10- to 20-mmHg reduction in systolic and diastolic arterial blood pressure, at similar exposures. CONCLUSIONS AND IMPLICATIONS: Acute pharmacological inhibition of NaV1.7 is antinociceptive, but also has the potential to impact the cardiovascular system. Further work is merited to understand the role of NaV1.7 in autonomic ganglia involved in the control of heart rate and blood pressure, and the effect of selective NaV1.7 inhibition on cardiovascular function.
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Canal de Sodio Activado por Voltaje NAV1.7 , Animales , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Masculino , Humanos , Femenino , Reflejo/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Relación Dosis-Respuesta a DrogaRESUMEN
NaV1.7 plays a crucial role in inducing and conducting action potentials in pain-transducing sensory nociceptor fibres, suggesting that NaV1.7 blockers could be effective non-opioid analgesics. While SCN9A is expressed in both sensory and autonomic neurons, its functional role in the autonomic system remains less established. Our single neuron rt-PCR analysis revealed that 82% of sympathetic neurons isolated from guinea-pig stellate ganglia expressed NaV1.7 mRNA, with NaV1.3 being the only other tetrodotoxin-sensitive channel expressed in approximately 50% of neurons. We investigated the role of NaV1.7 in conducting action potentials in postganglionic sympathetic nerves and in the sympathetic adrenergic contractions of blood vessels using selective NaV1.7 inhibitors. Two highly selective NaV1.7 blockers, GNE8493 and PF 05089771, significantly inhibited postganglionic compound action potentials by approximately 70% (P < 0.01), with residual activity being blocked by the NaV1.3 inhibitor, ICA 121431. Electrical field stimulation (EFS) induced rapid contractions in guinea-pig isolated aorta, pulmonary arteries, and human isolated pulmonary arteries via stimulation of intrinsic nerves, which were inhibited by prazosin or the NaV1 blocker tetrodotoxin. Our results demonstrated that blocking NaV1.7 with GNE8493, PF 05089771, or ST2262 abolished or strongly inhibited sympathetic adrenergic responses in guinea-pigs and human vascular smooth muscle. These findings support the hypothesis that pharmacologically inhibiting NaV1.7 could potentially reduce sympathetic and parasympathetic function in specific vascular beds and airways. KEY POINTS: 82% of sympathetic neurons isolated from the stellate ganglion predominantly express NaV1.7 mRNA. NaV1.7 blockers inhibit action potential conduction in postganglionic sympathetic nerves. NaV1.7 blockade substantially inhibits sympathetic nerve-mediated adrenergic contractions in human and guinea-pig blood vessels. Pharmacologically blocking NaV1.7 profoundly affects sympathetic and parasympathetic responses in addition to sensory fibres, prompting exploration into the broader physiological consequences of NaV1.7 mutations on autonomic nerve activity.
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Canal de Sodio Activado por Voltaje NAV1.7 , Animales , Cobayas , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/fisiología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Humanos , Masculino , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Fibras Simpáticas Posganglionares/fisiología , Fibras Simpáticas Posganglionares/efectos de los fármacos , Femenino , Arterias/fisiología , Arterias/efectos de los fármacos , Arterias/inervación , Bloqueadores de los Canales de Sodio/farmacología , Ganglio Estrellado/fisiología , Sistema Nervioso Simpático/fisiología , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
Pulpitis constitutes a significant challenge in clinical management due to its impact on peripheral nerve tissue and the persistence of chronic pain. Despite its clinical importance, the correlation between neuronal activity and the expression of voltage-gated sodium channel 1.7 (Nav1.7) in the trigeminal ganglion (TG) during pulpitis is less investigated. The aim of this study was to examine the relationship between experimentally induced pulpitis and Nav1.7 expression in the TG and to investigate the potential of selective Nav1.7 modulation to attenuate TG abnormal activity associated with pulpitis. Acute pulpitis was induced at the maxillary molar (M1) using allyl isothiocyanate (AITC). The mice were divided into three groups: control, pulpitis model, and pulpitis model treated with ProTx-II, a selective Nav1.7 channel inhibitor. After three days following the surgery, we conducted a recording and comparative analysis of the neural activity of the TG utilizing in vivo optical imaging. Then immunohistochemistry and Western blot were performed to assess changes in the expression levels of extracellular signal-regulated kinase (ERK), c-Fos, collapsin response mediator protein-2 (CRMP2), and Nav1.7 channels. The optical imaging result showed significant neurological excitation in pulpitis TGs. Nav1.7 expressions exhibited upregulation, accompanied by signaling molecular changes suggestive of inflammation and neuroplasticity. In addition, inhibition of Nav1.7 led to reduced neural activity and subsequent decreases in ERK, c-Fos, and CRMP2 levels. These findings suggest the potential for targeting overexpressed Nav1.7 channels to alleviate pain associated with pulpitis, providing practical pain management strategies.
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Canal de Sodio Activado por Voltaje NAV1.7 , Pulpitis , Animales , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Ratones , Masculino , Pulpitis/metabolismo , Pulpitis/patología , Ganglio del Trigémino/metabolismo , Neuronas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Modelos Animales de Enfermedad , Péptidos y Proteínas de Señalización IntercelularAsunto(s)
Potenciales de Acción , Canal de Sodio Activado por Voltaje NAV1.7 , Sistema Nervioso Simpático , Canal de Sodio Activado por Voltaje NAV1.7/fisiología , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Animales , Potenciales de Acción/fisiología , Sistema Nervioso Simpático/fisiología , HumanosRESUMEN
BACKGROUND: Dravet syndrome is an infantile-onset developmental and epileptic encephalopathy (DEE) characterized by drug resistance, intractable seizures, and developmental comorbidities. This article focuses on manifestations in two Indonesian children with Javanese ethnicity who experienced Dravet syndrome with an SCN1A gene mutation, presenting genetic analysis findings using next-generation sequencing. CASE PRESENTATION: We present a case series involving two Indonesian children with Javanese ethnicity whom had their first febrile seizure at the age of 3 months, triggered after immunization. Both patients had global developmental delay and intractable seizures. We observed distinct genetic findings in both our cases. The first patient revealed heterozygous deletion mutation in three genes (TTC21B, SCN1A, and SCN9A). In our second patient, previously unreported mutation was discovered at canonical splice site upstream of exon 24 of the SCN1A gene. Our patient's outcomes improved after therapeutic evaluation based on mutation findings When comparing clinical manifestations in our first and second patients, we found that the more severe the genetic mutation discovered, the more severe the patient's clinical manifestations. CONCLUSION: These findings emphasize the importance of comprehensive genetic testing beyond SCN1A, providing valuable insights for personalized management and tailored therapeutic interventions in patients with Dravet syndrome. Our study underscores the potential of next-generation sequencing in advancing genotype-phenotype correlations and enhancing diagnostic precision for effective disease management.
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Epilepsias Mioclónicas , Canal de Sodio Activado por Voltaje NAV1.1 , Humanos , Epilepsias Mioclónicas/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Masculino , Femenino , Lactante , Canal de Sodio Activado por Voltaje NAV1.7/genética , Indonesia , Anticonvulsivantes/uso terapéutico , Mutación , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , PreescolarRESUMEN
Nociceptive sensory neurons convey pain-related signals to the CNS using action potentials. Loss-of-function mutations in the voltage-gated sodium channel NaV1.7 cause insensitivity to pain (presumably by reducing nociceptor excitability) but clinical trials seeking to treat pain by inhibiting NaV1.7 pharmacologically have struggled. This may reflect the variable contribution of NaV1.7 to nociceptor excitability. Contrary to claims that NaV1.7 is necessary for nociceptors to initiate action potentials, we show that nociceptors can achieve similar excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8. Selectively blocking one of those NaV subtypes reduces nociceptor excitability only if the other subtypes are weakly expressed. For example, excitability relies on NaV1.8 in acutely dissociated nociceptors but responsibility shifts to NaV1.7 and NaV1.3 by the fourth day in culture. A similar shift in NaV dependence occurs in vivo after inflammation, impacting ability of the NaV1.7-selective inhibitor PF-05089771 to reduce pain in behavioral tests. Flexible use of different NaV subtypes exemplifies degeneracy - achieving similar function using different components - and compromises reliable modulation of nociceptor excitability by subtype-selective inhibitors. Identifying the dominant NaV subtype to predict drug efficacy is not trivial. Degeneracy at the cellular level must be considered when choosing drug targets at the molecular level.
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Analgésicos , Bencenosulfonamidas , Nociceptores , Éteres Fenílicos , Animales , Analgésicos/farmacología , Nociceptores/metabolismo , Nociceptores/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Ratones , Potenciales de Acción/efectos de los fármacos , Dolor/tratamiento farmacológico , Humanos , Canales de Sodio/metabolismo , Canales de Sodio/genética , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8/genéticaAsunto(s)
Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Humanos , Enfermedades del Sistema Nervioso Autónomo/fisiopatología , Bloqueadores de los Canales de Sodio/farmacología , Bloqueadores de los Canales de Sodio/uso terapéutico , Masculino , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/uso terapéuticoRESUMEN
ETHNOPHARMACOLOGY RELEVANCE: Zanthoxylum bungeanum Maxim. (Z. bungeanum), a member of the Rutaceae family, has a rich history of traditional use in Asia for treating arthritis and toothache conditions. As characteristic chemical components, numerous kinds of alkaloids have been extracted from plants and their diverse biological activities have been reported. However, research on the isoquinoline alkaloid, a specific type of alkaloids, in Z. bungeanum was scarce. AIM OF THE STUDY: The study aimed to isolate a novel isoquinoline alkaloid from Z. bungeanum and explore its pharmacological activity in vitro and analgesic activity in vivo. MATERIALS AND METHODS: Isoquinoline alkaloid isolation and identification from Z. bungeanum were conducted using chromatographic and spectroscopic methods. The whole-cell patch-clamp technique was applied to assess its impact on neuronal excitability, and endogenous voltage-gated potassium (Kv) and sodium (Nav) currents in acutely isolated mouse small-diameter dorsal root ganglion (DRG) neurons. Its inhibitory impacts on channels were further validated with HEK293 cells stably expressing Nav1.7 and Nav1.8, and Chinese hamster ovary (CHO) cells transiently expressing Kv2.1. The formalin inflammatory pain model was utilized to evaluate the potential analgesic activity in vivo. RESULTS: A novel isoquinoline alkaloid named HJ-69 (N-13-(3-methoxyprop-1-yl)rutaecarpine) was isolated and identified from Z. bungeanum for the first time. HJ-69 significantly suppressed the firing frequency and amplitudes of action potentials in DRG neurons. Consistently, it state-dependently inhibited endogenous Nav currents of DRG neurons, with half maximal inhibitory concentration (IC50) values of 13.06 ± 2.06 µM and 30.19 ± 2.07 µM for the inactivated and resting states, respectively. HJ-69 significantly suppressed potassium currents in DRG neurons, which notably inhibited the delayed rectifier potassium (IK) currents (IC50 = 6.95 ± 1.29 µM) and slightly affected the transient outward potassium (IA) currents (IC50 = 523.50 ± 39.16 µM). Furtherly, HJ-69 exhibited similar potencies on heterologously expressed Nav1.7, Nav1.8, and Kv2.1 channels, which correspondingly represent the main components in neurons. Notably, intraperitoneal administration of 30 mg/kg and 100 mg/kg HJ-69 significantly alleviated pain behaviors in the mouse inflammatory pain model induced by formalin. CONCLUSION: The study concluded that HJ-69 is a novel and active isoquinoline alkaloid, and the inhibition of Nav and Kv channels contributes to its analgesic activity. HJ-69 may be a promising prototype for future analgesic drug discovery based on the isoquinoline alkaloid.