RESUMO
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.
Assuntos
Antitussígenos , Tosse , Regulação para Baixo , Flores , Ginsenosídeos , Canal de Sódio Disparado por Voltagem NAV1.7 , Panax notoginseng , Canais de Cátion TRPV , Animais , Canais de Cátion TRPV/metabolismo , Cobaias , Flores/química , Tosse/tratamento farmacológico , Ginsenosídeos/farmacologia , Antitussígenos/farmacologia , Masculino , Camundongos , Panax notoginseng/química , Regulação para Baixo/efeitos dos fármacos , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Células HEK293 , Simulação de Acoplamento Molecular , Cricetulus , Modelos Animais de Doenças , Células CHO , Saponinas/farmacologia , OvalbuminaRESUMO
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.
Assuntos
Inteligência Artificial , Descoberta de Drogas , Simulação de Acoplamento Molecular , Descoberta de Drogas/métodos , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/química , Ligação Proteica , Cristalografia por Raios X , Ligantes , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodosRESUMO
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.
Assuntos
Dor , Células Receptoras Sensoriais , Animais , Células Receptoras Sensoriais/metabolismo , Camundongos , Masculino , Feminino , Dor/metabolismo , Biossíntese de Proteínas , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Glutamato Descarboxilase/metabolismo , Glutamato Descarboxilase/genética , Polirribossomos/metabolismo , Camundongos Endogâmicos C57BL , Gânglios Espinais/metabolismoRESUMO
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.
Assuntos
Analgésicos , Gânglios Espinais , Canal de Sódio Disparado por Voltagem NAV1.7 , Neurônios , Tetrodotoxina , Animais , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/citologia , Neurônios/efeitos dos fármacos , Camundongos , Tetrodotoxina/farmacologia , Analgésicos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Venenos de Crotalídeos/toxicidade , Venenos de Crotalídeos/farmacologia , Masculino , Crotalus , Potenciais da Membrana/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , PeptídeosRESUMO
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.
Assuntos
Membrana Celular , Colesterol , Simulação de Dinâmica Molecular , Canal de Sódio Disparado por Voltagem NAV1.7 , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Humanos , Colesterol/metabolismo , Colesterol/química , Membrana Celular/metabolismo , Membrana Celular/química , Células HEK293 , Ativação do Canal IônicoRESUMO
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.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Bloqueadores do Canal de Sódio Disparado por Voltagem , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Dor do Câncer/tratamento farmacológico , Dor do Câncer/metabolismo , Analgésicos/química , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Relação Estrutura-Atividade , Manejo da Dor/métodos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/uso terapêuticoRESUMO
(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.
Assuntos
Atractylodes , Modelos Animais de Doenças , Síndrome do Intestino Irritável , Canais de Cátion TRPV , Zimosan , Animais , Síndrome do Intestino Irritável/tratamento farmacológico , Síndrome do Intestino Irritável/induzido quimicamente , Canais de Cátion TRPV/metabolismo , Camundongos , Atractylodes/química , Masculino , Extratos Vegetais/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos dos fármacosRESUMO
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.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Animais , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Masculino , Humanos , Feminino , Reflexo/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Relação Dose-Resposta a DrogaRESUMO
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.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Pulpite , Animais , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Camundongos , Masculino , Pulpite/metabolismo , Pulpite/patologia , Gânglio Trigeminal/metabolismo , Neurônios/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
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.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Canal de Sódio Disparado por Voltagem NAV1.8 , Canal de Sódio Disparado por Voltagem NAV1.9 , Bloqueadores dos Canais de Sódio , Humanos , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Animais , Ratos , Canal de Sódio Disparado por Voltagem NAV1.9/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Bloqueadores dos Canais de Sódio/farmacologia , Células HEK293 , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Gânglios Espinais/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/citologiaRESUMO
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.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Animais , Cobaias , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Humanos , Masculino , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Fibras Simpáticas Pós-Ganglionares/fisiologia , Fibras Simpáticas Pós-Ganglionares/efeitos dos fármacos , Feminino , Artérias/fisiologia , Artérias/efeitos dos fármacos , Artérias/inervação , Bloqueadores dos Canais de Sódio/farmacologia , Gânglio Estrelado/fisiologia , Sistema Nervoso Simpático/fisiologia , Sistema Nervoso Simpático/efeitos dos fármacosRESUMO
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.
Assuntos
Dependovirus , Canal de Sódio Disparado por Voltagem NAV1.7 , Células Receptoras Sensoriais , Animais , Ratos , Dependovirus/genética , Células Receptoras Sensoriais/metabolismo , Masculino , Humanos , Feminino , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Gânglios Espinais/metabolismo , Ratos Sprague-Dawley , Neuralgia/metabolismo , Neuralgia/genética , Neuralgia/tratamento farmacológico , AnalgesiaAssuntos
Potenciais de Ação , Canal de Sódio Disparado por Voltagem NAV1.7 , Sistema Nervoso Simpático , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Animais , Potenciais de Ação/fisiologia , Sistema Nervoso Simpático/fisiologia , HumanosRESUMO
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.
Assuntos
Analgésicos , Gânglios Espinais , Dor , Zanthoxylum , Animais , Zanthoxylum/química , Humanos , Células HEK293 , Analgésicos/farmacologia , Analgésicos/química , Analgésicos/isolamento & purificação , Analgésicos/uso terapêutico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Camundongos , Masculino , Dor/tratamento farmacológico , Isoquinolinas/farmacologia , Isoquinolinas/isolamento & purificação , Isoquinolinas/química , Alcaloides/farmacologia , Alcaloides/isolamento & purificação , Alcaloides/química , Alcaloides/uso terapêutico , Bloqueadores dos Canais de Potássio/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Inflamação/tratamento farmacológico , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/química , Camundongos Endogâmicos C57BL , CricetulusRESUMO
Botulinum neurotoxin type A BoNT/A is used off-label as a third line therapy for neuropathic pain. However, the mechanism of action remains unclear. In recent years, the role of voltage-gated sodium channels (Nav) in neuropathic pain became evident and it was suggested that block of sodium channels by BoNT/A would contribute to its analgesic effect. We assessed sodium channel function in the presence of BoNT/A in heterologously expressed Nav1.7, Nav1.3, and the neuronal cell line ND7/23 by high throughput automated and manual patch-clamp. We used both the full protein and the isolated catalytic light chain LC/A for acute or long-term extracellular or intracellular exposure. To assess the toxin's effect in a human cellular system, we differentiated induced pluripotent stem cells (iPSC) into sensory neurons from a healthy control and a patient suffering from a hereditary neuropathic pain syndrome (inherited erythromelalgia) carrying the Nav1.7/p.Q875E-mutation and carried out multielectrode-array measurements. Both BoNT/A and the isolated catalytic light chain LC/A showed limited effects in heterologous expression systems and the neuronal cell line ND7/23. Spontaneous activity in iPSC derived sensory neurons remained unaltered upon BoNT/A exposure both in neurons from the healthy control and the mutation carrying patient. BoNT/A may not specifically be beneficial in pain syndromes linked to sodium channel variants. The favorable effects of BoNT/A in neuropathic pain are likely based on mechanisms other than sodium channel blockage and new approaches to understand BoNT/A's therapeutic effects are necessary.
Assuntos
Toxinas Botulínicas Tipo A , Células-Tronco Pluripotentes Induzidas , Canal de Sódio Disparado por Voltagem NAV1.7 , Neuralgia , Humanos , Neuralgia/tratamento farmacológico , Toxinas Botulínicas Tipo A/farmacologia , Toxinas Botulínicas Tipo A/uso terapêutico , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Analgésicos/farmacologia , Animais , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células HEK293 , Linhagem CelularRESUMO
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.
Assuntos
Analgésicos , Benzenossulfonamidas , Nociceptores , Éteres Fenílicos , Animais , Analgésicos/farmacologia , Nociceptores/metabolismo , Nociceptores/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Camundongos , Potenciais de Ação/efeitos dos fármacos , Dor/tratamento farmacológico , Humanos , Canais de Sódio/metabolismo , Canais de Sódio/genética , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/genéticaAssuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Humanos , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Masculino , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Canal de Sódio Disparado por Voltagem NAV1.7 , Células Receptoras Sensoriais , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação , Sistemas CRISPR-CasRESUMO
Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.