RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Chronic pain management represents a serious healthcare problem worldwide. The use of opioid analgesics for pain has always been hampered by their side effects; in particular, the addictive liability associated with chronic use. Finding a morphine replacement has been a long-standing goal in the field of analgesia. In traditional Chinese medicine, processed Buthus martensii Karsch (BmK) scorpion has been used as a painkiller to treat chronic inflammatory arthritis and spondylitis, so called "Scorpio-analgesia". However, the molecular basis and the underline mechanism for the Scorpio-analgesia are still unclear. AIM OF THE STUDY: The study aims to investigate the molecular basis of "Scorpio analgesia" and identify novel analgesics from BmK scorpion. MATERIALS AND METHODS: In this study, the analgesic abilities were determined using formalin-, acetic acid- and complete Freund's adjuvant-induced pain models. The effect of BmK venom and processed BmK venom on Nav1.7 were detected by whole-cell voltage-clamp recordings on HEK293-hNav1.7 stable cell line. Action potentials in Dorsal root ganglion (DRG) neurons induced by Makatoxin-3-R58A were recorded in current-clamp mode. The content of Makatoxin-3 was detected using competitive enzyme-linked immunosorbent assay based on the Makatoxin-3 antibody. High performance liquid chromatography, western blot and circular dichroism spectroscopy were used to analysis the stability of Makatoxin-3. RESULTS: Here we demonstrate that Makatoxin-3, an α-like toxin in BmK scorpion venom targeting Nav1.7 is the critical component in Scorpio-analgesia. The analgesic effect of Makatoxin-3 could not be reversed by naloxone and is more potent than Nav1.7-selective inhibitors and non-steroidal anti-inflammatory drugs in inflammatory models. Moreover, a R58A mutant of Makatoxin-3 is capable of eliciting analgesia effect without inducing pain response. CONCLUSIONS: This study advances ion channel biology and proposes Nav1.7 agonists, rather than the presumed Nav1.7-only blockers, for non-narcotic relief of chronic pain.
Assuntos
Analgésicos/farmacologia , Inflamação/tratamento farmacológico , Dor/tratamento farmacológico , Venenos de Escorpião/farmacologia , Potenciais de Ação/efeitos dos fármacos , Analgésicos/isolamento & purificação , Animais , Modelos Animais de Doenças , Adjuvante de Freund , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Dor/patologia , Agonistas do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Agonistas do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Dor/tratamento farmacológico , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/farmacologia , Venenos de Aranha/síntese química , Animais , Degranulação Celular/efeitos dos fármacos , Cistina/química , Desenho de Fármacos , Temperatura Alta , Mastócitos/efeitos dos fármacos , Modelos Moleculares , Medição da Dor/efeitos dos fármacos , Ratos , Venenos de Aranha/farmacologiaRESUMO
Gating modifier toxins (GMTs) isolated from venomous organisms such as Protoxin-II (ProTx-II) and Huwentoxin-IV (HwTx-IV) that inhibit the voltage-gated sodium channel NaV1.7 by binding to its voltage-sensing domain II (VSDII) have been extensively investigated as non-opioid analgesics. However, reliably predicting how a mutation to a GMT will affect its potency for NaV1.7 has been challenging. Here, we hypothesize that structure-based computational methods can be used to predict such changes. We employ free-energy perturbation (FEP), a physics-based simulation method for predicting the relative binding free energy (RBFE) between molecules, and the cryo electron microscopy (cryo-EM) structures of ProTx-II and HwTx-IV bound to VSDII of NaV1.7 to re-predict the relative potencies of forty-seven point mutants of these GMTs for NaV1.7. First, FEP predicted these relative potencies with an overall root mean square error (RMSE) of 1.0 ± 0.1 kcal/mol and an R2 value of 0.66, equivalent to experimental uncertainty and an improvement over the widely used molecular-mechanics/generalized born-surface area (MM-GB/SA) RBFE method that had an RMSE of 3.9 ± 0.8 kcal/mol. Second, inclusion of an explicit membrane model was needed for the GMTs to maintain stable binding poses during the FEP simulations. Third, MM-GB/SA and FEP were used to identify fifteen non-standard tryptophan mutants at ProTx-II[W24] predicted in silico to have a at least a 1 kcal/mol gain in potency. These predicted potency gains are likely due to the displacement of high-energy waters as identified by the WaterMap algorithm for calculating the positions and thermodynamic properties of water molecules in protein binding sites. Our results expand the domain of applicability of FEP and set the stage for its prospective use in biologics drug discovery programs involving GMTs and NaV1.7.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Peptídeos/toxicidade , Venenos de Aranha/toxicidade , Bloqueadores do Canal de Sódio Disparado por Voltagem/toxicidade , Sítios de Ligação , Simulação por Computador , Microscopia Crioeletrônica , Modelos Moleculares , Mutação , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Relação Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismoRESUMO
Human voltage-gated sodium channel Nav1.7 (hNav1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNav1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Nav selectivity of hNav1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNav1.7. Site-directed mutagenesis analysis indicated key residues on hNav1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNav1.7 with a 30-fold improved potency (IC50 0.007 ± 0.001 µM) and >1000-fold selectivity against Nav1.4 and Nav1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Nav subtypes with robust analgesia.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/genética , Dor Nociceptiva/tratamento farmacológico , Peptídeos/genética , Venenos de Aranha/química , Animais , Humanos , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.5/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Dor Nociceptiva/genética , Dor Nociceptiva/patologia , Peptídeos/química , Peptídeos/farmacologia , Venenos de Aranha/genéticaRESUMO
ETHNOPHARMACOLOGY RELEVANCE: Pain is an unpleasant sensory and emotional experience, often accompanied by the occurrence of a variety of diseases. More than 800 kinds of traditional Chinese medicines (TCM) has now been reported for pain relief and several monomers have been developed into novel analgesic drugs. Bupleurum chinense and Angelica biserrata were representatives of the TCM that are currently available for the treatment of pain. AIM OF THE STUDY: The study aims to detect the potential analgesic activity of each monomer of Bupleurum chinense and Angelica biserrata and to explore whether Nav1.7 is one of the targets for its analgesic activity. MATERIALS AND METHODS: In this study, five monomers from Bupleurum chinense (Saikosaponin A, Saikosaponin B1, Saikosaponin B2, Saikosaponin C, Saikosaponin D) and five monomers from the Angelica biserrata (Osthole, Xanthotoxin, Imperatorin, Isoimperatorin, Psoralen) were examined by whole-cell patch-clamp on Nav1.7, which was closely associated with pain. Classical mouse pain models were also used to further verify the analgesic activity in vivo. RESULTS: The results showed that monomers of Saikosaponins and Angelica biserrata all inhibited the peak currents of Nav1.7, indicating that Nav1.7 might be involved in the analgesic mechanism of Saikosaponins and Angelica biserrata. Among them, Saikosaponin A and Imperatorin showed the strongest inhibitory effect on Nav1.7. Furthermore, both Saikosaponin A and Imperatorin showed inhibitory effects on thermal pain and formalin-induced pain in phase II in vivo. CONCLUSION: The results provide valuable information for future studies on the potential of TCM in alleviating pain.
Assuntos
Analgésicos/farmacologia , Angelica/química , Bupleurum/química , Medicamentos de Ervas Chinesas/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Dor/tratamento farmacológico , Analgésicos/química , Analgésicos/uso terapêutico , Animais , Células CHO , Cricetulus , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Formaldeído/toxicidade , Furocumarinas/farmacologia , Furocumarinas/uso terapêutico , Temperatura Alta/efeitos adversos , Masculino , Medicina Tradicional Chinesa , Camundongos , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Dor/etiologia , Raízes de Plantas/química , Saponinas/farmacologia , Saponinas/uso terapêutico , Sódio/fisiologiaRESUMO
Voltage-gated sodium channels are critical for the generation and propagation of action potentials. Gating modifier toxins from spider venom can modulate the gating mechanism of sodium channels and thus have potential as drug leads. Here, we established expression of the gating modifier toxin PaurTx-3, a sodium channel inhibitor found in the venom of the spider Phrixotrichus auratus. Whole-cell voltage-clamp recordings indicated that recombinant PaurTx-3 (rPaurTx-3) inhibited Nav1.4, Nav1.5, and Nav1.7 currents with IC50 values of 61 nM, 72 nM, and 25 nM, respectively. Furthermore, rPaurTx-3 irreversibly inhibited Nav1.7 currents, but had 60-70% recovery in Nav1.4 and Nav1.5 after washing with a bath solution. rPaurTx-3 also hyperpolarized the voltage-dependent steady-state inactivation curve and significantly slowed recovery from fast inactivation of Nav1.7. Current-clamp recordings showed that rPaurTx-3 suppressed small DRG neuron activity. The biological activity assay findings for rPaurTx-3 support its potent pharmacological effect in Nav1.7 and small DRG neurons.
Assuntos
Proteínas de Artrópodes/toxicidade , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Venenos de Aranha/toxicidade , Bloqueadores do Canal de Sódio Disparado por Voltagem/toxicidade , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Gânglios Espinais/citologia , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Técnicas de Patch-Clamp , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidade , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Alinhamento de Sequência , Canais de Sódio/genética , Canais de Sódio/metabolismo , Venenos de Aranha/genética , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
PURPOSE: This work describes a staged approach to the application of pharmacokinetic-pharmacodynamic (PK-PD) modeling in the voltage-gated sodium ion channel (NaV1.7) inhibitor drug discovery effort to address strategic questions regarding in vitro to in vivo translation of target modulation. METHODS: PK-PD analysis was applied to data from a functional magnetic resonance imaging (fMRI) technique to non-invasively measure treatment mediated inhibition of olfaction signaling in non-human primates (NHPs). Initial exposure-response was evaluated using single time point data pooled across 27 compounds to inform on in vitro to in vivo correlation (IVIVC). More robust effect compartment PK-PD modeling was conducted for a subset of 10 compounds with additional PD and PK data to characterize hysteresis. RESULTS: The pooled compound exposure-response facilitated an early exploration of IVIVC with a limited dataset for each individual compound, and it suggested a 2.4-fold in vitro to in vivo scaling factor for the NaV1.7 target. Accounting for hysteresis with an effect compartment PK-PD model as compounds advanced towards preclinical development provided a more robust determination of in vivo potency values, which resulted in a statistically significant positive IVIVC with a slope of 1.057 ± 0.210, R-squared of 0.7831, and p value of 0.006. Subsequent simulations with the PK-PD model informed the design of anti-nociception efficacy studies in NHPs. CONCLUSIONS: A staged approach to PK-PD modeling and simulation enabled integration of in vitro NaV1.7 potency, plasma protein binding, and pharmacokinetics to describe the exposure-response profile and inform future study design as the NaV1.7 inhibitor effort progressed through drug discovery.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Algoritmos , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Circulação Cerebrovascular , Desenho de Fármacos , Descoberta de Drogas , Células HEK293 , Humanos , Técnicas In Vitro , Macaca mulatta , Imageamento por Ressonância Magnética , Modelos Biológicos , Olfato/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacocinéticaRESUMO
Background: Women with temporomandibular disorders (TMDs) experience some amelioration of pain during pregnancy. Progesterone increases dramatically and steadily during pregnancy. Sodium channel 1.7 (Nav1.7) plays a prominent role in pain perceptions, as evidenced by deletion of Nav1.7 alone leading to a complete loss of pain. In a previous study, we showed that Nav1.7 in trigeminal ganglion (TG) is involved in allodynia of inflamed temporomandibular joint (TMJ). Whether progesterone modulates allodynia of inflamed TMJ through Nav1.7 in TG remains to be investigated. Methods: The effects of progesterone on sodium currents of freshly isolated TG neurons were examined using whole-cell recording. Female rats were ovariectomized and treated with increasing doses of progesterone for 10 days. Complete Freund's adjuvant was administered intra-articularly to induce TMJ inflammation. TMJ nociceptive responses were evaluated by head withdrawal thresholds. Real-time PCR and Western blotting were used to examine Nav1.7 mRNA and protein expression in TG. Immunohistofluorescence was used to examine the colocalization of progesterone receptors (PRα/ß) and Nav1.7 in TG. Results: Whole-cell recording showed that progesterone could attenuate sodium currents. Moreover, progesterone dose-dependently downregulated Nav1.7 mRNA expression and reduced the sensitivity of TMJ nociception in ovariectomized rats. Furthermore, treatment with progesterone attenuated allodynia of inflamed TMJ in a dose-dependent manner and repressed inflammation-induced Nav1.7 mRNA and protein expression in ovariectomized rats. The progesterone receptor antagonist, RU-486, partially reversed the effect of progesterone on allodynia of inflamed TMJ and TMJ inflammation-induced Nav1.7 mRNA and protein expression. Conclusion: Progesterone, by modulating trigeminal ganglionic Nav1.7, may represent a promising agent to prevent allodynia of inflamed TMJ.
Assuntos
Hiperalgesia/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Progesterona/farmacologia , Transtornos da Articulação Temporomandibular/metabolismo , Gânglio Trigeminal/efeitos dos fármacos , Animais , Feminino , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nociceptividade/efeitos dos fármacos , Gravidez , Progesterona/metabolismo , Ratos , Ratos Sprague-Dawley , Gânglio Trigeminal/metabolismoRESUMO
BACKGROUND: Voltage-gated sodium channel Nav1.7 has been validated as a perspective target for selective inhibitors with analgesic and anti-itch activity. The objective of this study was to discover new candidate compounds with Nav1.7 inhibitor properties. The authors hypothesized that their approach would yield at least one new compound that inhibits sodium currents in vitro and exerts analgesic and anti-itch effects in mice. METHODS: In silico structure-based similarity search of 1.5 million compounds followed by docking to the Nav1.7 voltage sensor of Domain 4 and molecular dynamics simulation was performed. Patch clamp experiments in Nav1.7-expressing human embryonic kidney 293 cells and in mouse and human dorsal root ganglion neurons were conducted to test sodium current inhibition. Formalin-induced inflammatory pain model, paclitaxel-induced neuropathic pain model, histamine-induced itch model, and mouse lymphoma model of chronic itch were used to confirm in vivo activity of the selected compound. RESULTS: After in silico screening, nine compounds were selected for experimental assessment in vitro. Of those, four compounds inhibited sodium currents in Nav1.7-expressing human embryonic kidney 293 cells by 29% or greater (P < 0.05). Compound 9 (3-(1-benzyl-1H-indol-3-yl)-3-(3-phenoxyphenyl)-N-(2-(pyrrolidin-1-yl)ethyl)propanamide, referred to as DA-0218) reduced sodium current by 80% with a 50% inhibition concentration of 0.74 µM (95% CI, 0.35 to 1.56 µM), but had no effects on Nav1.5-expressing human embryonic kidney 293 cells. In mouse and human dorsal root ganglion neurons, DA-0218 reduced sodium currents by 17% (95% CI, 6 to 28%) and 22% (95% CI, 9 to 35%), respectively. The inhibition was greatly potentiated in paclitaxel-treated mouse neurons. Intraperitoneal and intrathecal administration of the compound reduced formalin-induced phase II inflammatory pain behavior in mice by 76% (95% CI, 48 to 100%) and 80% (95% CI, 68 to 92%), respectively. Intrathecal administration of DA-0218 produced acute reduction in paclitaxel-induced mechanical allodynia, and inhibited histamine-induced acute itch and lymphoma-induced chronic itch. CONCLUSIONS: This study's computer-aided drug discovery approach yielded a new Nav1.7 inhibitor that shows analgesic and anti-pruritic activity in mouse models.
Assuntos
Analgésicos/uso terapêutico , Desenho de Fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Prurido/tratamento farmacológico , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Goshajinkigan (GJG), a traditional Japanese Kampo formula, has been shown to exhibit several pharmacological actions, including antinociceptive effects. Processed aconite root (PA), which is considered to be an active ingredient of GJG, has also been demonstrated to have an ameliorative effect on pain, such as diabetic peripheral neuropathic pain. We recently identified neoline as the active ingredient of both GJG and PA that is responsible for its effects against oxaliplatin-induced neuropathic pain in mice. AIM OF THE STUDY: In the present study, we investigated whether GJG, PA, and neoline could inhibit Nav1.7 voltage-gated sodium channel (VGSC) current and whether neoline could ameliorate mechanical hyperalgesia in diabetic mice. MATERIALS AND METHODS: To assess the electrophysiological properties of GJG extract formulation, powdered PA, and neoline on Nav1.7 VGSCs, whole-cell patch clamp recording was performed using human HEK293 cells expressing Nav1.7 VGSCs. In addition, the ameliorative effects of neoline on diabetic peripheral neuropathic pain were evaluated using the von Frey test in streptozotocin (STZ)-induced diabetic model mice. RESULTS: GJG extract formulation significantly inhibited Nav1.7 VGSC peak current. Powdered PA also inhibited Nav1.7 VGSC peak current. Like GJG and PA, neoline could inhibit Nav1.7 VGSC current. When diabetic mice were treated with neoline by intraperitoneal acute administration, the mechanical threshold was increased in diabetic mice, but not in non-diabetic mice, in a behavioral study. CONCLUSION: These results suggest that neoline might be a novel active ingredient of GJG and PA that is one of responsible ingredients for ameliorating mechanical hyperalgesia in diabetes via the inhibition of Nav1.7 VGSC current at least.
Assuntos
Aconitina/análogos & derivados , Aconitum , Analgésicos/farmacologia , Neuropatias Diabéticas/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Hiperalgesia/prevenção & controle , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Raízes de Plantas , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Aconitina/isolamento & purificação , Aconitina/farmacologia , Aconitum/química , Analgésicos/isolamento & purificação , Animais , Comportamento Animal/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/fisiopatologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Células HEK293 , Humanos , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Potenciais da Membrana , Camundongos Endogâmicos ICR , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Limiar da Dor/efeitos dos fármacos , Raízes de Plantas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificaçãoRESUMO
The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.
Assuntos
Ensaios de Triagem em Larga Escala , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Técnicas de Patch-Clamp , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Animais , Células CHO , Cricetulus , Fenômenos Eletrofisiológicos , Eletrofisiologia , Células HEK293 , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/genéticaRESUMO
Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of "tiger" spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product's diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.
Assuntos
Ácido Aspártico/química , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Neurotoxinas/farmacologia , Venenos de Aranha/farmacologia , Agonistas do Canal de Sódio Disparado por Voltagem/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Descoberta de Drogas , Humanos , Isomerismo , Potenciais da Membrana , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Neurotoxinas/química , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Venenos de Aranha/química , Relação Estrutura-Atividade , Agonistas do Canal de Sódio Disparado por Voltagem/químicaRESUMO
Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (NaV1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for NaV1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human NaV1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for NaV1.7, examined alongside off-target NaVs, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at NaV1.7 and affinity for lipid bilayers. In a murine NaV1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target NaV1.7 and with an in vivo efficacy similar to that of native HwTx-IV.
Assuntos
Bicamadas Lipídicas/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Nociceptividade/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Venenos de Escorpião/toxicidadeRESUMO
Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S-Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 µm, and Nav1.5 is inactive at 10 µm We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.
Assuntos
Analgésicos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Venenos de Aranha/química , Venenos de Aranha/genética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Sequência de Aminoácidos/genética , Desenvolvimento de Medicamentos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Mutagênese , Canal de Sódio Disparado por Voltagem NAV1.2/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/tratamento farmacológico , Biblioteca de Peptídeos , Mutação Puntual , Engenharia de Proteínas , Isoformas de Proteínas , Proteínas Recombinantes , Venenos de Aranha/isolamento & purificaçãoRESUMO
It has been reported that Heteropodatoxin3 (HpTx3), a peptidic neurotoxin purified from the venom of the spider species Heteropoda venatoria, could inhibit Kv4.2 channels. Our present study newly found that HpTx3 also has potent and selective inhibitory action on Nav1.7, with an IC50 of 135.61 ± 12.98 nM. Without effect on the current-voltage (I-V) relationship of Nav1.7, HpTx3 made minor alternation in the voltage-dependence of activation and steady-state inactivation of Nav1.7 (4.15 mV and 7.29 mV, respectively) by interacting with the extracellular S3-S4 loop (S3b-S4 sequence) in domain II and the domain IV of the Nav channel subtype, showing the characteristics of both pore blocker and gate modifier toxin. During the interaction of HpTx3 with the S3b-S4 sequence of Nav1.7, the amino acid residue D in the sequence played a key role. When administered intraperitoneally or intramuscularly, HpTx3 displayed potent analgesic activity in a dose-dependent manner in different mouse pain models induced by formalin, acetic acid, complete Freund's adjuvant, hot plate, or spared nerve injury, demonstrating that acute, inflammatory, and neuropathic pains were all effectively inhibited by the toxin. In most cases HpTx3 at doses of ≥ 1mg/kg could produce the analgesic effect comparable to that of 1 mg/kg morphine. These results suggest that HpTx3 not only can be used as a molecular probe to investigate ion channel function and pain mechanism, but also has potential in the development of the drugs that treat the Nav1.7 channel-related pain.
Assuntos
Analgésicos não Narcóticos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Venenos de Aranha/farmacologia , Analgésicos Opioides/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Relação Dose-Resposta a Droga , Injeções Intramusculares , Injeções Intraperitoneais , Camundongos , Morfina/farmacologia , Neuralgia/tratamento farmacológico , Medição da Dor/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVE: Current pain therapies often do not provide adequate pain relief and have dose-limiting adverse effects. Genetic evidence indicates that NaV1.7 sodium channels are required for pain transduction and therefore represent an important therapeutic target. GDC-0276 is a novel NaV1.7 inhibitor developed for the treatment of pain. This first-in-human trial evaluated the safety, tolerability, and pharmacokinetics of orally administered GDC-0276 in healthy subjects. METHODS: This phase I, randomized, double-blind, placebo-controlled study assessed GDC-0276 as powder-in-capsule (PIC) or cyclodextrin solution (CD) single doses (SDs) of 2-270 mg (seven cohorts) and 45-540 mg (five cohorts), respectively. Multiple (MD) PIC doses were administered as total daily doses of 15-540 mg divided into two or three doses/day, up to 10 or 14 days. Safety was assessed by monitoring adverse events (AEs), vital signs, physical examinations, electrocardiograms, and laboratory tests for up to 15 days after the last day of dosing. GDC-0276 plasma pharmacokinetics were also determined. RESULTS: Three stages included 183 randomized subjects. GDC-0276 plasma exposure increased with dose level for all stages. Exposure was higher in the SD-CD cohorts compared with the equivalent SD-PIC dose levels. SDs were adequately tolerated up to 270 mg (SD-PIC) and 360 mg (SD-CD). Hypotension limited tolerability in the 540-mg SD-CD cohort. Multiple PIC doses were tolerated up to 270 mg twice daily, however liver transaminase elevations were frequently observed. No deaths or serious AEs occurred. CONCLUSION: GDC-0276 exhibited a safety and pharmacokinetic profile that supports its future investigation as a potential therapeutic for pain.
Assuntos
Azetidinas , Benzamidas , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Dor/tratamento farmacológico , Bloqueadores dos Canais de Sódio , Adolescente , Adulto , Azetidinas/efeitos adversos , Azetidinas/farmacocinética , Azetidinas/farmacologia , Benzamidas/efeitos adversos , Benzamidas/farmacocinética , Benzamidas/farmacologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Placebos , Bloqueadores dos Canais de Sódio/administração & dosagem , Bloqueadores dos Canais de Sódio/efeitos adversos , Bloqueadores dos Canais de Sódio/farmacocinética , Adulto JovemRESUMO
Screening of 100 acylsulfonamides from the Bristol-Myers Squibb compound collection identified the C3-cyclohexyl indole 6 as a potent Nav1.7 inhibitor. Replacement of the C2 furanyl ring of 6 with a heteroaryl moiety or truncation of this group led to the identification of 4 analogs with hNav1.7 IC50 values under 50â¯nM. Fluorine substitution of the truncated compound 12 led to 34 with improved potency and isoform selectivity. The inverted indole 36 also maintained good activity. Both 34 and 36 exhibited favorable CYP inhibition profiles, good membrane permeability and a low efflux ratio and, therefore, represent new leads in the search for potent and selective Nav1.7 inhibitors to treat pain.
Assuntos
Descoberta de Drogas , Indóis/química , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Sulfonamidas/farmacologia , Humanos , Concentração Inibidora 50 , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Fenamates are N-substituted anthranilic acid derivatives, clinically used as nonsteroidal anti-inflammatory drugs (NSAIDs) in fever, pain and inflammation treatments. Previous studies have shown that they are also modulators of diverse ion channels, exhibiting either activation or inhibitory effects. However, the effects of fenamates on sodium channel subtypes are still unknown. In this study, fenamates, including mefenamic acid, flufenamic acid and tolfenamic acid, were examined by whole-cell patch clamp techniques on the sodium channels hNav1.7 and hNav1.8, which are closely associated with pain. The results showed that the mefenamic acid, flufenamic acid, and tolfenamic acid inhibited the peak currents of hNav1.7 and hNav1.8 in CHO cells stably expressing hNav1.7 and hNav1.8. However, much lighter inhibition effects of hNav1.8 were registered in the experimental system. Furthermore, the mefenamic acid, flufenamic acid and tolfenamic acid significantly affected the inactivation processes of hNav1.7 and hNav1.8 with I-V curves left-shifted to hyperpolarized direction. These data indicate that the inhibition effects of Nav1.7 and Nav1.8 by mefenamic acid, flufenamic acid and tolfenamic acid might contribute to their analgesic activity in addition to their inhibition of cyclooxygenase. These findings provide a basis for further studies in the discovery of other potential targets for NSAIDs.
Assuntos
Fenamatos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.8/efeitos dos fármacos , Dor/tratamento farmacológico , Analgésicos/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus/metabolismo , Humanos , ortoaminobenzoatos/farmacologiaRESUMO
BACKGROUND: This study aimed to describe recruitment challenges encountered during a phase IIa study of vixotrigine, a state and use-dependent Nav1.7 channel blocker, in individuals with trigeminal neuralgia. METHODS: This was an international, multicenter, placebo-controlled, randomized withdrawal study that included a 7-day run-in period, a 21-day open-label phase, and a 28-day double-blind phase in which patients (planned n = 30) were randomized to vixotrigine or placebo. Before recruitment, all antiepileptic drugs had to be stopped, except for gabapentin or pregabalin. After the trial, patients returned to their original medications. Patient recruitment was expanded beyond the original five planned (core) centers in order to meet target enrollment (total recruiting sites N = 25). Core sites contributed data related to patient identification for study participation (prescreening data). Data related to screening failures and study withdrawal were also analyzed using descriptive statistics. RESULTS: Approximately half (322/636; 50.6%) of the patients who were prescreened at core sites were considered eligible for the study and 56/322 (17.4%) were screened. Of those considered eligible, 26/322 (8.1%) enrolled in the study and 6/322 (1.9%) completed the study. In total, 125 patients were screened across all study sites and 67/125 (53.6%) were enrolled. At prescreening, reasons for noneligibility varied by site and were most commonly diagnosis change (78/314; 24.8%), age > 80 years (75/314; 23.9%), language/distance/mobility (61/314; 19.4%), and noncardiac medical problems (53/314; 16.9%). At screening, frequently cited reasons for noneligibility included failure based on electrocardiogram, insufficient pain, and diagnosis change. CONCLUSIONS: Factors contributing to recruitment challenges encountered in this study included diagnosis changes, anxiety over treatment changes, and issues relating to distance, language, and mobility. Wherever possible, future studies should be designed to address these challenges. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01540630 . EudraCT, 2010-023963-16. 07 Aug 2015.
Assuntos
Analgésicos/uso terapêutico , Seleção de Pacientes , Doenças Raras/tratamento farmacológico , Bloqueadores dos Canais de Sódio/uso terapêutico , Nervo Trigêmeo/efeitos dos fármacos , Neuralgia do Trigêmeo/tratamento farmacológico , Adulto , Idoso , Analgésicos/efeitos adversos , Método Duplo-Cego , Europa (Continente) , Humanos , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Medição da Dor , Doenças Raras/diagnóstico , Doenças Raras/metabolismo , Doenças Raras/fisiopatologia , Tamanho da Amostra , Bloqueadores dos Canais de Sódio/efeitos adversos , África do Sul , Fatores de Tempo , Resultado do Tratamento , Nervo Trigêmeo/metabolismo , Nervo Trigêmeo/fisiopatologia , Neuralgia do Trigêmeo/diagnóstico , Neuralgia do Trigêmeo/metabolismo , Neuralgia do Trigêmeo/fisiopatologia , Adulto JovemRESUMO
Voltage-gated sodium channel Nav1.7 is a threshold channel in peripheral dorsal root ganglion (DRG), trigeminal ganglion, and sympathetic ganglion neurons. Gain-of-function mutations in Nav1.7 have been shown to increase excitability in DRG neurons and have been linked to rare Mendelian and more common pain disorders. Discovery of Nav1.7 variants in patients with pain disorders may expand the spectrum of painful peripheral neuropathies associated with a well-defined molecular target, thereby providing a basis for more targeted approaches for treatment. We screened the genome of a patient with adult-onset painful peripheral neuropathy characterized by severe burning pain and report here the new Nav1.7-V810M variant. Voltage-clamp recordings were used to assess the effects of the mutation on biophysical properties of Nav1.7 and the response of the mutant channel to treatment with carbamazepine (CBZ), and multi-electrode array (MEA) recordings were used to assess the effects of the mutation on the excitability of neonatal rat pup DRG neurons. The V810M variant increases current density, shifts activation in a hyperpolarizing direction, and slows kinetics of deactivation, all gain-of-function attributes. We also show that DRG neurons that express the V810M variant become hyperexcitable. The patient responded to treatment with CBZ. Although CBZ did not depolarize activation of the mutant channel, it enhanced use-dependent inhibition. Our results demonstrate the presence of a novel gain-of-function variant of Nav1.7 in a patient with adult-onset painful peripheral neuropathy and the responsiveness of that patient to treatment with CBZ, which is likely due to the classical mechanism of use-dependent inhibition.