RESUMO
Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1-1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Ciguatoxinas/toxicidade , Gânglios Espinais/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Células Cultivadas , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Isoformas de Proteínas/metabolismoRESUMO
The µO-conotoxins MrVIA, MrVIB, and MfVIA inhibit the voltage-gated sodium channel NaV1.8, a well described target for the treatment of pain; however, little is known about the residues or structural elements that define this activity. In this study, we determined the three-dimensional structure of MfVIA, examined its membrane binding properties, performed alanine-scanning mutagenesis, and identified residues important for its activity at human NaV1.8. A second round of mutations resulted in (E5K,E8K)MfVIA, a double mutant with greater positive surface charge and greater affinity for lipid membranes compared with MfVIA. This analogue had increased potency at NaV1.8 and was analgesic in the mouse formalin assay.
Assuntos
Analgésicos/farmacologia , Membrana Celular/metabolismo , Conotoxinas/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Dor/prevenção & controle , Sequência de Aminoácidos , Animais , Comportamento Animal/efeitos dos fármacos , Cristalografia por Raios X , Eletrofisiologia , Células HEK293 , Humanos , Lipossomos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.8/química , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Dor/induzido quimicamente , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition.
Assuntos
Evolução Biológica , Conotoxinas/genética , Caramujo Conus/fisiologia , Camundongos/fisiologia , Dor , Comportamento Predatório , Sequência de Aminoácidos , Animais , Conotoxinas/metabolismo , Conotoxinas/farmacologia , Caramujo Conus/genética , Masculino , Camundongos Endogâmicos C57BL , Alinhamento de SequênciaRESUMO
Selective activation of peripheral κ opioid receptors (KORs) may overcome the dose-limiting adverse effects of conventional opioid analgesics. We recently developed a vicinal disulfide-stabilized class of peptides with subnanomolar potency at the KOR. The aim of this study was to assess the analgesic effects of one of these peptides, named conorphin-1, in comparison with the prototypical KOR-selective small molecule agonist U-50488, in several rodent pain models. Surprisingly, neither conorphin-1 nor U-50488 were analgesic when delivered peripherally by intraplantar injection at local concentrations expected to fully activate the KOR at cutaneous nerve endings. While U-50488 was analgesic when delivered at high local concentrations, this effect could not be reversed by coadministration with the selective KOR antagonist ML190 or the nonselective opioid antagonist naloxone. Instead, U-50488 likely mediated its peripheral analgesic effect through nonselective inhibition of voltage-gated sodium channels, including peripheral sensory neuron isoforms NaV1.8 and NaV1.7. Our study suggests that targeting the KOR in peripheral sensory nerve endings innervating the skin is not an alternative analgesic approach.
Assuntos
Terminações Nervosas/metabolismo , Dor/patologia , Peptídeos/uso terapêutico , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Pele/inervação , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/uso terapêutico , Analgésicos/uso terapêutico , Analgésicos não Narcóticos/uso terapêutico , Animais , Carragenina/toxicidade , Cisplatino/toxicidade , Modelos Animais de Doenças , Adjuvante de Freund/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Inflamação/induzido quimicamente , Inflamação/complicações , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naloxona/farmacologia , Naloxona/uso terapêutico , Terminações Nervosas/efeitos dos fármacos , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Dor/induzido quimicamente , Dor/tratamento farmacológico , Medição da Dor , Peptídeos/farmacologia , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Ratos , Ratos WistarRESUMO
The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1ß (IL-1ß) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle-Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Síndromes Periódicas Associadas à Criopirina/tratamento farmacológico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Inflamassomos/antagonistas & inibidores , Interleucina-1beta/efeitos dos fármacos , Esclerose Múltipla , Sulfonas/uso terapêutico , Animais , Modelos Animais de Doenças , Furanos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Indenos , Inflamação , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Sulfonamidas , Sulfonas/farmacologiaRESUMO
MrIC is a recently described selective agonist of endogenously expressed α7 nAChR. In this study, we further characterize the pharmacological activity of MrIC using Ca(2+) imaging approaches in SH-SY5Y cells endogenously expressing α7 nAChR and demonstrate that MrIC exclusively activates α7 nAChR modulated by type II positive allosteric modulators, including PNU120596. MrIC was a full agonist at PNU120596-modulated α7 nAChR compared with choline, albeit with slower kinetics, but failed to elicit a Ca(2+) response in the absence of PNU120596. Interestingly, the NMR structure of MrIC showed a typical 4/7 α-conotoxin fold, indicating that its unusual pharmacological activity is likely sequence-dependent. Overall, our results suggest that MrIC acts as a biased agonist that can only activate α7 nAChR modified by type II positive allosteric modulators, and thus represents a valuable tool to probe the pharmacological properties of this important ion channel.
Assuntos
Conotoxinas/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Linhagem Celular Tumoral , Conotoxinas/química , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
A new α-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of α3ß2, α3α5ß2 and α7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10 nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized α-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at α7 and α3ß2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at α3ß2 nAChRs but reduced potency 3-fold at α7 nAChRs. This study gives further insight into α-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology.
Assuntos
Conotoxinas/isolamento & purificação , Caramujo Conus/química , Antagonistas Nicotínicos/isolamento & purificação , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Cromatografia de Fase Reversa , Conotoxinas/síntese química , Conotoxinas/química , Conotoxinas/farmacologia , Corantes Fluorescentes , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Antagonistas Nicotínicos/síntese química , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Relação Quantitativa Estrutura-Atividade , Especificidade por SubstratoRESUMO
Ciguatera, the most common form of nonbacterial ichthyosarcotoxism, is caused by consumption of fish that have bioaccumulated the polyether sodium channel activator ciguatoxin. The neurological symptoms of ciguatera include distressing, often persistent sensory disturbances such as paraesthesias and the pathognomonic symptom of cold allodynia. We show that intracutaneous administration of ciguatoxin in humans elicits a pronounced axon-reflex flare and replicates cold allodynia. To identify compounds able to inhibit ciguatoxin-induced Nav responses, we developed a novel in vitro ciguatoxin assay using the human neuroblastoma cell line SH-SY5Y. Pharmacological characterisation of this assay demonstrated a major contribution of Nav1.2 and Nav1.3, but not Nav1.7, to ciguatoxin-induced Ca2+ responses. Clinically available Nav inhibitors, as well as the Kv7 agonist flupirtine, inhibited tetrodotoxin-sensitive ciguatoxin-evoked responses. To establish their in vivo efficacy, we used a novel animal model of ciguatoxin-induced cold allodynia. However, differences in the efficacy of these compounds to reverse ciguatoxin-induced cold allodynia did not correlate with their potency to inhibit ciguatoxin-induced responses in SH-SY5Y cells or at heterologously expressed Nav1.3, Nav1.6, Nav1.7, or Nav1.8, indicating cold allodynia might be more complex than simple activation of Nav channels. These findings highlight the need for suitable animal models to guide the empiric choice of analgesics, and suggest that lamotrigine and flupirtine could be potentially useful for the treatment of ciguatera.
Assuntos
Analgésicos/uso terapêutico , Ciguatoxinas/toxicidade , Temperatura Baixa/efeitos adversos , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Ciguatoxinas/isolamento & purificação , Enguias , Humanos , Hiperalgesia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
Venomous animals produce a diverse range of peptides and small molecules that are of both therapeutic and pharmacologic value. One such animal, the cone snail, produces peptides known as conotoxins, which may be of interest to those studying the mammalian immune system. Conotoxins are a family of venom peptides that display extraordinary diversity and often exquisite specificity for membrane protein targets, especially voltage and ligand activated ion channels. Conopeptides are proving to be important pharmacological tools to probe human physiology, with some showing promise as therapeutics for conditions such as neuropathic pain. The potential of these peptides to interact and modulate the human immune system has not been investigated despite literature suggesting that conotoxins could be valuable research tools and potential therapeutics in area of immunology. Known pharmacological targets of conopeptides expressed by immunocompetent cells include voltage-gated potassium channel (Kv), voltage-gated calcium channel (Cav), nicotinic and acetylcholine receptors. In addition, the 5-HT3, GABAB and NMDA receptors that are not considered classic immunomodulators but may play a secondary role in modulating immune responses. This review highlights venom peptides with potential to act at immunological targets within the mammalian immune system.