RESUMO
Late sodium current (INa) inhibitors are a new subclass of antiarrhythmic agents. To overcome the drawbacks, e.g., low efficacy and inhibition effect on K+ current, of the FDA-approved late INa inhibitor ranolazine, chain amide 6a-6q, 1,4-disubstituted piperazin-2-ones 7a-7s, and their derivatives 8a-8n were successively designed, synthesized, and evaluated in vitro on the NaV1.5-transfected HEK293T cells by the whole-cell patch clamp recording assay at the concentration of 40 µM. Among the new skeleton compounds, 7d showed the highest efficacy (IC50 = 2.7 µM) and good selectivity (peak/late ratio >30 folds), as well as excellent pharmacokinetics properties in mice (T1/2 of 3.5 h, F = 90%, 3 mg/kg, po). It exhibited low hERG inhibition and was able to reverse the ATX-II-induced augmentation of late INa phenotype of LQT3 model in isolated rabbit hearts. These results suggest the application potentials of 7d in the treatments of arrhythmias related to the enhancement of late INa.
Assuntos
Piperazinas , Animais , Coelhos , Células HEK293 , Humanos , Piperazinas/farmacologia , Piperazinas/química , Piperazinas/síntese química , Piperazinas/farmacocinética , Antiarrítmicos/farmacologia , Antiarrítmicos/química , Antiarrítmicos/farmacocinética , Antiarrítmicos/síntese química , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/farmacocinética , Camundongos , Síndrome do QT Longo/induzido quimicamente , Relação Estrutura-Atividade , Masculino , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Coração/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Doença do Sistema de Condução CardíacoRESUMO
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
The discovery of novel analgesic agents with high potency, low toxicity and low addictive properties remain a priority. This study aims to identify the analgesic potential of quinoline derived α-trifluoromethylated alcohols (QTA) and their mechanism of action. We synthesized and characterized several compounds of QTAs and screened them for antiepileptic and analgesic activity using zebrafish larvae in high thorough-put behavior analyses system. Toxicity and behavioral screening of 9 compounds (C1-C9) identified four candidates (C2, C3, C7 and C9) with antiepileptic properties that induces specific and reversible reduction in photomotor activity. Importantly, compounds C2 and C3 relieved the thermal pain response in zebrafish larvae indicating analgesic property. Further, using novel inâ vivo CoroNa green assay, we show that compounds C2 and C3 block sodium channels and reduce inflammatory sodium signals released by peripheral nerve and tissue damage. Thus, we have identified novel QTA compounds with antiepileptic and analgesic properties which could alleviate neuropathic pain.
Assuntos
Analgésicos/farmacologia , Anticonvulsivantes/farmacologia , Metanol/análogos & derivados , Quinolinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Analgésicos/síntese química , Analgésicos/química , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Metanol/síntese química , Metanol/química , Metanol/farmacologia , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.
Assuntos
Amidas , Analgésicos , Dor/tratamento farmacológico , Bloqueadores dos Canais de Sódio , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Analgésicos/síntese química , Analgésicos/química , Analgésicos/farmacologia , Animais , Avaliação de Medicamentos , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Dor/induzido quimicamente , Dor/metabolismo , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their NaV -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuroâ 2A in both cell-based and electrophysiological analyses, with EC50 and IC50 values of 8.5 and 30.7â nm, respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of NaV 1.5, with an IC50 value of 94.1â nm. Derivatives 3 a-d and 3 f showed low recovery rates from NaV 1.2 subtype (ca 45-79 %) compared to natural dcSTX (2), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with NaV in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp1717 .
Assuntos
Saxitoxina/química , Bloqueadores dos Canais de Sódio/síntese química , Canais de Sódio Disparados por Voltagem/metabolismo , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Técnicas de Patch-Clamp , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Teoria Quântica , Saxitoxina/metabolismo , Saxitoxina/farmacologia , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/química , Tetrodotoxina/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
µ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of µ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of µ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical µ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (NaV), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.
Assuntos
Conotoxinas/química , Espectroscopia de Ressonância Magnética , Sequência de Aminoácidos , Conotoxinas/síntese química , Conotoxinas/farmacologia , Dissulfetos/química , Leucina/química , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/química , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/química , Canais de Sódio/metabolismo , Relação Estrutura-AtividadeRESUMO
The physicochemical properties of 4-substituted carbamazepine derivatives were investigated. It was elucidated that the 4-substitution is not effective in reducing the rotations (E/Z) about the N-C1' axes around the outer carbamoyl moiety. However, the atropisomers were isolated with high stereochemical stability, meaning that the 4-substitution reduced the butterfly motion of the tricyclic ring system efficiently. The Cl/CH3-substituted carbamazepine derivatives showed greater inhibitory effects on hNav1.2 channel currents compared with carbamazepine, although no difference in the activity between enantiomers was observed.
Assuntos
Carbamazepina/análogos & derivados , Carbamazepina/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Células CHO , Carbamazepina/síntese química , Carbamazepina/química , Cricetulus , Humanos , Conformação Molecular , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Estereoisomerismo , Temperatura , TermodinâmicaRESUMO
The lupin alkaloid sparteine is a well-known chiral diamine with a range of applications in asymmetric synthesis, as well as a blocker of voltage-gated sodium channels (VGSCs). However, there is only scarce information on the VGSC-blocking activity of sparteine derivatives where the structure of the parent alkaloid is retained. Building on the recent renewed availability of sparteine and derivatives we report herein how modification of sparteine at positionâ 2 produces irreversible blockers of VGSCs. These compounds could be clinically envisaged as long-lasting local anesthetics.
Assuntos
Bloqueadores dos Canais de Sódio/farmacologia , Esparteína/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Estrutura Molecular , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Esparteína/síntese química , Esparteína/química , Relação Estrutura-AtividadeRESUMO
Nav 1.1 (SCN1A) channels primarily located in gamma-aminobutyric acid (GABA)ergic fast-spiking interneurons are pivotal for action potential generation and propagation in these neurons. Inappropriate function of fast-spiking interneurons, leading to disinhibition of pyramidal cells and network desynchronization, correlates with decreased cognitive capability. Further, reduced functionality of Nav 1.1 channels is linked to various diseases in the central nervous system. There is, at present, however no subtype selective pharmacological activators of Nav 1.1 channels available for studying pharmacological modulation of interneuron function. In the current study, we identified a small molecule Nav 1.1 activator, 3-amino-5-(4-methoxyphenyl)thiophene-2-carboxamide, named AA43279, and provided an in vitro to in vivo characterization of the compound. In HEK-293 cells expressing human Nav 1.1 channels, AA43279 increased the Nav 1.1-mediated current in a concentration-dependent manner mainly by impairing the fast inactivation kinetics of the channels. In rat hippocampal brain slices, AA43279 increased the firing activity of parvalbumin-expressing, fast-spiking GABAergic interneurons and increased the spontaneous inhibitory post-synaptic currents (sIPSCs) recorded from pyramidal neurons. When tested in vivo, AA43279 had anti-convulsive properties in the maximal electroshock seizure threshold test. AA43279 was tested for off-target effects on 72 different proteins, including Nav 1.2, Nav 1.4, Nav 1.5, Nav 1.6 and Nav 1.7 and exhibited reasonable selectivity. Taken together, AA43279 might constitute a valuable tool compound for revealing biological functions of Nav 1.1 channels.
Assuntos
Anticonvulsivantes/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Convulsões/tratamento farmacológico , Bloqueadores dos Canais de Sódio/farmacologia , Tiofenos/farmacologia , Potenciais de Ação , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/uso terapêutico , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/fisiologia , Potenciais Pós-Sinápticos Excitadores , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Células HEK293 , Humanos , Interneurônios/metabolismo , Interneurônios/fisiologia , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/uso terapêuticoRESUMO
The enantioselective total synthesis of (-)-tetrodotoxin [(-)-TTX] and 4,9-anhydrotetrodotoxin, which are selective blockers of voltage-gated sodium channels, was accomplished from the commercially available p-benzoquinone. This synthesis was based on efficient stereocontrol of the six contiguous stereogenic centers on the core cyclohexane ring through Ogasawara's method, [3,3]-sigmatropic rearrangement of an allylic cyanate, and intramolecular 1,3-dipolar cycloaddition of a nitrile oxide. Our synthetic route was applied to the synthesis of the tetrodotoxin congeners 11-norTTX-6(R)-ol and 4,9-anhydro-11-norTTX-6(R)-ol through late-stage modification of the common intermediate. Neutral deprotection at the final step enabled easy purification of tetrodotoxin and 11-norTTX-6(R)-ol without competing dehydration to their 4,9-anhydro forms.
Assuntos
Bloqueadores dos Canais de Sódio/síntese química , Tetrodotoxina/análogos & derivados , Tetrodotoxina/síntese química , Benzoquinonas/síntese química , Benzoquinonas/química , Técnicas de Química Sintética , Bloqueadores dos Canais de Sódio/química , Estereoisomerismo , Tetrodotoxina/químicaRESUMO
A novel family of small molecule inhibitors of voltage-gated sodium channels (NaVs) based on the structure of batrachotoxin (BTX), a well-known channel agonist, is described. Protein mutagenesis and electrophysiology experiments reveal the binding site as the inner pore region of the channel, analogous to BTX, alkaloid toxins, and local anesthetics. Homology modeling of the eukaryotic channel based on recent crystallographic analyses of bacterial NaVs suggests a mechanism of action for ion conduction block.
Assuntos
Batraquiotoxinas/análise , Batraquiotoxinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Batraquiotoxinas/síntese química , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Modelos Moleculares , Estrutura Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Técnicas de Patch-Clamp , Ratos , Bloqueadores dos Canais de Sódio/síntese química , Canais de Sódio/genética , Canais de Sódio/metabolismo , Relação Estrutura-AtividadeRESUMO
The α-aminoamide family of sodium ion channel blockers have exhibited analgesic effects on neuropathic pain. Here, a series of novel α-aminoamides containing an indole ring were designed and synthesized. These compounds were evaluated in mice using a formalin test and they exhibited significant anti-allodynia activities. However, the analgesic mechanism of these compounds remains unclear; a subset of the synthesized compounds can only moderately inhibit the sodium ion channel, Nav1.7, in a whole-cell patch clamp assay. Overall, these results suggest that introduction of an indole moiety to α-aminoamide derivatives can significantly improve their bioactivity and further study is warranted.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Analgésicos/síntese química , Analgésicos/farmacologia , Indóis/química , Amidas/química , Analgésicos/química , Animais , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
The paralytic shellfish poisons are a collection of guanidine-containing natural products that are biosynthesized by prokaryote and eukaryote marine organisms. These compounds bind and inhibit isoforms of the mammalian voltage-gated Na(+) ion channel at concentrations ranging from 10(-11) to 10(-5) M. Here, we describe the de novo synthesis of three paralytic shellfish poisons, gonyautoxin 2, gonyautoxin 3, and 11,11-dihydroxysaxitoxin. Key steps include a diastereoselective Pictet-Spengler reaction and an intramolecular amination of an N-guanidyl pyrrole by a sulfonyl guanidine. The IC50's of GTX 2, GTX 3, and 11,11-dhSTX have been measured against rat NaV1.4, and are found to be 22 nM, 15 nM, and 2.2 µM, respectively.
Assuntos
Toxinas Marinhas/síntese química , Saxitoxina/análogos & derivados , Saxitoxina/síntese química , Aminas/química , Animais , Ciclização , Toxinas Marinhas/farmacologia , Proteínas Musculares/antagonistas & inibidores , Pirróis/química , Ratos , Saxitoxina/farmacologia , Frutos do Mar , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio , EstereoisomerismoRESUMO
Previously we disclosed the discovery of potent Late INa current inhibitor 2 (GS-458967, IC50 of 333nM) that has a good separation of late versus peak Nav1.5 current, but did not have a favorable CNS safety window due to high brain penetration (3-fold higher partitioning into brain vs plasma) coupled with potent inhibition of brain sodium channel isoforms (Nav1.1, 1.2, 1.3). We increased the polar surface area from 50 to 84Å(2) by adding a carbonyl to the core and an oxadiazole ring resulting in 3 GS-462808 that had lower brain penetration and serendipitously lower activity at the brain isoforms. Compound 3 has an improved CNS window (>20 rat and dog) relative to 2, and improved anti-ischemic potency relative to ranolazine. The development of 3 was not pursued due to liver lesions in 7day rat toxicology studies.
Assuntos
Azóis/farmacologia , Descoberta de Drogas , Coração/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Piridinas/farmacologia , Ranolazina/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Animais , Azóis/síntese química , Azóis/química , Cães , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Haplorrinos , Humanos , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Coelhos , Ranolazina/síntese química , Ranolazina/química , Ratos , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Relação Estrutura-AtividadeRESUMO
Two series of new derivatives of pyrrolidine-2,5-dione were synthesized and evaluated for their anticonvulsant properties. Initial screening for their anticonvulsant properties was performed in mice after intraperitoneal administration, using the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and 6-Hz seizure tests. Quantitative pharmacological research revealed that the highest level of protection was demonstrated by compound N-[{4-methylpiperazin-1-yl}-methyl]-3-(1-phenylethyl)-pyrrolidine-2,5-dione monohydrochloride (22) which was effective both in the scPTZ test (ED50=39 mg/kg) and in the 6-Hz test (ED50=36 mg/kg). This molecule showed higher potency than reference antiepileptic drugs such as ethosuximide, lacosamide and valproic acid. With the aim of explaining the possible mechanism of action of the selected molecule, its influence on sodium and calcium channels as well as NMDA and GABAA receptors binding properties were evaluated in vitro.
Assuntos
Anticonvulsivantes/farmacologia , Bases de Mannich/farmacologia , Piperazinas/farmacologia , Pirrolidinas/farmacologia , Succinimidas/farmacologia , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Antagonistas de Receptores de GABA-A/síntese química , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacologia , Bases de Mannich/síntese química , Bases de Mannich/química , Camundongos , Piperazinas/síntese química , Pirrolidinas/síntese química , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Relação Estrutura-Atividade , Succinimidas/síntese química , Succinimidas/químicaRESUMO
We started with a medium throughput screen of heterocyclic compounds without basic amine groups to avoid hERG and ß-blocker activity and identified [1,2,4]triazolo[4,3-a]pyridine as an early lead. Optimization of substituents for Late INa current inhibition and lack of Peak INa inhibition led to the discovery of 4h (GS-458967) with improved anti-arrhythmic activity relative to ranolazine. Unfortunately, 4h demonstrated use dependent block across the sodium isoforms including the central and peripheral nervous system isoforms that is consistent with its low therapeutic index (approximately 5-fold in rat, 3-fold in dog). Compound 4h represents our initial foray into a 2nd generation Late INa inhibitor program and is an important proof-of-concept compound. We will provide additional reports on addressing the CNS challenge in a follow-up communication.
Assuntos
Descoberta de Drogas , Coração/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Piridinas/farmacologia , Ranolazina/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Triazóis/farmacologia , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Macaca fascicularis , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Coelhos , Ranolazina/síntese química , Ranolazina/química , Ratos , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin µO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin µO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of µO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic µO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ß-strands stabilized by three disulfide bridges. The loop region linking the ß-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of µO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of µO§-GVIIJ on a distinct surface of the peptide.
Assuntos
Conotoxinas/química , Dissulfetos/química , Proteínas Musculares/química , Canal de Sódio Disparado por Voltagem NAV1.2/química , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Conotoxinas/síntese química , Cristalografia por Raios X , Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Caramujos/química , Bloqueadores dos Canais de Sódio/síntese química , Canais de Sódio/genética , Canais de Sódio/metabolismo , Técnicas de Síntese em Fase Sólida , Relação Estrutura-AtividadeRESUMO
We recently reported the chemical synthesis and identification of the genetically predicted biosynthetic intermediates of saxitoxin (STX), including a 2-aminoimidazole-bearing monoguanidine compound (Int-C'2) in two paralytic shellfish toxin (PST)-producing microorganisms. In this study, we achieved the direct conversion of Int-C'2 into a tricyclic bisguanidine compound (called Cyclic-C'), which is structurally related to STX, through oxidative intramolecular guanidine transfer to 2-aminoimidazole catalyzed by Pd/C under basic conditions in air. By using HPLC-MS analysis, Cyclic-C' was also identified in the PST-producing microorganisms, suggesting that Cyclic-C' is either another biosynthetic intermediate or a shunt product of PSTs. In addition, a weak inhibitory activity of Cyclic-C' to the voltage-gated sodium channels was detected by using a cell-based assay.
Assuntos
Guanidina/análogos & derivados , Toxinas Marinhas/química , Saxitoxina/química , Animais , Catálise , Linhagem Celular , Ciclização , Dinoflagellida/química , Guanidina/síntese química , Guanidina/farmacologia , Imidazóis/química , Toxinas Marinhas/síntese química , Toxinas Marinhas/farmacologia , Camundongos , Oxirredução , Frutos do Mar/análise , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismoRESUMO
Tetrodotoxin (TTX) is a potent neurotoxin found in a number of marine creatures including the pufferfish, where it is synthesized by bacteria and accumulated through the food chain. It is a potent and selective blocker of some types of voltage-gated Na+ channel (NaV channel). 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX) was purified from a crude mixture of TTX analogues (such as TTX, 4-epiTTX, 6-epiTTX, 11-oxoTTX and 11-deoxyTTX) by the use of liquid chromatography-fluorescence detection (LC-FLD) techniques. Recently, it has been reported that 4,9-anhydroTTX selectively blocks the activity of NaV1.6 channels with a blocking efficacy 40-160 times higher than that for other TTX-sensitive NaV1.x channel isoforms. However, little attention has been paid to the molecular properties of the α-subunit in NaV1.6 channels and the characteristics of binding of 4,9-anhydroTTX. From a functional point of view, it is important to determine the relative expression of NaV1.6 channels in a wide variety of tissues. The aim of this review is to discuss briefly current knowledge about the pharmacology of 4,9-anhydroTTX, and provide an analysis of the molecular structure of native NaV1.6 channels. In addition, chemical aspects of 4,9-anhydroTTX are briefly covered.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.6/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/análogos & derivados , Animais , Humanos , Camundongos , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Bloqueadores dos Canais de Sódio/síntese química , Tetrodotoxina/síntese química , Tetrodotoxina/farmacologiaRESUMO
We have identified a new series of N-aryl azacycles as sodium channel blockers, which showed good potency on Nav1.7 in FLIPR-based and electrophysiological functional assays. Analogs from this series possessed selectivity over hERG, reasonable oral exposure in rat PK studies and are predicted to have limited CNS penetration.