RESUMO
The nonselective cation channel TRPA1 (ANKTM1, p120) is a potential mediator of pain, and selective pharmacological modulation of this channel may be analgesic. Although several TRPA1 activators exist, these tend to be either reactive or of low potency and/or selectivity. The aim of the present study, therefore, was to identify novel TRPA1 agonists. Using a combination of calcium fluorescent assays and whole-cell electrophysiology, we discovered several compounds that possess potent, selective TRPA1-activating activity, including several lipid compounds (farnesyl thiosalicylic acid, farnesyl thioacetic acid, 15-deoxy-Delta(12,14)-prostaglandin J(2), and 5,8,11,14-eicosatetraynoic acid), and two marketed drugs: disulfiram (Antabuse; a compound used in the treatment of alcohol abuse) and the antifungal agent chlordantoin. Farnesyl thiosalicylic acid activates the channel in excised patches and in the absence of calcium. Furthermore, using a quadruple TRPA1 mutant, we show that the mechanism of action of farnesyl thiosalicylic acid differs from that of the reactive electrophilic reagent allylisothiocyanate. As a TRPA1 agonist with a potentially novel mechanism of action, farnesyl thiosalicylic acid may be useful in the study of TRPA1 channels.
Assuntos
Canais de Cálcio/metabolismo , Farneseno Álcool/análogos & derivados , Ativação do Canal Iônico/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Salicilatos/farmacologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Cães , Eletrofisiologia , Farneseno Álcool/química , Farneseno Álcool/farmacologia , Fluorescência , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Isotiocianatos/farmacologia , Masculino , Proteínas do Tecido Nervoso/agonistas , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Rutênio Vermelho , Salicilatos/química , Canal de Cátion TRPA1 , Transfecção , Canais de Potencial de Receptor Transitório/agonistasRESUMO
Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing Förster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltage-sensitive dyes, simultaneously determining the resting membrane potential of a cell.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Antibacterianos/farmacologia , Calibragem , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Gramicidina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Rim/citologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Estrutura Molecular , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tiobarbitúricos/análise , Tiobarbitúricos/química , Tiobarbitúricos/metabolismo , Fatores de TempoRESUMO
As an integrator of multiple nociceptive and/or inflammatory stimuli, TRPV1 is an attractive therapeutic target for the treatment of various painful disorders. Several TRPV1 antagonists have been advanced into clinical trials and the initial observations suggest that TRPV1 antagonism may be associated with mild hyperthermia and thermal insensitivity in man. However, no clinical efficacy studies have been described to date, making an assessment of risk:benefit impossible. Furthermore, it is not clear whether these early observations are representative of all TRPV1 antagonists and whether additional clinical studies with novel TRPV1 antagonists are required in order to understand optimal compound characteristics. In the present study we describe 2-(2,6-dichloro-benzyl)-thiazolo[5,4-d]pyrimidin-7-yl]-(4-trifluoromethyl-phenyl)-amine (JNJ-39729309) as a novel, TRPV1 antagonist. JNJ-39729209 displaced tritiated resiniferotoxin binding to TRPV1 and prevented TRPV1 activation by capsaicin, protons and heat. In-vivo, JNJ-39729209 blocked capsaicin-induced hypotension, induced a mild hyperthermia and inhibited capsaicin-induced hypothermia in a dose dependent manner. JNJ-39729209 showed significant efficacy against carrageenan- and CFA-evoked thermal hyperalgesia and exhibited significant anti-tussive activity in a guinea-pig model of capsaicin-induced cough. In pharmacokinetic studies, JNJ-39729209 was found to have low clearance, a moderate volume of distribution, good oral bioavailability and was brain penetrant. On the basis of these findings, JNJ-39729209 represents a structurally novel TRPV1 antagonist with potential for clinical development. The advancement of JNJ-39729209 into human clinical trials could be useful in further understanding the analgesic potential of TRPV1 antagonists.
Assuntos
Pirimidinas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Temperatura Corporal/efeitos dos fármacos , Linhagem Celular , Ensaios Clínicos como Assunto , Tosse/tratamento farmacológico , Cães , Feminino , Cobaias , Humanos , Hiperalgesia/tratamento farmacológico , Hipotensão/tratamento farmacológico , Masculino , Camundongos , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Ratos , Tiazóis/farmacocinética , Tiazóis/uso terapêuticoRESUMO
Hyperpolarization- and Cyclic Nucleotide-gated (HCN) channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. The HCN family consists of four members (HCN1-4). HCN channels represent the molecular correlates of I(h) (also known as 'funny' I(f) and 'queer' I(q)), a hyperpolarization-activated current best known for its role in controlling heart rate and in the regulation of neuronal resting membrane potential and excitability. A significant body of molecular and pharmacological evidence is now emerging to support a role for these channels in the function of sensory neurons and pain sensation, particularly pain associated with nerve or tissue injury. As such, HCN channels may represent valid targets for novel analgesic agents. This evidence will be reviewed in this article. We will then summarize our efforts to develop and validate methods for screening for novel HCN channel blockers.