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OBJECTIVE: To screen a library of potential therapeutic compounds for a woman with Lennox-Gastaut syndrome due to a Y302C GABRB3 (c.905A>G) mutation. METHODS: We compared the electrophysiological properties of cells with wild-type or the pathogenic GABRB3 mutation. RESULTS: Among 1320 compounds, multiple candidates enhanced GABRB3 channel conductance in cell models. Vinpocetine, an alkaloid derived from the periwinkle plant with anti-inflammatory properties and the ability to modulate sodium and channel channels, was the lead candidate based on efficacy and safety profile. Vinpocetine was administered as a dietary supplement over 6 months, reaching a dosage of 20 mg three times per day, and resulted in a sustained, dose-dependent reduction in spike-wave discharge frequency on electroencephalograms. Improved language and behavior were reported by family, and improvements in global impression of change surveys were observed by therapists blinded to intervention. SIGNIFICANCE: Vinpocetine has potential efficacy in treating patients with this mutation and possibly other GABRB3 mutations or other forms of epilepsy. Additional studies on pharmacokinetics, potential drug interactions, and safety are needed.
Assuntos
Síndrome de Lennox-Gastaut/tratamento farmacológico , Síndrome de Lennox-Gastaut/genética , Mutação/genética , Medicina de Precisão/métodos , Receptores de GABA-A/genética , Alcaloides de Vinca/uso terapêutico , Adulto , Relação Dose-Resposta a Droga , Eletroencefalografia/efeitos dos fármacos , Eletroencefalografia/métodos , Feminino , Células HEK293 , Humanos , Síndrome de Lennox-Gastaut/diagnóstico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Alcaloides de Vinca/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
TRPA1, a member of the transient receptor potential channel (TRP) family, is genetically linked to pain in humans, and small molecule inhibitors are efficacious in preclinical animal models of inflammatory pain. These findings have driven significant interest in development of selective TRPA1 inhibitors as potential analgesics. The majority of TRPA1 inhibitors characterized to date have been reported to interact with the S5 transmembrane helices forming part of the pore region of the channel. However, the development of many of these inhibitors as clinical drug candidates has been prevented by high lipophilicity, low solubility, and poor pharmacokinetic profiles. Identification of alternate compound interacting sites on TRPA1 provides the opportunity to develop structurally distinct modulators with novel structure-activity relationships and more desirable physiochemical properties. In this paper, we have identified a previously undescribed potent and selective small molecule thiadiazole structural class of TRPA1 inhibitor. Using species ortholog chimeric and mutagenesis strategies, we narrowed down the site of interaction to ankyrinR #6 within the distal N-terminal region of TRPA1. To identify the individual amino acid residues involved, we generated a computational model of the ankyrinR domain. This model was used predictively to identify three critical amino acids in human TRPA1, G238, N249, and K270, which were confirmed by mutagenesis to account for compound activity. These findings establish a small molecule interaction region on TRPA1, expanding potential avenues for developing TRPA1 inhibitor analgesics and for probing the mechanism of channel gating.
Assuntos
Bibliotecas de Moléculas Pequenas/química , Canal de Cátion TRPA1/química , Canal de Cátion TRPA1/metabolismo , Sequência de Aminoácidos , Animais , Repetição de Anquirina , Humanos , Modelos Moleculares , Ligação Proteica , Ratos , Alinhamento de Sequência , Bibliotecas de Moléculas Pequenas/metabolismo , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/genéticaRESUMO
OBJECTIVE: Many previous studies of drug repurposing have relied on literature review followed by evaluation of a limited number of candidate compounds. Here, we demonstrate the feasibility of a more comprehensive approach using high-throughput screening to identify inhibitors of a gain-of-function mutation in the SCN8A gene associated with severe pediatric epilepsy. METHODS: We developed cellular models expressing wild-type or an R1872Q mutation in the Nav 1.6 sodium channel encoded by SCN8A. Voltage clamp experiments in HEK-293 cells expressing the SCN8A R1872Q mutation demonstrated a leftward shift in sodium channel activation as well as delayed inactivation; both changes are consistent with a gain-of-function mutation. We next developed a fluorescence-based, sodium flux assay and used it to assess an extensive library of approved drugs, including a panel of antiepileptic drugs, for inhibitory activity in the mutated cell line. Lead candidates were evaluated in follow-on studies to generate concentration-response curves for inhibiting sodium influx. Select compounds of clinical interest were evaluated by electrophysiology to further characterize drug effects on wild-type and mutant sodium channel functions. RESULTS: The screen identified 90 drugs that significantly inhibited sodium influx in the R1872Q cell line. Four drugs of potential clinical interest-amitriptyline, carvedilol, nilvadipine, and carbamazepine-were further investigated and demonstrated concentration-dependent inhibition of sodium channel currents. SIGNIFICANCE: A comprehensive drug repurposing screen identified potential new candidates for the treatment of epilepsy caused by the R1872Q mutation in the SCN8A gene.
Assuntos
Anticonvulsivantes/uso terapêutico , Reposicionamento de Medicamentos/métodos , Epilepsia/tratamento farmacológico , Epilepsia/genética , Ensaios de Triagem em Larga Escala/métodos , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Anticonvulsivantes/farmacologia , Criança , Relação Dose-Resposta a Droga , Epilepsia/diagnóstico , Feminino , Células HEK293 , Humanos , Masculino , Mutação/efeitos dos fármacos , Mutação/genéticaRESUMO
A series of TRPA1 antagonists is described which has as its core structure an indazole moiety. The physical properties and in vitro DMPK profiles are discussed. Good in vivo exposure was obtained with several analogs, allowing efficacy to be assessed in rodent models of inflammatory pain. Two compounds showed significant activity in these models when administered either systemically or topically. Protein chimeras were constructed to indicate compounds from the series bound in the S5 region of the channel, and a computational docking model was used to propose a binding mode for example compounds.
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A prerequisite for a successful target-based drug discovery program is a robust data set that increases confidence in the validation of the molecular target and the therapeutic approach. Given the significant time and resource investment required to carry a drug to market, early selection of targets that can be modulated safely and effectively forms the basis for a strong portfolio and pipeline. In this article we present some of the more useful scientific approaches that can be applied toward the validation of ion channel targets, a molecular family with a history of clinical success in therapeutic areas such as cardiovascular, respiratory, pain and neuroscience.
Assuntos
Descoberta de Drogas/métodos , Canais Iônicos/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Animais , Genoma Humano , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Moduladores de Transporte de Membrana/química , Estudos de Validação como AssuntoRESUMO
BACKGROUND AND PURPOSE: NaV 1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Nav 1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACH: The inhibition of Nav 1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTS: PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50 : 331 nM) and in recombinantly expressed h Nav 1.8 channels (IC50 : 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav 1.5 channels (IC50 : â¼10 µM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50 : â¼10-18 µM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav 1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONS: Using PF-01247324, we have confirmed a role for Nav 1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav 1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.
Assuntos
Nociceptividade/efeitos dos fármacos , Ácidos Picolínicos/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Administração Oral , Animais , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.8/efeitos dos fármacos , Medição da Dor/efeitos dos fármacos , Ácidos Picolínicos/administração & dosagem , Ácidos Picolínicos/farmacocinética , Ratos , Tetrodotoxina/antagonistas & inibidores , Tetrodotoxina/farmacologiaRESUMO
Current marketed antiepileptic drugs (AEDs) consist of a variety of structural classes with different mechanisms of action. These agents typically have non-overlapping efficacy and side-effect profiles presenting multiple treatment options for the patient population. However, approximately 30% of seizure sufferers fail to respond to current therapies often because poorly tolerated side-effects limit adequate dosing. The scope of this review is to summarize selected advances in 2nd and 3rd generation AEDs as well as compounds in development with novel mechanisms of action.
RESUMO
Human ether-à-go-go-related gene (hERG) potassium channel activity helps shape the cardiac action potential and influences its duration. In this study, we report the discovery of 3-nitro-N-(4-phenoxyphenyl) benzamide (ICA-105574), a potent and efficacious hERG channel activator with a unique mechanism of action. In whole-cell patch-clamp studies of recombinant hERG channels, ICA-105574 steeply potentiated current amplitudes more than 10-fold with an EC(50) value of 0.5 +/- 0.1 microM and a Hill slope (n(H)) of 3.3 +/- 0.2. The effect on hERG channels was confirmed because the known hERG channel blockers, N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide, 2HCl (E-4031) and BeKm-1, potently blocked the stimulatory effects of ICA-105574. The primary mechanism by which ICA-105574 potentiates hERG channel activity is by removing hERG channel inactivation, because ICA-105574 (2 microM) shifts the midpoint of the voltage-dependence of inactivation by >180 mV from -86 to +96 mV. In addition to the effects on inactivation, greater concentrations of ICA-105574 (3 microM) produced comparatively small hyperpolarizing shifts (up to 11 mV) in the voltage-dependence of channel activation and a 2-fold slowing of channel deactivation. In isolated guinea pig ventricular cardiac myocytes, ICA-105574 induced a concentration-dependent shortening of action potential duration (>70%, 3 microM) that could be prevented by preincubation with E-4031. In conclusion, we identified a novel agent that can prevent the inactivation of hERG potassium channels. This compound may provide a useful tool to further understand the mechanism by which hERG channels inactivate and affect cardiac function in addition to the role of hERG channels in other cell systems.
Assuntos
Canais de Potássio Éter-A-Go-Go/agonistas , Bloqueadores dos Canais de Potássio/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Cobaias , Humanos , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Piridinas/farmacologiaRESUMO
The mammalian KCNQ (Kv7) gene family is composed of five members (KCNQ1-5). KCNQ2, Q4 and Q5 (KCNQ2-5) channels co-express with KCNQ3 to form heterotetrameric voltage-gated K(+) (KCNQ2-5/3) channels that underlie the endogenous M-current and regulate neuronal excitability in CNS and PNS neurons. Openers of one or a mixture of these channels may be an attractive therapeutic agent for epilepsy and pain. Non-selective KCNQ2-5/3 activators have shown efficacy in pre-clinical and clinical studies. However, more selective pharmacological profiles, including greater KCNQ sub-type-selective activation, could provide efficacy with fewer side effects. One such compound, ICA-27243, sub-type selectively enhances the activation of KCNQ2/3 channels and also exhibits efficacy in pre-clinical anticonvulsant models; Roeloffs et al. (2008) [15]; Wickenden et al. (2008) [27]. The binding site of non-selective KCNQ2-5/3 openers maps to the S5-S6 pore domain and is altered by mutation of a tryptophan residue (Trp236 in KCNQ2, Trp265 in KCNQ3) conserved among KCNQ2-5 channels; Schenzer et al. (2005) [19]; Wuttke et al. (2005) [30]. Here we report that the activity of the KCNQ2/3 selective opener ICA-27243 is not affected by these Trp mutations and does not map to the S5-S6 domain. Rather, the selective activity of ICA-27243 is determined by a novel site within the S1-S4 voltage-sensor domain (VSD) of KCNQ channels. The sub-type-selective activity of ICA-27243 may arise from greater sequence diversity of KCNQ family members within the ICA-27243 binding pocket, allowing for more selective small molecule-protein interactions.
Assuntos
Benzamidas/farmacologia , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Piridinas/farmacologia , Sequência de Aminoácidos , Animais , Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Benzamidas/química , Sítios de Ligação/genética , Células CHO , Carbamatos/química , Carbamatos/farmacologia , Cricetinae , Cricetulus , Humanos , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ3/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Moduladores de Transporte de Membrana/química , Dados de Sequência Molecular , Mutação , Fenilenodiaminas/química , Fenilenodiaminas/farmacologia , Piridinas/química , Alinhamento de Sequência , Triptofano/genética , Triptofano/metabolismoRESUMO
The activation of the slow afterhyperpolarization (sAHP) in CA1 neurons was studied using whole-cell recordings in the presence of inhibitors of the fast and medium-duration AHPs. The amplitude of the slow afterhyperpolarization current (IsAHP) increased as a function of duration and magnitude of the depolarizing voltage pulse reflecting graded increases in Ca(2+) influx through voltage-dependent Ca(2+) channels. Therefore, the time constant for activation, tau(max), determined from a family of IsAHPs as a function of pulse duration, was voltage dependent decreasing several-fold within the range of -20 to 20 mV and was dependent on extracellular [Ca(2+)]. The IsAHP displayed a pronounced rising phase that was well fit by a single exponential with a time constant, tau(rise), that was invariant of pulse duration, voltage, IsAHP amplitude, or external [Ca(2+)] and was significantly slower than the tau(max). In current clamp, the magnitude of the sAHP increased with the number of evoked action potentials, yet tau(rise) of the sAHP was invariant of action potential number and was similar to the tau(rise) of the IsAHP recorded in voltage-clamp. The results suggest that there are two components to the development of the IsAHP, a rapid, voltage- and Ca(2+)-dependent step, the magnitude and rate of which reflects the voltage dependence of the Ca(2+) channels, that triggers a second rate-limiting, voltage-independent process that dictates the slow IsAHP rise kinetics.
Assuntos
Hipocampo/fisiologia , Neurônios/fisiologia , Animais , Apamina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Tamanho Celular , Eletrofisiologia , Potenciais Evocados/fisiologia , Hipocampo/citologia , Técnicas In Vitro , Indóis/farmacologia , Cinética , Masculino , Potenciais da Membrana/fisiologia , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Piridinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu(406) (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27 degrees C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.