RESUMEN
Voltage-gated sodium channels are known to be expressed in neurons and other excitable cells. Recently, voltage-gated sodium channels have been found to be expressed in human prostate cancer cells. α-Hydroxy-α-phenylamides are a new class of small molecules that have demonstrated potent inhibition of voltage-gated sodium channels. The hydroxyamide motif, an isostere of a hydantoin ring, provides an active scaffold from which several potent racemic sodium channel blockers have been derived. With little known about chiral preferences, the development of chiral syntheses to obtain each pure enantiomer for evaluation as sodium channel blockers is important. Using Seebach and Frater's chiral template, cyclocondensation of (R)-3-chloromandelic acid with pivaldehyde furnished both the cis- and trans-2,5-disubsituted dioxolanones. Using this chiral template, we synthesized both enantiomers of 2-(3-chlorophenyl)-2-hydroxynonanamide, and evaluated their ability to functionally inhibit hNa(v) isoforms, human prostate cancer cells and xenograft. Enantiomers of lead demonstrated significant ability to reduce prostate cancer in vivo.
Asunto(s)
Amidas/química , Amidas/uso terapéutico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/uso terapéutico , Amidas/síntesis química , Animales , Antineoplásicos/síntesis química , Línea Celular , Línea Celular Tumoral , Técnicas de Química Sintética/métodos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Isomerismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Próstata/efectos de los fármacos , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Bloqueadores de los Canales de Sodio/síntesis química , Canales de Sodio/química , Canales de Sodio/metabolismoRESUMEN
N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.
Asunto(s)
Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo N/metabolismo , Indoles/uso terapéutico , Dolor/tratamiento farmacológico , Animales , Bloqueadores de los Canales de Calcio/farmacocinética , Bloqueadores de los Canales de Calcio/farmacología , Perros , Haplorrinos , Humanos , Indoles/farmacocinética , Indoles/farmacología , RatasRESUMEN
A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.
Asunto(s)
Compuestos de Bifenilo/química , Neuralgia/tratamiento farmacológico , Pirazoles/química , Bloqueadores de los Canales de Sodio/química , Canales de Sodio/química , Animales , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/uso terapéutico , Perros , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Actividad Motora/efectos de los fármacos , Pirazoles/farmacocinética , Pirazoles/uso terapéutico , Ratas , Bloqueadores de los Canales de Sodio/farmacocinética , Bloqueadores de los Canales de Sodio/uso terapéutico , Canales de Sodio/metabolismoRESUMEN
Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. A series of low molecular weight biaryl substituted pyrazole carboxamides were identified with good in-vitro potency and in-vivo efficacy. Compound 26, a Nav1.7 blocker has excellent efficacy in the Chung model of neuropathic pain.
Asunto(s)
Neuralgia/tratamiento farmacológico , Pirazoles/química , Pirazoles/uso terapéutico , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/uso terapéutico , Canales de Sodio/metabolismo , Animales , Perros , Haplorrinos , Humanos , Microsomas Hepáticos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7 , Pirazoles/farmacocinética , Pirazoles/farmacología , Ratas , Bloqueadores de los Canales de Sodio/farmacocinética , Bloqueadores de los Canales de Sodio/farmacología , Relación Estructura-ActividadRESUMEN
Voltage-gated sodium channels have been shown to play a critical role in neuropathic pain. With a goal to develop potent peripherally active sodium channel blockers, a series of low molecular weight biaryl substituted imidazoles, oxazoles, and thiazole carboxamides were identified with good in vitro and in vivo potency.
Asunto(s)
Neuralgia/tratamiento farmacológico , Oxazoles/uso terapéutico , Bloqueadores de los Canales de Sodio/uso terapéutico , Canales de Sodio/metabolismo , Tiazoles/uso terapéutico , Animales , Perros , Humanos , Imidazoles/química , Imidazoles/metabolismo , Imidazoles/farmacología , Imidazoles/uso terapéutico , Microsomas Hepáticos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7 , Oxazoles/química , Oxazoles/metabolismo , Oxazoles/farmacología , Ratas , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Tiazoles/química , Tiazoles/metabolismo , Tiazoles/farmacologíaRESUMEN
Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50) > 20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.
Asunto(s)
Analgésicos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/fisiología , Indoles/farmacología , Triazoles/farmacología , Analgésicos/efectos adversos , Analgésicos/farmacocinética , Animales , Barorreflejo/efectos de los fármacos , Disponibilidad Biológica , Bloqueadores de los Canales de Calcio/efectos adversos , Bloqueadores de los Canales de Calcio/farmacocinética , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo R/fisiología , Proteínas de Transporte de Catión/fisiología , Línea Celular , Perros , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Hiperalgesia/tratamiento farmacológico , Hipotensión Ortostática/inducido químicamente , Indoles/efectos adversos , Indoles/farmacocinética , Masculino , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/fisiología , Dolor/tratamiento farmacológico , Dolor/etiología , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Triazoles/efectos adversos , Triazoles/farmacocinéticaRESUMEN
Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.
Asunto(s)
Isoxazoles/química , Isoxazoles/farmacología , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Animales , Isoxazoles/uso terapéutico , Modelos Moleculares , Estructura Molecular , Dolor/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/uso terapéutico , Nervios Espinales/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
A series of novel isoxazole voltage gated sodium channel blockers have been synthesized and evaluated. Substitutions on the benzylic position of benzamide were investigated to determine their effect on Na(v)1.7 inhibitory potency. The spirocyclobutyl substitution had the most significant enhancement on Na(v)1.7 inhibitory activity.
Asunto(s)
Isoxazoles/uso terapéutico , Dolor/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/uso terapéutico , Canales de Sodio/metabolismo , Animales , Línea Celular , Enfermedad Crónica , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Dolor/inmunología , Ratas , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Nervios Espinales/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.
Asunto(s)
Anticolesterolemiantes/farmacología , Azetidinas/farmacología , Colesterol en la Dieta/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Perros , Ezetimiba , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/genética , Proteínas de Transporte de Membrana/genética , Ratones , Mutación , Polimorfismo Genético , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Proteómica/métodosRESUMEN
Secondary active glucose transport is mediated by at least four members of the solute-linked carrier 5 gene family (sodium/glucose transporter [SGLT] 1-4). Human genetic disorders of SGLTs including glucose-galactose malabsorption and familial renal glucosuria have increased attention on members of this family of transporters as putative drug targets. Using human SGLT1 (hSGLT1) as a paradigm, we developed a functional assay that should be adaptable to ultra-high-throughput operation and to other SGLTs. Human embryonic kidney (HEK) 293 cells stably expressing hSGLT1 (hSGLT1/HEK293 cells) display a Na(+)-dependent, phlorizin-sensitive alpha-methyl-D-[(14)C]glucopyranoside flux with expected kinetic parameters. In electrophysiological studies with hSGLT1/HEK293 cells, substrate-dependent changes in membrane potential were observed, consistent with the electrogenic operation of hSGLT1. With the use of voltage-sensitive dyes, a membrane potential, fluorescence resonance energy transfer-based functional assay on a voltage/ion probe reader platform has been established for SGLT1. This high-capacity functional assay displays similar characteristics in terms of substrate specificity and phlorizin sensitivity to those determined by more traditional approaches and should provide a means to identify novel and selective SGLT inhibitors.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Transportador 1 de Sodio-Glucosa/análisis , Barbitúricos , Línea Celular , Colorantes , Cumarinas , Interpretación Estadística de Datos , Electrofisiología , Etanolaminas , Humanos , Isoxazoles , Cinética , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Florizina/farmacología , Transportador 1 de Sodio-Glucosa/metabolismo , TiobarbitúricosRESUMEN
A series of imidazopyridines were evaluated as potential sodium channel blockers for the treatment of neuropathic pain. Several members were identified with good hNa(v)1.7 potency and excellent rat pharmacokinetic profiles. Compound 4 had good efficacy (52% and 41% reversal of allodynia at 2 and 4h post-dose, respectively) in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain when dosed orally at 10mg/kg.
Asunto(s)
Piridinas/química , Piridinas/farmacología , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Analgésicos/química , Analgésicos/farmacología , Animales , Inflamación/tratamiento farmacológico , Estructura Molecular , Canal de Sodio Activado por Voltaje NAV1.7 , Dolor/tratamiento farmacológico , Ratas , Bloqueadores de los Canales de Sodio/farmacocinética , Relación Estructura-ActividadRESUMEN
A series of 3-amino-1,5-benzodiazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of this series displayed subnanomolar, state-dependent sodium channel block, and was orally efficacious in a mouse model of epilepsy.
Asunto(s)
Anticonvulsivantes/farmacología , Benzodiazepinonas/farmacología , Epilepsia/tratamiento farmacológico , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Bloqueadores de los Canales de Sodio/farmacología , Animales , Anticonvulsivantes/síntesis química , Anticonvulsivantes/farmacocinética , Benzodiazepinonas/síntesis química , Benzodiazepinonas/farmacocinética , Electrofisiología , Electrochoque , Epilepsia/metabolismo , Canales de Potasio Éter-A-Go-Go/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ratones , Estructura Molecular , Ratas , Bloqueadores de los Canales de Sodio/síntesis química , Bloqueadores de los Canales de Sodio/farmacocinéticaRESUMEN
Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.
Asunto(s)
Azetidinas/metabolismo , Colesterol/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Azetidinas/química , Células CACO-2 , Línea Celular , Clonación Molecular , Perros , Ezetimiba , Humanos , Proteínas de Transporte de Membrana/metabolismo , Reproducibilidad de los Resultados , Sitoesteroles/metabolismo , Sulfonamidas/química , Transfección , Tritio , beta-Lactamas/metabolismoRESUMEN
Voltage-gated sodium channels (Nav1) transmit pain signals from peripheral nociceptive neurons, and blockers of these channels have been shown to ameliorate a number of pain conditions. Because these drugs can have adverse effects that limit their efficacy, more potent and selective Nav1 inhibitors are being pursued. Recent human genetic data have provided strong evidence for the involvement of the peripheral nerve sodium channel subtype, Nav1.7, in the signaling of nociceptive information, highlighting the importance of identifying selective Nav1.7 blockers for the treatment of chronic pain. Using a high-throughput functional assay, novel Nav1.7 blockers, namely, the 1-benzazepin-2-one series, have recently been identified. Further characterization of these agents indicates that, in addition to high-affinity inhibition of Nav1.7 channels, selectivity against the Nav1.5 and Nav1.8 subtypes can also be achieved within this structural class. The most potent, nonselective member of this class of Nav1.7 blockers has been radiolabeled with tritium. [3H]BNZA binds with high affinity to rat brain synaptosomal membranes (Kd = 1.5 nM) and to membranes prepared from HEK293 cells stably transfected with hNav1.5 (Kd = 0.97 nM). In addition, and for the first time, high-affinity binding of a radioligand to hNav1.7 channels (Kd = 1.6 nM) was achieved with [3H]BNZA, providing an additional means for identifying selective Nav1.7 channel inhibitors. Taken together, these data suggest that members of the novel 1-benzazepin-2-one structural class of Nav1 blockers can display selectivity toward the peripheral nerve Nav1.7 channel subtype, and with appropriate pharmacokinetic and drug metabolism properties, these compounds could be developed as analgesic agents.
Asunto(s)
Benzazepinas/química , Bloqueadores de los Canales de Sodio/química , Canales de Sodio/fisiología , Animales , Benzazepinas/metabolismo , Benzazepinas/farmacología , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Electrofisiología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Estructura Molecular , Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.8 , Ensayo de Unión Radioligante , Ratas , Bloqueadores de los Canales de Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Sinaptosomas/metabolismoRESUMEN
A series of benzazepinones were synthesized and evaluated as hNa(v)1.7 sodium channel blockers. Several compounds from this series displayed good oral bioavailability and exposure and were efficacious in a rat model of neuropathic pain.
Asunto(s)
Benzodiazepinonas/síntesis química , Benzodiazepinonas/uso terapéutico , Neuralgia/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/síntesis química , Bloqueadores de los Canales de Sodio/uso terapéutico , Canales de Sodio/efectos de los fármacos , Animales , Benzodiazepinonas/farmacocinética , Disponibilidad Biológica , Modelos Animales de Enfermedad , Perros , Evaluación Preclínica de Medicamentos , Estructura Molecular , Canal de Sodio Activado por Voltaje NAV1.7 , Ratas , Bloqueadores de los Canales de Sodio/farmacocinética , Canales de Sodio/químicaRESUMEN
A series of benzodiazepines and benzazepinones were synthesized and evaluated as potential sodium channel blockers in a functional, membrane potential-based assay. One member of the benzazepinone series, compound 47, displayed potent, state-dependent block of hNa(v)1.7, and was orally efficacious in a rat model of neuropathic pain.
Asunto(s)
Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Dolor/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/clasificación , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Animales , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Compuestos Heterocíclicos con 3 Anillos/farmacología , Estructura Molecular , Canal de Sodio Activado por Voltaje NAV1.7 , Ratas , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/uso terapéutico , Relación Estructura-ActividadRESUMEN
Novel cyclopentane-based 3-phenyl-1-hydroxypropyl compounds were evaluated for inhibitory activity against the peripheral nerve sodium channel Na(V)1.7 and off-target activity against the cardiac potassium channel hERG. The stereochemistry of the hydroxyl group and substitution on the phenyl rings with either fluorinated O-alkyl or alkyl groups were found to be critical for conferring potency against Na(V)1.7. A benchmark compound from this series displayed efficacy in rat models of inflammatory and neuropathic pain.
Asunto(s)
Ciclopentanos/química , Ciclopentanos/farmacología , Bloqueadores de los Canales de Sodio/síntesis química , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Animales , Ciclopentanos/síntesis química , Ciclopentanos/farmacocinética , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Ratas , Bloqueadores de los Canales de Sodio/química , Bloqueadores de los Canales de Sodio/farmacocinética , Relación Estructura-ActividadRESUMEN
A new series of voltage-gated sodium channel blockers with potential for treatment of chronic pain is reported. Systematic structure-activity relationship studies, starting with compound 1, led to identification of potent analogs that displayed use-dependent block of sodium channels, were efficacious in pain models in vivo, and most importantly, were devoid of activity against the cardiac potassium channel hERG.
Asunto(s)
Dolor/tratamiento farmacológico , Bloqueadores de los Canales de Sodio/uso terapéutico , Enfermedad Crónica , Humanos , Conformación Molecular , Sondas Moleculares , Bloqueadores de los Canales de Sodio/químicaRESUMEN
The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.
Asunto(s)
Colorantes/análisis , Transferencia Resonante de Energía de Fluorescencia/métodos , Canales de Sodio/análisis , Canales de Sodio/fisiología , Línea Celular , Colorantes/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas Musculares/análisis , Proteínas Musculares/fisiología , Canal de Sodio Activado por Voltaje NAV1.5 , Canal de Sodio Activado por Voltaje NAV1.7 , Bloqueadores de los Canales de Sodio/farmacología , Veratridina/farmacologíaRESUMEN
Sodium channel blockers are used clinically to treat a number of neuropathic pain conditions, but more potent and selective agents should improve on the therapeutic index of currently used drugs. In a high-throughput functional assay, a novel sodium channel (Na(V)) blocker, N-[[2'-(aminosulfonyl)biphenyl-4-yl]methyl]-N'-(2,2'-bithien-5-ylmethyl)succinamide (BPBTS), was discovered. BPBTS is 2 orders of magnitude more potent than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain. Resembling block by these agents, block of Na(V)1.2, Na(V)1.5, and Na(V)1.7 by BPBTS was found to be voltage- and use-dependent. BPBTS appeared to bind preferentially to open and inactivated states and caused a dose-dependent hyperpolarizing shift in the steady-state availability curves for all sodium channel subtypes tested. The affinity of BPBTS for the resting and inactivated states of Na(V)1.2 was 1.2 and 0.14 microM, respectively. BPBTS blocked Na(V)1.7 and Na(V)1.2 with similar potency, whereas block of Na(V)1.5 was slightly more potent. The slow tetrodotoxin-resistant Na(+) current in small-diameter DRG neurons was also potently blocked by BPBTS. [(3)H]BPBTS bound with high affinity to a single class of sites present in rat brain synaptosomal membranes (K(d) = 6.1 nM), and in membranes derived from HEK cells stably expressing Na(V)1.5 (K(d) = 0.9 nM). BPBTS dose-dependently attenuated nociceptive behavior in the formalin test, a rat model of tonic pain. On the basis of these findings, BPBTS represents a structurally novel and potent sodium channel blocker that may be used as a template for the development of analgesic agents.