RESUMEN
Herpes simplex virus (HSV) infection induces a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), which plays a critical role in facilitating viral entry. T-type calcium channel blockers and EGTA, a chelate of extracellular Ca2+, suppress HSV-2 infection. But the cellular mechanisms mediating HSV infection-activated Ca2+ signaling have not been completely defined. In this study we investigated whether the TRPV4 channel was involved in HSV-2 infection in human vaginal epithelial cells. We showed that the TRPV4 channel was expressed in human vaginal epithelial cells (VK2/E6E7). Using distinct pharmacological tools, we demonstrated that activation of the TRPV4 channel induced Ca2+ influx, and the TRPV4 channel worked as a Ca2+-permeable channel in VK2/E6E7 cells. We detected a direct interaction between the TRPV4 channel protein and HSV-2 glycoprotein D in the plasma membrane of VK2/E6E7 cells and the vaginal tissues of HSV-2-infected mice as well as in phallic biopsies from genital herpes patients. Pretreatment with specific TRPV4 channel inhibitors, GSK2193874 (1-4 µM) and HC067047 (100 nM), or gene silence of the TRPV4 channel not only suppressed HSV-2 infectivity but also reduced HSV-2-induced cytokine and chemokine generation in VK2/E6E7 cells by blocking Ca2+ influx through TRPV4 channel. These results reveal that the TRPV4 channel works as a Ca2+-permeable channel to facilitate HSV-2 infection in host epithelial cells and suggest that the design and development of novel TRPV4 channel inhibitors may help to treat HSV-2 infections.
Asunto(s)
Infecciones por Herpesviridae , Herpesvirus Humano 2 , Canales Catiónicos TRPV , Animales , Femenino , Humanos , Ratones , Señalización del Calcio/genética , Señalización del Calcio/fisiología , Células Epiteliales/metabolismo , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/metabolismo , Transducción de Señal/fisiología , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/fisiologíaRESUMEN
OBJECTIVE: To study the effects of total ginsenosides (TG) extract from Panax ginseng on neural stem cell (NSC) proliferation and differentiation and their underlying mechanisms. METHODS: The migration of NSCs after treatment with various concentrations of TG extract (50, 100, or 200 µ g/mL) were monitored. The proliferation of NSCs was examined by a combination of cell counting kit-8 and neurosphere assays. NSC differentiation mediated by TG extract was evaluated by Western blotting and immunofluorescence staining to monitor the expression of nestin and microtubule associated protein 2 (MAP2). The GSK-3ß/ß-catenin pathway in TG-treated NSCs was examined by Western blot assay. The NSCs with constitutively active GSK-3ß mutant were made by adenovirus-mediated gene transfection, then the proliferation and differentiation of NSCs mediated by TG were further verified. RESULTS: TG treatment significantly enhanced NSC migration (P<0.01 or P<0.05) and increased the proliferation of NSCs (P<0.01 or P<0.05). TG mediation also significantly upregulated MAP2 expression but downregulated nestin expression (P<0.01 or P<0.05). TG extract also significantly induced GSK-3ß phosphorylation at Ser9, leading to GSK-3ß inactivation and, consequently, the activation of the GSK-3ß/ß-catenin pathway (P<0.01 or P<0.05). In addition, constitutive activation of GSK-3ß in NSCs by the transfection of GSK-3ß S9A mutant was found to significantly suppress TG-mediated NSC proliferation and differentiation (P<0.01 or P<0.05). CONCLUSION: TG promoted NSC proliferation and neuronal differentiation by inactivating GSK-3ß.
Asunto(s)
Ginsenósidos , Células-Madre Neurales , Panax , Animales , Diferenciación Celular , Proliferación Celular , Ginsenósidos/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células-Madre Neurales/metabolismo , Extractos Vegetales/farmacología , Ratas , beta Catenina/metabolismoRESUMEN
Dysregulation of NLRP3 inflammasome results in uncontrolled inflammation, which participates in various chronic diseases. TWIK2 potassium channel mediates potassium efflux that has been reported to be an essential upstream mechanism for ATP-induced NLRP3 inflammasome activation. Thus, TWIK2 potassium channel could be a potential drug target for NLRP3-related inflammatory diseases. In the present study we investigated the effects of known K2P channel modulators on TWIK2 channel expressed in a heterologous system. In order to increase plasma membrane expression and thus TWIK2 currents, a mutant channel with three mutations (TWIK2I289A/L290A/Y308A) in the C-terminus was expressed in COS-7 cells. TWIK2 currents were assessed using whole-cell voltage-clamp recording. Among 6 known K2P channel modulators tested (DCPIB, quinine, fluoxetine, ML365, ML335, and TKDC), ML365 was the most potent TWIK2 channel blocker with an IC50 value of 4.07 ± 1.5 µM. Furthermore, ML365 selectively inhibited TWIK2 without affecting TWIK1 or THIK1 channels. We showed that ML365 (1, 5 µM) concentration-dependently inhibited ATP-induced NLRP3 inflammasome activation in LPS-primed murine BMDMs, whereas it did not affect nigericin-induced NLRP3, or non-canonical, AIM2 and NLRC4 inflammasomes activation. Knockdown of TWIK2 significantly impaired the inhibitory effect of ML365 on ATP-induced NLRP3 inflammasome activation. Moreover, we demonstrated that pre-administration of ML365 (1, 10, 25 mg/kg, ip) dose-dependently ameliorated LPS-induced endotoxic shock in mice. In a preliminary pharmacokinetic study conducted in rats, ML365 showed good absolute oral bioavailability with F value of 22.49%. In conclusion, ML365 provides a structural reference for future design of selective TWIK2 channel inhibitors in treating related inflammatory diseases.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Adenosina Trifosfato/metabolismo , Animales , Proteínas de Unión al ADN , Inflamasomas/metabolismo , Inflamación , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , RatasRESUMEN
Urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) are important targets for the development of uric acid-lowering drugs. We previously showed that the flexible linkers of URAT1 inhibitors could enhance their potency. In this study we designed and synthesized CDER167, a novel RDEA3710 analogue, by introducing a linker (methylene) between the naphthalene and pyridine rings to increase flexibility, and characterized its pharmacological and pharmacokinetics properties in vitro and in vivo. We showed that CDER167 exerted dual-target inhibitory effects on both URAT1 and GLUT9: CDER167 concentration-dependently inhibited the uptake of [14C]-uric acid in URAT1-expressing HEK293 cells with an IC50 value of 2.08 ± 0.31 µM, which was similar to that of RDEA3170 (its IC50 value was 1.47 ± 0.23 µM). Using site-directed mutagenesis, we demonstrated that CDER167 might interact with URAT1 at S35 and F365. In GLUT9-expressing HEK293T cells, CDER167 concentration-dependently inhibited GLUT9 with an IC50 value of 91.55 ± 15.28 µM, whereas RDEA3170 at 100 µM had no effect on GLUT9. In potassium oxonate-induced hyperuricemic mice, oral administration of CDER167 (10 mg·kg-1 · d-1) for 7 days was more effective in lowering uric acid in blood and significantly promoted uric acid excretion in urine as compared with RDEA3170 (20 mg·kg-1 · d-1) administered. The animal experiment proved the safety of CDER167. In addition, CDER167 displayed better bioavailability than RDEA3170, better metabolic stability and no hERG toxicity at 100 µM. These results suggest that CDER167 deserves further investigation as a candidate antihyperuricemic drug targeting URAT1 and GLUT9.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa , Hiperuricemia , Transportadores de Anión Orgánico , Proteínas de Transporte de Catión Orgánico , Humanos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Células HEK293 , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/metabolismo , Estructura Molecular , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Relación Estructura-ActividadRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in controlling pacemaker activity in both heart and nervous system. Developing HCN channel inhibitors has been proposed to be an important strategy for the treatment of pain, heart failure, arrhythmias, and epilepsy. One HCN channel inhibitor, ivabradine, has been clinically approved for the treatment of angina pectoris and heart failure. In this study, we designed and synthesized eight alkanol amine derivatives, and assessed their effects on HCN channels expressed in COS7 cells using a whole-cell patch clamp method. Among them, compound 4e displayed the most potent inhibitory activity with an IC50 of 2.9 ± 1.2 µM at - 120 mV on HCN2 channel expressed in COS7 cells. Further analysis revealed that application of compound 4e (10 µM) caused a slowing of activation and a hyperpolarizing shift (ΔV1/2 = - 30.2 ± 2.9 mV, n = 5) in the voltage dependence of HCN2 channel activation. The inhibitory effect of compound 4e on HCN1 and HCN4 channel expressed in COS7 cells was less potent with IC50 of 17.2 ± 1.3 and 7.3 ± 1.2 µM, respectively. Besides, we showed that application of compound 4e (10 µM) inhibited Ih and action potential firing in acutely dissociated mouse small dorsal root ganglion neurons. Our study provides a new strategy for the design and development of potent HCN channel inhibitors.
Asunto(s)
Amino Alcoholes/farmacología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Moduladores del Transporte de Membrana/farmacología , Potenciales de Acción/efectos de los fármacos , Amino Alcoholes/síntesis química , Amino Alcoholes/química , Animales , Células COS , Chlorocebus aethiops , Humanos , Masculino , Moduladores del Transporte de Membrana/síntesis química , Moduladores del Transporte de Membrana/química , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Canales de PotasioRESUMEN
AIM: Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies. METHODS: A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System. RESULTS: A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC50 value=0.10 ± 0.05 µmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 µmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 µmol/L) did not affect the kinetics of channel activation and deactivation. CONCLUSION: The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.
Asunto(s)
Anticonvulsivantes/farmacología , Carbamatos/farmacología , Canal de Potasio KCNQ2/antagonistas & inhibidores , Fenilendiaminas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Animales , Antracenos/farmacología , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Células CHO , Carbamatos/síntesis química , Carbamatos/química , Cricetinae , Cricetulus , Diseño de Fármacos , Concentración 50 Inhibidora , Técnicas de Placa-Clamp , Fenilendiaminas/síntesis química , Fenilendiaminas/química , Bloqueadores de los Canales de Potasio/síntesis química , Bloqueadores de los Canales de Potasio/química , Relación Estructura-ActividadRESUMEN
AIM: This study was conducted to test the selectivity of DC031050 on cardiac and neuronal potassium channels. METHODS: Human ether-à-go-go related gene (hERG), KCNQ and Kv1.2 channels were expressed in CHO cells. The delayed rectifier potassium current (I(K)) was recorded from dissociated hippocampal pyramidal neurons of neonatal rats. Whole-cell voltage patch clamp was used to record the voltage-activated potassium currents. Drug-containing solution was delivered using a RSC-100 Rapid Solution Changer. RESULTS: Both DC031050 and dofetilide potently inhibited hERG currents with IC(50) values of 2.3 ± 1.0 and 17.9 ± 1.2 nmol/L, respectively. DC031050 inhibited the I(K) current with an IC(50) value of 2.7 ± 1.5 µmol/L, which was >1000 times the concentration required to inhibit hERG current. DC031050 at 3 µmol/L did not significantly affect the voltage-dependence of the steady activation, steady inactivation of I(K), or the rate of I(K) from inactivation. Intracellular application of DC031050 (5 µmol/L) was insufficient to inhibit I(K). DC031050 up to 10 µmol/L had no effects on KCNQ2 and Kv1.2 channel currents. CONCLUSION: DC031050 is a highly selective hERG potassium channel blocker with a substantial safety margin of activity over neuronal potassium channels, thus holds significant potential for therapeutic application as a class III antiarrhythmic agent.
Asunto(s)
Antiarrítmicos/farmacología , Canales de Potasio Éter-A-Go-Go/metabolismo , Canales de Potasio KCNQ/metabolismo , Canal de Potasio Kv.1.2/metabolismo , Fenetilaminas/farmacología , Células Piramidales/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Antiarrítmicos/química , Células CHO , Cricetinae , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/genética , Expresión Génica , Hipocampo/citología , Hipocampo/efectos de los fármacos , Humanos , Canales de Potasio KCNQ/antagonistas & inhibidores , Canales de Potasio KCNQ/genética , Canal de Potasio Kv.1.2/antagonistas & inhibidores , Canal de Potasio Kv.1.2/genética , Técnicas de Placa-Clamp , Fenetilaminas/química , Potasio/metabolismo , Células Piramidales/metabolismo , Ratas , Ratas Sprague-Dawley , Sulfonamidas/químicaRESUMEN
Human ether-a-go-go related gene (hERG) potassium (K(+)) channels play a critical role in cardiac action potential repolarization. Mutations that reduce hERG conductance or surface expression may cause congenital long QT syndrome (LQTS). However, the channels can be inhibited by structurally diverse small molecules, resulting in an acquired form of LQTS. Consequently, small molecules that increase the hERG current may be of value for treatment for LQTS. So far, nine hERG activators have been reported. The aim of this review is to discuss recent advances concerning the identification and action mechanism of hERG activators.