RESUMEN
Pendrin (SLC26A4) is a Cl(-)/anion exchanger expressed in the epithelium of inflamed airways where it is thought to facilitate Cl(-) absorption and HCO3 (-) secretion. Studies using pendrin knockout mice and airway epithelial cells from hearing-impaired subjects with pendrin loss of function suggest involvement of pendrin in inflammatory lung diseases, including cystic fibrosis (CF), perhaps by regulation of airway surface liquid (ASL) volume. Here we identified small-molecule pendrin inhibitors and demonstrated their efficacy in increasing ASL volume. A cell-based, functional high-throughput screen of â¼36,000 synthetic small molecules produced 3 chemical classes of inhibitors of human pendrin. After structure-activity studies, tetrahydropyrazolopyridine and pyrazolothiophenesulfonamide compounds reversibly inhibited pendrin-facilitated Cl(-) exchange with SCN(-), I(-), NO3 (-), and HCO3 (-) with drug concentration causing 50% inhibition down to â¼2.5 µM. In well-differentiated primary cultures of human airway epithelial cells from non-CF and CF subjects, treatment with IL-13, which causes inflammation with strong pendrin up-regulation, strongly increased Cl(-)/HCO3 (-) exchange and the increase was blocked by pendrin inhibition. Pendrin inhibition significantly increased ASL depth (by â¼8 µm) in IL-13-treated non-CF and CF cells but not in untreated cells. These studies implicate the involvement of pendrin-facilitated Cl(-)/HCO3 (-) in the regulation of ASL volume and suggest the utility of pendrin inhibitors in inflammatory lung diseases, including CF.-Haggie, P. M., Phuan, P.-W., Tan, J.-A., Zlock, L., Finkbeiner, W. E., Verkman, A. S. Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis.
Asunto(s)
Antiportadores de Cloruro-Bicarbonato/metabolismo , Fibrosis Quística/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Piridinas/farmacología , Sulfonamidas/farmacología , Animales , Células Cultivadas , Antiportadores de Cloruro-Bicarbonato/antagonistas & inhibidores , Antiportadores de Cloruro-Bicarbonato/genética , Chlorocebus aethiops , Células Epiteliales/efectos de los fármacos , Humanos , Interleucina-13/farmacología , Proteínas de Transporte de Membrana/genética , Piridinas/química , Ratas , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismo , Relación Estructura-Actividad , Transportadores de Sulfato , Sulfonamidas/químicaRESUMEN
Dry eye disorders, including Sjögren's syndrome, constitute a common problem in the aging population, with limited effective therapeutic options available. The cAMP-activated Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is a major prosecretory channel at the ocular surface. We investigated whether compounds that target CFTR can correct the abnormal tear film in dry eye. Small-molecule activators of human wild-type CFTR identified by high-throughput screening were evaluated in cell culture and in vivo assays, to select compounds that stimulate Cl(-)-driven fluid secretion across the ocular surface in mice. An aminophenyl-1,3,5-triazine, CFTRact-K089, fully activated CFTR in cell cultures with EC50 â¼250 nM and produced an â¼8.5 mV hyperpolarization in ocular surface potential difference. When delivered topically, CFTRact-K089 doubled basal tear volume for 4 h and had no effect in CF mice. CFTRact-K089 showed sustained tear film bioavailability without detectable systemic absorption. In a mouse model of aqueous-deficient dry eye produced by lacrimal ablation, topical administration of 0.1 nmol CFTRact-K089 3 times daily restored tear volume to basal levels, preventing corneal epithelial disruption when initiated at the time of surgery and reversing it when started after development of dry eye. Our results support the potential utility of CFTR-targeted activators as a novel prosecretory treatment for dry eye.-Flores, A. M., Casey, S. D., Felix, C. M., Phuan, P. W., Verkman, A. S., Levin, M. H. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/agonistas , Síndromes de Ojo Seco/prevención & control , Regulación de la Expresión Génica/efectos de los fármacos , Lágrimas/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Mutación , Ratas , Ratas Endogámicas F344RESUMEN
The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.
Asunto(s)
Acuaporina 1/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Acuaporina 1/metabolismo , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Cricetinae , Cricetulus , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Fluoresceínas/metabolismo , Hemoglobinas/metabolismo , Humanos , Ósmosis/efectos de los fármacos , Ratas Wistar , Bibliotecas de Moléculas Pequeñas/química , Agua/metabolismoRESUMEN
Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC50 of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC50 of ~0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC50 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC50>15 vs. 2.8 mM).
Asunto(s)
Moduladores del Transporte de Membrana/farmacología , Proteínas de Transporte de Membrana/efectos de los fármacos , Tiourea/farmacología , Urea/metabolismo , Animales , Perros , Relación Dosis-Respuesta a Droga , Células de Riñón Canino Madin Darby , Moduladores del Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Estructura Molecular , Ratas , Relación Estructura-Actividad , Tiourea/análogos & derivados , Tiourea/química , Transfección , Transportadores de UreaRESUMEN
Water channel aquaporin-1 (AQP1) is expressed at epithelial cell plasma membranes in renal proximal tubules and thin descending limb of Henle. Recently, AQP1 was reported to interact with ß-catenin. Here we investigated the relationship between AQP1 and Wnt signaling in in vitro and in vivo models of autosomal dominant polycystic kidney disease (PKD). AQP1 overexpression decreased ß-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with ß-catenin, glycogen synthase kinase 3ß, LRP6, and Axin1. AQP1 inhibited cyst development and promoted branching in matrix-grown MDCK cells. In embryonic kidney cultures, AQP1 deletion increased cyst development by up to â¼ 40%. Kidney size and cyst number were significantly greater in AQP1-null PKD mice than in AQP1-expressing PKD mice, with the difference mainly attributed to a greater number of proximal tubule cysts. Biochemical analysis revealed decreased ß-catenin phosphorylation and increased ß-catenin expression in AQP1-null PKD mice, suggesting enhanced Wnt signaling. These results implicate AQP1 as a novel determinant in renal cyst development that may involve inhibition of Wnt signaling by an AQP1-macromolecular signaling complex.
Asunto(s)
Acuaporina 1/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/patología , Vía de Señalización Wnt , Animales , Acuaporina 1/deficiencia , Acuaporina 1/genética , Adhesión Celular , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Perros , Riñón/embriología , Riñón/metabolismo , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , beta Catenina/metabolismoRESUMEN
We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by â¼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP.
Asunto(s)
Adenosina Trifosfato/metabolismo , Unión Competitiva/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Pirimidinas/metabolismo , Sitios de Unión/fisiología , Unión Competitiva/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Oxazinas/química , Oxazinas/metabolismo , Oxazinas/farmacología , Estructura Secundaria de Proteína , Pirimidinas/química , Pirimidinas/farmacologíaRESUMEN
Combination drug therapies under development for cystic fibrosis caused by the ∆F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a "corrector" to improve its cellular processing and a "potentiator" to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces ∆F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with ∆F508-CFTR stability or corrector action. High-throughput screening and structure-activity analysis identified several classes of potentiators that do not impair corrector action, including tetrahydrobenzothiophenes, thiooxoaminothiazoles, and pyrazole-pyrrole-isoxazoles. The most potent compounds have an EC(50) for ∆F508-CFTR potentiation down to 18 nM and do not reduce corrector efficacy in heterologous ∆F508-CFTR-expressing cells or primary cultures of ∆F508/∆F508 human bronchial epithelia. The ΔF508-CFTR potentiators also activated wild-type and G551D CFTR, albeit weakly. The efficacy of combination therapy for cystic fibrosis caused by the ∆F508 mutation may be improved by replacement of Ivacaftor with a potentiator that does not interfere with corrector action.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activación del Canal Iónico/fisiología , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacología , Animales , Benzodioxoles/química , Benzodioxoles/metabolismo , Benzodioxoles/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/agonistas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Relación Estructura-ActividadRESUMEN
The astrocyte water channel aquaporin-4 (AQP4) regulates extracellular space (ECS) K(+) concentration ([K(+)]e) and volume dynamics following neuronal activation. Here, we investigated how AQP4-mediated changes in [K(+)]e and ECS volume affect the velocity, frequency, and amplitude of cortical spreading depression (CSD) depolarizations produced by surface KCl application in wild-type (AQP4(+/+)) and AQP4-deficient (AQP4(-/-)) mice. In contrast to initial expectations, both the velocity and the frequency of CSD were significantly reduced in AQP4(-/-) mice when compared with AQP4(+/+) mice, by 22% and 32%, respectively. Measurement of [K(+)]e with K(+)-selective microelectrodes demonstrated an increase to â¼35 mM during spreading depolarizations in both AQP4(+/+) and AQP4(-/-) mice, but the rates of [K(+)]e increase (3.5 vs. 1.5 mM/s) and reuptake (t1/2 33 vs. 61 s) were significantly reduced in AQP4(-/-) mice. ECS volume fraction measured by tetramethylammonium iontophoresis was greatly reduced during depolarizations from 0.18 to 0.053 in AQP4(+/+) mice, and 0.23 to 0.063 in AQP4(-/-) mice. Analysis of the experimental data using a mathematical model of CSD propagation suggested that the reduced velocity of CSD depolarizations in AQP4(-/-) mice was primarily a consequence of the slowed increase in [K(+)]e during neuronal depolarization. These results demonstrate that AQP4 effects on [K(+)]e and ECS volume dynamics accelerate CSD propagation.
Asunto(s)
Acuaporina 4/metabolismo , Encéfalo/fisiología , Depresión de Propagación Cortical/genética , Ratones Transgénicos/metabolismo , Análisis de Varianza , Animales , Acuaporina 4/genética , Fraccionamiento Celular , Depresión de Propagación Cortical/efectos de los fármacos , Estimulación Eléctrica , Espacio Extracelular/metabolismo , Electrodos de Iones Selectos , Ratones , Ratones Transgénicos/genética , Modelos Teóricos , Potasio/farmacologíaRESUMEN
Aquaporin 1 (AQP1) is a plasma membrane water-transporting protein expressed strongly in tumor microvascular endothelia. We previously reported impaired angiogenesis in implanted tumors in AQP1-deficient mice and reduced migration of AQP1-deficient endothelial cells in vitro. Here, we investigated the consequences of AQP1 deficiency in mice that spontaneously develop well-differentiated, luminal-type breast adenomas with lung metastases [mouse mammary tumor virus-driven polyoma virus middle T oncogene (MMTV-PyVT)]. AQP1(+/+) MMTV-PyVT mice developed large breast tumors with total tumor mass 3.5 ± 0.5 g and volume 265 ± 36 mm(3) (SE, 11 mice) at age 98 d. Tumor mass (1.6±0.2 g) and volume (131±15 mm(3), 12 mice) were greatly reduced in AQP1(-/-) MMTV-PyVT mice (P<0.005). CD31 immunofluorescence showed abnormal microvascular anatomy in tumors of AQP1(-/-) MMTV-PyVT mice, with reduced vessel density. HIF-1α expression was increased in tumors in AQP1(-/-) MMTV-PyVT mice. The number of lung metastases (5±1/mouse) was much lower than in AQP1(+/+) MMTV-PyVT mice (31±8/mouse, P<0.005). These results implicate AQP1 as an important determinant of tumor angiogenesis and, hence, as a potential drug target for adjuvant therapy of solid tumors.
Asunto(s)
Acuaporina 1/genética , Neoplasias de la Mama/patología , Eliminación de Gen , Neoplasias Pulmonares/secundario , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/genética , Ratones , Ratones Noqueados , Neovascularización Patológica/genéticaRESUMEN
Aquaporin-4 (AQP4), the principal water channel in astrocytes, is involved in brain water movement, inflammation, and neuroexcitation. In this study, there was strong neuroprotection in mice lacking AQP4 in a model of global cerebral ischemia produced by transient, bilateral carotid artery occlusion (BCAO). Survival and neurological outcome were greatly improved in the AQP4(-/-) vs. AQP4(+/+) mice after occlusion, with large and robust differences in both outbred (CD1) and inbred (C57bl/6) mouse strains without or with mechanical ventilation. Improved survival was also seen in mice lacking the scaffold protein α-syntrophin, which manifest reduced astrocyte water permeability secondary to defective AQP4 plasma membrane targeting. Intracranial pressure elevation and brain water accumulation were much reduced in the AQP4(-/-) vs. AQP4(+/+) mice after carotid artery occlusion, as were blood-brain barrier (BBB) disruption and neuronal loss. Brain slices from AQP4(-/-) mice showed significantly reduced cell swelling and cytotoxicity in response to oxygen-glucose deprivation, compared with slices from AQP4(+/+) mice. Our findings suggest that the neuroprotective effect of AQP4 deletion in global cerebral ischemia involves reduced astrocyte swelling and brain water accumulation, resulting in reduced BBB disruption, inflammation, and neuron death. AQP4 water transport inhibition may improve survival and neurological outcome after cardiac arrest and in other conditions associated with global cerebral ischemia.
Asunto(s)
Acuaporina 4/metabolismo , Isquemia Encefálica/metabolismo , Animales , Acuaporina 4/genética , Isquemia Encefálica/genética , Isquemia Encefálica/fisiopatología , Hipocampo/metabolismo , Hipocampo/patología , Inmunohistoquímica , Presión Intracraneal/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to â¼1 µM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by â¼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.
Asunto(s)
Transporte Biológico/efectos de los fármacos , Diuresis/efectos de los fármacos , Capacidad de Concentración Renal/efectos de los fármacos , Proteínas de Transporte de Membrana/química , Bibliotecas de Moléculas Pequeñas/farmacología , Orina/química , Animales , Cromatografía Liquida , Diuresis/fisiología , Perros , Ensayos Analíticos de Alto Rendimiento , Células de Riñón Canino Madin Darby , Masculino , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Concentración Osmolar , Ratas , Ratas Wistar , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Cloruro de Sodio , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Distribución Tisular , Sistema Urinario/efectos de los fármacos , Sistema Urinario/metabolismo , Transportadores de UreaRESUMEN
Pseudomonas aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-κB transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-κB activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restore NF-κB-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermalC12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy.
Asunto(s)
4-Butirolactona/análogos & derivados , Antibacterianos/aislamiento & purificación , Pseudomonas aeruginosa/fisiología , Quinolinas/aislamiento & purificación , Factores de Virulencia/antagonistas & inhibidores , 4-Butirolactona/antagonistas & inhibidores , 4-Butirolactona/metabolismo , 4-Butirolactona/toxicidad , Animales , Antibacterianos/farmacología , Células Cultivadas , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , FN-kappa B/metabolismo , Pseudomonas aeruginosa/inmunología , Quinolinas/farmacología , Transducción de Señal , Factores de Virulencia/metabolismo , Factores de Virulencia/toxicidadRESUMEN
BACKGROUND: Rotavirus is the most common cause of severe secretory diarrhoea in infants and young children globally. The rotaviral enterotoxin, NSP4, has been proposed to stimulate calcium-activated chloride channels (CaCC) on the apical plasma membrane of intestinal epithelial cells. We previously identified red wine and small molecule CaCC inhibitors. OBJECTIVE: To investigate the efficacy of a red wine extract and a synthetic small molecule, CaCCinh-A01, in inhibiting intestinal CaCCs and rotaviral diarrhoea. DESIGN: Inhibition of CaCC-dependent current was measured in T84 cells and mouse ileum. The effectiveness of an orally administered wine extract and CaCCinh-A01 in inhibiting diarrhoea in vivo was determined in a neonatal mouse model of rotaviral infection. RESULTS: Screening of â¼150 red wines revealed a Cabernet Sauvignon that inhibited CaCC current in T84 cells with IC50 at a â¼1:200 dilution, and higher concentrations producing 100% inhibition. A >1 kdalton wine extract prepared by dialysis, which retained full inhibition activity, blocked CaCC current in T84 cells and mouse intestine. In rotavirus-inoculated mice, oral administration of the wine extract prevented diarrhoea by inhibition of intestinal fluid secretion without affecting rotaviral infection. The wine extract did not inhibit the cystic fibrosis chloride channel (CFTR) in cell cultures, nor did it prevent watery stools in neonatal mice administered cholera toxin, which activates CFTR-dependent fluid secretion. CaCCinh-A01 also inhibited rotaviral diarrhoea. CONCLUSIONS: Our results support a pathogenic role for enterocyte CaCCs in rotaviral diarrhoea and demonstrate the antidiarrhoeal action of CaCC inhibition by an alcohol-free, red wine extract and by a synthetic small molecule.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Diarrea/prevención & control , Enterocitos/metabolismo , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Infecciones por Rotavirus/prevención & control , Vino , Animales , Línea Celular , Diarrea/metabolismo , Diarrea/virología , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Infecciones por Rotavirus/metabolismoRESUMEN
Quantum dots are bright, photostable fluorophores used extensively to investigate biological processes. In this study, we report that bromocresol green (BCG) at low micromolar concentrations rapidly, efficiently and reversibly quenches the fluorescence of commercial quantum dots having a wide range of functionalities. The broad utility of BCG quenching of quantum dots in cell biology is showed in quantitative assays of trafficking of the ß(2) -adrenergic receptor (ß(2) AR) and the cystic fibrosis transmembrane conductance regulator (CFTR).
Asunto(s)
Verde de Bromocresol/química , Transporte de Proteínas , Puntos Cuánticos , Animales , Línea Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Endocitosis , Exocitosis , Haplorrinos , Humanos , Indicadores y Reactivos , Microscopía Fluorescente/métodos , Estructura Molecular , Tamaño de la Partícula , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Espectrometría de Fluorescencia/métodos , Resonancia por Plasmón de Superficie/métodos , TransfecciónRESUMEN
The supramolecular assembly of aquaporin-4 (AQP4) in orthogonal arrays of particles (OAPs) involves N-terminus interactions of the M23-AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4-transfected cells, (ii) photobleaching recovery of green fluorescent protein-AQP4 chimeras in live cells and (iii) freeze-fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes, but not in the Golgi, was not related to AQP4 density, pH, membrane lipid composition, C-terminal PDZ domain interactions or α-syntrophin expression. Remarkably, however, fusion of AQP4-containing Golgi vesicles with (AQP4-free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature-dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to â¼110 nm diameter, but not to â¼220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post-Golgi phenomenon involving plasma membrane-specific factor(s). Post-Golgi and membrane curvature-dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4-facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica.
Asunto(s)
Acuaporina 4/química , Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Multimerización de Proteína , Animales , Acuaporina 4/genética , Acuaporina 4/ultraestructura , Astrocitos/metabolismo , Astrocitos/ultraestructura , Encéfalo/metabolismo , Encéfalo/ultraestructura , Células CHO , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Membrana Celular/ultraestructura , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Recuperación de Fluorescencia tras Fotoblanqueo , Técnica de Fractura por Congelación , Aparato de Golgi/ultraestructura , Humanos , Immunoblotting , Microscopía Electrónica , Microscopía Fluorescente , Estructura Terciaria de Proteína , Fracciones Subcelulares , TransfecciónRESUMEN
The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.
Asunto(s)
Aminopiridinas/farmacología , Benzodioxoles/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Línea Celular , Cloruros/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Perros , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Células de Riñón Canino Madin Darby , Mutación/efectos de los fármacos , Mutación/genética , Permeabilidad/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Mucosa Respiratoria/metabolismo , Relación Estructura-ActividadRESUMEN
Brain edema and associated astrocyte swelling leading to increased intracranial pressure are hallmarks of acute liver failure (ALF). Elevated blood and brain levels of ammonia have been implicated in the development of brain edema in ALF. Cultured astrocytes treated with ammonia have been shown to undergo cell swelling and such swelling was associated with an increase in the plasma membrane expression of aquaporin-4 (AQP4) protein. Further, silencing the AQP4 gene in cultured astrocytes was shown to prevent the ammonia-induced cell swelling. Here, we examined the evolution of brain edema in AQP4-null mice and their wild type counterparts (WT-mice) in different models of ALF induced by thioacetamide (TAA) or acetaminophen (APAP). Induction of ALF with TAA or APAP significantly increased brain water content in WT mice (by 1.6% ± 0.3 and 2.3 ± 0.4%, respectively). AQP4 protein was significantly increased in brain plasma membranes of WT mice with ALF induced by either TAA or APAP. In contrast to WT-mice, brain water content did not increase in AQP4-null mice. Additionally, AQP4-null mice treated with either TAA or APAP showed a remarkably lesser degree of neurological deficits as compared to WT mice; the latter displayed an inability to maintain proper gait, and demonstrated a markedly reduced exploratory behavior, with the mice remaining in one corner of the cage with its head tilted downwards. These results support a central role of AQP4 in the brain edema associated with ALF.
Asunto(s)
Acuaporina 4/deficiencia , Encefalopatías/etiología , Edema Encefálico/etiología , Regulación de la Expresión Génica/genética , Fallo Hepático Agudo/complicaciones , Acetaminofén/toxicidad , Análisis de Varianza , Animales , Acuaporina 4/genética , Encefalopatías/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 1/metabolismo , Ratones , Ratones Transgénicos , Tioacetamida/toxicidad , Factores de TiempoRESUMEN
The aquaporins are a family of membrane water channels, some of which also transport glycerol. They are involved in a wide range of physiological functions (including water/salt homeostasis, exocrine fluid secretion, and epidermal hydration) and human diseases (including glaucoma, cancer, epilepsy, and obesity). At the cellular level, aquaporin-mediated osmotic water transport across cell plasma membranes facilitates transepithelial fluid transport, cell migration, and neuroexcitation; aquaporin-mediated glycerol transport regulates cell proliferation, adipocyte metabolism, and epidermal water retention. Genetic diseases caused by loss-of-function mutations in aquaporins include nephrogenic diabetes insipidus and congenital cataracts. The neuroinflammatory demyelinating disease neuromyelitis optica is marked by pathogenic autoantibodies against astrocyte water channel aquaporin-4. There remain broad opportunities for the development of aquaporin-based diagnostics and therapeutics. Disease-relevant aquaporin polymorphisms are beginning to be explored. There is great promise in the development of small-molecule aquaporin modulators for therapy of some types of refractory edema, brain swelling, neuroinflammation, glaucoma, epilepsy, cancer, pain, and obesity.
Asunto(s)
Acuaporinas/fisiología , Edema Encefálico/fisiopatología , Neuromielitis Óptica/fisiopatología , Obesidad/fisiopatología , Equilibrio Hidroelectrolítico/fisiología , Acuaporinas/genética , Edema Encefálico/genética , Humanos , Neuromielitis Óptica/genética , Obesidad/genéticaRESUMEN
Aquaporin-4 (AQP4) is a water channel expressed in astrocytes, skeletal muscle and epithelial cells that forms supramolecular aggregates in plasma membranes called orthogonal arrays of particles (OAPs). AQP4 is expressed as a short isoform (M23) that forms large OAPs, and a long isoform (M1) that does not form OAPs by itself but can mingle with M23 to form relatively small OAPs. AQP4 OAPs were imaged with ~20 nm spatial precision by photoactivation localization microscopy (PALM) in cells expressing chimeras of M1- or M23-AQP4 with photoactivatable fluorescent proteins. Native AQP4 was imaged by direct stochastic optical reconstruction microscopy (dSTORM) using a primary anti-AQP4 antibody and fluorescent secondary antibodies. We found that OAP area increased from 1878±747 to 3647±958 nm(2) with decreasing M1:M23 ratio from 1:1 to 1:3, and became elongated. Two-color dSTORM indicated that M1 and M23 co-assemble in OAPs with a M1-enriched periphery surrounding a M23-enriched core. Native AQP4 in astrocytes formed OAPs with an area of 2142±829 nm(2), which increased to 5137±1119 nm(2) with 2-bromopalmitate. PALM of AQP4 OAPs in live cells showed slow diffusion (average ~10(-12) cm(2)/s) and reorganization. OAP area was not altered by anti-AQP4 IgG autoantibodies (NMO-IgG) that cause the neurological disease neuromyelitis optica. Super-resolution imaging allowed elucidation of novel nanoscale structural and dynamic features of OAPs.
Asunto(s)
Acuaporina 4/química , Acuaporina 4/metabolismo , Membrana Celular/metabolismo , Imagenología Tridimensional/métodos , Animales , Astrocitos/metabolismo , Células CHO , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Análisis por Conglomerados , Cricetinae , Cricetulus , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Isoformas de Proteínas/metabolismo , Estructura Cuaternaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Diarrheal diseases constitute a significant global health burden and are a major cause of childhood mortality and morbidity. Treatment of diarrheal disease has centered on the replacement of fluid and electrolyte losses using oral rehydration solutions. Although oral rehydration solutions have been highly successful, significant mortality and morbidity due to diarrheal disease remains. Secretory diarrheas, such as those caused by bacterial and viral enterotoxins, result from activation of cyclic nucleotide and/or Ca(2+) signaling pathways in intestinal epithelial cells, enterocytes, which increase the permeability of Cl(-) channels at the lumen-facing membrane. Additionally, there is often a parallel reduction in intestinal Na(+) absorption. Inhibition of enterocyte Cl(-) channels, including the cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, represents an attractive strategy for antisecretory drug therapy. High-throughput screening of synthetic small-molecule collections has identified several classes of Cl(-) channel inhibitors that show efficacy in animal models of diarrhea but remain to be tested clinically. In addition, several natural product extracts with Cl(-) channel inhibition activity have shown efficacy in diarrhea models. However, a number of challenges remain to translate the promising bench science into clinically useful therapeutics, including efficiently targeting orally administered drugs to enterocytes during diarrhea, funding development costs, and carrying out informative clinical trials. Nonetheless, Cl(-) channel inhibitors may prove to be effective adjunctive therapy in a broad spectrum of clinical diarrheas, including acute infectious and drug-related diarrheas, short bowel syndrome, and congenital enteropathies.