RESUMEN
Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.
Asunto(s)
Conexinas/química , Conexinas/metabolismo , Microscopía por Crioelectrón , Activación del Canal Iónico , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Adenosina Trifosfato/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Apoptosis , Sitios de Unión/efectos de los fármacos , Carbenoxolona/química , Carbenoxolona/metabolismo , Carbenoxolona/farmacología , Caspasa 7/metabolismo , Línea Celular , Conexinas/ultraestructura , Uniones Comunicantes , Glicosilación , Humanos , Activación del Canal Iónico/efectos de los fármacos , Modelos Moleculares , Mutación , Proteínas del Tejido Nervioso/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Células Sf9RESUMEN
Carbenoxolone is a derivative of glycyrrhetinic acid found in the root of Glycyrrhiza glabra, colloquially known as licorice. It has been used as a treatment for peptic and oral ulcers. In recent years, carbenoxolone has been utilized in basic research for its ability to block gap junctional communication. Better understanding the distribution of carbenoxolone after systemic administration can lead to a better understanding of its potential sites of action. Presented is an ultra high-performance liquid chromatography tandem mass spectrometer (UHPLC-MS/MS) method for the identification and quantification of carbenoxolone in mouse blood and brain tissue. Twenty mice were injected intraperitoneally with 25 mg/kg carbenoxolone and brain tissue and blood were collected for analysis. Blood concentrations (mean ± SD) at 15, 30, 60 and 120 min were determined to be (n = 5) 5394 ± 778, 2636 ± 836, 1564 ± 541 and 846 ± 252 ng/mL, respectively. Brain concentrations (mean ± SD) at 15, 30, 60 and 120 mins were determined to be (n = 5) 171 ± 62, 102 ± 35, 55 ± 10 and 27 ± 9 ng/g, respectively. The analysis of these specimens at the four different time points resulted in blood and brain half-lives in mice of ~43 and 41 min, respectively. The UHPLC-MS/MS method was determined to be sensitive and robust for quantification of carbenoxolone.
Asunto(s)
Química Encefálica/fisiología , Carbenoxolona/análisis , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Carbenoxolona/administración & dosificación , Carbenoxolona/química , Carbenoxolona/farmacocinética , Estabilidad de Medicamentos , Inyecciones Intraperitoneales , Límite de Detección , Modelos Lineales , Masculino , Ratones , Reproducibilidad de los ResultadosRESUMEN
Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.
Asunto(s)
Aniones/metabolismo , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Aniones/química , Carbenoxolona/química , Carbenoxolona/farmacología , Espacio Extracelular/química , Espacio Extracelular/efectos de los fármacos , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Humanos , Técnicas In Vitro , Cinética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Neurotransmisores/química , Neurotransmisores/farmacología , Oocitos/química , Oocitos/metabolismo , Concentración Osmolar , Permeabilidad , Multimerización de Proteína , Relación Estructura-Actividad , Taurina/química , Taurina/metabolismo , Agua/química , XenopusRESUMEN
We examined cell-to-cell interaction between pre-osteoblasts and osteocytes using MC3T3-E1 and MLO-Y4, respectively. First, GFP expressing MC3T3-E1 (E1-GFP) cells were generated to isolate the cells from co-culture with MLO-Y4. No changes were observed in the expression of osteogenic transcription factors Runx2, Osterix, Dlx5 and Msx2, but expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP) in E1-GFP co-cultured with MLO-Y4 was 300-400-fold greater than that in mono-cultured E1-GFP. In addition, mineralized nodule formation was drastically increased in co-cultured E1-GFP cells compared to mono-cultured cells. Patch clamp assay showed the presence of gap junctions between E1-GFP and MLO-Y4. Furthermore, when the gap junction inhibitor carbenoxolone (CBX) was added to the culture, increased expression of ALP and BSP in E1-GFP co-cultured with MLO-Y4 was suppressed. These results suggest that gap junction detected between pre-osteoblasts and osteocytes plays an important role on the terminal differentiation of pre-osteoblasts.
Asunto(s)
Uniones Comunicantes/fisiología , Regulación de la Expresión Génica , Osteoblastos/citología , Osteocitos/citología , Células 3T3 , Fosfatasa Alcalina/metabolismo , Animales , Carbenoxolona/química , Proteínas Portadoras/metabolismo , Comunicación Celular , Ciclo Celular , Diferenciación Celular , Técnicas de Cocultivo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Sialoproteína de Unión a Integrina/metabolismo , Ratones , Osteocitos/metabolismo , Técnicas de Placa-Clamp , ARN Mensajero/metabolismoRESUMEN
We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ß-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ((10)Panx1) decreased [ATP]e by 75-84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of â¼1 pmol × (10(6) cells)(-1). A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (10(6) cells)(-1), followed by a slower exponential decay (t(½) = 3.7 min) to a constant value of 1.3 pmol × (10(6) cells)(-1). Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (â¼28 fmol × (10(6) cells min)(-1)), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.
Asunto(s)
Adenosina Trifosfato/química , Eritrocitos/metabolismo , Adenilil Ciclasas/química , Animales , Carbenoxolona/química , Colforsina/farmacología , AMP Cíclico/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Homeostasis , Humanos , Hidrólisis , Isoproterenol/farmacología , Cinética , Luciferasas/metabolismo , Microscopía Fluorescente/métodos , Papaverina/farmacología , Péptidos/química , XenopusRESUMEN
The objective of this study was to prepare and evaluate the pectin-based dosage form for buccal adhesion. Carbenoxolone sodium, which is used for the treatment of aphthous ulcers in oral cavity, was used as a model drug. The pectin buccal discs were prepared by direct compression. The water uptake and erosion of pectin disc increased progressively with the swelling time. The bioadhesion of dried pectin discs decreased when either the discs were hydrated or the buccal tissue was wet with a small volume of medium. The influencing factors such as pectin type, pectin to lactose ratio, and sweetener type on the formulations were investigated. The results demonstrated that buccal discs prepared from pectin with a high degree of esterification (DE) showed a weaker and more friable characteristic than that with low DE. Decreasing pectin to lactose ratio resulted in the high dissolution rate with low bioadhesive properties. Addition of sweetener in the formulations also affected the hardness, friability, and bioadhesive properties of the discs. The pectin discs containing sweetening agent showed a higher drug release than those without sweetener. The results suggested that pectin-based bioadhesive discs could be used to deliver carbenoxolone sodium in oral cavity.
Asunto(s)
Carbenoxolona/administración & dosificación , Carbenoxolona/química , Portadores de Fármacos/química , Pectinas/química , Estomatitis Aftosa/tratamiento farmacológico , Adhesivos Tisulares/química , Administración Oral , Animales , Antiulcerosos/administración & dosificación , Antiulcerosos/química , Materiales Biomiméticos/química , Humanos , Mucosa Bucal/química , Edulcorantes/químicaRESUMEN
The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19 µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other "FOXO-resistant" cancers.
Asunto(s)
Carbenoxolona/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Proteína Forkhead Box O3/antagonistas & inhibidores , Proteína Forkhead Box O3/metabolismo , Neuroblastoma/patología , Bibliotecas de Moléculas Pequeñas , Carbenoxolona/química , Muerte Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Humanos , Peso Molecular , Estadificación de Neoplasias , Transcripción Genética/efectos de los fármacosRESUMEN
Nanotechnology based antimicrobial drugs are developed to enhance their properties to combat multidrug resistant microbes. Carbenoxolone (CBX) is a semi-synthetic derivate of a natural substance from the licorice plant, with anti- (inflammatory, fungal, viral, microbial, fibrotic and cancer) properties. Though used to treat gastric ulcers, its low aqueous stability, low bioavailability and toxicity limited the drug's utility. To enhance its antimicrobial activity and reduce cytotoxicity, a controlled release nanoformulation was developed using natural biodegradable polymer chitosan (CS) as a carrier which is biocompatible, nontoxic with placid antimicrobial property. UV-visible spectroscopy, electron microscopy, and Fourier transform infrared spectroscopy were used for characterization of the resultant CS-CBX nanoparticles (NPs). They were spherical with uniform dispersion, ~200 nm in size with surface charge of +18.6 mV and drug encapsulation of >80%. Drug release kinetics exhibited a controlled release of 86% over 36 h following zero order kinetics. The anti-microbial activity against common pathogenic Gram -ve and +ve bacteria and yeast increased ~2-fold with a concomitant 4-fold reduction in cytotoxicity assessed using human lung adeno carcinoma (A549) cells. This study demonstrates the affirmative aspects of CS-CBX NPs as a promising antibacterial agent and may facilitate repositioning of the drug for diverse applications.
Asunto(s)
Carbenoxolona/química , Quitosano/química , Nanopartículas/química , Antiinfecciosos/farmacología , Candida albicans/efectos de los fármacos , Carbenoxolona/farmacología , Carbenoxolona/toxicidad , Línea Celular Tumoral , Quitosano/farmacología , Quitosano/toxicidad , Portadores de Fármacos , Liberación de Fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Ensayo de Materiales , Pruebas de Sensibilidad Microbiana , Nanopartículas/toxicidad , Espectroscopía Infrarroja por Transformada de Fourier , Electricidad EstáticaRESUMEN
The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to the active glucocorticoid cortisol, thereby acting as a cellular switch to mediate glucocorticoid action in many tissues. Several studies have indicated that 11beta-HSD1 plays a crucial role in the onset of type 2 diabetes and central obesity. As a consequence, selective inhibition of 11beta-HSD1 in humans might become a new and promising approach for lowering blood glucose concentrations and for counteracting the accumulation of visceral fat and its related metabolic abnormalities in type 2 diabetes. In this study, we present the synthesis and the biological evaluation of ursan or oleanan type triterpenoids which may act as selective 11beta-HSD1 inhibitors in liver as well as in peripheral tissues, like adipocytes and muscle cells. In order to rationalise the outcomes of the inhibition data, docking simulations of the ligands were performed on the experimentally determined structure of 11beta-HSD1. Furthermore, we discuss the structural determinants that confer enzymatic specificity. From our investigation, valuable information has been obtained to design selective 11beta-HSD1 blockers based on the oleanan and ursan scaffold.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácido Oleanólico/análogos & derivados , Triterpenos/farmacología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/antagonistas & inhibidores , Carbenoxolona/química , Carbenoxolona/farmacología , Dominio Catalítico , Humanos , Concentración 50 Inhibidora , Ácido Oleanólico/química , Ácido Oleanólico/farmacología , Triterpenos/químicaRESUMEN
BACKGROUND: Asthma is a chronic respiratory disease, which needs a safer medication preferably in inhalation form. In view of this, we have evaluated the effect of inhaled carbenoxolone (CBX), a herbal-derived compound, on asthma in a mouse model. METHODS: Mice were sensitized and challenged with ovalbumin (OVA) to develop certain characteristic features of asthma such as airway hyperreactivity (AHR), airway eosinophilia, lung inflammation and mucus hypersecretion. To evaluate the effect of CBX on the above asthmatic features, CBX (2.5, 5 and 10 mg/ml, 3 ml) or vehicle (water) was given by inhalation. AHR was determined using whole-body plethysmography. Infiltration of eosinophils was estimated by microscopy. Lung inflammation and mucus hypersecretion were assessed using hematoxylin and eosin, and periodic acid-Schiff staining, respectively. Th-2 cytokines, IL-4 and IL-5 were measured in bronchoalveolar lavage (BAL) fluid and IgE in sera. To identify the possible mode of CBX action, we measured corticosterone levels in the BAL fluid and 5-lipoxygenase (5-LO) expression in the lungs. RESULTS: CBX (5 mg/ml) inhalation markedly alleviated AHR (p = 0.0032) and reduced lung inflammation and mucus hypersecretion. Also, it prevented the increase in IL-4 (p = 0.0192), IL-5 (p = 0.0116) and eosinophils (p < 0.0005) in the BAL fluid, and OVA-specific IgE levels (p = 0.00061) in sera. 5-LO expression was also markedly reduced. However, corticosterone levels were not affected. CONCLUSIONS: Inhaled CBX alleviates the asthmatic features in mice and could be a potent nebulized therapy in clinical asthma.
Asunto(s)
Asma/prevención & control , Hiperreactividad Bronquial/prevención & control , Carbenoxolona/uso terapéutico , Administración por Inhalación , Animales , Asma/inmunología , Hiperreactividad Bronquial/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Carbenoxolona/administración & dosificación , Carbenoxolona/química , Modelos Animales de Enfermedad , Inmunoglobulina E/sangre , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Nebulizadores y Vaporizadores , Ovalbúmina/inmunologíaRESUMEN
Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain.
Asunto(s)
Conexina 43/química , Osteocitos/metabolismo , Prostaglandinas/metabolismo , Animales , Biotinilación , Western Blotting , Huesos/metabolismo , Carbenoxolona/química , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Pollos , Dendritas/metabolismo , Uniones Comunicantes , Ácido Glicirretínico/química , Ratones , Microscopía Fluorescente , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacología , Ratas , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X7 , Estrés MecánicoRESUMEN
OBJECTIVE: To study the analgesic effects and sites of oxymatrine-carbenoxolone sodium complex (OCSC). METHOD: Adopting formalin test, warm water tail-flick test and intracerebroventricularly (icv) injection to observe the analgesic effects of OCSC in mice. RESULT: Intraperitoneally injecting (ip) OCSC (75, 150 mg x kg(-1)) remarkedly inhibited the pain of mice in the formalin test and prolonged latent phases of tail-shrinking of mice, icy OCSC (1.875, 3.75, 7.5 mg x kg(-1)) significantly prolonged latent phases of tail-shrinking of mice, it had dose-dependent effect with concentration. CONCLUSION: The result indicated that OCSC has obvious analgesic effects and its mechanism may be involved in central nervous system (CNS).
Asunto(s)
Alcaloides/química , Analgésicos/química , Analgésicos/farmacología , Carbenoxolona/química , Carbenoxolona/farmacología , Quinolizinas/química , Analgésicos/administración & dosificación , Analgésicos/uso terapéutico , Animales , Carbenoxolona/administración & dosificación , Carbenoxolona/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Dolor/tratamiento farmacológicoRESUMEN
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11beta-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11beta-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18beta-glycyrrhetinic acid (18beta-GA) derivatives (2-5) and their inhibitory activities against rat hepatic11beta-HSD1 and rat renal 11beta-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds' ability to inhibit human 11beta-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18beta-GA derivatives 2 and 3 with apparent selectivity for rat 11beta-HSD1 showed a high percentage inhibition for human microsomal 11beta-HSD1 at 10 microM and exhibited IC50 values of 400 and 1100 nM, respectively. The side chain modified 18beta-GA derivatives 4 and 5, although showing selectivity for rat 11beta-HSD1 inhibited human microsomal 11beta-HSD1 with IC50 values in the low micromolar range.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacología , Animales , Carbenoxolona/análogos & derivados , Carbenoxolona/química , Carbenoxolona/farmacología , Ácido Glicirretínico/química , Humanos , Técnicas In Vitro , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Modelos Biológicos , Modelos Moleculares , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: Bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD) reversibly oxidizes the 3 alpha and 20 beta hydroxyl groups of androstanes and pregnanes and uses nicotinamide adenine dinucleotide as a cofactor. 3 alpha, 20 beta-HSD belongs to a family of short-chain dehydrogenases that has a highly conserved Tyr-X-X-X-Lys sequence. The family includes mammalian enzymes involved in hypertension, digestion, fertility and spermatogenesis. Several members of the enzyme family, including 3 alpha, 20 beta-HSD, are competitively inhibited by glycyrrhizic acid, a steroidal compound found in licorice, and its derivative, carbenoxolone, an anti-inflammatory glucocorticoid. RESULTS: The three-dimensional structure of the enzyme-carbenoxolone complex has been determined and refined at 2.2 A resolution to a crystallographic R-factor of 19.4%. The hemisuccinate side chain of carbenoxolone makes a hydrogen bond with the hydroxyl group of the conserved residue Tyr152 and occupies the position of the nicotinamide ring of the cofactor. The occupancies of the inhibitor in four independent catalytic sites refine to 100%, 95%, 54% and 36%. CONCLUSIONS: The steroid binds at the catalytic site in a mode much like the previously proposed mode of binding of the substrate cortisone. No bound cofactor molecules were found. The varying occupancy of steroid molecules observed in the four catalytic sites is either due to packing differences or indicates a cooperative effect among the four sites. The observed binding accounts for the inhibition of 3 alpha, 20 beta-HSD.
Asunto(s)
Carbenoxolona/farmacología , Cortisona Reductasa/antagonistas & inhibidores , Sitios de Unión , Carbenoxolona/química , Cortisona Reductasa/química , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , NAD/química , Conformación Proteica , Streptomyces/enzimologíaRESUMEN
The crystal structures of three ester derivatives of glycyrrhetinic acid (GE) are reported. X-ray crystallography revealed that despite differences in the size of the ester substituents (ethyl, isopropyl and 2-morpholinoethyl) the scheme of molecular self-assembly is similar in all three cases but differs significantly from that observed in other known GE esters. According to our analysis, the two basic patterns of self-assembly of GE esters observed in their unsolvated crystals correspond to two distinct orientations of the ester groups relative to the triterpene backbone. Moreover, comparison of the self-assembly modes of GE esters in their unsolvated forms with the supramolecular organization of GE and carbenoxolone in their solvated crystals revealed that ester substituents replace solvent molecules hydrogen bonded to the COOH group at the triterpene skeleton, resulting in similar packing arrangements of these compounds.
Asunto(s)
Ácido Glicirretínico/análogos & derivados , Carbenoxolona/química , Cristalografía por Rayos X , Esterificación , Ésteres/química , Enlace de Hidrógeno , Modelos Moleculares , Triterpenos/químicaRESUMEN
Carbenoxolone (CBXH2), a pharmaceutically relevant derivative of glycyrrhetinic acid, was studied by X-ray crystallography. The crystal structures of its unsolvated form, propionic acid and dimethoxyethane solvates and a solvated cocrystal of the free acid with its monobasic sodium salt CBXH2·CBXHNa·(butan-2-one)2·2H2O reveal that the recurring motif of supramolecular architecture in all crystal forms is a one-dimensional ribbon with closely packed triterpene fragments. It does not result from strong specific interactions but solely from van der Waals interactions. The ribbons are further arranged into diverse layer-type aggregates with a hydrophobic interior (triterpene skeletons) and hydrophilic surfaces covered with carboxylic/carboxylate groups. Solvent molecules included at the interface between the layers influence hydrogen-bonding interactions between the carbenoxolone molecules and organization of the ribbons within the layer. Comparison of crystal structures of carbenoxolone, glycyrrhizic acid and its aglycone-glycyrrhetinic acid have shown the impact of the size and hydrophilic character of the substituent at the triterpene C3 atom on the supramolecular architecture of these three closely related molecules.
Asunto(s)
Carbenoxolona/química , Ácido Glicirretínico/química , Ácido Glicirrínico/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Molecular , Estructura MolecularRESUMEN
Gap junctional communication is likely one means by which neurons can endure glutamate cytotoxicity associated with CNS insults (i.e. ischemia). To examine this neuroprotective role of gap junctions, we employed gap junctional blockers to neuronal and astrocytic co-cultures during exposure to a high concentration of extracellular glutamate. Co-cultures were treated with the blocking agents carbenoxolone (CBX; 25 microM), 18alpha-glycyrrhetinic acid (AGA; 10 microM), vehicle or the inactive blocking analogue glycyrrhizic acid (GZA; 25 microM). Twenty-four hours following the insult, cell mortality was analyzed and quantified by the release of lactate dehydrogenase (LDH) into the media, the cells' inability to exclude propidium iodide, and terminal dUTP nick end labeling (TUNEL). Measurement of LDH release revealed that the glutamate insult was detrimental to the co-cultures when gap junctions were blocked with CBX and AGA. Based on propidium iodide and TUNEL labeling, the glutamate insult caused significant cell death compared to sham vehicle and mortality was amplified in the presence of CBX and AGA. Since blockers were not themselves toxic and did not affect astrocytic uptake of glutamate, it is likely that blocked gap junctions lead to the increased glutamate cytotoxicity. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.
Asunto(s)
Astrocitos/efectos de los fármacos , Comunicación Celular/fisiología , Uniones Comunicantes/metabolismo , Ácido Glutámico/farmacología , Neuronas/efectos de los fármacos , Animales , Apoptosis/fisiología , Astrocitos/metabolismo , Biomarcadores , Carbenoxolona/química , Carbenoxolona/farmacología , Células Cultivadas , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero , Colorantes Fluorescentes/metabolismo , Uniones Comunicantes/efectos de los fármacos , Ácido Glutámico/metabolismo , Ácido Glicirretínico/química , Ácido Glicirretínico/farmacología , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , L-Lactato Deshidrogenasa/metabolismo , Ratones , Neuronas/metabolismoRESUMEN
Endogenous glucocorticoids (GC) can be metabolized through the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD); in the rat, corticosterone (B) is converted to its inactive metabolite 11-dehydrocorticosterone (A). Since increased tissue concentrations of GCs may affect blood pressure by potentiating the vasoactive effects of alpha-adrenergic agonists and possibly other pressors, we studied the metabolism of corticosterone in freshly dissected aortae and cultured vascular smooth muscle cells (VSMC). Incubations were generally conducted for 60 min with 10(-8) M steroid; steroids were isolated and identified by HPLC. In aortic minces stripped of endothelium, the oxo-reductase reaction of A back to B was nearly 4 times greater than the dehydrogenase reaction of B to A (2.8 +/- 0.5 x 10(-11) versus 7.3 +/- 1.0 x 10(-12) mol/mg protein). This pattern was also seen in cultured VSMC during growth and quiescent states (growth A to B 3.2 +/- 0.4 x 10(-12) versus B to A 9.7 +/- 0.9 x 10(-13) mol/mg protein; quiescent A to B 8.8 +/- 0.1 x 10(-12) versus B to A 1.2 +/- 0.2 x 10(-12) mol/mg protein). Enzyme activity in either direction was less during growth, correlating with a decrease in mRNA for 11 beta-OHSD. In cell homogenates containing 200 microM NADP(H), the enzyme functioned equally in either direction at pH 7.4 with an apparent Km for corticosterone of approximately 2 x 10(-7) M. Carbenoxolone, an inhibitor of 11 beta-OHSD, suppressed the dehydrogenase reaction to a greater degree than the reverse oxo-reductase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Corticosterona/análogos & derivados , Corticosterona/farmacología , Hidroxiesteroide Deshidrogenasas/metabolismo , Músculo Liso Vascular/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasas , Animales , Aorta , Carbenoxolona/química , División Celular , Células Cultivadas , Corticosterona/metabolismo , Expresión Génica , Hidroxiesteroide Deshidrogenasas/biosíntesis , Cinética , Músculo Liso Vascular/citología , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
The specific properties, chemical transformations, and biological activity of glycyrrhizic acid and its derivatives are discussed. Particular attention is paid to investigations that hold promise for the development of important drugs for clinical use.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Antiinflamatorios/uso terapéutico , Antifúngicos/uso terapéutico , Antivirales/uso terapéutico , Ácido Glicirrínico/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Administración Tópica , Animales , Antiinflamatorios/química , Antiinflamatorios no Esteroideos/química , Antiulcerosos/química , Antiulcerosos/uso terapéutico , Antifúngicos/química , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Antivirales/química , Carbenoxolona/química , Carbenoxolona/uso terapéutico , Glucuronatos/química , Glucuronatos/uso terapéutico , Ácido Glucurónico , Ácido Glicirretínico/química , Ácido Glicirretínico/uso terapéutico , Ácido Glicirrínico/análogos & derivados , Ácido Glicirrínico/química , Hepatitis Crónica/tratamiento farmacológico , Humanos , RatasRESUMEN
Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 ß-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.