RESUMEN
Asymmetric reduction of 2-chloro-1-(6-fluorochroman-2-yl)ethan-1-one (NEB-7) into 2-chloro-1-(6-fluorochroman-2-yl)ethan-1-ol (NEB-8) is the crucial step for synthesis of liposoluble ß1 receptor blocker nebivolol. Four efficient and stereoselective alcohol dehydrogenases were identified, enabling the stereoselective synthesis of all enantiomers of NEB-8 at a substrate loading of 137 g·L-1 with ee values of >99% and high space-time yields. This study provides novel biocatalysts for the efficient synthesis of nebivolol precursors and uncovers the molecular basis for enantioselectivity manipulation by parametrization of Prelog's rule.
Asunto(s)
Biocatálisis , Nebivolol , Nebivolol/química , Estereoisomerismo , Estructura Molecular , Antagonistas de Receptores Adrenérgicos beta 1/química , Antagonistas de Receptores Adrenérgicos beta 1/síntesis química , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/metabolismo , Alcohol Deshidrogenasa/químicaRESUMEN
Silymarin is known for its hepatoprotective effects. Although there is solid evidence for its protective effects against Amanita phalloides intoxication, only inconclusive data are available for alcoholic liver damage. Since silymarin flavonolignans have metal-chelating activity, we hypothesized that silymarin may influence alcoholic liver damage by inhibiting zinc-containing alcohol dehydrogenase (ADH). Therefore, we tested the zinc-chelating activity of pure silymarin flavonolignans and their effect on yeast and equine ADH. The most active compounds were also tested on bovine glutamate dehydrogenase, an enzyme blocked by zinc ions. Of the six flavonolignans tested, only 2,3-dehydroderivatives (2,3-dehydrosilybin and 2,3-dehydrosilychristin) significantly chelated zinc ions. Their effect on yeast ADH was modest but stronger than that of the clinically used ADH inhibitor fomepizole. In contrast, fomepizole strongly blocked mammalian (equine) ADH. 2,3-Dehydrosilybin at low micromolar concentrations also partially inhibited this enzyme. These results were confirmed by in silico docking of active dehydroflavonolignans with equine ADH. Glutamate dehydrogenase activity was decreased by zinc ions in a concentration-dependent manner, and this inhibition was abolished by a standard zinc chelating agent. In contrast, 2,3-dehydroflavonolignans blocked the enzyme both in the absence and presence of zinc ions. Therefore, 2,3-dehydrosilybin might have a biologically relevant inhibitory effect on ADH and glutamate dehydrogenase.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Quelantes/farmacología , Flavonolignanos/farmacología , Silimarina/farmacología , Zinc/aislamiento & purificación , Animales , Glutamato Deshidrogenasa/antagonistas & inhibidores , Caballos , Silibina/farmacología , Levaduras/efectos de los fármacos , Zinc/metabolismoRESUMEN
A methylimidizolium ionic liquid (M8OI) was recently found to be contaminating the environment and to be related to and/or potentially a component of an environmental trigger for the autoimmune liver disease primary biliary cholangitis (PBC). The aims of this study were to investigate human exposure to M8OI, hepatic metabolism and excretion. PBC patient and control sera were screened for the presence of M8OI. Human livers were perfused with 50µM M8OI in a closed circuit and its hepatic disposition examined. Metabolism was examined in cultured human hepatocytes and differentiated HepaRG cells by the addition of M8OI and metabolites in the range 10-100 µM. M8OI was detected in the sera from 5/20 PBC patients and 1/10 controls. In perfused livers, M8OI was cleared from the plasma with its appearance - primarily in the form of its hydroxylated (HO8IM) and carboxylated (COOH7IM) products - in the bile. Metabolism was reflected in cultured hepatocytes with HO8IM production inhibited by the cytochrome P450 inhibitor ketoconazole. Further oxidation of HO8IM to COOH7IM was sequentially inhibited by the alcohol and acetaldehyde dehydrogenase inhibitors 4-methyl pyrazole and disulfiram respectively. Hepatocytes from 1 donor failed to metabolise M8OI to COOH7IM over a 24 h period. These results demonstrate exposure to M8OI in the human population, monooxygenation by cytochromes P450 followed by alcohol and acetaldehyde dehydrogenase oxidation to a carboxylic acid that are excreted, in part, via the bile in human liver.
Asunto(s)
Eliminación Hepatobiliar , Imidazoles/sangre , Imidazoles/farmacocinética , Adulto , Anciano , Alcohol Deshidrogenasa/antagonistas & inhibidores , Aldehído Oxidorreductasas/antagonistas & inhibidores , Células Cultivadas , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hidroxilación , Técnicas In Vitro , Cetoconazol/farmacología , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Adulto JovenRESUMEN
The aim of the present study was to elucidate how fructose is able to increase the rate of ethanol metabolism in the liver, an observation previously termed the fructose effect. Previous studies suggest that an increase in ATP consumption driven by glucose synthesis from fructose stimulates the oxidation of NADH in the mitochondrial respiratory chain, allowing faster oxidation of ethanol by alcohol dehydrogenase; however, this idea has been frequently challenged. We tested the effects of fructose, sorbose and tagatose both in vitro and in vivo. Both ethanol and each sugar were either added to isolated hepatocytes or injected intraperitoneally in the rat. In the in vitro experiments, samples were taken from the hepatocyte suspension in a time-dependent manner and deproteinized with perchloric acid. In the in vivo experiments, blood samples were taken every 15 min and the metabolites were determined in the plasma. These metabolites include ethanol, glucose, glycerol, sorbitol, lactate, fructose and sorbose. Ethanol oxidation by rat hepatocytes was increased by more than 50% with the addition of fructose. The stimulation was accompanied by increased glucose, glycerol, lactate and sorbitol production. A similar effect was observed with sorbose, while tagatose had no effect. The same pattern was observed in the in vivo experiments. This effect was abolished by inhibiting alcohol dehydrogenase with 4-methylpyrazole, whereas inhibition of the respiratory chain with cyanide did not affect the fructose effect. In conclusion, present results provide evidence that, by reducing glyceraldehyde and glycerol and fructose to sorbitol, respectively, NADH is consumed, allowing an increase in the elimination of ethanol. Hence, this effect is not linked to a stimulation of mitochondrial re-oxidation of NADH driven by ATP consumption.
Asunto(s)
Etanol/metabolismo , Fructosa/administración & dosificación , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Tasa de Depuración Metabólica/efectos de los fármacos , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Inyecciones Intraperitoneales , Masculino , Tasa de Depuración Metabólica/fisiología , RatasRESUMEN
Alcohol dehydrogenases (ADHs) are most known for their roles in oxidation and elimination of ethanol. Although less known, ADHs also play a critical role in the metabolism of a number of drugs and metabolites that contain alcohol functional groups, such as abacavir (HIV/AIDS), hydroxyzine (antihistamine), and ethambutol (antituberculosis). ADHs consist of 7 gene family numbers and several genetic polymorphic forms. ADHs are cytosolic enzymes that are most abundantly found in the liver, although also present in other tissues including gastrointestinal tract and adipose. Marked species differences exist for ADHs including genes, proteins, enzymatic activity, and tissue distribution. The active site of ADHs is relatively small and cylindrical in shape. This results in somewhat narrow substrate specificity. Secondary alcohols are generally poor substrates for ADHs. In vitro-in vivo correlations for ADHs have not been established, partly due to insufficient clinical data. Fomepizole (4-methylpyrazole) is a nonspecific ADH inhibitor currently being used as an antidote for the treatment of methanol and ethylene glycol poisoning. Fomepizole also has the potential to treat intoxication of other substances of abuse by inhibiting ADHs to prevent formation of toxic metabolites. ADHs are inducible through farnesoid X receptor (FXR) and other transcription factors. Drug-drug interactions have been observed in the clinic for ADHs between ethanol and therapeutic drugs, and between fomepizole and ADH substrates. Future research in this area will provide additional insights about this class of complex, yet fascinating enzymes.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Fármacos Anti-VIH/farmacocinética , Antituberculosos/farmacocinética , Etanol/metabolismo , Antagonistas de los Receptores Histamínicos H1/farmacocinética , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/genética , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/química , Antituberculosos/administración & dosificación , Antituberculosos/química , Didesoxinucleósidos/administración & dosificación , Didesoxinucleósidos/química , Didesoxinucleósidos/farmacocinética , Interacciones Farmacológicas , Etambutol/administración & dosificación , Etambutol/química , Etambutol/farmacocinética , Etanol/química , Fomepizol/farmacología , Antagonistas de los Receptores Histamínicos H1/administración & dosificación , Antagonistas de los Receptores Histamínicos H1/química , Humanos , Hidroxizina/administración & dosificación , Hidroxizina/química , Hidroxizina/farmacocinética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Oxidación-Reducción/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
The nitric oxide (NO) metabolome and the NO metabolite-based neurovascular protective pathways are dysregulated after stroke. The major NO metabolite S-nitrosoglutahione (GSNO) is essential for S-nitrosylation-based signaling events and the inhibition of S-nitrosoglutahione (GSNO)-metabolizing enzyme GSNO reductase (GSNOR) provides protective effects following cardiac ischemia. However, the role of GSNOR and GSNOR inhibition-mediated increased GSNO/S-nitrosylation is not understood in neurovascular diseases such as stroke. Because age is the major risk factor of stroke and recovery in aged stroke patients is low and slow, we investigated the efficacy of GSNOR inhibition using a GSNOR selective inhibitor N6022 in a clinically relevant middle-aged cerebral ischemia and reperfusion (IR) mouse model of stroke. N6022 (5 mg/kg; iv) treatment of IR mice at 2 h after reperfusion followed by the treatment of the same dose daily for 3 days reduced the infarct volume and decreased the neurological score. Daily treatment of IR animals with N6022 for 2 weeks significantly improved neurological score, brain infarctions/atrophy, survival rate, motor (measured by cylinder test) and cognitive (evaluated by novel object recognition test) functions which paralleled the decreased activity of GSNOR, reduced levels of peroxynitrite and decreased neurological score. These results are the first evidence of a new pathway for the treatment of stroke via the inhibition of GSNOR. Based on the efficacy of N6022 in the stroke animal model and its use in human therapeutic studies without toxicity, we submit that GSNOR is a druggable target, and N6022 is a promising drug candidate for human stroke therapy.
Asunto(s)
Envejecimiento/efectos de los fármacos , Alcohol Deshidrogenasa/antagonistas & inhibidores , Benzamidas/administración & dosificación , Modelos Animales de Enfermedad , Pirroles/administración & dosificación , Recuperación de la Función/efectos de los fármacos , Accidente Cerebrovascular/tratamiento farmacológico , Envejecimiento/metabolismo , Alcohol Deshidrogenasa/metabolismo , Animales , Sistemas de Liberación de Medicamentos/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Recuperación de la Función/fisiología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
A review of literature shows certain phytochemicals, phyllanthin, hypophyllanthin and gallic acid have beneficial effects in experimental animals for improving liver metabolism in alcoholic liver disease. We investigated the ability of these chemicals to exhibit the inhibitory effect on the enzyme, alcohol dehydrogenase active site. The software used were CASTp, AutoDock and Molinspiration in Windows platform. We observed the phytochemicals, phyllanthin (-2.37 kcal/mol), hypophyllanthin (-3.23 kcal/mol) and gallic acid (-5.85 kcal/mol) in the order of increasing binding efficiency, which was as good as 4-methyl pyrazole (-4.18 kcal/mol).
Asunto(s)
Alcohol Deshidrogenasa/química , Ácido Gálico/química , Lignanos/química , Simulación del Acoplamiento Molecular , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/metabolismo , Animales , Dominio Catalítico , Ácido Gálico/metabolismo , Ácido Gálico/farmacología , Humanos , Lignanos/metabolismo , Lignanos/farmacología , Unión Proteica , Programas InformáticosRESUMEN
BACKGROUND: The nitric oxide (NO)-producing activity of endothelial nitric oxide synthase (eNOS) plays a significant role in maintaining endothelial function and protecting against the stroke injury. However, the activity of the eNOS enzyme and the metabolism of major NO metabolite S-nitrosoglutathione (GSNO) are dysregulated after stroke, causing endothelial dysfunction. We investigated whether an administration of exogenous of GSNO or enhancing the level of endogenous GSNO protects against neurovascular injury in wild-type (WT) and eNOS-null (endothelial dysfunction) mouse models of cerebral ischemia-reperfusion (IR). METHODS: Transient cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) for 60 minutes in male adult WT and eNOS null mice. GSNO (0.1 mg/kg body weight, intravenously) or N6022 (GSNO reductase inhibitor, 5.0 mg/kg body weight, intravenously) was administered 30 minutes before MCAO in preinjury and at the reperfusion in postinjury studies. Brain infarctions, edema, and neurobehavioral functions were evaluated at 24 hours after the reperfusion. RESULTS: eNOS-null mice had a higher degree (P< .05) of injury than WT. Pre- or postinjury treatment with either GSNO or N6022 significantly reduced infarct volume, improved neurological and sensorimotor function in both WT and eNOS-null mice. CONCLUSION: Reduced brain infarctions and edema, and improved neurobehavioral functions by pre- or postinjury GSNO treatment of eNOS knock out mice indicate that GSNO can attenuate IR injury, likely by mimicking the eNOS-derived NO-dependent anti-ischemic and anti-inflammatory functions. Neurovascular protection by GSNO/N6022 in both pre- and postischemic injury groups support GSNO as a promising drug candidate for the prevention and treatment of stroke injury.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Benzamidas/farmacología , Encéfalo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Pirroles/farmacología , S-Nitrosoglutatión/farmacología , Alcohol Deshidrogenasa/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/embriología , Encéfalo/patología , Edema Encefálico/enzimología , Edema Encefálico/patología , Edema Encefálico/prevención & control , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/enzimología , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/deficiencia , Óxido Nítrico Sintasa de Tipo III/genéticaRESUMEN
Neuronal nitric oxide synthase (nNOS) plays a crucial role in the maintenance of correct skeletal muscle function due, at least in part, to S-nitrosylation of specific protein targets. Similarly, we recently provided evidence for a muscular phenotype in mice lacking the denitrosylase S-nitrosoglutathione reductase (GSNOR). Here, we demonstrate that nNOS and GSNOR are concomitantly expressed during differentiation of C2C12. They colocalizes at the sarcolemma and co-immunoprecipitate in cells and in myofibers. We also provide evidence that GSNOR expression decreases in mouse models of muscular dystrophies and of muscle atrophy and wasting, i.e., aging and amyotrophic lateral sclerosis, suggesting a more general regulatory role of GSNOR in skeletal muscle homeostasis.
Asunto(s)
Envejecimiento/genética , Alcohol Deshidrogenasa/genética , Homeostasis/genética , Desarrollo de Músculos/genética , Distrofias Musculares/genética , Óxido Nítrico Sintasa de Tipo I/genética , Envejecimiento/metabolismo , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/deficiencia , Animales , Diferenciación Celular , Línea Celular Transformada , Modelos Animales de Enfermedad , Proteínas Asociadas a la Distrofina/genética , Proteínas Asociadas a la Distrofina/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Mioblastos/citología , Mioblastos/enzimología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Sarcolema/enzimología , Transducción de Señal , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Methanol is the simplest alcohol. Compared to ethanol that is fully detoxified by metabolism. Methanol gets activated in toxic products by the enzymes, alcohol dehydrogenase and aldehyde dehydrogenase. Paradoxically, the same enzymes convert ethanol to harmless acetic acid. This review is focused on a discussion and overview of the literature devoted to methanol toxicology and antidotal therapy. Regarding the antidotal therapy, three main approaches are presented in the text: 1) ethanol as a competitive inhibitor in alcohol dehydrogenase; 2) use of drugs like fomepizole inhibiting alcohol dehydrogenase; 3) tetrahydrofolic acid and its analogues reacting with the formate as a final product of methanol metabolism. All the types of antidotal therapies are described and how they protect from toxic sequelae of methanol is explained.
Asunto(s)
Antídotos/farmacología , Inhibidores Enzimáticos/farmacología , Metanol/antagonistas & inhibidores , Metanol/envenenamiento , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/metabolismo , Antídotos/química , Inhibidores Enzimáticos/química , Humanos , Estructura MolecularRESUMEN
1,4-Butanediol (BDO)-used as solvent and abused for its euphoric effects-is converted to gamma-hydroxybutyrate (GHB) by the enzyme alcohol dehydrogenase. This double-blind, placebo-controlled crossover study with six healthy volunteers is the first to date investigating the role of the ADH inhibitor fomepizole (4-methylpyrazole (4MP)) in moderating this conversion in humans. Participants received on two different days either intravenous placebo or 15 mg/kg 4MP followed by oral administration of 25 mg/kg BDO. Pretreatment with 4MP resulted in significantly higher BDO maximal plasma concentration (P = 0.001) and area under the concentration-time curve (AUC; P = 0.028), confirming that ADH is the primary pathway for the conversion of BDO to GHB in humans. With 4MP, the mean arterial pressure was significantly lower at 105 minutes compared to baseline (P = 0.003), indicating that blood pressure lowering, observed not with a temporal relationship to 4MP administration but after the maximum BDO concentration was reached, may be an intrinsic effect of BDO.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Butileno Glicoles/farmacocinética , Fomepizol , Oxibato de Sodio/farmacocinética , Adulto , Estudios Cruzados , Método Doble Ciego , Monitoreo de Drogas/métodos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Fomepizol/administración & dosificación , Fomepizol/farmacocinética , Voluntarios Sanos , Humanos , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Psicotrópicos/farmacocinética , Solventes/farmacocinética , Resultado del TratamientoRESUMEN
RATIONALE: Protein S-nitros(yl)ation (SNO) has been implicated as an essential mediator of nitric oxide-dependent cardioprotection. Compared with males, female hearts exhibit higher baseline levels of protein SNO and associated with this, reduced susceptibility to myocardial ischemia-reperfusion injury. Female hearts also exhibit enhanced S-nitrosoglutathione reductase (GSNO-R) activity, which would typically favor decreased SNO levels as GSNO-R mediates SNO catabolism. OBJECTIVE: Because female hearts exhibit higher SNO levels, we hypothesized that GSNO-R is an essential component of sex-dependent cardioprotection in females. METHODS AND RESULTS: Male and female wild-type mouse hearts were subjected to ex vivo ischemia-reperfusion injury with or without GSNO-R inhibition (N6022). Control female hearts exhibited enhanced functional recovery and decreased infarct size versus control males. Interestingly, GSNO-R inhibition reversed this sex disparity, significantly reducing injury in male hearts, and exacerbating injury in females. Similar results were obtained with male and female GSNO-R-/- hearts using ex vivo and in vivo models of ischemia-reperfusion injury. Assessment of SNO levels using SNO-resin assisted capture revealed an increase in total SNO levels with GSNO-R inhibition in males, whereas total SNO levels remained unchanged in females. However, we found that although GSNO-R inhibition significantly increased SNO at the cardioprotective Cys39 residue of nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 3 in males, SNO-NADH dehydrogenase subunit 3 levels were surprisingly reduced in N6022-treated female hearts. Because GSNO-R also acts as a formaldehyde dehydrogenase, we examined postischemic formaldehyde levels and found that they were nearly 2-fold higher in N6022-treated female hearts compared with nontreated hearts. Importantly, the mitochondrial aldehyde dehydrogenase 2 activator, Alda-1, rescued the phenotype in GSNO-R-/- female hearts, significantly reducing infarct size. CONCLUSIONS: These striking findings point to GSNO-R as a critical sex-dependent mediator of myocardial protein SNO and formaldehyde levels and further suggest that different therapeutic strategies may be required to combat ischemic heart disease in males and females.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Corazón/efectos de los fármacos , Daño por Reperfusión Miocárdica/metabolismo , Alcohol Deshidrogenasa/antagonistas & inhibidores , Animales , Benzamidas/farmacología , Benzamidas/uso terapéutico , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocardio/metabolismo , Estrés Oxidativo , Pirroles/farmacología , Pirroles/uso terapéutico , Factores SexualesRESUMEN
Previous studies indicate women have a higher blood alcohol (i.e., ethanol) and acetaldehyde concentration after consuming an equivalent amount of alcohol, and that women are more susceptible to the long-term negative health effects of alcohol. However, there is a paucity of data pertaining to whether there is a sexual dimorphic response in skeletal muscle to alcohol. Adult male and female Sprague-Dawley rats were used and the primary endpoint was in vivo determined muscle (gastrocnemius) protein synthesis (MPS). The initial study indicated MPS did not differ in female rats during proestrus, estrus, metestrus, or diestrus; hence, subsequent studies used female rats irrespective of estrus cycle phase. There was no difference in MPS between male and female rats under basal fasted conditions, and the time- and dose-responsiveness of both groups to the inhibitory effect of acute alcohol did not differ. The ability of alcohol to suppress MPS was comparable in male and female rats pretreated with alcohol dehydrogenase inhibitor 4-methylpyrazol. Chronic alcohol feeding for 6 weeks decreased MPS in male but not in female rats; however, MPS was reduced in both sexes at 14 weeks. Finally, oral gavage of leucine increased MPS similarly in male and female rats and chronic alcohol feeding for 14 weeks prevented the anabolic effect in both sexes. These data suggest normal fluctuations in ovarian hormones do not significantly alter MPS in female rats, and that there is no sexual dimorphic response to the effects of acute alcohol intoxication on MPS. While chronic alcohol consumption appeared to decrease MPS at an early time point in male compared to female rats, there was no sex difference in the suppressive effect of alcohol at a later time point. Overall, these data do not support the prevailing belief that females are more susceptible than males to alcohol's catabolic effect on MPS.
Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Proteínas Musculares/metabolismo , Músculo Esquelético/efectos de los fármacos , Alcohol Deshidrogenasa/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/farmacología , Estrógenos/sangre , Femenino , Fomepizol/farmacología , Leucina/farmacología , Masculino , Metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Ratas , Ratas Sprague-Dawley , Factores SexualesRESUMEN
In plant cells, many stresses, including low oxygen availability, result in a higher production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can lead to redox-dependent post-translational modification of proteins Cys residues. Here, we studied the effect of different redox modifications on alcohol dehydrogenase (ADH) from Arabidopsis thaliana. ADH catalyzes the last step of the ethanol fermentation pathway used by plants to cope with energy deficiency during hypoxic stress. Arabidopsis suspension cell cultures showed decreased ADH activity upon exposure to H2O2, but not to the thiol oxidizing agent diamide. We purified recombinant ADH and observed a significant decrease in the enzyme activity by treatments with H2O2 and diethylamine NONOate (DEA/NO). Treatments leading to the formation of a disulfide bond between ADH and glutathione (protein S-glutathionylation) had no negative effect on the enzyme activity. LC-MS/MS analysis showed that Cys47 and Cys243 could make a stable disulfide bond with glutathione, suggesting redox sensitivity of these residues. Mutation of ADH Cys47 to Ser caused an almost complete loss of the enzyme activity while the Cys243 to Ser mutant had increased specific activity. Incubation of ADH with NAD+ or NADH prevented inhibition of the enzyme by H2O2 or DEA/NO. These results suggest that binding of ADH with its cofactors may limit availability of Cys residues to redox modifications. Our study demonstrates that ADH from A. thaliana is subject to different redox modifications. Implications of ADH sensitivity to ROS and RNS during hypoxic stress conditions are discussed.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Alcohol Deshidrogenasa/antagonistas & inhibidores , Alcohol Deshidrogenasa/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/genética , Línea Celular , Cisteína/química , Cisteína/genética , Disulfuro de Glutatión/química , Disulfuro de Glutatión/metabolismo , Hidrazinas/farmacología , Peróxido de Hidrógeno/farmacología , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en TándemRESUMEN
INTRODUCTION: In the 1980s-1990s numerous studies were performed on H2-receptor antagonist inhibition of ethanol first-pass metabolism. Blood alcohol concentrations warranting possible driving under the influence citations in the United States have subsequently dropped from ≥100 mg/dL to 50 mg/dL (Utah in 2019) (30 mg/dL or zero tolerance in some parts of the world). A reexamination of these studies seemed important. AREAS COVERED: Papers were compiled that addressed the effect of cimetidine, ranitidine, famotidine, and nizatidine on ethanol metabolism first from a PubMed search and then from citations within these papers. Studies were tabulated for fasting versus fed, ethanol and H2-receptor antagonist dose and a summary of pharmacokinetic changes. EXPERT OPINION: At doses of 0.15-0.30 mg/kg in the postprandial state (primarily after breakfast), the H2-receptor antagonists: cimetidine, ranitidine, famotidine, and nizatidine have all been found to increase the first-pass metabolism of ethanol. With cimetidine, there were sufficient studies to suggest it might be inhibitory outside these restricted states. While the role of inhibition of alcohol dehydrogenase has not been clearly defined, there is circumstantial evidence to support this mechanism. Further studies are required to elucidate the ability of H2-receptor antagonists to inhibit first-pass metabolism of ethanol.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Etanol/farmacocinética , Antagonistas de los Receptores H2 de la Histamina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Antagonistas de los Receptores H2 de la Histamina/administración & dosificación , Humanos , Periodo PosprandialRESUMEN
4-MP is a potent competitive inhibitor of ADH activity with an affinity about a 1000 times more than toxic alcohols. 4-MP was shown to reduce the formation of toxic metabolites in lethal methanol and ethylene glycol poisoning in animal models and in methanol poisoning in humans. 4-MP has long-lasting gastroprotective effect against ethanol and other chemically induced acute gastric mucosa lesions in rats. We showed, for the first time, that 4-MP also provides significant protection of the human stomach against alcohol induced acute mucosal injury.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Mucosa Gástrica/efectos de los fármacos , Pirazoles/farmacología , Alcohol Deshidrogenasa/metabolismo , Alcoholes/antagonistas & inhibidores , Alcoholes/farmacología , Animales , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , HumanosRESUMEN
We previously reported that S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, attenuated TH17-mediated immune responses in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Cellular GSNO homeostasis is regulated via its synthesis by reaction between nitric oxide and glutathione and its enzymatic catabolism by GSNO reductase (GSNOR). In this study, we evaluated potential of reversible inhibitor of GSNOR (N6022) in comparison with exogenous GSNO in immunopathogenesis of EAE. Daily treatment of EAE mice with N6022 or exogenous GSNO significantly attenuated the clinical disease of EAE, but N6022 treatment showed greater efficacy than GSNO. Both N6022 and exogenous GSNO treatments increased the spleen levels of GSNO, as documented by increased protein-associated S-nitrosothiols, and inhibited polarization and CNS effector function of proinflammatory TH17 cells while inducing the polarization and CNS effector function of anti-inflammatory CD4+ CD25+ FOXP3- regulatory T (Treg) cells. Moreover, N6022 further attenuated TH1 while inducing TH2 and CD4+ CD25+ FOXP3+ Treg in their polarization and CNS effector functions. Similar to GSNO, the N6022 treatment protected against the EAE disease induced demyelination. However, neither exogenous GSNO nor N6022 treatment did not cause significant systemic lymphopenic effect as compared to FTY720. Taken together, these data document that optimization of cellular GSNO homeostasis by GSNOR inhibitor (N6022) in NO metabolizing cells attenuates EAE disease via selective inhibition of pro-inflammatory subsets of CD4+ cells (TH1/TH17) while upregulating anti-inflammatory subsets of CD4+ cells (TH2/Treg) without causing lymphopenic effects and thus offers a potential treatment option for MS/EAE.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Benzamidas/farmacología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Pirroles/farmacología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Proteína S/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/enzimología , Células TH1/efectos de los fármacos , Células TH1/enzimología , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Acetaldehyde dehydrogenase (AdhE) is a bifunctional acetaldehyde-coenzyme A (CoA) dehydrogenase and alcohol dehydrogenase involved in anaerobic metabolism in gram-negative bacteria. This enzyme was recently found to be a key regulator of the type three secretion (T3S) system in Escherichia coli. AdhE inhibitors can be used as tools to study bacterial virulence and a starting point for discovery of novel antibacterial agents. We developed a robust enzymatic assay, based on the acetaldehyde-CoA dehydrogenase activity of AdhE using both absorption and fluorescence detection models (Z' > 0.7). This assay was used to screen ~11,000 small molecules in 384-well format that resulted in three hits that were confirmed by resynthesis and validation. All three compounds are noncompetitive with respect to acetaldehyde and display a clear dose-response effect with hill slopes of 1-2. These new inhibitors will be used as chemical tools to study the interplay between metabolism and virulence and the role of AdhE in T3S regulation in gram-negative bacteria, and as starting points for the development of novel antibacterial agents.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Aldehído Oxidorreductasas/antagonistas & inhibidores , Antibacterianos/farmacología , Evaluación Preclínica de Medicamentos , Escherichia coli Enterohemorrágica/efectos de los fármacos , Escherichia coli Enterohemorrágica/enzimología , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Alcohol Deshidrogenasa/genética , Alcohol Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Animales , Antibacterianos/química , Línea Celular , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Escherichia coli Enterohemorrágica/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Ratones , Flujo de TrabajoRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Decoctions of Plectranthus species are traditionally ingested after large meals for treatment of food digestion and alcohol abuse. AIM OF THE STUDY: This study aims at associating the digestion-related ethno-uses of Plectranthus species decoctions to molecular mechanism that might explain them: easing digestion (AChE inhibition) and treating hangover (ADH inhibition) MATERIAL AND METHODS: Decoctions from Plectranthus species were analysed for their alcohol dehydrogenase (ADH) inhibition and acetylcholinesterase (AChE) inhibition, related with alcohol metabolism and intestinal motility, respectively. Identification of the active components was carried out by LC-MS/MS and the docking studies were performed with AChE and the bioactive molecules detected. RESULTS: All decoctions inhibited ADH activity. This inhibition was correlated with their rosmarinic acid (RA) content, which showed an IC50 value of 19⯵g/mL, similar to the reference inhibitor CuCl2. The presence of RA also leads to most decoctions showing AChE inhibiting capacity. P. zuluensis decoction with an IC50 of 80⯵g/mL presented also medioresinol, an even better inhibitor of AChE, as indicated by molecular docking studies. Furthermore, all decoctions tested showed no toxicity towards two human cell lines, and a high capacity to quench free radicals (DPPH), which also play a helpful in the digestive process, related with their RA content. CONCLUSIONS: All activities presented by the RA-rich Plectranthus decoctions support their use in treating digestion disorders and P. barbatus could explain its use also for alleviating hangover symptoms. Medioresinol, which is present in P. zuluensis, exhibited a significant AChE inhibition and may provide, in the future, a new lead for bioactive compounds.
Asunto(s)
Alcohol Deshidrogenasa/antagonistas & inhibidores , Inhibidores de la Colinesterasa/farmacología , Extractos Vegetales/farmacología , Plectranthus/química , Acetilcolinesterasa/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Intoxicación Alcohólica/tratamiento farmacológico , Línea Celular , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/aislamiento & purificación , Cromatografía Liquida , Cinamatos/química , Cinamatos/aislamiento & purificación , Cinamatos/farmacología , Depsidos/química , Depsidos/aislamiento & purificación , Depsidos/farmacología , Enfermedades del Sistema Digestivo/tratamiento farmacológico , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/aislamiento & purificación , Depuradores de Radicales Libres/farmacología , Humanos , Concentración 50 Inhibidora , Medicina Tradicional/métodos , Simulación del Acoplamiento Molecular , Extractos Vegetales/administración & dosificación , Extractos Vegetales/toxicidad , Espectrometría de Masas en Tándem , Ácido RosmarínicoRESUMEN
Alcohol dehydrogenases (ADH) are key enzymes of ethanol metabolism that mediate its oxidation to acetaldehyde. ADHs are also able to oxidize some types of neurotransmitters such as dopamine, serotonin and norepinephrine. Increased level of ADHs activity, induced by chronic alcohol consumption, is presumably associated with disturbed neurotransmitters metabolism that leads to stable alcohol craving. As earlier reported, intraperitoneal administration of 4-methilpirasole (non-specific inhibitor of ADHs) has shown to provide a short-term anti-alcoholic effect, but individual roles of ADH isoforms in this process were still unclear. The aim of this work was to study the roles of brain and serum ADH isoforms in alcohol consumption and neurotransmitter metabolism in the rats. In the study we used specific-pathogen-free (SPF) Wistar rats chronically alcoholized with 15% ethanol. 4-methilpirasole intranasal administration in small doses led to local inhibition of ADH III activity in the brain estimated by spectrophotometric assay. It correlated with dose-dependent reduction of dopamine concentration and increased level of its metabolic products in the brain but did not influence alcohol consumption. These data allowed us to propose an important role of brain ADHs (predominantly ADH III) in metabolism of dopamine in chronically alcoholized rats but not in regulation of alcohol consumption. To evaluate the role of serum ADH isoforms we immunized the rats with recombinant horse ADH that led to production of high levels of cross-reactive anti-ADH antibodies verified by ELISA assay. Immunization led to 30% decrease in alcohol consumption and recovery of general behavioral parameters such as motor activity, anxiety and depression level. At the same time active immunization did not cause any impairments in animal blood composition. We can conclude that immunization against ADHs appeared to be a safe way to decrease alcohol consumption that could be possibly associated with neurotransmitters metabolism correction.