RESUMEN
OBJECTIVES: Retinoic acid plays diverse physiological and pathophysiological roles in reproduction, immune function, energy metabolism and carcinogenesis. Because of the potential benefits of inhibiting retinoic acid biosynthesis in certain disease states, efforts are underway to develop inhibitors of retinoic acid biosynthesis via inhibition of the aldehyde dehydrogenase-1 A (ALDH1A) family of enzymes. However, many potential ALDH1A inhibitors also inhibit the related ALDH2 enzyme that plays a role in the metabolism of ethanol. Accurate in vitro assessment of ALDH2 inhibition is problematic, and to date, there are no published in vivo assays to determine inhibition of ALDH2 by candidate ALDH1A inhibitors. STUDY DESIGN: To address this, we developed a novel gas-chromatography-mass-spectrometry ethanol clearance assay in mice using orally administered ethanol and serial measurement of ethanol over time. We then used this assay to determine pharmacological inhibition of ALDH2 by candidate ALDH1A inhibitors. RESULTS: Ethanol clearance in untreated male mice occurs within sixty minutes. Male mice treated with WIN 18,446, a known ALDH1A inhibitor that also inhibits ALDH2, demonstrated significant inhibition of ethanol clearance compared to untreated controls. Novel pyrazole and piperazine ALDH1A inhibitors were then tested with the piperazine inhibitor demonstrating ALDH2 inhibition via impaired ethanol clearance while the pyrazole inhibitor did not interfere with ethanol metabolism, suggesting a lack of ALDH2 inhibition. CONCLUSIONS: Inhibition of ethanol clearance is a useful in vivo method of inferring pharmacologic inhibition of hepatic ALDH2. This assay may be useful in the development of novel ALDH1A specific inhibitors for a variety of therapeutic indications.
Asunto(s)
Etanol , Tretinoina , Ratones , Masculino , Animales , Aldehído Deshidrogenasa Mitocondrial/metabolismo , Pirazoles/farmacología , PiperazinasRESUMEN
BACKGROUND: Retinoic acid (RA) controls diverse physiological functions including weight regulation and energy metabolism. It has been reported that mice lacking ALDH1A1, one of the aldehyde dehydrogenases (ALDH) that synthesize RA, are healthy and resistant to weight gain, raising the possibility that inhibiting this enzyme might treat obesity. We previously demonstrated that treatment with a pan-ALDH1A enzyme inhibitor, WIN18446, suppressed weight gain in mice fed a high-fat diet (HFD), but caused increased hepatic lipidosis and reversible male infertility. METHODS: A series of piperazine compounds that inhibited ALDH1A1 were identified and their inhibitory activity was characterized in vitro using purified recombinant enzymes and cell-based assay systems. One potent compound, FSI-TN42 (N42) was examined for its oral bioavailability and pharmacodynamic effects. In addition, its effect on weight gain was investigated by daily oral administration to C57BL/6 male mice receiving a HFD, and compared with mice receiving WIN18446 or vehicle alone (n = 6/group, 200 mg compound/kg body weight) for 5 weeks. Body weights were measured weekly, and a glucose tolerance test was performed after 4 weeks of treatment. Tissues were collected to determine changes in adipose weight, hepatic lipidosis, retinoid metabolism, and expression of genes associated with RA and lipid metabolism. RESULTS: N42 irreversibly binds and inhibits ALDH1A1 in vitro with a low nM IC50 and 800-fold specificity for ALDH1A1 compared to ALDH1A2. Daily oral administration of N42 significantly suppressed weight gain (P < 0.05) and reduced visceral adiposity (p < 0.05) in mice fed a HFD without the hepatic lipidosis observed with WIN18446 treatment. CONCLUSIONS: We developed a potent and specific inhibitor of ALDH1A1 that suppressed weight gain in mice fed a HFD. These findings demonstrate that inhibition of ALDH1A1 is a feasible target for drug development to treat and/or prevent obesity.
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Familia de Aldehído Deshidrogenasa 1/antagonistas & inhibidores , Obesidad/metabolismo , Piperazinas/farmacología , Retinal-Deshidrogenasa/antagonistas & inhibidores , Aumento de Peso/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Administración Oral , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Piperazinas/administración & dosificación , Piperazinas/químicaRESUMEN
Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types.
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Aldehído Deshidrogenasa/metabolismo , Testículo/metabolismo , Anciano , Anciano de 80 o más Años , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Cromatografía Liquida , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Espectrometría de Masas en Tándem , Tretinoina/metabolismoRESUMEN
Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives.
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Diaminas/farmacología , Retinoides/metabolismo , Espermatogénesis/efectos de los fármacos , Tretinoina/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Animales , Biocatálisis/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Ésteres/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Retinal-Deshidrogenasa/metabolismo , Retinoides/sangre , Espermatocitos/efectos de los fármacos , Espermatocitos/metabolismo , Testículo/enzimología , Testículo/metabolismo , Tretinoina/farmacología , Vitamina A/sangre , Vitamina A/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
OBJECTIVES: We examined the return to fertility and transgenerational impact of treatment with WIN 18,446, an experimental male contraceptive, in mice. STUDY DESIGN: We paired male mice treated with WIN 18,446 for 4 weeks to suppress spermatogenesis, followed by a 9-week recovery, and mated them with normal females to assess fertility. F1 generation mice were subsequently mated to ascertain any transgenerational impact of treatment on fertility. Testes were examined histologically. RESULTS: WIN 18,446-treated mice and their progeny produced normally sized litters (6.5 pups per litter after treatment and 7.3 pups per litter from the progeny). However, testes histology revealed rare residual intratesticular foci of mineralization after treatment. CONCLUSIONS: Fertility normalizes after WIN 18,446 treatment, and progeny also have normal fertility.
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Anticonceptivos Masculinos , Humanos , Femenino , Ratones , Animales , Masculino , Anticonceptivos Masculinos/farmacología , Testículo , Fertilidad , Espermatogénesis , ReproducciónRESUMEN
The most common heritable genetic disease in the United States, cystic fibrosis (CF), is caused by mutations in the CF transmembrane conductance regulator (CFTR), a chloride channel that interacts with and regulates a number of other proteins. The bacteria Pseudomonas aeruginosa infects 80% of patients causing decreased pulmonary function and life expectancy. It is not known how malfunction of the chloride channel allows for preferential colonization of patients by a single pathogen. The hypothesis that CFTR interacts with toll-like receptor 4 (TLR4) to phagocytize bacteria was tested. A competitive antagonist of TLR4, MKLPS, was studied for its effect in gentamicin-protection-based bacterial invasion assays. Pre-incubation (15 min 50 microg/mL) with MKLPS did not alter the rate of phagocytosis of P. aeruginosa by cultured epithelia. However, further studies with GFP-transfected P. aeruginosa revealed prominent antibiotic resistant microcolonies were formed. If CFTR is involved in phagocytosis of the bacteria, then internalization was predicted to decrease in iodide efflux. Surprisingly, cultured epithelia exposed to P. aeruginosa for 15 min showed increased cAMP-activated iodide efflux through CFTR. In addition, 15-min exposure to bacterial cell wall component, LPS, purified from P. aeruginosa also increased CFTR iodide efflux in a dose-dependent manner (50, 100 and 200 microg/mL LPS had 25%, 37% and 47% increase). In a reversal of this phenomenon, shorter 5-min exposure to 100 microg/mL LPS resulted in a 25% decrease in forskolin-activated CFTR channel activity compared to controls. This data is consistent with a model in which CFTR is removed from the plasma membrane during phagocytosis of P. aeruginosa followed by recruitment of channels to the membrane to replace those removed during phagocytosis. More studies are needed to confirm this model, but this is the first report of a bacterial product causing a biphasic time-dependent and a dose-dependent alteration of CFTR channel activity.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/inmunología , Yoduros/metabolismo , Lipopolisacáridos/inmunología , Pseudomonas aeruginosa/inmunología , Receptor Toll-Like 4/metabolismo , Bioensayo , Línea Celular , Fibrosis Quística/microbiología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Gentamicinas/farmacología , Humanos , FagocitosisRESUMEN
Many inflammatory bowel disease (IBD) patients require surgical intervention due to limited pharmacological treatment options. Antibodies targeting α4ß7, a gut-homing integrin, are one of the most promising IBD treatments. As retinoic acid (RA) regulates expression of gut-homing proteins including α4ß7 integrin, we tested if ALDH1A enzymes in the RA synthesis pathway could be targeted for IBD treatment using a potent inhibitor, WIN 18,446. Age- and sex-matched Smad3-/- mice were fed a diet with and without WIN 18,446 for 3 weeks before triggering inflammation with Helicobacter bilis infection. Colitis was evaluated by histopathology one week following the IBD trigger, and T cell subsets were evaluated before and after the IBD trigger. WIN 18,446 treatment significantly reduced IBD severity in Smad3-/- mice and reduced expression of α4ß7 integrin on multiple activated CD4+ T cell subsets. This change was associated with increased ratios of induced regulatory T cells to Th17 cells during the inflammatory response in the draining lymph nodes. These studies indicate that RA reduction via ALDH1A enzyme inhibition is a potential new target for IBD treatment. Further studies are needed to examine its effects on other types of immune cells, to evaluate the efficacy window for this target, and to determine its efficacy in other animal models of IBD.
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Familia de Aldehído Deshidrogenasa 1/metabolismo , Colitis/tratamiento farmacológico , Helicobacter/metabolismo , Integrina alfa4/genética , Activación de Linfocitos/efectos de los fármacos , Retinal-Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1/antagonistas & inhibidores , Animales , Colitis/etiología , Colitis/microbiología , Diaminas/farmacología , Modelos Animales de Enfermedad , Femenino , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/tratamiento farmacológico , Integrina alfa4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Retinal-Deshidrogenasa/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Vitamin A deficient females have reduced fertility due to decreased retinoic acid production. WIN 18,446 inhibits retinoic acid biosynthesis and functions as a contraceptive in males. We tested whether WIN 18,446 treatment would suppress fertility in female mice. STUDY DESIGN: Female mice were treated with WIN 18,446 and mated. Pregnancy rates were compared using Fisher's exact test. RESULTS: WIN 18,446 reduced pregnancy compared with control (p=.03). However, one animal became pregnant with malformed embryos. CONCLUSIONS: WIN 18,446 treatment significantly reduces fecundity, but teratogenicity in the setting of contraceptive failure limits the appeal of this approach to female contraception.
RESUMEN
BACKGROUND: Retinoic acid (RA) is known to play a role in weight regulation. Because mice without ALDH1A1, a major RA synthesizing enzyme, are resistant to diet-induced obesity, we tested a hypothesis that pharmacological inhibition of RA synthesis can suppress weight gain in a murine model of diet-induced obesity. METHODS: C57BL/6J male mice were fed a high fat diet (HFD) for 8 weeks to induce obesity and then randomized to a HFD with or without WIN 18,446, an RA synthesis inhibitor, for an additional 9 weeks. Body weight, body composition, energy expenditure, activity, and food intake were measured. Levels of retinoids, lipids, and genes involved in the metabolism of retinoid and lipids were also determined. RESULT: s Mice treated with WIN 18,446 gained significantly less weight and had decreased adipose tissue weight, adipocyte size, and macrophage infiltration in adipose tissue. In addition, we observed higher UCP1 expression in adipose tissues and decreased expression of RA responsive genes and genes involved in fatty acid synthesis in the livers and lungs of mice treated with WIN 18,446. CONCLUSIONS: Pharmacological suppression of RA synthesis via inhibition of ALDH1A1 may be a potential target for treatment of obesity.