RESUMEN
Precise control of the canonical Wnt pathway is crucial in embryogenesis and all stages of life, and dysregulation of this pathway is implicated in many human diseases including cancers and birth defect disorders. A key aspect of canonical Wnt signaling is the cytoplasmic to nuclear translocation of ß-catenin, a process that remains incompletely understood. Here we report the identification of a previously undescribed component of the canonical Wnt signaling pathway termed Custos, originally isolated as a Dishevelled-interacting protein. Custos contains casein kinase phosphorylation sites and nuclear localization sequences. In Xenopus, custos mRNA is expressed maternally and then widely throughout embryogenesis. Depletion or overexpression of Custos produced defective anterior head structures by inhibiting the formation of the Spemann-Mangold organizer. In addition, Custos expression blocked secondary axis induction by positive signaling components of the canonical Wnt pathway and inhibited ß-catenin/TCF-dependent transcription. Custos binds to ß-catenin in a Wnt responsive manner without affecting its stability, but rather modulates the cytoplasmic to nuclear translocation of ß-catenin. This effect on nuclear import appears to be the mechanism by which Custos inhibits canonical Wnt signaling. The function of Custos is conserved as loss-of-function and gain-of-function studies in zebrafish also demonstrate a role for Custos in anterior head development. Our studies suggest a role for Custos in fine-tuning canonical Wnt signal transduction during embryogenesis, adding an additional layer of regulatory control in the Wnt-ß-catenin signal transduction cascade.
Asunto(s)
Desarrollo Embrionario , Cabeza/embriología , Proteínas de Homeodominio/metabolismo , Vertebrados/embriología , Vertebrados/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Pez Cebra/metabolismo , beta Catenina/metabolismo , Animales , Tipificación del Cuerpo , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Vía de Señalización Wnt , Xenopus laevis/embriología , Pez Cebra/embriologíaRESUMEN
A promising therapeutic approach to diminish pathological inflammation is to inhibit the synthesis and/or biological activity of macrophage migration inhibitory factor (MIF). Prior studies have shown that intraperitoneal administration of small-molecule inhibitors targeting the catalytic pocket of MIF (e.g., ISO-1) elicits a therapeutic effect in mouse inflammation models. However, it remains to be elucidated whether these tautomerase activity inhibitors block the synthesis and/or biological activity of MIF. In this study, we investigated and compared the activity of representative MIF inhibitors from isoxazole series (fluorinated analog of ISO-1; ISO-F) and substituted quinoline series (compound 7E; 7E). Our results demonstrate that ISO-F is a more potent MIF inhibitor than 7E. Both ISO-F and 7E do not inhibit MIF synthesis but "bind-onto" MIF thereby blocking its recognition. However, in contrast to 7E, ISO-F docks well in the active site of MIF and also has a stronger binding affinity towards MIF. In line with these observations, ISO-F, but not 7E, robustly inhibits the biological function of MIF. Most importantly, ISO-F, when administered orally in a therapeutic regimen, significantly suppresses dextran sulphate sodium (DSS)-induced murine colitis. This study, which provides mechanistic insights into the anti-inflammatory efficacy of ISO-F, is the first documented report of in vivo anti-inflammatory efficacy of a MIF inhibitor upon oral administration. Moreover, the findings from this study reinforce the potential of catalytic site of MIF as a target for eliciting therapeutic effect in inflammatory disorders. Compounds (e.g., ISO-F) that block not only the recognition but also the biological function of MIF are potentially attractive for reducing pathological inflammation.
Asunto(s)
Antiinflamatorios/farmacología , Colitis/tratamiento farmacológico , Isoxazoles/farmacología , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Administración Oral , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/química , Línea Celular , Colitis/fisiopatología , Sulfato de Dextran , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Isoxazoles/administración & dosificación , Isoxazoles/química , Factores Inhibidores de la Migración de Macrófagos/biosíntesis , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Quinolinas/administración & dosificación , Quinolinas/química , Quinolinas/farmacologíaRESUMEN
Natural products have immense therapeutic potential not only due to their structural variation and complexity but also due to their range of biological activities. Research based on natural products has led to the discovery of molecules with biomedical and pharmaceutical applications in different therapeutic areas like cancer, inflammation responses, diabetes, and infectious diseases. There are still several challenges to be overcome in natural product drug discovery research programs and the challenge of high throughput screening of natural substances is one of them. Bioactivity screening is an integral part of the drug discovery process and several in vitro and in vivo biological models are now available for this purpose. Among other well-reported biological models, the zebrafish (Danio rerio) is emerging as an important in vivo model for preclinical studies of synthetic molecules in different therapeutic areas. Zebrafish embryos have a short reproductive cycle, show ease of maintenance at high densities in the laboratory and administration of drugs is a straightforward procedure. The embryos are optically transparent, allowing for the visualization of drug effects on internal organs during the embryogenesis process. In this review, we illustrate the importance of using zebrafish as an important biological model in the discovery of bioactive drugs from natural sources.