RESUMO
A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Antivirais/uso terapêutico , Inibidores de Glicosídeo Hidrolases , Vírus da Hepatite B da Marmota/efeitos dos fármacos , Hepatite B Crônica/veterinária , Doenças dos Roedores/terapia , 1-Desoxinojirimicina/uso terapêutico , Animais , Transporte Biológico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Glucosídeos/sangue , Glicosilação , Hepatite B Crônica/terapia , Manosídeos/sangue , Marmota , Oligossacarídeos/sangue , Dobramento de Proteína , Replicação Viral/efeitos dos fármacosAssuntos
Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Animais , Aspergillus/enzimologia , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Café/enzimologia , Fabaceae/enzimologia , Fígado/enzimologia , Manosidases/isolamento & purificação , Manosidases/metabolismo , Dados de Sequência Molecular , Neuraminidase/isolamento & purificação , Neuraminidase/metabolismo , Vírus da Doença de Newcastle/enzimologia , Plantas Medicinais , Especificidade por Substrato , Suínos , alfa-Galactosidase/isolamento & purificação , alfa-Galactosidase/metabolismo , alfa-Glucosidases/isolamento & purificação , alfa-Glucosidases/metabolismo , alfa-Manosidase , beta-Galactosidase/isolamento & purificação , beta-Galactosidase/metabolismo , beta-N-Acetil-Hexosaminidases/isolamento & purificação , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
A sensitive and reproducible high performance chromatographic procedure is described for the assay of jack bean beta-galactosidase in which the reaction products are separated on a Dionex AS6 ion exchange column under alkaline conditions and detected by triple-pulsed amperometry. Quantition of the enzyme-released galactose is accomplished by using either fucose or lactose, the substrate, as an internal standard. The validity of the procedure as a general method for the assay and kinetic characterization of exoglycosidases was demonstrated by performing parallel measurements of galactose using an established coupled-enzyme assay, and using these values to calculate Km and Vmax values against lactose. Additional data are presented which establish the applicability of using a similar HPLC approach for the assay of glycosyltransferases.