RESUMEN
In order to understand the binding modes of human DNA polymerase alpha (pol alpha) inhibitors on a molecular level, a 3D homology model of the active site of the enzyme was proposed based on the application of molecular modelling methods and molecular dynamic simulations using available crystal coordinates of pol alpha relatives. Docking results for a series of known nucleotide analogue inhibitors were consistent with reported experimental binding data and offered the possibility to elucidate structure-activity relationships via investigations of active site-inhibitor interactions. Furthermore, the study could explain, at least partially, the inhibitory effect of aphidicolin on pol alpha. In molecular dynamics simulations, aphidicolin occupied the catalytic centre, but acted in a not truly competitive manner with respect to nucleotides. It destabilized the replicating "closed" form of the pol alpha and transferred the enzyme into the inactive "open" conformation. This result is consistent with recent experiments on the binding mode of aphidicolin.
Asunto(s)
División Celular/efectos de los fármacos , ADN Polimerasa I/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Queratinocitos/enzimología , Adenina/análogos & derivados , Adenina/farmacología , Antivirales/farmacología , Afidicolina/farmacología , Sitios de Unión , ADN Polimerasa I/química , Humanos , Queratinocitos/efectos de los fármacos , Ligandos , Modelos Biológicos , Modelos Moleculares , Organofosfonatos/farmacología , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
BACKGROUND AND AIM: Alpha-lipoic acid has cytoprotective potential which has previously been explained by its antioxidant properties. The aim of this study was to assess LA-induced-specific cytoprotective signalling pathways in hepatocytes. METHODS: Apoptosis of rat hepatocytes was induced by actinomycinD/TNF-alpha. Caspase-3-like activity was determined by a fluorometric; LDH by an enzymatic assay; and phosphorylation of the insulin receptor, Akt, and Bad by Western blot (after immunoprecipitation). Protein kinase and insulin receptor activities were measured by in vitro phosphorylation. Computer modeling studies were performed by using the program GRID. RESULTS: Alpha-lipoic acid decreased actinomycinD/TNF-alpha-induced apoptosis, as did the antioxidants Trolox and N-acetylcysteine. The activation of PI3-kinase/Akt involving phosphorlyation of Bad markedly contributed to the cytoprotective action of alpha-lipoic acid. Alpha-lipoic acid but not other antioxidants protected against actinomycinD/TNF-alpha-induced apoptosis via phosphorylation of the insulin receptor. Computer modeling studies revealed a direct binding site for alpha-lipoic acid at the tyrosine kinase domain of the insulin receptor, suggesting a stabilizing function in loop A that is involved in ATP binding. Treatment of immunoprecipitated insulin receptor with LA induced substrate phosphorylation. CONCLUSIONS: Alpha-lipoic acid mediates its antiapoptotic action via activation of the insulin receptor/PI3-kinase/Akt pathway. We show for the first time a direct binding site for alpha-lipoic acid at the insulin receptor tyrosine kinase domain, which might make alpha-lipoic acid a model substance for the development of insulin mimetics.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Hepatocitos/citología , Receptor de Insulina/metabolismo , Ácido Tióctico/farmacología , Animales , Células Cultivadas , Dactinomicina/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Insulina/metabolismo , Masculino , Modelos Moleculares , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Ácido Tióctico/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Activation of caspases represents one of the earliest biochemical indicators for apoptotic cell death. Therefore, measurement of caspase activity is a widely used and generally accepted method to determine apoptosis in a wide range of in vivo and in vitro settings. Numerous publications characterize the role of the heme-catabolizing enzyme heme oxygenase-1 (HO-1) in regulating apoptotic processes. Different metalloporphyrins representing inducers and inhibitors of this enzyme are often used, followed by assessment of apoptotic cell death. In the present work, we found that caspase-3-like activity, as well as activity of caspase-8 measured in either Fas (CD95) ligand-treated Jurkat T-lymphocytes or by the use of recombinant caspase-3 or -8, was inhibited by different metalloporphyrins (cobalt(III) protoporphyrin IX, tin and zinc(II) protoporphyrin-IX). Moreover, employing the mouse model of Fas-induced liver apoptosis these properties of porphyrins could also be demonstrated in vivo. The metalloporphyrins were shown to inhibit caspase-3-mediated PARP cleavage. Molecular modeling studies demonstrated that porphyrins can occupy the active site of caspase-3 in an energetically favorable manner and in a binding mode similar to that of known inhibitors. The data shown here introduce metalloporphyrins as direct inhibitors of caspase activity. This finding points to the need for careful employment of metalloporphyrins as modulators of HO-1.