RESUMEN
Neurofibrillary tangles are composed of insoluble aggregates of microtubule-associated protein Tau. In the pathology of Alzheimer's disease (AD), accumulation of hyperphosphorylated Tau results in formation of paired helical filaments. One of the main candidate to hyperphosphorylate Tau in AD is glycogen synthase kinase 3beta (GSK-3beta). Here we introduce a non-neuronal cell line, stably co-expressing human Tau and GSK-3beta proteins, where the effect of potential kinase inhibitors on Tau phosphorylation can be monitored. The aim of our study was to establish a new flow-cytometry-based method to quantitatively analyze the changing of Tau phosphorylation, which is a suitable alternative to the well-accepted but non-quantitative Western blot technique. Our results demonstrate that the flow cytometry-based method is a convenient tool to analyze the effect of GSK-3beta inhibitors on Tau phosphorylation. This new approach provides appropriate throughput for screening purposes in preclinical research for characterization of GSK-3beta inhibitors, as potential drug candidate to cure Alzheimer's disease.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Citometría de Flujo/métodos , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/uso terapéutico , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Humanos , Ovillos Neurofibrilares/efectos de los fármacos , Ovillos Neurofibrilares/enzimología , Fosforilación , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteínas tau/genéticaRESUMEN
Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase that has recently emerged as a key target for neurodegenerative diseases and diabetes. As an initial step of our lead discovery program, we developed a virtual screen to discriminate known GSK-3beta inhibitors and inactive compounds using FlexX, FlexX-Pharm, and FlexE. The maximal enrichment factor (EF = 28) suggests that our protocol identifies potential GSK-3beta inhibitors effectively from large compound collections. The effectiveness of our screening protocol was further investigated by comparative experimental and virtual high-throughput screens (HTSs) performed for the same subset of our corporate library. Enrichment factors, the significantly higher hit rate of virtual screening (12.9%) than that of the HTS (0.55%), and also the comparison of active clusters suggest that our virtual screening protocol is an effective tool in GSK-3beta-based library focusing. Head-to-head comparison of true/false positives and negatives revealed the two approaches to be complementary rather than competitive.
Asunto(s)
Bases de Datos Factuales , Inhibidores Enzimáticos/química , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/química , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Cristalografía por Rayos X , Glucógeno Sintasa Quinasa 3 beta , Estructura MolecularRESUMEN
A readily automated solid-phase approach to the synthesis of diverse N-(phenylalkyl)cinnamides, analogues of the NR2B antagonist 2, is described. The procedure utilizes polymer supported N-(phenylalkyl)amines, (diethylphosphono)acetic acid and a wide range of commercially available hydroxybenzaldehydes. The key step, a Horner-Wadsworth-Emmons reaction is achieved under mild conditions and was found to be general for a large number of benzaldehydes. A 225-member focused library was synthesized using a Tecan Combitec synthesizer.