RESUMEN

A high-throughput chemiluminescence and ELISA-based biochemical assay to identify mTORC1/mTORC2 kinase inhibitors is described. These mTOR complexes were isolated from HeLa whole cell lysate using mTOR antibodies and in-well immunoprecipitation. The integrity and purity of the mTORC1 and mTORC2 immunocomplexes were confirmed by western blotting. Full-length recombinant 4E-BP1 was used as a substrate and the catalytic activity was measured by detection of p4E-BP1 [T37/46] by a chemiluminescence method. The performance of this assay that can be used to identify dual mTORC1 and mTORC2 kinase inhibitors in a high-throughput 384-well format is described.

Asunto(s)

Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Cromonas/farmacología , Evaluación Preclínica de Medicamentos/métodos , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Humanos , Inmunoprecipitación , Indicadores y Reactivos , Luminiscencia , Diana Mecanicista del Complejo 1 de la Rapamicina , Morfolinas/farmacología , Complejos Multiproteicos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas , Reproducibilidad de los Resultados , Especificidad por Sustrato , Serina-Treonina Quinasas TOR