RESUMO
Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.
Assuntos
Caseína Quinase Idelta/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteólise , Sequência de Aminoácidos , Animais , Caseína Quinase Idelta/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/química , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteínas Tirosina Quinases/química , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.