RESUMO
This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ,B-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.
Assuntos
Cisteína Endopeptidases/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Hepacivirus/metabolismo , Inibidores de Proteases/farmacologia , Automação , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Genes Reporter , Humanos , Células Jurkat , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes de Fusão/metabolismoAssuntos
Mentores , Ciência/educação , Bioquímica/educação , Bioquímica/história , Indústria Farmacêutica/tendências , História do Século XX , História do Século XXI , Humanos , Masculino , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/fisiologia , EnsinoRESUMO
An imidazolylpyrimidine was identified in a CXCR2 chemokine receptor antagonist screen and was optimized for potency, in vitro metabolic stability, and oral bioavailability. It was found that subtle structural modification within the series affected the oral bioavailability. Potent and orally available CXCR2 antagonists are herein reported.