RESUMO
Because of the essential roles of SARS-CoV-2 papain-like protease (PLpro) in the viral polyprotein processing and suppression of host immune responses, it is a crucial target for drug discovery against COVID-19. To develop robust biochemical methodologies for inhibitor screening against PLpro, extensive characterization of recombinant protein is important. Here we report cloning, expression, and purification of the recombinant SARS-CoV-2 PLpro, and explore various parameters affecting its stability and the catalytic activity. We also report the optimum conditions which should be used for high-throughput inhibitor screening using a fluorogenic tetrapeptide substrate.
Assuntos
Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/isolamento & purificação , Cumarínicos/química , Cumarínicos/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Dimetil Sulfóxido/química , Difusão Dinâmica da Luz , Ácido Edético/química , Estabilidade Enzimática , Fluorometria/métodos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Peptídeos/química , Peptídeos/metabolismo , TemperaturaRESUMO
The unprecedented scale of the ongoing COVID-19 pandemic has catalyzed an intense effort of the global scientific community to unravel different aspects of the disease in a short time. One of the crucial aspects of these developments is the determination of more than three hundred experimental structures of SARS-CoV-2 proteins in the last few months. These include structures of viral non-structural, structural, and accessory proteins and their complexes determined by either X-ray diffraction or cryo-electron microscopy. These structures elucidate the intricate working of different components of the viral machinery at the atomic level during different steps of the viral life cycle, including attachment to the host cell, viral genome replication and transcription, and genome packaging and assembly of the virion. Some of these proteins are also potential targets for drug development against the disease. In this review, we discuss important structural features of different SARS-CoV-2 proteins with their function, and their potential as a target for therapeutic interventions.