RESUMEN
The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos , Exorribonucleasas/antagonistas & inhibidores , Metiltransferasas/antagonistas & inhibidores , Caperuzas de ARN/metabolismo , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Animales , Antivirales/química , Clorobencenos/farmacología , Chlorocebus aethiops , Pruebas de Enzimas , Exorribonucleasas/genética , Exorribonucleasas/aislamiento & purificación , Exorribonucleasas/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Indazoles/farmacología , Indenos/farmacología , Indoles/farmacología , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Metiltransferasas/metabolismo , Nitrilos/farmacología , Fenotiazinas/farmacología , Purinas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Especificidad por Sustrato , Trifluperidol/farmacología , Células Vero , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/aislamiento & purificación , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5' cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3'-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.