RESUMO
Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.
Assuntos
Antivirais/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteases/farmacologia , Vírus de RNA/enzimologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Avaliação Pré-Clínica de Medicamentos , Vírus da Encefalite Transmitidos por Carrapatos/enzimologia , Corantes Fluorescentes/química , Humanos , RNA Helicases/antagonistas & inibidores , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Recent discoveries establish DNA and RNA as bona fide substrates for ADP-ribosylation. NADAR ("NAD- and ADP-ribose"-associated) enzymes reverse guanine ADP-ribosylation and serve as antitoxins in the DarT-NADAR operon. Although NADARs are widespread across prokaryotes, eukaryotes, and viruses, their specificity and broader physiological roles remain poorly understood. Using phylogenetic and biochemical analyses, we further explore de-ADP-ribosylation activity and antitoxin functions of NADAR domains. We demonstrate that different subfamilies of NADAR proteins from representative E. coli strains and an E. coli-infecting phage retain biochemical activity while displaying specificity in providing protection from toxic guanine ADP-ribosylation in cells. Furthermore, we identify a myxobacterial enzyme within the YbiA subfamily that functions as an antitoxin for its associated DarT-unrelated ART toxin, which we termed YarT, thus presenting a hitherto uncharacterised ART-YbiA toxin-antitoxin pair. Our studies contribute to the burgeoning field of DNA ADP-ribosylation, supporting its physiological relevance within and beyond bacterial toxin-antitoxin systems. Notably, the specificity and confinement of NADARs to non-mammals infer their potential as highly specific targets for antimicrobial drugs with minimal off-target effects.