The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 μM and 9.0 μM. In addition, three compounds inhibited PLpro with IC50 ranging from 1.9 μM to 3.3 μM. To verify the specificity of Mpro and PLpro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific Mpro and PLpro inhibitors. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.
- MeSH
- antivirové látky * farmakologie chemie MeSH
- COVID-19 MeSH
- inhibitory proteas * farmakologie chemie MeSH
- koronavirové proteasy 3C * antagonisté a inhibitory MeSH
- koronavirové proteasy podobné papainu * antagonisté a inhibitory MeSH
- lidé MeSH
- naftochinony * chemie farmakologie MeSH
- papain MeSH
- SARS-CoV-2 * účinky léků enzymologie MeSH
- simulace molekulového dockingu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
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.
- MeSH
- antivirové látky farmakologie MeSH
- COVID-19 MeSH
- farmakoterapie COVID-19 MeSH
- fluorescenční barviva chemie MeSH
- inhibitory proteas farmakologie MeSH
- koronavirové proteasy podobné papainu antagonisté a inhibitory chemie genetika metabolismus MeSH
- lidé MeSH
- preklinické hodnocení léčiv MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- RNA-helikasy antagonisté a inhibitory chemie genetika metabolismus MeSH
- RNA-viry enzymologie MeSH
- rychlé screeningové testy metody MeSH
- SARS-CoV-2 enzymologie MeSH
- serinové endopeptidasy chemie genetika metabolismus MeSH
- virové nestrukturální proteiny antagonisté a inhibitory chemie genetika metabolismus MeSH
- viry klíšťové encefalitidy enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH