High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses

Molecules. 2021 Jun 22;26(13):3792. doi: 10.3390/molecules26133792.

Abstract

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.

Keywords: SARS-CoV-2; TBEV; discovery; drug; flavivirus; high-throughput screening; papain-like; protease; virus.

MeSH terms

  • Antiviral Agents / pharmacology*
  • COVID-19 Drug Treatment
  • Coronavirus Papain-Like Proteases / antagonists & inhibitors
  • Coronavirus Papain-Like Proteases / chemistry
  • Coronavirus Papain-Like Proteases / genetics
  • Coronavirus Papain-Like Proteases / metabolism
  • Drug Evaluation, Preclinical
  • Encephalitis Viruses, Tick-Borne / enzymology
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry
  • High-Throughput Screening Assays / methods*
  • Humans
  • Protease Inhibitors / pharmacology*
  • RNA Helicases / antagonists & inhibitors
  • RNA Helicases / chemistry
  • RNA Helicases / genetics
  • RNA Helicases / metabolism
  • RNA Viruses / enzymology*
  • SARS-CoV-2 / enzymology
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism
  • Viral Nonstructural Proteins / antagonists & inhibitors
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism

Substances

  • Antiviral Agents
  • Fluorescent Dyes
  • NS3 protein, flavivirus
  • Protease Inhibitors
  • Viral Nonstructural Proteins
  • Serine Endopeptidases
  • Coronavirus Papain-Like Proteases
  • papain-like protease, SARS-CoV-2
  • RNA Helicases