High-Throughput Screening for Inhibitors of the SARS-CoV-2 Protease Using a FRET-Biosensor

Molecules. 2020 Oct 13;25(20):4666. doi: 10.3390/molecules25204666.


The global SARS-CoV-2 pandemic started late 2019 and currently continues unabated. The lag-time for developing vaccines means it is of paramount importance to be able to quickly develop and repurpose therapeutic drugs. Protein-based biosensors allow screening to be performed using routine molecular laboratory equipment without a need for expensive chemical reagents. Here we present a biosensor for the 3-chymotrypsin-like cysteine protease from SARS-CoV-2, comprising a FRET-capable pair of fluorescent proteins held in proximity by a protease cleavable linker. We demonstrate the utility of this biosensor for inhibitor discovery by screening 1280 compounds from the Library of Pharmaceutically Active Compounds collection. The screening identified 65 inhibitors, with the 20 most active exhibiting sub-micromolar inhibition of 3CLpro in follow-up EC50 assays. The top hits included several compounds not previously identified as 3CLpro inhibitors, in particular five members of a family of aporphine alkaloids that offer promise as new antiviral drug leads.

Keywords: 3CLPro; COVID-19; SARS-CoV-2; apomorphine; aporphine; cysteine protease; ebselen.

MeSH terms

  • Betacoronavirus / drug effects*
  • Betacoronavirus / enzymology
  • Betacoronavirus / isolation & purification
  • Biosensing Techniques / methods*
  • COVID-19
  • Coronavirus 3C Proteases
  • Coronavirus Infections / drug therapy*
  • Coronavirus Infections / virology
  • Cysteine Endopeptidases
  • Fluorescence Resonance Energy Transfer / methods*
  • High-Throughput Screening Assays
  • Humans
  • Pandemics
  • Pneumonia, Viral / drug therapy*
  • Pneumonia, Viral / virology
  • Protease Inhibitors / chemistry*
  • Protease Inhibitors / pharmacology*
  • SARS-CoV-2
  • Viral Nonstructural Proteins / antagonists & inhibitors*


  • Protease Inhibitors
  • Viral Nonstructural Proteins
  • Cysteine Endopeptidases
  • Coronavirus 3C Proteases