A Dual-Color Fluorescent Probe Allows Simultaneous Imaging of Main and Papain-like Proteases of SARS-CoV-2-Infected Cells for Accurate Detection and Rapid Inhibitor Screening

The main protease (MPpro) and papain-like protease (MPpro and PLpro) play critical roles in SARS-CoV-2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of MPpro and PLpro is extremely valuable for SARS-CoV-2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual-color probe (3MBP5) for the simultaneous detection of M-pro and PLpro by fluorescence (or Forster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by MPpro and PLpro. 3MBP5-activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid-transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells. Results from the dual-color probe first verified the simultaneous detection and intracellular distribution of SARS-CoV-2 MPpro and PLpro. This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.

Automated summary

Researchers have developed a dual-color probe for the simultaneous detection of the main protease (MPpro) and papain-like protease (PLpro) of SARS-CoV-2. This probe produces fluorescence from two different fluorophores, which can be used for rapid screening of antiviral inhibitors.