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

Abstract

The main protease (Mpro) and papain‐like protease (PLpro) play critical roles in SARS‐CoV‐2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro 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 Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro 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 Mpro and PLpro. This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.

Label‐free Sensing of Main Protease Activity of SARS‐CoV‐2 with an Aerolysin Nanopore

“…PL pro is essential non‐structural proteins (NSPs) for processing the viral precursor polyprotein to form functional proteins during viral replication and can cleave polypeptides containing RLRGG/K. [8] Ptyalase promotes starch digestion in the human mouth. [26] Trypsin can specifically digest peptide that contain R or K. As indicated in Figure 4 , S2R and S2E translocation blocks are solely produced in response to the M pro .…”

“… [7] Notably, the M pro can act as a natural SARS‐CoV‐2 biomarker, because it is not closely linked to any human protease. [8] While the fluorescent method enables the sensitive measurement of M pro activity and the colorimetric assay enables the visual detection of SARS‐CoV‐2, [9] they both require tedious labeling and cautious probe designs and have weak anti‐interference capabilities. In this context, the accurate, sensitive, and rapid evaluation of M pro activity will offer an efficient tool for the detection of SARS‐CoV‐2.…”

Management of fluorescent organic/inorganic nanohybrids for biomedical applications in the NIR-II region

“…Diseases are accompanied by the changes of multiple physiological parameters in biological tissues, for example, low pH value, high GSH, H 2 O 2 , and enzymes. 110 The currently reported NIR-II nanohybrids are mostly responsive to a single disease related stimuli for bio-sensing. However, in a complex and dynamic physiological environment, this method may suffer from non-specific responses and cause false positive results.…”

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

“…Detect caspase-3 and survivin mRNA [26] /PL pro and M pro in SARS-CoV-2 [27] /DNA topoisomerase, [34] conformational changes of ribosome [37] /ATP [32] /DNA methylation in genomic DNA [38] BHQ-3 672 620-730 42 700 FAM/FPR-675 (18 000; 675)/IRDye 800CW (270 000; 789)/Cy5…”

“…As concentrations of M pro and PL pro increased, the fluorescence intensity of 3MBP5 increased gradually under different excitation wavelengths. Based on this property, this probe is able to simultaneously detect PL pro and M pro in SARS‐CoV‐2 infected cells (Figure 5d) [27] . To the best of our knowledge, this is currently the only FRET‐based probe that uses one quencher to quench two or more intramolecular fluorophores.…”