Figure 2.

The covalent binding of hit compounds as determined by LC-MS and fluorescent staining assay. (A) Representative protein intact MS of hit compound covalently binding with SARS-CoV-2 M^pro. (B) Time-dependent increasing of the labeling percentage for compound 1e as determined by LC-MS. (C) Fluorescent analogs (Cy5 labeled) of compounds 1a and 2a were synthesized and incubated with SARS-CoV-2 Mpro. SDS-PAGE analysis of the resulting mixtures was then performed and theCy5 signal which overlaps with the protein bands (Coomassie blue) suggested the formation of covalent complex between protein and hit compounds. 1a-Fluo and 2a-Fluo represent compound 1a- and 2a-Fluorescence respectively. (D) Similar fluorescent staining assay as in 2C was performed for covalent binding in the presence of cell lysate. The Cy5 signal which is mainly corresponding to the target protein bands (Coomassie blue) suggested the formation of covalent complex between protein and hit compounds.