Figure 2.

Time-dependent inhibition of M^pro by alkyne 4d and nirmatrelvir (4g). (A) M^pro (0.5 μM) was incubated with increasing concentrations (up to 40 μM) of 4d or nirmatrelvir (4g) in the reaction buffer at 30 °C for 0 or 3 h. The reactions were then initiated by the addition of the FRET substrate (20 μM) followed by continuous measurements of fluorescence for 1 h. IC₅₀ values were determined in duplicates and shown as the mean ± SD. (B) Proposed irreversible thiol–alkyne addition of alkyne-containing inhibitors with M^pro to form a vinyl sulfide linkage. (C) Known reversible reaction of nitrile inhibitors, such as nirmatrelvir, with M^pro to form a thioimidate linkage.