The IC50 values of the
testing compounds against various SARS-CoV-2, EV-A71, and EV-D68 proteases
in the presence or in the absence of 4 mM DTT were measured with a
common protocol as the following: First, 100 μL of protease
(SARS-CoV-2 Mpro at 100 nM; SARS-CoV-2 PLpro at 200 nM; EV-A71 2Apro at 3 μM; EV-A71 3Cpro at 2 μM; EV-D68 2Apro at 1 μM; or
EV-D68 3Cpro at 100 nM) was incubated with various concentrations
of testing inhibitors at 30 °C for 30 min in its reaction buffer
in a 96-well plate, and then the reaction was initiated by adding
FRET substrate (SARS-CoV-2 Mpro and PLpro substrates
at 10 μM; EV-A71 and EV-D68 substrates at 20 μM). The
reaction was monitored for 2 h, and the initial velocity was calculated
using the data from the first 15 min by linear regression. The IC50 was calculated by plotting the initial velocity against
various concentrations of testing inhibitor by using a four parameters
dose–response curve in Prism (v8.0) software. The reaction
buffers used were as follows:

SARS-CoV-2 Mpro reaction buffer: 20 mM HEPES,
pH 6.5, 120 mM NaCl, 0.4 mM EDTA, and 20% glycerol

SARS-CoV-2 PLpro reaction buffer: 50 mM HEPES,
pH7.5, 0.01% triton X-100

EV-A71 2Apro reaction buffer: 50 mM Tris
pH 7.0, 150 mM NaCl, 10% glyceol

EV-A71
3Cpro reaction buffer: 50 mM Tris
pH 7.0, 150 mM NaCl, 1 mM EDTA, 10% glycerol

EV-D68 2Apro reaction buffer: same as EV-A71
2Apro reaction buffer

EV-D68
3Cpro reaction buffer: same as EV-A71
3Cpro reaction buffer