SARS-CoV-2 M pro . The enzyme was expressed and purified as previously described. The assay was performed at 37 °C with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The total volume per well was 50 μL. The assay buffer was 50 mM MOPS, pH 7.2 containing 10 mM NaCl, 1 mM EDTA, and 0.01% Triton X-100. The substrate Boc-Abu-Tle-Leu-Gln-AMC was prepared as a 2.5 mM stock solution in DMSO. The substrate stock solution was diluted 1+23 with assay buffer and pipetted into a well containing 1 μL of inhibitor solution. This mixture was kept at 37 °C for 5 min. A volume of 1 μL of an enzyme-containing solution was diluted 1+24 with assay buffer and added to start the reaction, which was monitored for 60 min. The final substrate concentration was 50 μM (= 1.03 Km), and the final DMSO content was 4% (v/v).