Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

Chunlong Ma; Brett Hurst; Yanmei Hu; Tommy Szeto; Bart Tarbet; Jun Wang

and 4 mM DTT and 20% glycerol. As such, all the following proteolytic assay was conducted using this pH 6.5 buffer. Next, we characterized the enzymatic activity of this SARS-CoV-2 M pro by measuring the Km and Vmax values. When 100 nM M pro was mixed with various concentration of FRET substrate (0 to 200 µM), the initial velocity was measured and plotted against substrate concentration. Curve fitting with Michaelis-Menton equation gave the best-fit values of Km and Vmax as 32.8 ± 3.5 µM and 29.4 ± 1.1 RFU/s, respectively (Fig. 1C) . The protease inhibitors are grouped based on their targets and mechanism of action and include proteasome inhibitors (1-8); HIV protease inhibitors (9-14); γ-secretase inhibitors (15) (16) (17) (18) (19) (20) (21) (22) ; HCV NS3-4A protease inhibitors (23) (24) (25) (26) (27) (28) (29) ; DPP-4 inhibitors (30) (31) (32) (33) (34) (35) ; miscellaneous serine protease inhibitors (36) (37) (38) (39) ; cathepsin and calpain protease inhibitors (40-43); miscellaneous cysteine protease inhibitors (44-48); matrix metalloprotease inhibitors (49-51); and miscellaneous protease inhibitors (52-55). The inhibitors were pre-incubated with 100 nM of M pro at 30 °C for 30 minutes in the presence of 4 mM 1,4-dithiothreitol (DTT) before the addition of 10 µM FRET substrate.