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

In light of the promising results of the calpain inhibitor MG-132 (43), we then pursued to testing other calpain and cathepsin inhibitors that are commercially available (56-63) ( Table 2 ). These compounds were not included in the initial library because they have not been advanced to clinical studies. Among this series of analogs, calpain inhibitor II (61) and XII (62) are the most potent We also included two well-known viral 3CL protease inhibitors GC-376 (64) and rupintrivir (65) in the secondary screening. GC-376 (64) is an investigational veterinary drug that is being developed for feline infectious peritonitis (FIP). 10,11 GC-376 (64) was designed to target the viral 3CL protease and had potent antiviral activity against multiple viruses including MERS, FIPV, and norovirus. 10, 12 Rupintrivir (65) was developed as a rhinovirus antiviral by targeting the viral 3CL protease, but it was discontinued in clinical trials due to side effects. 13 In our study, we found that GC-376 (64) was the most potent M pro inhibitor with an IC50 value of 0.03 µM. It shifted the melting curve of M pro by 18.30 o C upon binding. In contrast, rupintrivir (65) was not active against M pro (IC50 > 20 µM). Previous report also showed that rupintrivir was not active against the SARS-CoV 3CL pro (M pro ) (IC50 > 100 µM). 14 Both compounds 64 and 65 were not active against the EV-A71 2A protease, but showed potent inhibition against the EV-A71 3C protease, which is consistent with previously reported results. 12, 15, 16 When plotting the IC50 values (log scale) of the inhibitors against M pro from the FRET enzymatic assay with the melting temperature shifts (ΔTm) from thermal shift binding assay (Fig. 3A) , a linear correlation was observed, and the r 2 of the linear regression fitting is 0.94. This suggests that there is a direct correlation between the enzymatic inhibition and protein binding: a more potent enzyme inhibitor also binds to the protein with higher affinity. The stabilization of the M pro against thermal denaturation was also compound concentration dependent (Fig. 3B ). Correlation of inhibition efficacy (IC50) with ΔTm from thermal shift binding assay. Data in Table 2 were used for the plot. The r 2 of fitting is 0.94. (B) Dose-dependent melting temperature (Tm) shift.