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

Abstract

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 , started to circulate among humans around December 2019, and it is now widespread as a global pandemic.The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.4% as of April 15, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M pro ). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, calpain inhibitors II, XII, and MG-132 were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies and thermal shift binding assays. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known M pro inhibitors. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

Simeprevir potently suppresses SARS-CoV-2 replication and synergizes with remdesivir

“…Mechanistically, we found unexpected complexity in the inhibition of SARS-CoV-2 replication by simeprevir -it does not inhibit the SARS-CoV-2 viral proteins PL pro or RdRP at physiologically feasible concentrations. Simeprevir is a weak inhibitor of M pro at ~10 μM, in keeping with the IC50 at ~13.7 μM as determined in a parallel study (31). This potency of M pro inhibition unlikely accounts for the strong suppression of SARS-CoV-2 viral replication observed.…”

“…The inhibition assay was based on fluorescence resonance energy transfer (FRET) using a fluorescent-protein-based substrate previously developed for SARS-CoV M pro (30,38). 0.1 μM of purified SARS-CoV-2 M pro was pre-incubated with 0 -250 μM simeprevir in 20 mM HEPES pH 6.5, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT for 30 min before the reaction was initiated by addition of 10 μM protein substrate (31). Protease activity was followed at 25 °C by FRET with excitation and emission wavelengths of 430 nm and 530 nm, respectively, using a multi-plate reader as described (30,38).…”

Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors

“…Our previous study showed that when GC376 binds to the SARS-CoV-2 M pro , the covalent thioketal adduct can adapt both the S-and R-configuration. 11 The MD simulations showed that the complexes formed with GC376 in either the S-or R-configuration did not deviate from the starting structures, which is the X-ray structure, with an RMSD in protein and ligand positions from the X-ray structure smaller than c.a. 2 Å for the protein and c.a.…”

“…5B), suggesting a binding ratio of one drug per monomer, which is consistent with its mechanism of action revealed by X-ray crystallography. 11 The addition of 4 mM DTT shifted the equilibrium to one ligand per dimer (Fig. 5C).…”

“…Several crystal structures of M pro in complex with inhibitors have been solved, showing that the pyrrolidone forms multiple hydrogen bonds with the His163 and Glu166 side chains and the main chain of Phe140. [11][12][13][14] In addition to the classic pyrrolidonecontaining M pro inhibitors, several non-canonical M pro inhibitors have also been reported with both enzymatic inhibition and cellular antiviral activity. [11][12] In this study, we aim to validate six previously reported M pro inhibitors, ebselen, disulfiram, tideglusib, carmofur, shikonin, and PX-12 ( Fig.…”