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

Abstract

A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), 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 6.35% as of May 26, 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, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC 50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC 50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic M pro inhibitors. A complex crystal structure of SARS-CoV-2 M pro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M pro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

“…The highest tested concentration of GC376 (57.5 μM) exhibited a complete inhibition, where the fluorescent intensity was equal to the background substrate in the absence of enzyme. This IC50 value of GC376 is comparable to the reported value (Ma et al, 2020), indicating the reliability of this enzymatic assay (Figure 3a).…”

A simplified cell-based assay to identify coronavirus 3CL protease inhibitors

“…1a-b). We sought to determine if the observed growth defect could be rescued by incubating cells with GC376, a feline coronavirus inhibitor that was recently reported to have activity against SARS-CoV-2 3CLpro 20 . In comparison to untreated control cells, the addition of GC376 led to a robust increase in cell growth ( Fig.…”

Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2

“…Kinetics of SARS-CoV-2 M pro were measured as previously described. 5,7 Briefly, 100 nM M pro in reaction buffer (20mM Tris, 100 mM NaCl, 1 mM DTT, pH 7. inhibitors for M pro and its SARS-CoV relative. 7,42,43 In almost all cases the predicted poses for the 42 compounds from the different docking programs agreed well.…”

“…5,7 Briefly, 100 nM M pro in reaction buffer (20mM Tris, 100 mM NaCl, 1 mM DTT, pH 7. inhibitors for M pro and its SARS-CoV relative. 7,42,43 In almost all cases the predicted poses for the 42 compounds from the different docking programs agreed well. The poses from Glide were then subjected to extensive visual scrutiny to check for unsatisfied hydrogen-bonding sites, electrostatic mismatches, and unlikely conformation of the ligand.…”

“…5 Thus, M pro is viewed as a promising target for anti SARS-CoV-2 drug design; it has been the focus of several studies since the pandemic has emerged. 2,[4][5][6][7] An X-ray crystal structure of M pro reveals that it forms a homodimer with a 2fold crystallographic symmetry axis. 2,5 Each protomer, with a length of 306 residues, is made of three domains (I-III).…”