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 (Mpro). 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 IC50 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 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 substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro 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 Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.
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Data availability
The structure for SARS-CoV-2 Mpro has been deposited in the Protein Data Bank with accession number 6WTT.
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Acknowledgements
This research was partially supported by the National Institutes of Health (NIH) (Grant AI147325) and the Arizona Biomedical Research Centre Young Investigator grant (ADHS18-198859) to J.W., B.H. and B.T. thanks for the support from the Respiratory Diseases Branch, National Institute of Allergy and Infectious Diseases, NIH, USA (Contract N01-AI-30048). J.A.T. and M.T.M. were funded by the National Institute of General Medical Sciences, NIH (Grant R35 GM128624 to M.T.M.). We thank Michael Kemp for assistance with crystallization and X-ray diffraction data collection. We also thank the staff members of the Advanced Photon Source of Argonne National Laboratory, particularly those at the Structural Biology Center (SBC), with X-ray diffraction data collection. SBC-CAT is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357.
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J.W. and C.M. conceived and designed the study; C.M. expressed the Mpro with the assistance of T.S.; C.M. performed the primary screening, secondary IC50 determination, thermal shift binding assay, and enzymatic kinetic studies; M.D.S. carried out Mpro crystallization and structure determination with the assistance of X.Z., and analyzed the data with Y.C.; B.H. and B.T. performed the SARS-CoV-2 CPE and VYR assays; J.A.T. performed the native mass spectrometry experiments with the guidance from M.T.M.; Y.H. performed the plaque reduction assay with influenza A/California/07/2009 (H1N1) virus; J.W. and Y.C. secured funding and supervised the study; J.W., Y.C., and C.M. wrote the paper with the input from others.
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J.W. and C.M. are inventors of a pending patent that claims the use of the identified compounds for COVID-19.
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Ma, C., Sacco, M.D., Hurst, B. et al. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. Cell Res 30, 678–692 (2020). https://doi.org/10.1038/s41422-020-0356-z
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DOI: https://doi.org/10.1038/s41422-020-0356-z
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