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

Cell Res. 2020 Aug;30(8):678-692. doi: 10.1038/s41422-020-0356-z. Epub 2020 Jun 15.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • Animals
  • Antiviral Agents / chemistry
  • Antiviral Agents / pharmacology*
  • Betacoronavirus / enzymology*
  • COVID-19
  • Caco-2 Cells
  • Chlorocebus aethiops
  • Coronavirus 3C Proteases
  • Coronavirus Infections / metabolism*
  • Coronavirus Infections / virology
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / metabolism
  • Drug Discovery / methods
  • Glycoproteins / pharmacology*
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Pandemics
  • Pneumonia, Viral / metabolism*
  • Pneumonia, Viral / virology
  • Proline / analogs & derivatives*
  • Proline / pharmacology
  • Protein Conformation
  • Pyrrolidines / chemistry
  • Pyrrolidines / pharmacology*
  • SARS-CoV-2
  • Vero Cells
  • Viral Nonstructural Proteins / antagonists & inhibitors*
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / metabolism
  • Virus Replication / drug effects*

Substances

  • Antiviral Agents
  • GC376
  • Glycoproteins
  • Pyrrolidines
  • Viral Nonstructural Proteins
  • calpain inhibitors
  • N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide
  • Proline
  • Cysteine Endopeptidases
  • Coronavirus 3C Proteases