Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease

Biochem J. 2021 Jul 16;478(13):2517-2531. doi: 10.1042/BCJ20210244.

Abstract

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

Keywords: COVID-19; coronavirus; protease; ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Monophosphate / analogs & derivatives
  • Adenosine Monophosphate / pharmacology
  • Alanine / analogs & derivatives
  • Alanine / pharmacology
  • Aniline Compounds / pharmacology
  • Animals
  • Antiviral Agents / chemistry*
  • Antiviral Agents / pharmacology*
  • Benzamides / pharmacology
  • Chlorocebus aethiops
  • Coronavirus Papain-Like Proteases / antagonists & inhibitors*
  • Coronavirus Papain-Like Proteases / genetics
  • Coronavirus Papain-Like Proteases / isolation & purification
  • Coronavirus Papain-Like Proteases / metabolism
  • Drug Evaluation, Preclinical*
  • Drug Synergism
  • Enzyme Assays
  • Flavins / pharmacology
  • Fluorescence Resonance Energy Transfer
  • Furans / pharmacology
  • High-Throughput Screening Assays
  • Inhibitory Concentration 50
  • Naphthalenes / pharmacology
  • Phenanthrenes / pharmacology
  • Quinones / pharmacology
  • Reproducibility of Results
  • SARS-CoV-2 / drug effects
  • SARS-CoV-2 / enzymology*
  • SARS-CoV-2 / growth & development
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology*
  • Vero Cells
  • Virus Replication / drug effects

Substances

  • 2,3,4,10-tetrahydro-7,10-dimethyl-2,4-dioxobenzo(g)pteridine
  • 5-amino-2-methyl-N-((R)-1-(1-naphthyl)ethyl)benzamide
  • Aniline Compounds
  • Antiviral Agents
  • Benzamides
  • Flavins
  • Furans
  • Naphthalenes
  • Phenanthrenes
  • Quinones
  • Small Molecule Libraries
  • remdesivir
  • Adenosine Monophosphate
  • dihydrotanshinone I
  • Coronavirus Papain-Like Proteases
  • papain-like protease, SARS-CoV-2
  • Alanine