Substitutions in SARS-CoV-2 Mpro Selected by Protease Inhibitor Boceprevir Confer Resistance to Nirmatrelvir

Viruses. 2023 Sep 21;15(9):1970. doi: 10.3390/v15091970.

Abstract

Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration-response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration-response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.

Keywords: COVID-19; Mpro; Mpro inhibitor; SARS-CoV-2; antiviral resistance; boceprevir; nirmatrelvir; protease inhibitor.

Publication types

  • Review
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Infective Agents*
  • Antiviral Agents / pharmacology
  • COVID-19 Drug Treatment
  • COVID-19*
  • Enzyme Inhibitors
  • Hepatitis C, Chronic*
  • Humans
  • Lactams
  • Protease Inhibitors / pharmacology
  • SARS-CoV-2 / genetics

Substances

  • Protease Inhibitors
  • Antiviral Agents
  • 3C-like proteinase, SARS-CoV-2
  • 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
  • Enzyme Inhibitors
  • Anti-Infective Agents
  • Lactams

Grants and funding

This research was funded by PhD stipends from the Candys Foundation (to K.A.G., A.O., J.B. and J.M.G.) and the China Scholarship Council (to Y.Z. and J.M.G.) and by grants from the Amager and Hvidovre Hospital Research Foundation (to C.R.D.H. and J.M.G.), the Danish Agency for Science and Higher Education (to S.R., J.B. and J.M.G.), the Independent Research Fund Denmark (to J.B.), the Innovation Fund Denmark (to J.M.G.), the Novo Nordisk Foundation including a Distinguished Investigator Grant (to J.B.), the Mauritzen la Fontaine Fonden (to J.B.), the Mauritzen La Fontaine Familiefond (to J.B.), the Region H Foundation (to J.B. and J.M.G.), the Toyota Foundation (to J.M.G.), and the Weimann Foundation (to U.F.).