# Characterization of the Cross-Resistance of SARS-CoV‑2 Main Protease Inhibitors, Ibuzatrelvir, Ensitrelvir, and Nirmatrelvir

## TL;DR

This study compares how well three SARS-CoV-2 protease inhibitors work against virus mutations that may cause drug resistance.

## Contribution

The study reveals cross-resistance patterns of next-gen Mpro inhibitors against clinically relevant mutants.

## Key findings

- Ibuzatrelvir, nirmatrelvir, and ensitrelvir show cross-resistance against Q192, S144, H172, and E166 Mpro mutants.
- A triple mutant virus (L50F/E166A/L167F) is highly resistant to all three drugs in antiviral assays.
- E166 mutations pose a significant challenge to current Mpro inhibitors.

## Abstract

The emergence of resistance to SARS-CoV-2 main protease
(Mpro) inhibitors such as nirmatrelvir poses a significant
threat
to the long-term effectiveness of COVID-19 antivirals. Ibuzatrelvir
(PF-07817883) and ensitrelvir are next-generation Mpro inhibitors
with enhanced metabolic stability, eliminating the need for coadministration
with ritonavir, unlike nirmatrelvir. Ibuzatrelvir is currently in
Phase 3 clinical trials in the United States, and ensitrelvir is approved
in Japan. In this study, we assessed the cross-resistance of ibuzatrelvir,
nirmatrelvir, and ensitrelvir against a panel of clinically relevant
Mpro mutants using FRET-based enzymatic assays, thermal
shift binding assays, and cell-based antiviral plaque assays. Our
results reveal a cross-resistance pattern of ibuzatrelvir, nirmatrelvir,
and ensitrelvir against Q192, S144, H172, and E166 mutants. Notably,
the recombinant SARS-CoV-2 virus containing the Mpro L50F/E166A/L167F
triple mutant is highly resistant to all three drugs in the antiviral
plaque assay. These findings underscore the challenge posed by E166
mutations and highlight the need for resistance-resistant Mpro inhibitors as future therapeutics.