# Structural insights into the nirmatrelvir-resistant SARS-CoV-2 Mpro L50F/E166A/L167F triple mutant-inhibitor-complex reveal strategies for next generation coronaviral inhibitor design

## TL;DR

This study explores a nirmatrelvir-resistant SARS-CoV-2 variant and identifies a potent inhibitor that could guide the design of next-generation antiviral drugs.

## Contribution

The study provides structural insights into a resistant Mpro variant and identifies a new inhibitor for broad-spectrum coronavirus treatment.

## Key findings

- Compound 4 effectively inhibits the nirmatrelvir-resistant Mpro L50F/E166A/L167F variant at low nanomolar concentrations.
- A co-crystal structure reveals how mutated residues stabilize inhibitor binding, guiding future inhibitor design.
- The findings support strategies for developing pan-coronaviral inhibitors effective against emerging variants.

## Abstract

Drug-resistance is an eminent threat in antiviral therapy, and is currently a concern in nirmatrelvir-based therapy of SARS-CoV-2. Nirmatrelvir (antiviral component in Paxlovid) binds covalently to the active site cysteine of the main protease of SARS-CoV-2 (Mpro), thereby blocking enzyme activity and halting viral replication. In vitro passage experiments mimicking a multi-dosage nirmatrelvir treatment regime, identified Mpro variants with mutations in the active site and near the C-terminal dimerization interface with variable levels of nirmatrelvir resistance. One such variant harbors a triple mutation in Mpro, L50F/E166A/L167F, that displays decreased potency for nirmatrelvir (IC50 ∼ 850–1600 nM) and ibuzatrelvir while viral replication remained similar to that of the wildtype (WT) virus. We here confirm a previously developed short peptide aldehyde bisulfite compound 4 as potent inhibitor for SARS-CoV-2 Mpro L50F/E166A/L167F and related variants. A co-crystal structure reveals tight inhibitor binding that is stabilized by a network of hydrogen bonds formed by the mutated residues A166 and F167. This study provides the groundwork for optimized Mpro inhibitors against potential emerging variants of SARS-CoV-2, as well as strategies for broad-spectrum inhibitor design against variants of Mpro.

4 was confirmed as low nanomolar binder of several coronavirus main proteases, including a nirmatrelvir-resistant variant of concern (L50F/E166A/L167F). A co-crystal structure informs about future design strategies for pan-coronaviral inhibitors.