The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitorin vitroand confer resistance to nirmatrelvir

Abstract

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6- to 72-fold). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir

“…E166H had reduced enzymatic activity (17.5-fold lower in k cat /K m value) and showed drug resistance (368.7-fold increase in IC 50 ). In parallel to our study, two preprints reported nirmatrelvir resistant M pro mutants identified from serial viral passage experiments in cell culture ( 13, 14 ).…”

“…Although the results reassure that the efficacy of nirmatrelvir is not compromised by the current dominant SARS-CoV-2 variants, drug resistance to nirmatrelvir is anticipated given the experience from the clinic use of HIV and HCV protease inhibitors (11,12). Several studies have been conducted to evolve or predict the nirmatrelvir resistant M pro mutants (13)(14)(15)(16).…”

“…Jochmans et al discovered a triple mutant L50F/E166A/L167F with a 72-fold increase in the enzymatic assay and a 51-fold increase in the antiviral assay against nirmatrelvir ( 14 ). However, the triple mutant only had 5.3% of the enzymatic activity of the WT, indicating an impaired fitness of replication.…”

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

“…1 ) and Huh7-ACE2 cells ( Fig. 2 ), multiple lineages with non- overlapping mutations evolved under increasing drug pressure, consistent with what has been seen in similar small-scale studies 24,25,34,35 . Conducting selection at scale, however, revealed that there are multiple mutational pathways to nirmatrelvir resistance but with several common trajectories preferred ( Figs.…”

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations

“…Using this assay, we found that compared to wild-type, the E166R mutation conferred strong nirmatrelvir resistance ( K i > 1000-fold). As the E166 site appears to be a hot spot for drug resistance from in vitro viral evolution experiments 13,14 , we solved the structures of two substitution mutants M pro E166N and M pro E166R, revealing how E166 mutations may compromise activity versus drug resistance, respectively. Our results demonstrate the yeast system can be a reliable tool to determine the activity and drug responses of M pro mutants.…”