Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

Abstract

Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3-9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at scale, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~300-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to shed light on the design of next generation protease inhibitors.

Hotspot residues and resistance mutations in the nirmatrelvir-binding site of SARS-CoV-2 main protease: Design, identification, and correlation with globally circulating viral genomes

“…These mutants are reported to be nirmatrelvir resistant, with K i values increasing between 19.2 to 38.0-fold, therefore corroborating our findings and reflecting the accuracy of our design methodology [ 13 ]. Interestingly, the E166V mutant, experimentally shown to confer the strongest resistance (∼100-fold) [ 23 ], was found to be one of the most plausible resistance-causing mutants in our design experiments ( Fig. 3 ).…”

Nirmatrelvir-resistant SARS-CoV-2 is efficiently transmitted in Syrian hamsters

“…In a FRET-based assay, the enzymatic activities of recombinant 3CLpro proteins carrying the L50F, E166A and L167F subsstitutions alone or combined proved to be significantly lower, compared to that of the WT protein (16) . In another similar study, in vitro selection of resistant variants against nirmatrelvir resulted in identification of several resistance-associated subsstitutions with the E166V subsstitution resulting in the strongest resistance phenotype (17) . However, this subsstitution at position 166 resulted in loss of the virus replication fitness in vitro that was restored by compensatory subsstitutions such as L50F and T21I (17) .…”

“…Two of the selected substitutions (i.e. L50F and L167F) have been reported to be detected (at low frequencies) in SARS-CoV-2 viruses naturally circulating in the population (17). Moreover, the Paxlovid label indicates that the E166V subsititution is among the treatment-emergent substitution, which is more common in nirmatrelvir/ritonavir treated subjects relative to placebo-treated subjects.…”

Predicting antiviral resistance mutations in SARS-CoV-2 main protease with computational and experimental screening

“…During our study, multiple preprints have appeared, discussing certain SARS-CoV-2 M pro mutations and their effect on NTV. [27][28][29][30][31][32][33] The methodologies used in these studies to discover the mutations with resistance potential are diverse, which is reflected in their results. As is observed in our study, previously reported mutational hotspots include the active site of M pro , with special emphasis on the residues S144, E166, L167, and Q192.…”

“…Indeed, several recent preprints disclose experimentally found M pro variants which reduce the potency of NTV. [27][28][29][30][31][32][33] These studies encompassed a variety of methodologies, including data mining of SARS-CoV-2 sequences and passage experiments. Especially substitutions of substrate binding pocket residues such as S144, E166, L167, and Q192 resulted in loss of NTV inhibition (Supporting Information Table 3).…”