Nirmatrelvir Resistant SARS-CoV-2 Variants with High Fitness in Vitro

Zhou, Yuyong; Gammeltoft, Karen Anbro; Ryberg, Line Abildgaard; Pham, Long V.; Fahnøe, Ulrik; Binderup, Alekxander; Hernandez-Suarez, Carlos; Offersgaard, Anna; Fernandez-Antunez, Carlota; Peters, Günther H.J.; Ramírez, Santseharay; Bukh, Jens; Gottwein, Judith M.

doi:10.1101/2022.06.06.494921

Cited by 41 publications

(63 citation statements)

References 40 publications

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“…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 ).…”

Section: Identification Of Sars-cov-2 Mpro Mutants From Gisaid Sequen...supporting

confidence: 66%

“…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).…”

Section: Identification Of Sars-cov-2 M Pro Mutants From Gisaid Seque...mentioning

confidence: 99%

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

Lewandowski

Tan

et al. 2022

Preprint

110

170

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The SARS-CoV-2 main protease (Mpro) is a cysteine protease and a validated antiviral drug target. Paxlovid is an FDA-approved oral COVID-19 antiviral that contains the Mpro inhibitor nirmatrelvir and the metabolic booster ritonavir. The emergence of SARS-CoV-2 variants mutations in the Mpro raised the alarm of potential drug resistance. In this study, we aim to discover Mpro drug resistant mutants from naturally observed polymorphisms. Through analyzing the SARS-CoV-2 sequences deposited in Global initiative on Sharing Avian influenza Data (GISAID) database, we identified 66 prevalent Mpro mutations located at the nirmatrelvir binding site. The Mpro mutant proteins were expressed and characterized for enzymatic activity and nirmatrelvir inhibition. While the majority of the Mpro mutants had reduced enzymatic activity (kcat/Km >10-fold decrease), 11 mutants including S144M/F/A/G/Y, M165T, E166Q, H172Q/F, and Q192T/S/V showed comparable enzymatic activity as the wild-type (kcat/Km <10-fold change) and resistance to nirmatrelvir (Ki > 10-fold increase). We further demonstrate that the enzymatic activity and inhibitor resistance of these single mutations can be enhanced by additional substitutions in a double mutant. X-ray crystal structures were determined for six of the single mutants with and/or without GC-376/nirmatrelvir. The structures illustrate how mutations can reduce ligand binding by impacting the conformational stability of the active site. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance.

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Section: Identification Of Sars-cov-2 Mpro Mutants From Gisaid Sequen...supporting

confidence: 66%

Section: Identification Of Sars-cov-2 M Pro Mutants From Gisaid Seque...mentioning

confidence: 99%

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

Lewandowski

Tan

et al. 2022

Preprint

110

170

View full text Add to dashboard Cite

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“…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.…”

Section: Discussionsupporting

confidence: 85%

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

Iketani

Mohri

Culbertson

et al. 2022

Preprint

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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.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Lewandowski

et al. 2022

Preprint

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The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

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Nirmatrelvir Resistant SARS-CoV-2 Variants with High Fitness in Vitro

Cited by 41 publications

References 40 publications

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

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

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

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

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Nirmatrelvir Resistant SARS-CoV-2 Variants with High Fitness in Vitro

Cited by 41 publications

References 40 publications

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

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

Multiple pathways for SARS-CoV-2 resistance to nirmatrelvir

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

Contact Info

Product

Resources

About

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