Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

Greasley, S.E.; Noell, Stephen; Plotnikova, Olga; Ferre, Rose Ann; Liu, Wei; Bolaños, Ben; Fennell, Kimberly F.; Nicki, Jennifer; Craig, T.K.; Zhu, Yuao; Stewart, Albert; Steppan, Claire M.

doi:10.1016/j.jbc.2022.101972

Cited by 122 publications

(85 citation statements)

References 17 publications

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“…Similarly, the P132H mutant remained sensitive to nirmatrelvir based on our yeast assay, potentially even more sensitive with an RC50 ~2.8-fold less than WT. This is consistent with previous reports showing that the P132H mutant remains sensitive to nirmatrelvir in in vitro enzyme assays [25][26][27][28] . It appears that M pro mutants (i.e.…”

Section: Discussionsupporting

confidence: 93%

“…PHASER was used for molecular replacement using a previously solved SARS-CoV-2 M pro structure (PDB ID: 7LYH) as a reference model. The CCP4 suite, (23) Coot, (24) and the PDB REDO server (pdb-redo.eu) (25) were used to complete the model building and refinement. The PyMOL Molecular Graphics System (Schrödinger, LLC) was used to generate all images.…”

Section: Recombinant Mpro and Proteolytic Activity Assaysmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 93%

Section: Recombinant Mpro and Proteolytic Activity Assaysmentioning

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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“…Nirmatrelvir is also an oral drug against COVID-19, developed by Pfizer. Unlike Remdesivir and Molnupiriavir, Nirmatrelvir is a protease inhibitor targeting the Mpro of all coronaviruses known to infect humans [ 136 , 137 ]. The M pro , or 3CLpro, is one of the viral proteases that cleaves the two orf1 polyproteins, and thereby plays a critical role during viral replication (See details in SARS-CoV-2 Life Cycle).…”

Section: Beyond the Nature—antiviral Pharmaceuticalsmentioning

confidence: 99%

Coronaviral Infection and Interferon Response: The Virus-Host Arms Race and COVID-19

Liu

Chi

Dmytruk

et al. 2022

Viruses

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The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in unprecedented morbidity and mortality worldwide. The host cells use a number of pattern recognition receptors (PRRs) for early detection of coronavirus infection, and timely interferon secretion is highly effective against SARS-CoV-2 infection. However, the virus has developed many strategies to delay interferon secretion and disarm cellular defense by intervening in interferon-associated signaling pathways on multiple levels. As a result, some COVID-19 patients suffered dramatic susceptibility to SARS-CoV-2 infection, while another part of the population showed only mild or no symptoms. One hypothesis suggests that functional differences in innate immune integrity could be the key to such variability. This review tries to decipher possible interactions between SARS-CoV-2 proteins and human antiviral interferon sensors. We found that SARS-CoV-2 actively interacts with PRR sensors and antiviral pathways by avoiding interferon suppression, which could result in severe COVID-19 pathogenesis. Finally, we summarize data on available antiviral pharmaceutical options that have shown potential to reduce COVID-19 morbidity and mortality in recent clinical trials.

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“…Several biochemical studies, however, showed that the prevalent Mpro mutants in the Variants of Concern or Variants of Interest declared by the World Health Organization (WHO), such as G15S (Lambda), K90R (Beta), and P132H (Omicron), are still susceptible to nirmatrelvir, with IC50 values and catalytic efficiencies similar to the wild type (WT) Mpro. 6 – 8 Nevertheless, biochemical assays of several infrequent natural substitutions, e.g., H164N, H172Y, and Q189K, are associated with reduced activities of nirmatrelvir, among which H172Y caused the largest reduction in the inhibitory activity, with a 233-fold increase in the K i value of nirmatrelvir according to a disclosure by Pfizer. 1 Although H172Y is a rare mutation (found in only a few entries of the database GISAID 9 ), it may become favored in the future under the selection pressure of nirmatrelvir therapy.…”

Section: Introductionmentioning

confidence: 99%

An Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease

Oliveira

Ibrahim

Sun

et al. 2022

Preprint

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Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID-19 patients. Although the prevalent Mpro mutants in the SARS-CoV-2 Variants of Concern (e.g., Omicron) are still susceptible to nirmatrelvir, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the selective pressure of antiviral therapy may favor resistance mutations, there is an urgent need to understand the effect of the H172Y mutation on Mpro's structure, function, and drug resistance. Here we report the molecular dynamics (MD) simulations as well as the measurements of stability, enzyme kinetics of H172Y Mpro, and IC50 value of nirmatrelvir. Simulations showed that mutation disrupts the interactions between the S1 pocket and N terminus of the opposite protomer. Intriguingly, a native hydrogen bond (H-bond) between Phe140 and the N terminus is replaced by a transient H-bond between Phe140 and Tyr172. In the ligand-free simulations, strengthening of this nonnative H-bond is correlated with disruption of the conserved aromatic stacking between Phe140 and His163, leading to a partial collapse of the oxyanion loop. In the nirmatrelvir-bound simulations, the nonnative H-bond is correlated with the loss of an important H-bond between Glu166 and nirmatrelvir's lactam nitrogen at P1 position. These results are consistent with the newly reported X-ray structures of H172Y Mpro and suggest a mechanism by which the H172Y substitution perturbs the S1 pocket, leading to the decreased structural stability and binding affinity, which in turn explains the drastic reduction in catalytic activity and antiviral susceptibility.

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Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

Cited by 122 publications

References 17 publications

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

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

Coronaviral Infection and Interferon Response: The Virus-Host Arms Race and COVID-19

An Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease

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Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

Cited by 122 publications

References 17 publications

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

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

Coronaviral Infection and Interferon Response: The Virus-Host Arms Race and COVID-19

An Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease

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Product

Resources

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

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