Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 M
            <sup>pro</sup>
            /3CL
            <sup>pro</sup>
            in Living Cells

Moghadasi, Seyed Arad; Esler, Morgan A.; Otsuka, Yuka; Becker, Jordan T.; Moraes, Sofia N; Anderson, Constance B.; Chamakuri, Srinivas; Belica, Christopher; Wick, Chloe; Harki, Daniel A.; Young, Damian W.; Scampavia, Louis; Spicer, Timothy; Shi, Ke; Aihara, Hideki; Brown, William L.; Harris, Reuben S.

doi:10.1128/mbio.00784-22

Cited by 25 publications

(36 citation statements)

References 65 publications

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“…We cross-validated several of our mutants in different assays. We confirmed the resistance data of L167F with a previously published cellular assay (27) and the mutants Y54C, L167F, and Q192R with a biochemical assay (47). We showed also in a biochemical assay that the kinetic metrics of the mutants Y54C, L167F, and Q192R are attenuated to varying degrees.…”

Section: Discussionsupporting

confidence: 86%

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SARS-CoV-2 3CL ^pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

et al. 2023

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Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor against the SARS-CoV-2 protease 3CL pro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein G, the SARS-CoV-2 3CL pro , and the VSV polymerase L. Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CL pro and release of the functional viral polymerase L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in an additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.

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Section: Discussionsupporting

confidence: 86%

“…The resistance phenotype of L167F observed with gain- and loss-of-signal assays was confirmed using another recently published cellular system ( 27 ). In this complementary assay, a polyprotein of Src, 3CL pro with N-and C-terminal autocleavage sites, HIV Tat, and luciferase was used to repress transcription when 3CL pro was active ( fig.…”

Section: Resultssupporting

confidence: 62%

SARS-CoV-2 3CL ^pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

et al. 2023

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“…31 Given the relatively rapid rate at which SARS-CoV-2 mutates, M pro targeting drug resistance is a major concern. 8,32,33 A detailed understanding of how M pro recognises its natural substrates thus may aid the development of next-generation inhibitors. 12,34 Here, we describe the application of dynamical-nonequilibrium molecular dynamics (D-NEMD) [35][36][37][38] simulations to investigate the response of M pro to the instantaneous removal of a substrate in one of the active sites in the dimer.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dynamical nonequilibrium molecular dynamics simulations identify allosteric sites and positions associated with drug resistance in the SARS-CoV-2 main protease

Chan

Oliveira

Schofield

et al. 2022

Preprint

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The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalysing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behaviour of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.

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“…Of the results, out of six active compounds against SARS-CoV-2 M pro , ebselen exhibited promising antiviral activity in cellbased assays (Jin et al, 2020). The main protease, M pro activity, is critical for breaking the viral polyprotein into further protein units for virus replication and pathogenesis (Moghadasi et al, 2020). The position of SARS-CoV-2 M pro inhibitor in the prevention of SARS-Cov-2 propagation is shown in Figure 2 (Mengist et al, 2020).…”

Section: Prospects For Nano-se To Tackle Sars-cov-2 Variantsmentioning

confidence: 99%

Exploring nanoselenium to tackle mutated SARS-CoV-2 for efficient COVID-19 management

Singh

Singh²,

Kumar³

et al. 2022

Front. Nanotechnol.

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Despite ongoing public health measures and increasing vaccination rates, deaths and disease severity caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its new emergent variants continue to threaten the health of people around the world. Therefore, there is an urgent need to develop novel strategies for research, diagnosis, treatment, and government policies to combat the variant strains of SARS-CoV-2. Since the state-of-the-art COVID-19 pandemic, the role of selenium in dealing with COVID-19 disease has been widely discussed due to its importance as an essential micronutrient. This review aims at providing all antiviral activities of nanoselenium (Nano-Se) ever explored using different methods in the literature. We systematically summarize the studied antiviral activities of Nano-Se required to project it as an efficient antiviral system as a function of shape, size, and synthesis method. The outcomes of this article not only introduce Nano-Se to the scientific community but also motivate scholars to adopt Nano-Se to tackle any serious virus such as mutated SARS-CoV-2 to achieve an effective antiviral activity in a desired manner.

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Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 M ^pro /3CL ^pro in Living Cells

Abstract: The main protease, M pro , of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development.

Cited by 25 publications

References 65 publications

SARS-CoV-2 3CL ^pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

SARS-CoV-2 3CL ^pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

Dynamical nonequilibrium molecular dynamics simulations identify allosteric sites and positions associated with drug resistance in the SARS-CoV-2 main protease

Exploring nanoselenium to tackle mutated SARS-CoV-2 for efficient COVID-19 management

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Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 M pro /3CL pro in Living Cells

Abstract: The main protease, M pro , of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development.

Cited by 25 publications

References 65 publications

SARS-CoV-2 3CL pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

SARS-CoV-2 3CL pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

Dynamical nonequilibrium molecular dynamics simulations identify allosteric sites and positions associated with drug resistance in the SARS-CoV-2 main protease

Exploring nanoselenium to tackle mutated SARS-CoV-2 for efficient COVID-19 management

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Product

Resources

About

Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 M ^pro /3CL ^pro in Living Cells

SARS-CoV-2 3CL ^pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

SARS-CoV-2 3CL ^pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376