Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M pro and cathepsin L

Version published to 10.1126/sciadv.abe0751

Dec 11, 2020

SciScore for 10.1101/2020.07.27.223727: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement

not detected.

Randomization

The velocities were randomized and MD simulation for 12 ps was performed in the NPT ensemble and a Berendsen barostat55 with solute heavy atoms equally restrained at 10 K and another one at 300 K.

Blinding

not detected.

Power Analysis

not detected.

Sex as a biological variable

not detected.

Cell Line Authentication

not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific).	SARS-CoV-2 NP suggested: (Sino Biological Cat# 40588-R0004, RRID:AB_2857931) GTX635679 suggested: None anti-mouse suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Human rhabdomyosarcoma (RD), MDCK, Vero, Huh-7, and HCT-8 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium; Caco-2 and MRC-5 cell lines were maintained in Eagle's Minimum Essential Medium (EMEM) medium.	MDCK suggested: CLS Cat# 602280/p823_MDCK_(NBL-2), RRID:CVCL_0422 Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059 Huh-7 suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336 HCT-8 suggested: KCLB Cat# 10244, RRID:CVCL_2478 Caco-2 suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025 MRC-5 suggested: CLS Cat# 300395/NA, RRID:CVCL_0440
Vero E6 cells in 96-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in DMEM supplemented with 1% FBS.	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek)	Gen5 suggested: (Gen5, RRID:SCR_017317)
Rigid and restrained refinements were performed using REFMAC and model building was performed with COOT.	REFMAC suggested: (Refmac, RRID:SCR_014225) COOT suggested: (Coot, RRID:SCR_014222)
Protein structure figures were made using PyMOL (Schrödinger, LLC).	PyMOL suggested: (PyMOL, RRID:SCR_000305)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

Read the original source

SciScore for 10.1101/2020.07.27.223727: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	The velocities were randomized and MD simulation for 12 ps was performed in the NPT ensemble and a Berendsen barostat55 with solute heavy atoms equally restrained at 10 K and another one at 300 K.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary …

SciScore for 10.1101/2020.07.27.223727: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	The velocities were randomized and MD simulation for 12 ps was performed in the NPT ensemble and a Berendsen barostat55 with solute heavy atoms equally restrained at 10 K and another one at 300 K.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific).	SARS-CoV-2 NP suggested: None GTX635679 suggested: (GeneTex Cat# GTX635679, RRID:AB_2888553) anti-mouse suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell lines and viruses: Human rhabdomyosarcoma (RD), MDCK, Vero, Huh-7, and HCT-8 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium; Caco-2 and MRC-5 cell lines were maintained in Eagle’s Minimum Essential Medium (EMEM) medium.	MDCK suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422) Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059) Huh-7 suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336) HCT-8 suggested: ICLC Cat# HTL99005, RRID:CVCL_2478) Caco-2 suggested: None MRC-5 suggested: ICLC Cat# HL95001, RRID:CVCL_0440)
Plaque assay: Vero E6 cells in 6-well plates (Corning) were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at approximately 40 PFU per well.	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek).	Gen5 suggested: (Gen5, RRID:SCR_017317)
Rigid and restrained refinements were performed using REFMAC and model building was performed with COOT.	REFMAC suggested: (Refmac, RRID:SCR_014225) COOT suggested: (Coot, RRID:SCR_014222)
Protein structure figures were made using PyMOL (Schrödinger, LLC).	PyMOL suggested: (PyMOL, RRID:SCR_000305)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Read the original source

SciScore for 10.1101/2020.07.27.223727: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement

not detected.

Randomization

The velocities were randomized and MD simulation for 12 ps was performed in the NPT ensemble and a Berendsen barostat55 with solute heavy atoms equally restrained at 10 K and another one at 300 K.

Blinding

not detected.

Power Analysis

not detected.

Sex as a biological variable

not detected.

Cell Line Authentication

not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific).	SARS-CoV-2 NP suggested: (Sino Biological Cat# 40588-R0004, RRID:AB_2857931) GTX635679 suggested: None anti-mouse suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Human rhabdomyosarcoma (RD), MDCK, Vero, Huh-7, and HCT-8 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium; Caco-2 and MRC-5 cell lines were maintained in Eagle's Minimum Essential Medium (EMEM) medium.	MDCK suggested: CLS Cat# 602280/p823_MDCK_(NBL-2), RRID:CVCL_0422 Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059 Huh-7 suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336 HCT-8 suggested: KCLB Cat# 10244, RRID:CVCL_2478 Caco-2 suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025 MRC-5 suggested: CLS Cat# 300395/NA, RRID:CVCL_0440
Vero E6 cells in 6-well plates (Corning) were infected with SARS-CoV-2 (USAWA1/2020 isolate) at approximately 40 PFU per well.	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek)	Gen5 suggested: (Gen5, RRID:SCR_017317)
Rigid and restrained refinements were performed using REFMAC and model building was performed with COOT.	REFMAC suggested: (Refmac, RRID:SCR_014225) COOT suggested: (Coot, RRID:SCR_014222)
Protein structure figures were made using PyMOL (Schrödinger, LLC).	PyMOL suggested: (PyMOL, RRID:SCR_000305)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

Read the original source

SciScore for 10.1101/2020.07.27.223727: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific).	SARS-CoV-2 NP suggested: (Sino Biological Cat# 40588-R0004, AB_2857931) `<div style="margin-bottom:8px"> <div><b>GTX635679</b></div> <div>suggested: None</div> </div> <div style="margin-bottom:8px">`

SciScore for 10.1101/2020.07.27.223727: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies

Sentences Resources

The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific).

SARS-CoV-2 NP

suggested: (Sino Biological Cat# 40588-R0004, AB_2857931)

      <div style="margin-bottom:8px">
        <div><b>GTX635679</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-mouse</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Human rhabdomyosarcoma (RD), MDCK, Vero, Huh-7, and HCT-8 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium; Caco-2 and MRC-5 cell lines were maintained in Eagle's Minimum Essential Medium (EMEM) medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MDCK</b></div>
        <div>suggested: CLS Cat# 602280/p823_MDCK_(NBL-2), <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0422">CVCL_0422</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Vero</b></div>
        <div>suggested: CLS Cat# 605372/p622_VERO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0059">CVCL_0059</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Huh-7</b></div>
        <div>suggested: CLS Cat# 300156/p7178_HuH7, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0336">CVCL_0336</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>HCT-8</b></div>
        <div>suggested: KCLB Cat# 10244, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_2478">CVCL_2478</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Caco-2</b></div>
        <div>suggested: CLS Cat# 300137/p1665_CaCo-2, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0025">CVCL_0025</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>MRC-5</b></div>
        <div>suggested: CLS Cat# 300395/NA, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0440">CVCL_0440</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 cells in 6-well plates (Corning) were infected with SARS-CoV-2 (USAWA1/2020 isolate) at approximately 40 PFU per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Gen5</b></div>
        <div>suggested: (Gen5, <a href="https://scicrunch.org/resources/Any/search?q=SCR_017317">SCR_017317</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Rigid and restrained refinements were performed using REFMAC and model building was performed with COOT.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>REFMAC</b></div>
        <div>suggested: (Refmac, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014225">SCR_014225</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>COOT</b></div>
        <div>suggested: (Coot, <a href="https://scicrunch.org/resources/Any/search?q=SCR_014222">SCR_014222</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein structure figures were made using PyMOL (Schrödinger, LLC)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PyMOL</b></div>
        <div>suggested: (PyMOL, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000305">SCR_000305</a>)</div>
      </div>
    </td></tr></table>

Data from additional tools added to each annotation on a weekly basis.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

Read the original source

Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M ^pro and cathepsin L

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