A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors
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SciScore for 10.1101/2021.12.18.473303: (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 Antibodies and Virtual screening for anti-nsp5 compounds are described in the Supplementary Methods Cells and virus: HEK-293T cells (purchased from ATCC, # CRL-11268) and A549 cells stably expressing the human ACE2 receptor (A549-ACE2, kindly provided by O. Schwartz, Institut Pasteur) were grown in complete Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS). anti-nsp5suggested: (Grupo de la Dra. Susana Lopez; Instituto de Biotecnologia, UNAM. Cat# NSP5 (C-6, RRID:AB_2802098)A549-ACE2suggested: NoneImmunoblot membranes were incubated with primary antibodies … SciScore for 10.1101/2021.12.18.473303: (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 Antibodies and Virtual screening for anti-nsp5 compounds are described in the Supplementary Methods Cells and virus: HEK-293T cells (purchased from ATCC, # CRL-11268) and A549 cells stably expressing the human ACE2 receptor (A549-ACE2, kindly provided by O. Schwartz, Institut Pasteur) were grown in complete Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS). anti-nsp5suggested: (Grupo de la Dra. Susana Lopez; Instituto de Biotecnologia, UNAM. Cat# NSP5 (C-6, RRID:AB_2802098)A549-ACE2suggested: NoneImmunoblot membranes were incubated with primary antibodies directed against nsp5 [31], NS3/4A (ab21124, Abcam), FLAG-tag (F3165, Sigma) or GAPDH (MA5-15738, Invitrogen), and revealed with HRP-linked secondary antibodies (Jackson ImmunoResearch) using ECL2 substrate (Pierce). FLAG-tagsuggested: NoneGAPDHsuggested: NoneMA5-15738 , Invitrogen) ,suggested: NoneExperimental Models: Cell Lines Sentences Resources Antibodies and Virtual screening for anti-nsp5 compounds are described in the Supplementary Methods Cells and virus: HEK-293T cells (purchased from ATCC, # CRL-11268) and A549 cells stably expressing the human ACE2 receptor (A549-ACE2, kindly provided by O. Schwartz, Institut Pasteur) were grown in complete Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS). A549suggested: NoneTransfected HEK-293T cells were trypsinized at 6 hpt and distributed in the 384-well plates (2×104 cells per well in 50 µL). HEK-293Tsuggested: NoneAntiviral activity and cell viability assays: A549-ACE2 cells seeded in 384-well plates (1.5×103 cells per well) were incubated with the compound of interest at the indicated concentration in DMEM-2% FBS 2 h prior to infection. A549-ACE2suggested: NoneRecombinant DNA Sentences Resources These plasmids were used as templates to amplify the sequence encoding nsp5 (WT or C145A), and the resulting amplicons were subcloned into the pcDNA3.1 plasmid. pcDNA3.1suggested: RRID:Addgene_79663)The pLEX expression vectors for SARS-CoV-2, IBV and hCoV-229E nsp5 are described in [26] and were kindly provided by N. Heaton (Duke University). pLEXsuggested: RRID:Addgene_117987)The Hepatitis C Virus (HCV) NS3/4A expression vector was generated by subcloning a synthetic gene which was codon optimized for expression in human cells (Genscript) into the pCI plasmid. pCIsuggested: RRID:Addgene_74230)To generate the reverse-Nanoluciferase reporter plasmid Rev-Nluc-CoV, a synthetic codon-optimized nucleotide sequence encoding a permuted Nanoluciferase with the NGSVRLQSSLK linker between the N- and the C-terminal domains was cloned into the pLV-CMV-eGFP lentiviral vector (Duke vector core, Duke University) in place of the eGFP insert. reverse-Nanoluciferase reportersuggested: NonepLV-CMV-eGFPsuggested: NoneViral protease activity assays: HEK-293T cells were seeded in 96-well white opaque plates (Greiner Bio-One, 3×104 cells per well) one day before being transfected with 5 ng of the Rev-Nluc-CoV reporter plasmid and 95 ng of the pcDNA3.1-nsp4-5-6 WT, pcDNA-3.1-nsp4-5-6 C145A, pLEX-nsp5 or an empty control pCI plasmid, using polyethyleneimine (PEI-max, #24765-1 Polysciences Inc). pcDNA3.1-nsp4-5-6 WTsuggested: NonepcDNA-3.1-nsp4-5-6 C145Asuggested: NonepLEX-nsp5suggested: NoneTo measure NS3/4A activity, 5 ng of the Rev-Nluc-HCV reporter plasmid was co-transfected with 145 ng of the pCI-NS3/4A WT or pCI-NS3/4A S139A, using the same protocol. pCI-NS3/4Asuggested: NonepCI-NS3/4A S139Asuggested: NoneIn the 384-well plate setting, ∼107 HEK-293T cells were transfected in 50 cm2 dishes with 1 µg of the Rev-Nluc-CoV plasmid and 15 µg of the nsp4-5-6 WT or nsp4-5-6 C145A expression plasmids. Rev-Nluc-CoVsuggested: NoneSoftware and Algorithms Sentences Resources The Luna Universal One-Step RT-qPCR Kit (New England Biolabs) was used, with SARS-CoV-2 specific primers targeting the N gene region (5’-TAATCAGACAAGGAACTGATTA-3’ and 5’-CGAAGGTGTGACTTCCATG-3’) and with the following cycling conditions: 55 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 sec, followed by 60 °C for 1 min, in an Applied Biosystems QuantStudio 6 thermocycler. SARS-CoV-2suggested: (BioLegend Cat# 946101, RRID:AB_2892515)ChEMBL protocols [56]); Removal of chemical duplicates (Open Babel [57]); Selection of dominant tautomers (pH 7.4, ChemAxon Calculator Plugins version 19.0.9, https://chemaxon.com); Selection of species populating more than 25% or, if none, species that are present at more than 75% of the most frequent one, or at least the three most frequent ones; Generation of stereoisomers and low energy ring conformers generation (CORINA classic [58] version 3.4, https://www.mn-am.com); ChEMBLsuggested: (ChEMBL, RRID:SCR_014042)Molecules were kept in the sdf format for docking with FlexX [40] and converted into pdbqt files using MGLTools scripts [63]for docking with Smina [39]. MGLToolssuggested: NoneWe used Smina (version Dec 2020 based on Autodock Vina 1.1.2, exhaustiveness set to 12, 10 best score poses saved) to perform docking targeting all three PDB structures. Autodocksuggested: (AutoDock, RRID:SCR_012746)It also comprised 5RH8, which gave the best results with FlexX, allowing to diversify the screening with FlexX, while providing a basis for comparison between the two programs. FlexXsuggested: (FlexX, RRID:SCR_000186)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
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: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04960202 Recruiting EPIC-HR: Study of Oral PF-07321332/Ritonavir Compared With P… NCT05011513 Recruiting Evaluation of Protease Inhibition for COVID-19 in Standard-R… NCT05047783 Recruiting Masitinib in Patients With Symptomatic Mild to Moderate COVI… 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.
Results from scite Reference Check: We found no unreliable references.
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