Structure-based phylogeny identifies avoralstat as a TMPRSS2 inhibitor that prevents SARS-CoV-2 infection in mice
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SciScore for 10.1101/2021.01.04.425289: (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 Membranes were probed with mouse monoclonal anti-Flag antibody (1:1,000; Sigma-Aldrich; Cat. #F3165) for 16 hours at 4° C. anti-Flagsuggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)Blots were then washed three times with TBST (10 minutes/wash) and subsequently incubated with immunoglobulin-G labelled with horseradish peroxidase conjugated secondary anti-mouse antibody (1:5,000; Thermo Scientific™; Cat. #31432). anti-mousesuggested: (Thermo Fisher Scientific Cat# 31432, RRID:AB_228302)Experimental Models: Cell Lines Sentences Resources BL21 cells expressing TMPRSS2-S1P were induced with 0.5 mM IPTG. BL21s…SciScore for 10.1101/2021.01.04.425289: (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 Membranes were probed with mouse monoclonal anti-Flag antibody (1:1,000; Sigma-Aldrich; Cat. #F3165) for 16 hours at 4° C. anti-Flagsuggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)Blots were then washed three times with TBST (10 minutes/wash) and subsequently incubated with immunoglobulin-G labelled with horseradish peroxidase conjugated secondary anti-mouse antibody (1:5,000; Thermo Scientific™; Cat. #31432). anti-mousesuggested: (Thermo Fisher Scientific Cat# 31432, RRID:AB_228302)Experimental Models: Cell Lines Sentences Resources BL21 cells expressing TMPRSS2-S1P were induced with 0.5 mM IPTG. BL21suggested: RRID:CVCL_M639)TMPRSS2 autoproteolysis assay: HEK 293T cells (ATCC® Cat. # CRL-3216) were obtained from the Viral Vector Core Facility at the University of Iowa. HEK 293Tsuggested: NonePseudovirus transduction assay: HEK-293T cells were transfected to express either the SARS-CoV-2 spike protein (with the cytoplasmic tail removed; residues 1 - 1255) or the full-length Vesicular Stomatitis Virus (VSV)-G protein. HEK-293Tsuggested: NoneInfectious SARS-CoV-2 neutralization assay: The 2019n-CoV/USA-WA1/2019 strain of SARS-CoV-2 (Accession number: MT985325.1) used in these studies was passaged on Calu-3 2B4 cells and sequence verified. Calu-3 2B4suggested: RRID:CVCL_YZ47)Vero E6 cells in 12 well plates were inoculated at 37 ºC in 5% CO2 for 1 hour with gentle rocking every 15 minutes. Vero E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources The raw docking data, parameters, and 3DPhyloFold code are deposited to Mendeley Data (DOI:10.17632/h3pmycddwc.1 and 10.17632/kk3gkzdsbf.2.) Experimental model and subject details: mice, virus, and cells: Specific pathogen-free 6-week-old male and female BALB/c mice and were purchased from Envigo and maintained in the Animal Care Facilities at the University of Iowa. BALB/csuggested: RRID:IMSR_ORNL:BALB/cRl)Software and Algorithms Sentences Resources Database search and sequence alignment: We first searched the UniProt database for reviewed entries denoted as transmembrane serine proteases (containing an S1-peptidase domain). UniProtsuggested: (UniProtKB, RRID:SCR_004426)Structural modeling of TMPRSS2-S1P: Briefly, a BLAST search of human TMPRSS2-S1P against the Protein Data Bank (PDB) returned the structure of human Hepsin (PDB 1Z8G) as the top hit. BLASTsuggested: (BLASTX, RRID:SCR_001653)A TMPRSS2-S1P model was generated with the Hepsin template (41% sequence identity) using Phyre2, MODELLER, and SWISS-Model. MODELLERsuggested: (MODELLER, RRID:SCR_008395)31 The 600 sequences from our sequence-based phylogenetic analysis underwent MSA using MAFFT and conservation scores were calculated using the Bayesian method option in ConSurf. MAFFTsuggested: (MAFFT, RRID:SCR_011811)The TMPRSS2-S1P binding pocket was inferred by comparison to the structure of Hepsin bound to a peptidomimetic inhibitor (PDB 1Z8G) in PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.) PyMOLsuggested: (PyMOL, RRID:SCR_000305)We therefore searched the Pfam database for structures of mammalian peptidases and selected 74 representative structures (representing the wild-type protein) with an atomic resolution 3.2 Å or better (Supplementary Table 2). Pfamsuggested: (Pfam, RRID:SCR_004726)The phylogenetic tree was constructed using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method in MEGAX software as previously described. MEGAXsuggested: NoneKLKB1 (PDB 6O1S), and Factor VII (PDB 1W7X) were loaded into Maestro software (Schrödinger Release 2019-3). Maestrosuggested: (Maestro, RRID:SCR_016748)Kinetic parameters were then calculated by direct fitting to the Michaelis-Menten or Hill equation in GraphPad Prism 8 (GraphPad, San Diego, CA). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)TMPRSS2-FL cDNA (pcDNA3.1-SARS-2-S-C9; obtained from the Gallagher Laboratory, Loyola University Medical Center, Illinois). Gallagher Laboratorysuggested: NonePolyFect (20 μL) was added to the DNA solution followed by 10-minute incubation at room temperature. PolyFectsuggested: NoneData were analyzed by 1-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism 8.0. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04321096 Active, not recruiting The Impact of Camostat Mesilate on COVID-19 Infection 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.
- No funding statement was detected.
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