High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV-2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

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1. Version published to 10.1371/journal.pone.0250019

   Apr 22, 2021
2. 

   ScreenIT

   Jan 22, 2021

   SciScore for 10.1101/2021.01.21.427657: (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

   Experimental Models: Cell Lines
   Sentences	Resources
   CRL- 8155 human lymphocyte and HepG2 human hepatocyte cells (ATCC, Manassas, VA) were seeded in 96-well plates and incubated at 37°C for 48 h in the presence of test compound (serial-2 dilutions, in triplicate).	HepG2 suggested: None
   In vitro CPE assay in Vero cells: The antiviral activities of test compounds were evaluated in Vero 76 cells.	Vero 76 suggested: IZSLER Cat# BS CL 101, RRID:CVCL_0603)
   In vitro VYR assay in Caco-2 cells: As a follow-up to the CPE assay, a viral yield reduction assay was performed to evaluate the ability of test compounds to inhibit virus production in …

   SciScore for 10.1101/2021.01.21.427657: (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

   Experimental Models: Cell Lines
   Sentences	Resources
   CRL- 8155 human lymphocyte and HepG2 human hepatocyte cells (ATCC, Manassas, VA) were seeded in 96-well plates and incubated at 37°C for 48 h in the presence of test compound (serial-2 dilutions, in triplicate).	HepG2 suggested: None
   In vitro CPE assay in Vero cells: The antiviral activities of test compounds were evaluated in Vero 76 cells.	Vero 76 suggested: IZSLER Cat# BS CL 101, RRID:CVCL_0603)
   In vitro VYR assay in Caco-2 cells: As a follow-up to the CPE assay, a viral yield reduction assay was performed to evaluate the ability of test compounds to inhibit virus production in Caco-2 cells.	Caco-2 suggested: None
   In vitro antiviral assay in Calu3 cells: Ten thousand Calu-3 cells (HTB-55, ATCC) grown in Minimal Eagles Medium supplemented with 0.1% non-essential amino acids, 0.1% penicillin/streptomycin, and 10% FBS were plated in 384 well plates.	Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
   Calu3 cells were pretreated with controls and test drugs (in triplicate) for 2 h prior to infection.	Calu3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   Software and Algorithms
   Sentences	Resources
   GraphPad Prism (GraphPad Software Inc) was used to plot dose response curves and calculate IC50 values.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Spectra were analyzed manually in MassLynx v4.1 (Waters, Milford, MA)	MassLynx suggested: (MassLynx , RRID:SCR_014271)
   Mass deconvolution for monomers and hexamers in the CID/SID spectra was performed in UniDec v4.3.0 [42].	UniDec suggested: None
   Molecular docking simulations: Molecular docking simulations were carried out with AutoDock Qvina[28, 29] by restricting search space to the receptor binding pocket of the nsp15 crystal structure (PDB ID: 6XDH).	AutoDock suggested: (AutoDock, RRID:SCR_012746)
   Gels were stained with SYBR green II (Thermo Fisher) for 30 min and visualized on a Bio-Rad Gel Doc (Bio-Rad Laboratories, Hercules, CA).	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   Densitometry was performed using Bio-Rad Image Lab software.	Bio-Rad Image Lab suggested: None Image suggested: (NIH Image, RRID:SCR_003073)
   A non-linear regression curve fit analysis (GraphPad Prism 8) of POC Infection and cell viability versus the log10 transformed concentration values was used to calculate IC50 values for Infection and CC50 values for cell viability.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   One caveat for these experiments with Exebryl-1 was that the antiviral activity was sometimes close to the cytotoxicity (CC50), e.g. in Caco-2 cells. But clear separation of antiviral activity (EC50) from CC50 was observed in assays with Vero and Calu3 cells. In developing our HTS, we found that Mn2+ but not Mg2+ was essential for endoU activity. Conversely, Mg2+ but not Mn2+ led to a significant (3-4°C) positive shift in the DSF Tm of apo nsp15. This positive Tm in DSF often implies that a ligand complexes and stabilizes a protein structure. However, in this case, Mg2+ (or Mn2+) is not unambiguously present in any of the deposited crystal structures including structures with substrates (PDB IDs: 6WLC, 6X4I, 7K0R) or transition state analogs (PDB ID: 7K1L). Thus, the reason for the DSF shift with Mg2+ is not understood. Manganese has long been known to be important for nsp15 endoU function [12, 14, 19]. But to date, the catalytic role of Mn2+ is unclear. One hypothesis is that Mn2+ may maintain the conformation of the RNA substrate during catalysis [15]. An alternative hypothesis, supported by a possible Mg2+ density in their nsp15 apo structure (PDB ID 6VWW), postulates that a metal binding site near the active site is necessary for active site stability during the cleavage reaction [13]. Further experiments, and ideally structures, are necessary to address the mechanistic role of divalent cations in the activity of nsp15. CoV nsp15 forms a hexameric structure in solution an...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 41. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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3. 

   Version published to 10.1101/2021.01.21.427657v1 on bioRxiv

   Jan 21, 2021