Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for therapy

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   ScreenIT

   Mar 23, 2022

   SciScore for 10.1101/2022.03.18.484178: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were maintained in a humidified incubator with 5% CO2 at 37°C and were tested negative for mycoplasma. 2.2. Compounds: The following reagents were used: (5Z)-7-oxozeaenol (Cayman Chemical), sunitinib malate, (Selleckchem), erlotinib (LC Laboratories), gefitinib (Selleckchem), baricitinib (a gift from Dr. Chris Liang), SGC-GAK-1 and STK16-IN-1 (MedChemExpress).

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   HEK-293T (ATCC) cells were grown in DMEM supplemented with 10% FCS, 1%L-glutamine, and 1% …

   SciScore for 10.1101/2022.03.18.484178: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: Cells were maintained in a humidified incubator with 5% CO2 at 37°C and were tested negative for mycoplasma. 2.2. Compounds: The following reagents were used: (5Z)-7-oxozeaenol (Cayman Chemical), sunitinib malate, (Selleckchem), erlotinib (LC Laboratories), gefitinib (Selleckchem), baricitinib (a gift from Dr. Chris Liang), SGC-GAK-1 and STK16-IN-1 (MedChemExpress).

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   HEK-293T (ATCC) cells were grown in DMEM supplemented with 10% FCS, 1%L-glutamine, and 1% Pen-strep.	HEK-293T suggested: None
   Briefly, Vero E6 cells were transfected using Lipofectamine 2000 (Thermo Fisher) with 4 mg/well of pBeloBAC11-SARS-CoV-2/WT or – Nluc-2A.	Vero E6 suggested: None
   P1 virus was passaged twice in Vero E6-TMPRSS2 cells.	Vero E6-TMPRSS2 suggested: None
   Twenty-four hours later, culture supernatant was harvested, clarified by centrifugation, filtered (0.22 μm) and stored at −80°C. rVSV-SARS-CoV-2-S was titrated on Vero cells via luciferase assay, and TCID50 was determined.	Vero suggested: None
   Entry assays: Calu-3 cells were infected with WT rSARS-CoV-2/WT (MOI=1) or a high inoculum of rVSV-GP SARS-CoV-2.	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609)
   Briefly, HEK293 cells were transiently transfected with the NanoLuc® Fusion DNA and incubated at 37°C. 20 hours post-transfection, NanoBRET™ tracer reagent and RMC-242 were added to the cells and incubated at 37°C for 2 hours.	HEK293 suggested: None
   Recombinant DNA
   Sentences	Resources
   Briefly, Vero E6 cells were transfected using Lipofectamine 2000 (Thermo Fisher) with 4 mg/well of pBeloBAC11-SARS-CoV-2/WT or – Nluc-2A.	pBeloBAC11-SARS-CoV-2/WT suggested: None
   Software and Algorithms
   Sentences	Resources
   Kinase abundance was quantified label-free using MaxQuant software. 2.16.	MaxQuant suggested: (MaxQuant, RRID:SCR_014485)
   Statistical Analysis: All data were analyzed with GraphPad Prism software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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

   Our findings suggest that the benefit demonstrated with baricitinib clinically likely does not result from its predicted anti-NAK effect and point out a limitation of the artificial intelligence approach for drug discovery in predicting antiviral activity. Lastly, we show that combining compounds with anti-AAK1/BIKE and anti-GAK activities may provide a synergistic antiviral effect against SARS-CoV-2 in vitro. This finding is in agreement with our prior data with sunitinib/erlotinib combinations in HCV, DENV, and EBOV in vitro (Neveu et al., 2015; Bekerman et al., 2017) and the finding that their combination protected 85% and 50% of mice from DENV and EBOV challenges, respectively (Bekerman et al., 2017; Pu et al., 2018). AAK1/BIKE and GAK have partially overlapping functions (Zhang et al., 2005; Neveu et al., 2015; Pu et al., 2020), which may explain moderate or no antiviral effect with drug alone, yet synergistic activity upon treatment with sunitinib/erlotinib combinations. The observed synergistic effect may also result from inhibition of additional targets by these drugs. In summary, these findings validate NAKs as candidate druggable targets for antiviral therapy against SARS-CoV-2 infection and provide a proof of concept that anti-NAKs approaches may have a utility for treating SARS-CoV-2 and possibly other coronavirus infections.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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

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

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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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   Results from scite Reference Check: We found no unreliable references.

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2. 

   Version published to 10.1101/2022.03.18.484178v1 on bioRxiv

   Mar 20, 2022