Targeting the coronavirus nucleocapsid protein through GSK-3 inhibition

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1. Version published to 10.1073/pnas.2113401118

   Sep 30, 2021
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2021.02.17.21251933: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All work with infectious virus was performed in a Biosafety Level 3 laboratory and approved by the Institutional Biosafety Committee and Environmental Health and Safety. UCLA: Calu3 cells were seeded at 3x104 cells per well in 0.2 ml volumes in 96-well plates. IRB: EHR analysis for UPHS was performed under University of Pennsylvania Institutional Review Board (IRB) protocol #844360 and for MSMC under IRB#20-00338. Outcome: The primary outcome of our case-control EHR study was COVID-19 susceptibility where cases are defined by positive test results from RT-PCR of nasal samples and controls with negative test results.
   Randomization	not detected.
   Blinding	not …

   SciScore for 10.1101/2021.02.17.21251933: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All work with infectious virus was performed in a Biosafety Level 3 laboratory and approved by the Institutional Biosafety Committee and Environmental Health and Safety. UCLA: Calu3 cells were seeded at 3x104 cells per well in 0.2 ml volumes in 96-well plates. IRB: EHR analysis for UPHS was performed under University of Pennsylvania Institutional Review Board (IRB) protocol #844360 and for MSMC under IRB#20-00338. Outcome: The primary outcome of our case-control EHR study was COVID-19 susceptibility where cases are defined by positive test results from RT-PCR of nasal samples and controls with negative test results.
   Randomization	not detected.
   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
   Antibodies to the SARS-CoV-2 N protein were purchased from Invitrogen (#PA1- 41386).	the SARS-CoV-2 N protein suggested: None SARS-CoV-2 N protein suggested: (ABclonal Cat# A20021, RRID:AB_2862924)
   Antibodies from Cell Signaling included phospho-Glycogen Synthase (#3891), phospho-S6 (#4858), β-catenin (#9562), phospho-β-catenin (#9561), GAPDH (#2118), PKCα (#2056), PKCδ (#2058), PKCε (#2683), and phospho (Ser) substrate (#2261).	phospho-Glycogen Synthase suggested: (Cell Signaling Technology Cat# 3891, RRID:AB_2116390) phospho-S6 (#4858), suggested: (Cell Signaling Technology Cat# 4858, RRID:AB_916156) β-catenin suggested: (Cell Signaling Technology Cat# 9562, RRID:AB_331149) phospho-β-catenin suggested: None GAPDH suggested: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)
   Other antibodies included antibodies to GSK-3 (Calbiochem #368662), Tau (T14/46 antibodies provided by Virginia Lee, University of Pennsylvania), and β-actin (Sigma #A5441).	GSK-3 suggested: None T14/46 suggested: (Abcam Cat# T1446, RRID:AB_10704455) β-actin suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
   For immunoprecipitation/protein kinase assays, anti-Myc-tag antibody was incubated with Surebeads Protein G megnetic beads (Bio-Rad, #1614023) for 10 min, lysates were then added for an additional 1 hr at room temperature.	anti-Myc-tag suggested: None
   At 48 hours post infection (hpi), the cells were fixed with 4% paraformaldehyde and viral infection was examined by immunofluorescent analysis (IFA) using SARS-CoV Spike (S) antibody [BEI Resources: NR-616 Monoclonal Anti-24 SARS-CoV S Protein (Similar to 240C)].	Anti-24 SARS-CoV S Protein suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture, transfections, and CRISPR/Cas9 knockout: HEK293T cells (ATCC #CRL-1573) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO #11965) supplemented with 10% Fetal Bovine Serum (Hyclone #SH30071.03) and 1% penicillin/streptomycin (GIBCO #15140), and were maintained at 37°C and 5% CO2.	HEK293T suggested: None
   Two days after transduction, about 1,000 tranduced 293T cells were mixed with 1ml of methylcellulose (MethoCult H4034 Optimum, Stem Cell Technologies) into a 6-well plate and cultured at 37°C and 5% CO2 for two weeks.	293T suggested: None
   All work with infectious virus was performed in a Biosafety Level 3 laboratory and approved by the Institutional Biosafety Committee and Environmental Health and Safety. UCLA: Calu3 cells were seeded at 3x104 cells per well in 0.2 ml volumes in 96-well plates.	Calu3 suggested: None
   Software and Algorithms
   Sentences	Resources
   The total number of cells and the number of infected cells were measured using MetaXpress 5.3.3 cell scoring module and the percentage of infected cells was calculated.	MetaXpress suggested: (MetaXpress, RRID:SCR_016654)
   To minimize these differences, we used RxNorm – a resource of standardized nomenclature for drug names from the National Library of Medicine45.	RxNorm suggested: (RxNorm, RRID:SCR_006645)

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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:

   While the association of lithium therapy and reduced risk of COVID-19 across three health systems is both signficant and intriguing, observational studies have many limitations. A variety of factors with potential biases cannot be measured even after comparing the cases and controls in a manner that accounts for known confounding factors using a rigorous matching algorithm. For instance, details on medicine usage were derived from records of prescription orders, but information on compliance before SARS-CoV-2 PCR testing is not available. In addition, the collection of a non-random sample population can create a collider bias and lead to distorted associations. For example, the COVID-19 test was restricted particularly in the early pandemic to symptomatic patients so that many asymptomatic patients in the EHR were not tested. These findings should therefore be interpreted carefully and deeper investigation is required in a cohort with a larger sample size. Prior work has shown that lithium and the GSK-3 inhibitor Kenpaullone inhibit N phosphorylation and reduce viral titers in SARS-CoV and JHMV infected Vero6 cells8, 9 and GSK3 knockdown also impairs replication of IBV in Vero cells15. We also show that multiple small molecule GSK-3 inhibitors, including CHIR99021, BIM-I, AR-A014418, Enzastaurin, and Sotrastaurin block SARS-CoV-2 phosphorylation. These pharmacological studies are compelling evidence that GSK-3 is a critical host kinase for N protein, but these drugs may have ...

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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.
   • Thank you for including a protocol registration statement.

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3. 

   Version published to 10.1101/2021.02.17.21251933 on medRxiv

   Feb 22, 2021