The FDA-approved drug Alectinib compromises SARS-CoV-2 nucleocapsid phosphorylation and inhibits viral infection in vitro

Article activity feed

1. 

   ScreenIT

   Aug 15, 2020

   SciScore for 10.1101/2020.08.14.251207: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	Matrix processing and substrate scoring: The matrices were normalized by the sum of the 17 randomized amino acids (all amino acids expect for serine, threonine and cysteine), to yield a position specific scoring matrix.
   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
   For detection of SRPK1, membranes were incubated with primary antibody (BD cat. no. 611072) at 0.025 µg/mL in PBST containing 5% milk overnight at 4 °C.	SRPK1 suggested: None
   For detection of GAPDH, membranes were incubated with primary antibody (Cell …

   SciScore for 10.1101/2020.08.14.251207: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	Matrix processing and substrate scoring: The matrices were normalized by the sum of the 17 randomized amino acids (all amino acids expect for serine, threonine and cysteine), to yield a position specific scoring matrix.
   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
   For detection of SRPK1, membranes were incubated with primary antibody (BD cat. no. 611072) at 0.025 µg/mL in PBST containing 5% milk overnight at 4 °C.	SRPK1 suggested: None
   For detection of GAPDH, membranes were incubated with primary antibody (Cell Signaling, cat. no. 2118S) at a dilution of 1:10,000 in PBST containing 5% milk overnight at 4 °C.	GAPDH suggested: None
   Anti-mouse (Invitrogen cat. no. A16072) and anti-rabbit (Thermo Scientific cat. no. A16104) secondary antibodies were used at 1:20,000 and 1:10,000 dilutions, respectively, in PBST containing 5% milk for 1 hour at room temperature.	Anti-mouse suggested: None anti-rabbit suggested: None
   Cells were stained with either an anti-dsRNA antibody (J2, Sigma MABE1134) at a 1:125 dilution, or an anti-SARS-CoV-2 Spike RBD protein (ProSci #9087) at a 1:150 dilution.	anti-dsRNA suggested: (Millipore Cat# MABE1134, RRID:AB_2819101) J2 suggested: None anti-SARS-CoV-2 Spike RBD protein ( ProSci #9087 suggested: None
   The reactions were run phos-tag gel and blotted with monoclonal anti-N protein antibody (GeneTex).	anti-N protein suggested: None
   Samples were incubated with primary antibodies: Prosurfactant protein C (1:500, Milipore, cat# AB3786) and SARS-CoV-2 (1:500, Genetex cat# GTX632604) in blocking buffer at 4°C overnight.	SARS-CoV-2 suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture (Duke): All cells were obtained from ATCC and grown at 37°C in 5% CO2. 293T and A549 and Huh7 cells were grown in DMEM with 10% FBS, Glutamax, and Penicillin/Streptomycin.	293T suggested: None A549 suggested: None
   Calu-3 cells were grown in EMEM with 10% FBS and Penicillin/Streptomycin.	Calu-3 suggested: None
   HCoV-229E infections and titering: A stock of isolate HCoV-229E VR-740 (ATCC) was grown on Huh7 cells in complete media (DMEM supplemented with 10% FBS, 1% Pen/Strep, Glutamax).	Huh7 suggested: None
   For phosphoproteomic analysis, 6×107 Vero E6 or 6×107 A549-ACE2 cells were treated with 5 µM Alectinib or DMSO in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L-glutamine, 10 mM	Vero E6 suggested: None
   Lentiviruses were packaged as per standard protocols with a VSV-G envelope protein, A549 or A549-Cas9 cells were transduced and selected with puromycin at 2μg/mL. siRNA treatment of cells: A549-ACE2 cells were treated with 30μM siRNA (SRPK1: Horizon M-003982-02-0010; Non-targeting: Thermo 4390843) following the HiPerfect (Qiagen) Fast-Forward protocol and plated in 6 well plates.	A549-Cas9 suggested: None A549-ACE2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Differential expression analysis: Differential expression analysis of proteins and phosphorylation sites was done using Limma v3.42 package in R (81).	Limma suggested: (LIMMA, RRID:SCR_010943)
   A BLAST search was performed over each proteome using the SARS-CoV-2 nucleocapsid protein as the query; the top hit of each virus was taken to be the nucleocapsid.	BLAST suggested: (BLASTX, RRID:SCR_001653)
   Each designated nucleocapsid was then individually aligned to the SARS- CoV-2 nucleocapsid using MUSCLE.	MUSCLE suggested: (MUSCLE, RRID:SCR_011812)
   Bacterial colonies were then selected and plasmid DNA isolated using the Genejet Plasmid Miniprep kit (Thermo Fisher Cat# K0503).	Thermo Fisher Cat# suggested: None
   The SARS-CoV2 primer/probe set were synthesized using IDT DNA based on the sequences provided by the CDC “Research Use Only 2019-Novel Coronavirus (2019-nCov) Real-time RT-PCR Primers and Probes” set N1.	Probes” suggested: None
   Reactions were cycled on the Applied Biosystems QuantStudio 3 Real-Time PCR System and analyzed using QuantStudio software version v1.4.1.	QuantStudio suggested: None
   SRPIN340 and SPHINX were suspended in DMSO at 1000X the final concentration indicated, Alectinib at 500X.	SPHINX suggested: (SPHINX, RRID:SCR_000534)

   ---

   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: No clinical trial numbers were referenced.

   ---

   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.

   ---

   Read the original source
2. 

   Version published to 10.1101/2020.08.14.251207 on bioRxiv

   Aug 14, 2020