The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

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1. Version published to 10.1128/mbio.03705-21

   Apr 26, 2022
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   ScreenIT

   Mar 15, 2021

   SciScore for 10.1101/2021.03.09.434219: (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
   Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at RT and incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer + 0.2% Tween 20: α-β-actin (1:10,000), (Sigma-Aldrich, Saint Louis, MI, USA),	α-β-actin suggested: None
   After primary antibody incubation, membranes were washed three times with 0.1% PBS-Tween and incubated for 1 h with the following fluorescence-conjugated secondary antibodies: IRDye® 800CW Goat anti-Human IgG,	anti-Human IgG suggested: None
   Cells were then blocked in 2% milk (Roth) in PBS and incubated with primary antibodies in PBS (anti …

   SciScore for 10.1101/2021.03.09.434219: (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
   Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at RT and incubated overnight at 4°C with the following primary antibodies diluted in blocking buffer + 0.2% Tween 20: α-β-actin (1:10,000), (Sigma-Aldrich, Saint Louis, MI, USA),	α-β-actin suggested: None
   After primary antibody incubation, membranes were washed three times with 0.1% PBS-Tween and incubated for 1 h with the following fluorescence-conjugated secondary antibodies: IRDye® 800CW Goat anti-Human IgG,	anti-Human IgG suggested: None
   Cells were then blocked in 2% milk (Roth) in PBS and incubated with primary antibodies in PBS (anti dsRNA mouse monoclonal J2 antibody (Scicons) 1:2000 and convalescent SARS-CoV-2 patient serum 1:250).	anti dsRNA suggested: None J2 suggested: None
   Afterwards, cells were washed twice in PBS 0.02% Tween and incubated with secondary antibodies in PBS [1:1000 anti-mouse 568	anti-mouse suggested: None
   After washing, cells were stained with the primary rabbit polyclonal anti-SARS-CoV-2 spike glycoprotein antibody (1:1000, Abcam) for 1 h at RT or overnight at 4 °C.	anti-SARS-CoV-2 spike glycoprotein suggested: None
   After washing, cells were incubated with the secondary Alexa Fluor 488 goat anti-rabbit IgG antibody (1:500, Life Technologies) for 1 h at RT.	anti-rabbit IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Caco-2 (ATCC HTB-37),	Caco-2 suggested: None
   T84 (ATCC CCL-248) and VeroE6 (ATCC CRL-1586).	VeroE6 suggested: None
   The cell line condition filter was used to refine the analysis and include exclusively cell lines susceptible to SARS-CoV-2 infection (i.e. T84, Caco2, Calu-3 and A-549).	Caco2 suggested: None Calu-3 suggested: None A-549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
   Software and Algorithms
   Sentences	Resources
   Virtual screening and molecular docking: Identification of potentially active SARS-CoV-2 inhibitors with desirable Absorption, Distribution, Metabolism, Excretion and Toxicity (ADME-Tox) properties, was performed by structure-based virtual screening (SBVS) of Drugbank V.	Drugbank suggested: (DrugBank, RRID:SCR_002700)
   The qPCR reaction was performed on a CFX96/C1000 Touch qPCR system (Bio-Rad Laboratories, Hercules, CA, USA) using the following PCR program: polymerase activation/DNA denaturation 98°C for 3 min, followed by 45 cycles of denaturation at 98°C for 10s; annealing/extension at 60°C for 40s and a final extension step at the end of the program at 65°C for 30s.	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   Gene expression data in cell lines were retrieved from the aforementioned microarray dataset and from the RNAseq “mRNA Gene Level Homo sapiens (ref: Ensembl 75)” dataset.	Ensembl suggested: (Ensembl, RRID:SCR_002344)
   Quantification of infected cells (expressed as percentage of total cells imaged per well) was performed using a custom-made macro in ImageJ (85).	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Data analysis was conducted using GraphPad Prism v6 (GraphPad Software, San Diego, CA, USA)	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) 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:

   The use of pharmacoenhancers such as cobicistat (40) could help to overcome this limitation. While the present study exclusively focused on the combination of cobicistat and remdesivir, more than 30% of all drugs are metabolized by the main cellular targets of cobicistat (i.e. CYP3A4/5)(62). For example, the recently described SARS-CoV-2 inhibitor plitidepsin (63) is mainly metabolized by CYP3A4 in vitro (64). Therefore, it is conceivable that a synergistic effect similar to that described for remdesivir might be obtained by coupling cobicistat to other antiviral agents. In particular, other compounds tested in clinical trials of SARS-CoV-2 patients, such as chloroquine/hydroxychloroquine (65) and lopinavir (62) are well known substrates of CYP3A. The booster effect of cobicistat would be further complemented by the own antiviral activity of this drug, which was here proven on several models of SARS-CoV-2 infection. In line with this, we observed the strongest synergistic effect with remdesivir, when cobicistat was used at concentrations above its IC50 levels, suggesting a combination of pharmacokinetic and pharmacodynamic effects. Of note, the concentration range in which cobicistat could inhibit SARS-CoV-2 replication was higher than that achievable through standard dosages (i.e. 150mg/day) approved for treatment of HIV-1 infection (37). This potential drawback could be mitigated by the fact that cobicistat (as well as its “parent” drug, ritonavir) was previously shown to b...

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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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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3. 

   Version published to 10.1101/2021.03.09.434219v1 on bioRxiv

   Mar 9, 2021