Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir

Version published to 10.1021/acscentsci.0c01186

Apr 15, 2021

SciScore for 10.1101/2020.05.26.116020: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
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

Experimental Models: Cell Lines
Sentences	Resources
Vero E6 or A549-ACE2 cells were infected with SCoV2 at a multiplicity of infection (MOI) of 0.05 or 0.5, respectively, in the presence of varying concentrations and/or combinations of the test drugs.	A549-ACE2 suggested: None
Sample preparation for RNA-seq: Approximately 4 × 105 Vero E6 cells were seeded onto each well of 12-well plates, in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L glutamine, 25 mM HEPES and 1% …

SciScore for 10.1101/2020.05.26.116020: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
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

Experimental Models: Cell Lines
Sentences	Resources
Vero E6 or A549-ACE2 cells were infected with SCoV2 at a multiplicity of infection (MOI) of 0.05 or 0.5, respectively, in the presence of varying concentrations and/or combinations of the test drugs.	A549-ACE2 suggested: None
Sample preparation for RNA-seq: Approximately 4 × 105 Vero E6 cells were seeded onto each well of 12-well plates, in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L glutamine, 25 mM HEPES and 1% penicillin/streptomycin. Infections of SARS-CoV-2 were performed at a MOI of 1 for 24 hours, followed by drug treatment or 2% DMSO in triplicates.	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
AutoDock Vina v1.1.2 was employed to perform docking experiments45.	AutoDock Vina suggested: (AutoDock Vina, RRID:SCR_011958)
IC50 was determined from curve-fitting using the GraphPad Prism 8.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Quantification of product bands and analysis were performed using ImageQuant and Excel.	ImageQuant suggested: (ImageQuant, RRID:SCR_014246) Excel suggested: None
Bioinformatic Analyses: The raw reads quality was checked by the FastQC (0.11.7) and aligned to ChlSab1.1 (Chlorocebus sabaeus) reference genome by the STAR (2.5.0a) with default parameters.	Bioinformatic suggested: (QFAB Bioinformatics, RRID:SCR_012513) FastQC suggested: (FastQC, RRID:SCR_014583) STAR suggested: (STAR, RRID:SCR_015899)
The count matrix was generated by the featureCounts (as a component of Subread package 2.0.1) program.	featureCounts suggested: (featureCounts, RRID:SCR_012919) Subread suggested: (Subread, RRID:SCR_009803)
Differentially expressed genes (DEGs) were calculated by DESeq2 package (1.26.0) under R environment (3.6.1) and characterized for each sample (\|L2FC\| > 1, p-adjusted-value < 0.05)	DESeq2 suggested: (DESeq, RRID:SCR_000154)
Gene set enrichment analysis (GSEA) was performed as previously described using normalized counts with orthology gene converting to human gene by biomaRt package (2.42.1)50.	Gene set enrichment analysis suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199) biomaRt suggested: (biomaRt, RRID:SCR_019214)
Statistical Analyses: For in vitro experiments, four-parameter dose response curve fitting was performed with constraints: Top = 1, IC50 > 0 using GraphPad Prism 8.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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: We detected the following sentences addressing limitations in the study:

We note, however, there are also several limitations of simeprevir and the proposed simeprevir-remdesivir combination. Simeprevir requires dose adjustments in patients with Child-Pugh Class B or C cirrhosis, as well as in patients with East Asian ancestry37. In addition, simeprevir has been taken off the market since 2018 due to the emergence of next-generation HCV protease inhibitors, hence its supply may not be ramped up easily. Noteworthily, simeprevir is metabolized by the CYP3A4 enzyme with saturable kinetics37 while remdesivir itself is not only a substrate of CYP3A4 but also a CYP3A4 inhibitor. Whether such theoretical pharmacokinetic interaction will exacerbate liver toxicity or provide additional pharmacokinetic synergy (in addition to pharmacodynamic synergy) in vivo remains to be tested. Mechanistically, we found that simeprevir suppresses SARS-CoV-2 replication by targeting at least two viral proteins – it weakly inhibits Mpro at ~10 μM and unexpectedly inhibits RdRp at ~5 μM. The potency towards Mpro is consistent with the IC50 of ~13.7 μM as determined in a parallel study39. Our gel-based assay (Supplementary Fig. 9) suggested that simeprevir interferes with RNA-binding of RdRp because less probe was extended but to full length. This is also supported by the in silico docking results, in which simeprevir is docked to a highly conserved RNA binding site showing no amino acid polymorphism between SARS-CoV and SARS-CoV-2 (Supplementary Fig. 8A). This putative bindi...

Results from TrialIdentifier: No clinical trial numbers were referenced.

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).

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

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Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir

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