Development and optimization of a high‐throughput screening assay for in vitro anti‐SARS‐CoV‐2 activity: Evaluation of 5676 Phase 1 Passed Structures
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Abstract
Although vaccines are currently used to control the coronavirus disease 2019 (COVID‐19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS‐CoV‐2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS‐CoV‐2 and clinically successful against COVID‐19. As part of an immediate response to the COVID‐19 pandemic, a high‐throughput, high content imaging–based SARS‐CoV‐2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6‐eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG‐12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti–SARS‐CoV‐2 activity in VeroE6‐eGFP and/or Caco‐2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID‐19 treatment.
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SciScore for 10.1101/2022.02.02.478671: (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 Authentication: Data were analysed by non-linear curve fitting (four parameter fit) from a dose-response curve using GraphPad Prism to calculate CC50 (cytotoxic concentration of the compound that reduced cell viability to 50%). Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Virus stocks were inoculated and passaged first in HuH-7 cells and then 5 times in VeroE6-eGFP cells prior to storage at –80°C. HuH-7suggested: None40 µL of VeroE6-eGFP cells were seeded at 2000 cells/well in pre-spotted 384-well plates. VeroE6-eGFPsuggeste…SciScore for 10.1101/2022.02.02.478671: (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 Authentication: Data were analysed by non-linear curve fitting (four parameter fit) from a dose-response curve using GraphPad Prism to calculate CC50 (cytotoxic concentration of the compound that reduced cell viability to 50%). Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Virus stocks were inoculated and passaged first in HuH-7 cells and then 5 times in VeroE6-eGFP cells prior to storage at –80°C. HuH-7suggested: None40 µL of VeroE6-eGFP cells were seeded at 2000 cells/well in pre-spotted 384-well plates. VeroE6-eGFPsuggested: NoneAntiviral and toxicity assay in Caco-2 cells: After compounds with potential antiviral activity in VeroE6 cells were identified, confirmation of their inhibition of virus-induced CPE was performed in Caco-2 cells. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)Caco-2suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)Software and Algorithms Sentences Resources All passages were sequenced using a MinION platform (Oxford Nanopore). MinIONsuggested: (MinION, RRID:SCR_017985)After incubation, steps were followed according to the ATPlite™ manufacturer’s instructions. ATPlite™suggested: NoneData were analysed by non-linear curve fitting (four parameter fit) from a dose-response curve using GraphPad Prism to calculate CC50 (cytotoxic concentration of the compound that reduced cell viability to 50%). GraphPad Prismsuggested: (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: 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: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04960202 Active, not recruiting EPIC-HR: Study of Oral PF-07321332/Ritonavir Compared With P… NCT05011513 Active, not recruiting Evaluation of Protease Inhibition for COVID-19 in Standard-R… NCT05047601 Recruiting A Study of a Potential Oral Treatment to Prevent COVID-19 in… NCT04575597 Active, not recruiting Efficacy and Safety of Molnupiravir (MK-4482) in Non-Hospita… 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.
Results from scite Reference Check: We found no unreliable references.
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