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Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells

Adam Pickard , Ben C. Calverley, Joan Chang, Richa Garva, Sara Gago, Yinhui Lu, Karl E. Kadler

Abstract
COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease.

Introduction
The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is having a widespread impact on global health with substantial loss of life. SARS-CoV-2 infection in patients with COVID-19 can result in pulmonary distress, inflammation, and tropism to multiple organs.

Materials and methods

Generation of functional SARS-CoV-2 virus
DNA encoding the genome of SARS-CoV-2 and SARS-CoV-2-ΔOrf7a-NLuc were purchased from Vectorbuilder Inc. (Chicago, US). Transfection of DNA encoding the viruses failed to generate replicative virus when electroporated into 293T cells.

Virus production, maintenance and assessment of titer
Culture medium was collected from 293T cells 6 days after electroporation. This virus (P0) was used to infect cells of interest. As virus replication was slow in 293T cells, virus stocks were maintained by passage in Vero cells grown in DMEM supplemented with 2% FBS.

Electron microscopy
After 3 days infection with P4 virus, Vero cells were scrapped and pelleted. Cell pellets were fixed using 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer for 24 h, washed in ddH2O three times, 30 mins for each wash.

NLuc activity assay
Vero cells grown in 24-well plates (Corning, 3526) were assayed for NLuc activity by adding 1 μL of coelenterazine (final concentration 1.5 μM).

Drug screens
The DiscoveryProbe FDA-approved library of 1971 compounds (L1021, APExBIO Boston, US) was prepared as follows. After thawing the library for 4 hours at room temperature the library was arrayed into 96 well plates at 1 mM in DMSO and stored at -20°C. Stocks (1 mM) were thawed at room temperature for 2 hrs before compounds were added to cells.

Discussion
In this study we have shown that the SARS-CoV-2 virus infects and replicates in a range of human cells especially hepatocytes, kidney glomerulus, and proximal tubule cells of the kidney, and, that 9 drugs that have previously been shown to be safe in humans and approved by the FDA for clinical use are effective in inhibiting SARS-CoV-2 replication even after infection.

Acknowledgments: The authors thank Dr. Jennifer Cavet for providing assistance with working at containing level 3 and use of the facilities.

Citation: Pickard A, Calverley BC, Chang J, Garva R, Gago S, Lu Y, et al. (2021) Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells. PLoS Pathog 17(9): e1009840. https://doi.org/10.1371/journal.ppat.1009840

Editor: Andrew Pekosz, Johns Hopkins University Bloomberg School of Public Health, UNITED STATES

Received: May 27, 2021; Accepted: July 26, 2021; Published: September 9, 2021.

Copyright: © 2021 Pickard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The research was funded by Wellcome (London) (110126/Z/15/Z and 203128/Z/16/Z) to KEK. SG was funded by the NIHR Manchester Research Centre and the Fungal Infection Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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