key: cord-314546-fbddxbhd
authors: Ko, Meehyun; Jeon, Sangeun; Ryu, Wang‐Shick; Kim, Seungtaek
title: Comparative analysis of antiviral efficacy of FDA‐approved drugs against SARS‐CoV‐2 in human lung cells
date: 2020-08-16
journal: J Med Virol
DOI: 10.1002/jmv.26397
sha: 
doc_id: 314546
cord_uid: fbddxbhd

Drug repositioning represents an effective way to control the current COVID‐19 pandemic. Previously, we identified 24 FDA‐approved drugs which exhibited substantial antiviral effect against severe acute respiratory syndrome coronavirus 2 in Vero cells. Since antiviral efficacy could be altered in different cell lines, we developed an antiviral screening assay with human lung cells, which is more appropriate than Vero cell. The comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC(50) = 0.0022 µM).

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by a coronavirus. 1 The causative virus was named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because it is very similar to SARS-CoV (79.5%) and this virus belongs to the Betacoronavirus genus within the Coronaviridae family. 2 Both SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) also belong to the same Betacoronavirus genus.

Neither vaccine nor therapeutic has been developed for SARSand MERS-CoV and the current standard of care for the patients with COVID-19 is just supportive care. However, numerous clinical trials are ongoing globally with Food and Drug Administration (FDA)approved drugs as drug repositioning programs (https://www.covidtrials.org/). Among these drugs, (hydroxy)chloroquine, lopinavir/ ritonavir, and remdesivir are those that are the most frequently being tested worldwide due to the well-known in vitro antiviral effects on both MERS-and SARS-CoV and even on SARS-CoV-2. 3 Previously, we identified a total of 24 potential antiviral drug candidates from FDA-approved drugs using Vero cells. 4 Since antiviral efficacy could be altered in different cell lines, we developed a new image-based antiviral screening assay with Calu-3 cells, a well-known human lung cell line, 5 and compared the antiviral efficacy of the antiviral candidates in between Vero and Calu-3 cells.

Calu-3 used in this study is a clonal isolate, which shows higher growth rate compared with the parental Calu-3 obtained from the American Type Culture Collection (ATCCHTB-55). Calu-3 was maintained at 37°C with 5% CO 2 in Eagle's Minimum Essential Medium (EMEM, ATCC), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1X Antibiotic-Antimycotic solution (Gibco). SARS-CoV-2 (βCoV/KOR/KCDC03/2020) was provided by Korea Centers for Disease Control and Prevention (KCDC), and was propagated in Vero cells. Viral titers were determined by plaque assays in Vero cells. All experiments using SARS-CoV-2 were performed at Institut Pasteur Korea in compliance with the guidelines of the KNIH, using enhanced biosafety level 3 (BSL-3) containment procedures in laboratories approved for use by the KCDC. 

In our previous drug repositioning study, we identified a total of 24 potential antiviral drug candidates from FDA-approved drugs. 4 (Table 1) , the IC 50 of remdesivir rather decreased by 10 folds compared with that with Vero cells, perhaps due to the low metabolic capacity or prodrug activation rate in Vero cells 6 ( Table 2 ). These discrepancies might in part account for the different outcomes from numerous clinical trials using chloroquine, lopinavir, and remdesivir. So far, the treatment with (hydroxy) chloroquine or lopinavir/ritonavir did not show any promising results concerning the COVID-19 treatment 7-9 ; however remdesivir seems to be effective for treatment of patients with COVID-19 in certain clinical settings. 10 Interestingly, the IC 50 values of most drugs in our study increased in varying degrees in Calu-3 cells (Figures 2A,C) (Tables 1 and 3 ). Only six drugs showed decreases in IC 50 ( Figure 2B ) ( Table 2) : nafamostat mesylate, camostat mesylate, remdesivir, hydroxyprogesterone caproate, digitoxin, and cyclosporine. Although nafamostat mesylate and camostat mesylate were not selected as potent antiviral drug candidates in our earlier study, we compared the antiviral efficacy of these drugs at this time in between Vero and Calu-3 cells following the discovery that TMPRSS2, a host protease necessary for priming viral spike glycoprotein, could be a target for COVID-19 antiviral development. 11 The discrepancy in IC 50 was specifically remarkable with nafamostat mesylate; the IC 50 decreased by approximately 6000 folds when the drug was used in the SARS-CoV-2-infected Calu-3 cells perhaps due to the dominant role of TMPRSS2-dependent viral entry in the Calu-3 human lung epithelial cells. 12, 13 In addition, the IC 50 of nafamostat mesylate was exceptionally low (0.0022 µM), which indicates that nafamostat mesylate is approximately 600-fold more potent than remdesivir in Calu-3 cells. It became more apparent that blood clotting is one of the complicating manifestations in patients with COVID-19, 14, 15 and nafamostat mesylate may play dual roles not only as an antiviral to block viral entry but also as an anticoagulant to remove blood clots frequently associated with acute respiratory distress syndrome. A recent case report on the treatment of three patients with COVID-19 with nafamostat 16 and other in vitro studies 17,18 corroborated our findings. However, nafamostat has a short half-life in the serum, thus requires continuous intravenous injection, which disables convenient administration for a large group of patients. Solving this disadvantage will increase the accessibility of more COVID-19 patients to nafamostat treatment.

In summary, we compared antiviral efficacy of the potential antiviral drug candidates against SARS-CoV-2 in between Vero and Calu-3 cells and found that nafamostat mesylate is the most potent antiviral drug candidate in vitro. Importantly, nafamostat mesylate has been approved for human use in Japan and Korea for over a decade, thus it can be readily repurposed for COVID-19 following phase II-III clinical trials. Currently, a few clinical trials have been registered (https://clinicaltrials.gov/). According to our results, although in vivo animal models are preferred experimental systems for evaluating antiviral efficacy, in vitro testing using human lung cells is 

The authors declare that there are no conflict of interests. 

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Identification of antiviral drug candidates against SARS-CoV-2 from FDA-approved drugs

Cultured human airway epithelial cells (Calu-3): a model of human respiratory function, structure, and inflammatory responses

Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice

Observational study of hydroxychloroquine in hospitalized patients with COVID-19

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Remdesivir for the treatment of COVID-19-preliminary report

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Identification of nafamostat as a potent inhibitor of Middle East respiratory syndrome coronavirus S protein-mediated membrane fusion using the split-protein-based cellcell fusion assay

Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry

Incidence of thrombotic complications in critically ill ICU patients with COVID-19

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Three cases of treatment with nafamostat in elderly patients with COVID-19 pneumonia who need oxygen therapy

Nafamostat mesylate blocks activation of SARS-CoV-2: new treatment option for COVID-19

The anticoagulant nafamostat potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-typedependent manner

Comparative analysis of antiviral efficacy of FDA-approved drugs against SARS-CoV-2 in human lung cells

Conception: WSR and SK. Study design: MK, WSR, and SK. Participation in experiments and data collection: MK and SJ. Data acquisition, writing manuscript, and statistical analysis: MK and SK. All authors reviewed the manuscript and approved the final version.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Seungtaek Kim http://orcid.org/0000-0003-3954-5908