key: cord-0879930-4pywvaxi
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: Nafamostat is the most potent antiviral drug candidate
date: 2020-05-12
journal: bioRxiv
DOI: 10.1101/2020.05.12.090035
sha: 5f2f5b4585e2aaaca73195a6c1e4efd85ef4ebce
doc_id: 879930
cord_uid: 4pywvaxi

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 SARS-CoV-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. Comparative analysis of antiviral activities revealed that nafamostat is the most potent drug in human lung cells (IC50 = 0.0022µM).

4 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)(8); however remdesivir seems to be effective for treatment of patients with COVID-19 in certain clinical settings (https://www.nbcnews.com/health/health-news/coronavirus-drug-remdesivir-shows-promiselarge-trial-n1195171).

Interestingly, the IC50 values of most drugs in our study increased in varying degrees in Calu-3 cells ( Fig. 2A and C) ( Table 1 and 3). Only 5 drugs showed decreases in IC50 (Fig. 2B ) ( Table 2) :

Nafamostat mesylate, Remdesivir, Hydroxyprogesterone caproate, Digitoxin, Cyclosporine.

Although nafamostat mesylate was not included in our earlier study, we compared the antiviral efficacy of this drug 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 (9). The discrepancy in IC50 was remarkable with nafamostat mesylate; the IC50 decreased by ~ 6,000 fold when the drug was used in the SARS-CoV-2infected Calu-3 cells perhaps due to the dominant role of TMPRSS2-dependent viral entry in the Calu-3 human lung epithelial cells (10)(11). In addition, the IC50 of nafamostat mesylate was exceptionally low (0.0022 µM), which indicates that nafamostat mesylate is ~ 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 (12)(13), 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 (ARDS).

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 has 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 a viable option in addition to the commonly used Vero or VeroE6 cells for assessment of antiviral efficacy when the animal models are not readily available.

Calu-3 used in this study is a clonal isolate, which shows higher growth rate compared to the parental Calu-3 obtained from the American Type Culture Collection (ATCC CCL-81). Calu-3 was maintained at 37°C with 5% CO2 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. 

Ten-point DRCs were generated for each drug. Calu-3 cells were seeded at 2.0 × 10 4 cells per well in EMEM, supplemented with 10% FBS and 1X Antibiotic-Antimycotic solution (Gibco) in black, 384-well, μClear plates (Greiner Bio-One), 24 h prior to the experiment. Ten-point DRCs 6 were generated, with compound concentrations ranging from 0.1-50 μM. For viral infection, plates were transferred into the BSL-3 containment facility and SARS-CoV-2 was added at a multiplicity of infection (MOI) of 0.1. The cells were fixed at 24 hpi with 4% PFA and analyzed by immunofluorescence. The acquired images were analyzed using in-house software to quantify cell numbers and infection ratios, and antiviral activity was normalized to positive (mock) and negative (0.5% DMSO) controls in each assay plate. DRCs were generated in Prism7 SARS-CoV-2 infectivity was measured by immunofluorescence of SARS-CoV-2 N protein.

Each point is a mean of duplicate experiments ± standard deviation (SD). IC50, CC50 and selective index (SI) is noted below each graphs. 

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

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

The pathogen resource (NCCP43326) for this study was provided by the National Culture