The anticoagulant nafamostat potently inhibits SARS-CoV-2 infection in vitro : an existing drug with multiple possible therapeutic effects
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Abstract
Although infection by SARS-CoV-2, the causative agent of COVID-19, is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked MERS-CoV S protein-initiated cell fusion by targeting TMPRSS2, and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on SARS-CoV-2 S protein, ACE2 and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an EC 50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. These findings, together with accumulated clinical data regarding its safety, make nafamostat a likely candidate drug to treat COVID-19.
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SciScore for 10.1101/2020.04.22.054981: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The research protocol was approved by the Research Ethics Review Committee of the Institute of Medical Science of the University of Tokyo. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: The cells were incubated at 37 °C with 5% CO2, and regularly tested for mycoplasma contamination by using PCR and were confirmed to be mycoplasma-free. Table 2: Resources
Antibodies Sentences Resources The primary antibodies used were rabbit anti-ACE2 (1:1000, ab15348 Abcam, Cambridge, MA, USA) and anti-GAPDH (1:1000, sc-25778 Santa Cruz Biotechnology, Dallas, TX, USA). anti-AC…SciScore for 10.1101/2020.04.22.054981: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The research protocol was approved by the Research Ethics Review Committee of the Institute of Medical Science of the University of Tokyo. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication Contamination: The cells were incubated at 37 °C with 5% CO2, and regularly tested for mycoplasma contamination by using PCR and were confirmed to be mycoplasma-free. Table 2: Resources
Antibodies Sentences Resources The primary antibodies used were rabbit anti-ACE2 (1:1000, ab15348 Abcam, Cambridge, MA, USA) and anti-GAPDH (1:1000, sc-25778 Santa Cruz Biotechnology, Dallas, TX, USA). anti-ACE2suggested: (Abcam Cat# ab15348, RRID:AB_301861)anti-GAPDHsuggested: (Santa Cruz Biotechnology Cat# sc-25778, RRID:AB_10167668)Experimental Models: Cell Lines Sentences Resources For establishment of stable cell lines expressing the S protein of SARS-CoV-2, or MERS-CoV, recombinant pseudotype lentiviruses were produced using HEK293T cells with lentiviral transfer plasmid expressing S protein, psPAX2 packaging plasmid and vesicular stomatitis virus (VSV)-G-expressing plasmid. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Calu-3 cells (ATCC HTB-55) and H3255 cells (CVCL_6831), lung epithelial cell-derived immortalized cells established from human lung cancer, were used as target cells for the fusion and viral infection assays. H3255suggested: NCI-DTP Cat# NCI-H3255, RRID:CVCL_6831)Pooled Calu-3 cells infected with pseudotype viruses were selected with 1 μg/ml puromycin. Calu-3suggested: KCLB Cat# 30055, RRID:CVCL_0609)DSP assay to monitor membrane fusion: For the DSP assay using 293FT cells, effector cells expressing S protein with DSP8-11 and target cells expressing CD26 or ACE2, and TMPRSS2 with DSP1-7 were seeded in 12-well cell culture plates (2 x 105 cells/500 μl) one day before the assay. 293FTsuggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)The swabs were submitted to the Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, the University of Tokyo for virus isolation by inoculating with VeroE6 cells. VeroE6suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)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: No clinical trial numbers were referenced.
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.
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