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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-Type-Dependent Manner.
Viruses. 2020 06 10; 12(6)V

Abstract

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (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 Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (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 effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

Authors+Show Affiliations

Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Department of Virology 3, National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan.Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo 162-8655, Japan.Disease Control and Prevention Center, National Center for Global Health and Medicine, Tokyo 162-8655, Japan.Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53711, USA. Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Senior Professor Office, University of Tokyo, Tokyo 113-0033, Japan.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

32532094

Citation

Yamamoto, Mizuki, et al. "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-Type-Dependent Manner." Viruses, vol. 12, no. 6, 2020.
Yamamoto M, Kiso M, Sakai-Tagawa Y, et al. 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-Type-Dependent Manner. Viruses. 2020;12(6).
Yamamoto, M., Kiso, M., Sakai-Tagawa, Y., Iwatsuki-Horimoto, K., Imai, M., Takeda, M., Kinoshita, N., Ohmagari, N., Gohda, J., Semba, K., Matsuda, Z., Kawaguchi, Y., Kawaoka, Y., & Inoue, J. I. (2020). 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-Type-Dependent Manner. Viruses, 12(6). https://doi.org/10.3390/v12060629
Yamamoto M, et al. 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-Type-Dependent Manner. Viruses. 2020 06 10;12(6) PubMed PMID: 32532094.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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-Type-Dependent Manner. AU - Yamamoto,Mizuki, AU - Kiso,Maki, AU - Sakai-Tagawa,Yuko, AU - Iwatsuki-Horimoto,Kiyoko, AU - Imai,Masaki, AU - Takeda,Makoto, AU - Kinoshita,Noriko, AU - Ohmagari,Norio, AU - Gohda,Jin, AU - Semba,Kentaro, AU - Matsuda,Zene, AU - Kawaguchi,Yasushi, AU - Kawaoka,Yoshihiro, AU - Inoue,Jun-Ichiro, Y1 - 2020/06/10/ PY - 2020/04/29/received PY - 2020/06/08/revised PY - 2020/06/09/accepted PY - 2020/6/14/entrez PY - 2020/6/14/pubmed PY - 2020/7/1/medline KW - SARS-CoV-2 KW - TMPRSS2 KW - fusion inhibitor JF - Viruses JO - Viruses VL - 12 IS - 6 N2 - Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (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 Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (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 effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19. SN - 1999-4915 UR - https://www.unboundmedicine.com/medline/citation/32532094/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_Type_Dependent_Manner_ L2 - https://www.mdpi.com/resolver?pii=v12060629 DB - PRIME DP - Unbound Medicine ER -