title: Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay.
abstract: Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.
authors.affiliation: Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Mizuki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Shutoku
authors.initials: S
authors.lastname: Matsuyama
authors.affiliation: Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Takeda
authors.affiliation: Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.|Division of Molecular Virology, Department of Microbiology and Immunology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawaguchi
authors.affiliation: Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan jun-i@ims.u-tokyo.ac.jp zmatsuda@ims.u-tokyo.ac.jp.|Division of Cellular and Molecular Biology, Department of Cancer Biology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Jun-Ichiro
authors.initials: JI
authors.lastname: Inoue
authors.affiliation: Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan jun-i@ims.u-tokyo.ac.jp zmatsuda@ims.u-tokyo.ac.jp.|Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Zene
authors.initials: Z
authors.lastname: Matsuda
mesh_terms: D001550:Benzamidines; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D002459:Cell Fusion; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D018819:Dipeptidyl Peptidase 4 / Q000235:genetics / Q000378:metabolism; D045683:Furin / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006146:Guanidines / Q000494:pharmacology*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D054884:Host-Pathogen Interactions; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects*; D065207:Middle East Respiratory Syndrome Coronavirus / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D064370:Spike Glycoprotein, Coronavirus / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D065147:Viral Fusion Protein Inhibitors / Q000494:pharmacology*
chemical_list: D001550:Benzamidines; D006146:Guanidines; D064370:Spike Glycoprotein, Coronavirus; D065147:Viral Fusion Protein Inhibitors; D008156:Luciferases; D002403:Cathepsins; C042807:DPP4 protein, human; D018819:Dipeptidyl Peptidase 4; D012697:Serine Endopeptidases; C421305:TMPRSS2 protein, human; D045683:Furin; C032855:nafamostat