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.
Identifieur interne : 001214 ( Main/Exploration ); précédent : 001213; suivant : 001215Identification 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.
Auteurs : Mizuki Yamamoto [Japon] ; Shutoku Matsuyama [Japon] ; Xiao Li [République populaire de Chine] ; Makoto Takeda [Japon] ; Yasushi Kawaguchi [Japon] ; Jun-Ichiro Inoue [Japon] ; Zene Matsuda [Japon]Source :
- Antimicrobial agents and chemotherapy [ 1098-6596 ] ; 2016.
Descripteurs français
- KwdFr :
- Cathepsines (génétique), Cathepsines (métabolisme), Cellules HEK293, Coronavirus du syndrome respiratoire du Moyen-Orient (), Coronavirus du syndrome respiratoire du Moyen-Orient (croissance et développement), Coronavirus du syndrome respiratoire du Moyen-Orient (génétique), Dipeptidyl peptidase 4 (génétique), Dipeptidyl peptidase 4 (métabolisme), Furine (génétique), Furine (métabolisme), Fusion cellulaire, Fusion membranaire (), Glycoprotéine de spicule des coronavirus (antagonistes et inhibiteurs), Glycoprotéine de spicule des coronavirus (génétique), Glycoprotéine de spicule des coronavirus (métabolisme), Guanidines (pharmacologie), Gènes rapporteurs, Humains, Inhibiteurs des protéines de fusion virale (pharmacologie), Interactions hôte-pathogène, Luciferases (génétique), Luciferases (métabolisme), Membrane cellulaire (), Membrane cellulaire (métabolisme), Membrane cellulaire (virologie), Membranes intracellulaires (), Membranes intracellulaires (métabolisme), Membranes intracellulaires (virologie), Protéolyse, Régulation de l'expression des gènes, Serine endopeptidases (génétique), Serine endopeptidases (métabolisme), Tests de criblage à haut débit.
- MESH :
- antagonistes et inhibiteurs : Glycoprotéine de spicule des coronavirus.
- croissance et développement : Coronavirus du syndrome respiratoire du Moyen-Orient.
- génétique : Cathepsines, Coronavirus du syndrome respiratoire du Moyen-Orient, Dipeptidyl peptidase 4, Furine, Glycoprotéine de spicule des coronavirus, Luciferases, Serine endopeptidases.
- métabolisme : Cathepsines, Dipeptidyl peptidase 4, Furine, Glycoprotéine de spicule des coronavirus, Luciferases, Membrane cellulaire, Membranes intracellulaires, Serine endopeptidases.
- pharmacologie : Guanidines, Inhibiteurs des protéines de fusion virale.
- virologie : Membrane cellulaire, Membranes intracellulaires.
- Cellules HEK293, Coronavirus du syndrome respiratoire du Moyen-Orient, Fusion cellulaire, Fusion membranaire, Gènes rapporteurs, Humains, Interactions hôte-pathogène, Membrane cellulaire, Membranes intracellulaires, Protéolyse, Régulation de l'expression des gènes, Tests de criblage à haut débit.
English descriptors
- KwdEn :
- Cathepsins (genetics), Cathepsins (metabolism), Cell Fusion, Cell Membrane (drug effects), Cell Membrane (metabolism), Cell Membrane (virology), Dipeptidyl Peptidase 4 (genetics), Dipeptidyl Peptidase 4 (metabolism), Furin (genetics), Furin (metabolism), Gene Expression Regulation, Genes, Reporter, Guanidines (pharmacology), HEK293 Cells, High-Throughput Screening Assays, Host-Pathogen Interactions, Humans, Intracellular Membranes (drug effects), Intracellular Membranes (metabolism), Intracellular Membranes (virology), Luciferases (genetics), Luciferases (metabolism), Membrane Fusion (drug effects), Middle East Respiratory Syndrome Coronavirus (drug effects), Middle East Respiratory Syndrome Coronavirus (genetics), Middle East Respiratory Syndrome Coronavirus (growth & development), Proteolysis, Serine Endopeptidases (genetics), Serine Endopeptidases (metabolism), Spike Glycoprotein, Coronavirus (antagonists & inhibitors), Spike Glycoprotein, Coronavirus (genetics), Spike Glycoprotein, Coronavirus (metabolism), Viral Fusion Protein Inhibitors (pharmacology).
- MESH :
- chemical , antagonists & inhibitors : Spike Glycoprotein, Coronavirus.
- chemical , genetics : Cathepsins, Dipeptidyl Peptidase 4, Furin, Luciferases, Serine Endopeptidases, Spike Glycoprotein, Coronavirus.
- chemical , metabolism : Cathepsins, Dipeptidyl Peptidase 4, Furin, Luciferases, Serine Endopeptidases, Spike Glycoprotein, Coronavirus.
- drug effects : Cell Membrane, Intracellular Membranes, Membrane Fusion, Middle East Respiratory Syndrome Coronavirus.
- genetics : Middle East Respiratory Syndrome Coronavirus.
- growth & development : Middle East Respiratory Syndrome Coronavirus.
- metabolism : Cell Membrane, Intracellular Membranes.
- chemical , pharmacology : Guanidines, Viral Fusion Protein Inhibitors.
- virology : Cell Membrane, Intracellular Membranes.
- Cell Fusion, Gene Expression Regulation, Genes, Reporter, HEK293 Cells, High-Throughput Screening Assays, Host-Pathogen Interactions, Humans, Proteolysis.
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.
DOI: 10.1128/AAC.01043-16
PubMed: 27550352
Affiliations:
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Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay.</title><author><name sortKey="Yamamoto, Mizuki" sort="Yamamoto, Mizuki" uniqKey="Yamamoto M" first="Mizuki" last="Yamamoto">Mizuki Yamamoto</name><affiliation wicri:level="4"><nlm:affiliation>Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo</wicri:regionArea><placeName><settlement type="city">Tokyo</settlement><region type="région">Région de Kantō</region></placeName><orgName type="university">Université de Tokyo</orgName></affiliation></author><author><name sortKey="Matsuyama, Shutoku" sort="Matsuyama, Shutoku" uniqKey="Matsuyama S" first="Shutoku" last="Matsuyama">Shutoku Matsuyama</name><affiliation wicri:level="3"><nlm:affiliation>Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Virology III, National Institute of Infectious Diseases, Tokyo</wicri:regionArea><placeName><settlement type="city">Tokyo</settlement><region type="région">Région de Kantō</region></placeName></affiliation></author><author><name sortKey="Li, Xiao" sort="Li, Xiao" uniqKey="Li X" first="Xiao" last="Li">Xiao Li</name><affiliation wicri:level="3"><nlm:affiliation>Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People's Republic of China.</nlm:affiliation><country xml:lang="fr">République populaire de Chine</country><wicri:regionArea>Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing</wicri:regionArea><placeName><settlement type="city">Pékin</settlement></placeName></affiliation></author><author><name sortKey="Takeda, Makoto" sort="Takeda, Makoto" uniqKey="Takeda M" first="Makoto" last="Takeda">Makoto Takeda</name><affiliation wicri:level="3"><nlm:affiliation>Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan.</nlm:affiliation><country xml:lang="fr">Japon</country><wicri:regionArea>Department of Virology III, National Institute of Infectious Diseases, Tokyo</wicri:regionArea><placeName><settlement type="city">Tokyo</settlement><region type="région">Région de Kantō</region></placeName></affiliation></author><author><name sortKey="Kawaguchi, Yasushi" sort="Kawaguchi, Yasushi" uniqKey="Kawaguchi Y" first="Yasushi" last="Kawaguchi">Yasushi Kawaguchi</name><affiliation wicri:level="4"><nlm:affiliation>Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.</nlm:affiliation><country 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de Kantō</region></placeName><orgName type="university">Université de Tokyo</orgName></affiliation></author><author><name sortKey="Matsuda, Zene" sort="Matsuda, Zene" uniqKey="Matsuda Z" first="Zene" last="Matsuda">Zene Matsuda</name><affiliation wicri:level="4"><nlm: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.</nlm:affiliation><country wicri:rule="url">Japon</country><wicri:regionArea>Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, Tokyo</wicri:regionArea><placeName><settlement type="city">Tokyo</settlement><region type="région">Région de Kantō</region></placeName><orgName type="university">Université de Tokyo</orgName></affiliation></author></analytic><series><title level="j">Antimicrobial agents and chemotherapy</title><idno type="eISSN">1098-6596</idno><imprint><date when="2016" 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(virologie)</term><term>Protéolyse</term><term>Régulation de l'expression des gènes</term><term>Serine endopeptidases (génétique)</term><term>Serine endopeptidases (métabolisme)</term><term>Tests de criblage à haut débit</term></keywords><keywords scheme="MESH" type="chemical" qualifier="antagonists & inhibitors" xml:lang="en"><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cathepsins</term><term>Dipeptidyl Peptidase 4</term><term>Furin</term><term>Luciferases</term><term>Serine Endopeptidases</term><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cathepsins</term><term>Dipeptidyl Peptidase 4</term><term>Furin</term><term>Luciferases</term><term>Serine Endopeptidases</term><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" qualifier="antagonistes et inhibiteurs" 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type="abstract" xml:lang="en">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.</div></front></TEI><affiliations><list><country><li>Japon</li><li>République populaire de Chine</li></country><region><li>Région de Kantō</li></region><settlement><li>Pékin</li><li>Tokyo</li></settlement><orgName><li>Université de Tokyo</li></orgName></list><tree><country name="Japon"><region name="Région de Kantō"><name sortKey="Yamamoto, Mizuki" sort="Yamamoto, Mizuki" uniqKey="Yamamoto M" first="Mizuki" last="Yamamoto">Mizuki Yamamoto</name></region><name sortKey="Inoue, Jun Ichiro" sort="Inoue, Jun Ichiro" uniqKey="Inoue J" first="Jun-Ichiro" last="Inoue">Jun-Ichiro Inoue</name><name sortKey="Kawaguchi, Yasushi" sort="Kawaguchi, Yasushi" uniqKey="Kawaguchi Y" first="Yasushi" last="Kawaguchi">Yasushi Kawaguchi</name><name sortKey="Matsuda, Zene" sort="Matsuda, Zene" uniqKey="Matsuda Z" first="Zene" last="Matsuda">Zene Matsuda</name><name sortKey="Matsuyama, Shutoku" sort="Matsuyama, Shutoku" uniqKey="Matsuyama S" first="Shutoku" last="Matsuyama">Shutoku Matsuyama</name><name sortKey="Takeda, Makoto" sort="Takeda, Makoto" uniqKey="Takeda M" first="Makoto" last="Takeda">Makoto Takeda</name></country><country name="République populaire de Chine"><noRegion><name sortKey="Li, Xiao" sort="Li, Xiao" uniqKey="Li X" first="Xiao" last="Li">Xiao Li</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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