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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Safe, high-throughput screening of natural compounds of MERS-CoV entry inhibitors using a pseudovirus expressing MERS-CoV spike protein</title><author><name sortKey="Kim, Ji Yeun" sort="Kim, Ji Yeun" uniqKey="Kim J" first="Ji Yeun" last="Kim">Ji Yeun Kim</name><affiliation><nlm:aff id="aff0001">Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Young Il" sort="Kim, Young Il" uniqKey="Kim Y" first="Young Il" last="Kim">Young Il Kim</name><affiliation><nlm:aff id="aff0002">College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju-si, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Park, So Jung" sort="Park, So Jung" uniqKey="Park S" first="So Jung" last="Park">So Jung Park</name><affiliation><nlm:aff id="aff0003">Biomedical Research Center, Asan Institute for Life Sciences, Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, In Ki" sort="Kim, In Ki" uniqKey="Kim I" first="In Ki" last="Kim">In Ki Kim</name><affiliation><nlm:aff id="aff0003">Biomedical Research Center, Asan Institute for Life Sciences, Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Choi, Young Ki" sort="Choi, Young Ki" uniqKey="Choi Y" first="Young Ki" last="Choi">Young Ki Choi</name><affiliation><nlm:aff id="aff0002">College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju-si, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Sung Han" sort="Kim, Sung Han" uniqKey="Kim S" first="Sung-Han" last="Kim">Sung-Han Kim</name><affiliation><nlm:aff id="aff0001">Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">29772395</idno><idno type="pmc">7125825</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7125825</idno><idno type="RBID">PMC:7125825</idno><idno type="doi">10.1016/j.ijantimicag.2018.05.003</idno><date when="2018">2018</date><idno type="wicri:Area/Pmc/Corpus">000868</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000868</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Safe, high-throughput screening of natural compounds of MERS-CoV entry inhibitors using a pseudovirus expressing MERS-CoV spike protein</title><author><name sortKey="Kim, Ji Yeun" sort="Kim, Ji Yeun" uniqKey="Kim J" first="Ji Yeun" last="Kim">Ji Yeun Kim</name><affiliation><nlm:aff id="aff0001">Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Young Il" sort="Kim, Young Il" uniqKey="Kim Y" first="Young Il" last="Kim">Young Il Kim</name><affiliation><nlm:aff id="aff0002">College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju-si, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Park, So Jung" sort="Park, So Jung" uniqKey="Park S" first="So Jung" last="Park">So Jung Park</name><affiliation><nlm:aff id="aff0003">Biomedical Research Center, Asan Institute for Life Sciences, Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, In Ki" sort="Kim, In Ki" uniqKey="Kim I" first="In Ki" last="Kim">In Ki Kim</name><affiliation><nlm:aff id="aff0003">Biomedical Research Center, Asan Institute for Life Sciences, Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Choi, Young Ki" sort="Choi, Young Ki" uniqKey="Choi Y" first="Young Ki" last="Choi">Young Ki Choi</name><affiliation><nlm:aff id="aff0002">College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju-si, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Sung Han" sort="Kim, Sung Han" uniqKey="Kim S" first="Sung-Han" last="Kim">Sung-Han Kim</name><affiliation><nlm:aff id="aff0001">Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea</nlm:aff></affiliation></author></analytic><series><title level="j">International Journal of Antimicrobial Agents</title><idno type="ISSN">0924-8579</idno><idno type="eISSN">1872-7913</idno><imprint><date when="2018">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="De Groot, Rj" uniqKey="De Groot R">RJ De Groot</name></author><author><name sortKey="Baker, Sc" uniqKey="Baker S">SC Baker</name></author><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author><author><name sortKey="Brown, Cs" uniqKey="Brown C">CS Brown</name></author><author><name sortKey="Drosten, C" uniqKey="Drosten C">C Drosten</name></author><author><name sortKey="Enjuanes, L" uniqKey="Enjuanes L">L Enjuanes</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Millet, Jk" uniqKey="Millet J">JK Millet</name></author><author><name sortKey="Whittaker, Gr" uniqKey="Whittaker G">GR Whittaker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Otto, Hh" uniqKey="Otto H">HH Otto</name></author><author><name sortKey="Schirmeister, T" uniqKey="Schirmeister T">T Schirmeister</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Park, J Y" uniqKey="Park J">J-Y Park</name></author><author><name sortKey="Kim, Jh" uniqKey="Kim J">JH Kim</name></author><author><name sortKey="Kim, Ym" uniqKey="Kim Y">YM Kim</name></author><author><name sortKey="Jeong, Hj" uniqKey="Jeong H">HJ Jeong</name></author><author><name sortKey="Kim, Dw" uniqKey="Kim D">DW Kim</name></author><author><name sortKey="Park, Kh" uniqKey="Park K">KH Park</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="letter"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Antimicrob Agents</journal-id><journal-id journal-id-type="iso-abbrev">Int. J. Antimicrob. Agents</journal-id><journal-title-group><journal-title>International Journal of Antimicrobial Agents</journal-title></journal-title-group><issn pub-type="ppub">0924-8579</issn><issn pub-type="epub">1872-7913</issn><publisher><publisher-name>Elsevier B.V. and International Society of Chemotherapy.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">29772395</article-id><article-id pub-id-type="pmc">7125825</article-id><article-id pub-id-type="publisher-id">S0924-8579(18)30134-1</article-id><article-id pub-id-type="doi">10.1016/j.ijantimicag.2018.05.003</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Safe, high-throughput screening of natural compounds of MERS-CoV entry inhibitors using a pseudovirus expressing MERS-CoV spike protein</article-title></title-group><contrib-group><contrib contrib-type="author" id="au0001"><name><surname>Kim</surname><given-names>Ji Yeun</given-names></name><xref rid="aff0001" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au0002"><name><surname>Kim</surname><given-names>Young Il</given-names></name><xref rid="aff0002" ref-type="aff">b</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au0003"><name><surname>Park</surname><given-names>So Jung</given-names></name><xref rid="aff0003" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au0004"><name><surname>Kim</surname><given-names>In Ki</given-names></name><xref rid="aff0003" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au0005"><name><surname>Choi</surname><given-names>Young Ki</given-names></name><email>chiki55@chungbuk.ac.kr</email><xref rid="aff0002" ref-type="aff">b</xref><xref rid="cor0002" ref-type="corresp">⁎⁎</xref></contrib><contrib contrib-type="author" id="au0006"><name><surname>Kim</surname><given-names>Sung-Han</given-names></name><email>kimsunghanmd@hotmail.com</email><xref rid="aff0001" ref-type="aff">a</xref><xref rid="cor0001" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff0001"><label>a</label>Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea</aff><aff id="aff0002"><label>b</label>College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju-si, Republic of Korea</aff><aff id="aff0003"><label>c</label>Biomedical Research Center, Asan Institute for Life Sciences, Seoul, Republic of Korea</aff><author-notes><corresp id="cor0001"><label>⁎</label>Corresponding author: Sung-Han Kim, MD, Department of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Republic of Korea. <email>kimsunghanmd@hotmail.com</email></corresp><corresp id="cor0002"><label>⁎⁎</label>Co-Corresponding author: Young Ki Choi, PhD, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju-si, Republic of Korea. <email>chiki55@chungbuk.ac.kr</email></corresp><fn id="fn1"><label>1</label><p id="notep0001">These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>9</day><month>10</month><year>2018</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>11</month><year>2018</year></pub-date><pub-date pub-type="epub"><day>9</day><month>10</month><year>2018</year></pub-date><volume>52</volume><issue>5</issue><fpage>730</fpage><lpage>732</lpage><history><date date-type="received"><day>22</day><month>11</month><year>2017</year></date><date date-type="rev-recd"><day>30</day><month>4</month><year>2018</year></date><date date-type="accepted"><day>8</day><month>5</month><year>2018</year></date></history><permissions><copyright-statement>© 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.</copyright-statement><copyright-year>2018</copyright-year><copyright-holder>Elsevier Ltd</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions></article-meta></front><body><p content-type="salutation">Sir,</p><p id="para0003">Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the <italic>Coronaviridae</italic> family of enveloped, positive-sense, single-stranded RNA viruses <xref rid="bib0001" ref-type="bibr">[1]</xref>. MERS-CoV is mainly endemic in the Middle East but can spread outside this region, as shown by the 2015 outbreak in South Korea in which there were 38 deaths <xref rid="bib0002" ref-type="bibr">[2]</xref>. MERS-CoV remains a threat, with 250 cases and 77 deaths reported in 2017; however, there are no effective antiviral reagents.</p><p id="para0004">The spike (S) protein of MERS-CoV is the main determinant of virus entry as it plays an important role in both dipeptidyl peptidase 4 receptor binding and virus-cell membrane fusion <xref rid="bib0003" ref-type="bibr">[3]</xref>. Here, we produced a pseudovirus expressing the S protein of MERS-CoV (MERS-PV) and firefly luciferase. Using MERS-PV, we safely screened 502 compounds derived from natural products for their ability to block MERS-CoV entry in a BSL-2 laboratory. We then verified their antiviral activity against authentic MERS-CoV in a BSL-3 laboratory using an isolate from a patient infected with MERS during the 2015 Korean outbreak.</p><p id="para0005">We used the MERS-PV assay to screen for compounds showing ≥50% inhibition to identify those with entry-blocking effects. We then conducted a secondary screen for compounds showing <20% inhibition of pseudotyped vesicular stomatitis virus (VSV-PV) to verify the specificity of the effect. Cell viability assays were performed to exclude cytotoxic compounds and those with >90% viability were retained (Supplementary methods). The effects were reconfirmed at five concentrations (0.25–4 µg/mL). Three compounds, dihydrotanshinone, E-64-C, and E-64-D, met the screening criteria at a low concentration (1 µg/mL; <xref rid="fig0001" ref-type="fig">Fig. 1</xref>).<fig id="fig0001"><label>Fig. 1</label><caption><p><bold>Dose-response confirmation of the three selected natural compounds.</bold> The relative infectivities of MERS-PV (filled circles) and VSV-PV (filled squares) in the presence of the three hit compounds (a) dihydrotanshinone, (b) E-64-C, and (c) E-64-D were dose-dependently evaluated (at 0.25 to 4 µg/mL). The viability of cells (filled triangles) in the presence of the hit compounds at the same concentrations was also determined. The experiment was repeated three times, and data are presented as mean (%) and standard deviation. One-way ANOVA with Tukey's post-test was used to compare the variables. *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ***<italic>P</italic> < 0.001; ns, not significant.</p></caption><alt-text id="alt0001">Fig. 1</alt-text><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="para0006">We then evaluated the inhibitory mechanisms of the hit compounds (at 1 µg/mL) in pre- and post-attachment assays. In the pre-attachment assay, the compounds were incubated with MERS-PV for 2 h, then mixed with host cells. In the post-attachment assay, MERS-PV was mixed with host cells, and the compounds were added 2 h later. While the relative infectivity of MERS-PV treated with hit compounds was approximately 80% in the post-attachment assay, the relative infectivity in the pre-attachment and during attachment assays was significantly less (≤50%; Supplementary <xref rid="fig0001" ref-type="fig">Fig. 1</xref>), which indicates that the hit compounds may block virus entry. However, this assay could not evaluate post-attachment events, such as intracellular processes and viral RNA replication.</p><p id="para0007">The three selected compounds were also evaluated for inhibitory effects on MERS-CoV to assess their potential as antiviral agents in pre- and post-attachment assays in a BSL-3 laboratory at Chungbuk National University, as approved by the Korean Centers for Disease Control and Prevention (KCDC-14-3-07). Although E-64-C did not show a consistent reduction in infection in the pre-attachment assay, dihydrotanshinone and E-64-D showed antiviral activities in the pre-attachment assay (dihydrotanshinone, 6.5 to 5.5 Log TCID<sub>50</sub>/mL at 2 µg/mL; E-64-D, 6.5 to 5.1 Log TCID<sub>50</sub>/mL at 0.5 µg/mL; Supplementary Fig. 2a). E-64-D irreversibly and selectively inhibits cysteine proteases, such as papain, calpain, and cathepsin, by modifying the thiol group to the thioester form <xref rid="bib0004" ref-type="bibr">[4]</xref>. The critical factors for MERS-CoV entry include host cell proteases, such as the pH-dependent endosomal cysteine proteases cathepsin B and L <xref rid="bib0003" ref-type="bibr">[3]</xref>. We showed that E-64-D reduced the titer in the pre-attachment assay, which could be explained by inhibition of viral entry; hence, the antiviral effect might be associated with cathepsin inhibition.</p><p id="para0008">In the post-attachment assay, E-64-C and E-64-D did not show antiviral effects on MERS-CoV at <4 µg/mL. Interestingly, dihydrotanshinone showed a decimal reduction at 0.5 µg/mL, and excellent antiviral effects at ≥2 µg/mL, with a reduction in titer from 6.5 Log to 1.8 Log TCID<sub>50</sub>/mL (Supplementary Fig. 2B). Dihydrotanshinone is a major lipophilic compound extracted from the root of <italic>Salvia miltiorrhiza</italic> Bunge (known as Dansam in Korean) that is commonly used in traditional Asian medicine. Tanshinones derived from <italic>S. miltiorrhiza</italic> showed specific inhibitory activity against SARS-CoV 3CL<sup>pro</sup> and PL<sup>pro</sup> proteases, which are coronavirus cysteine proteases involved in initiation of viral replication <xref rid="bib0005" ref-type="bibr">[5]</xref>. Therefore, the inhibitory effect of dihydrotanshinone in the post-attachment assay could be associated with inhibition of coronavirus replication. This study demonstrated that dihydrotanshinone had inhibitory effects against viral entry in the MERS-CoV and MERS-PV assays. Therefore, dihydrotanshinone may have dual inhibitory effects, blocking virus entry and replication in association with viral proteases. However, further studies, including mechanistic analyses of dihydrotanshinone, are needed.</p><p id="para0009">In conclusion, the results of this study indicate that a high-throughput assay using MERS-PV is useful for screening antiviral compounds against MERS-CoV. Dihydrotanshinone warrants further studies as a candidate for prophylaxis or treatment of MERS-CoV infection.</p></body><back><ref-list id="cebibl1"><title>References</title><ref id="bib0001"><label>1</label><element-citation publication-type="journal" id="sbref0001"><person-group person-group-type="author"><name><surname>De Groot</surname><given-names>RJ</given-names></name><name><surname>Baker</surname><given-names>SC</given-names></name><name><surname>Baric</surname><given-names>RS</given-names></name><name><surname>Brown</surname><given-names>CS</given-names></name><name><surname>Drosten</surname><given-names>C</given-names></name><name><surname>Enjuanes</surname><given-names>L</given-names></name></person-group><article-title>Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group</article-title><source>J Virol</source><volume>87</volume><year>2013</year><fpage>7790</fpage><lpage>7792</lpage><pub-id pub-id-type="pmid">23678167</pub-id></element-citation></ref><ref id="bib0002"><label>2</label><element-citation publication-type="book" id="sbref0002"><person-group person-group-type="author"><collab>Ministry of Health and Welfare</collab></person-group><chapter-title>The 2015 MERS outbreak in the Republic of Korea: Learning from MERS</chapter-title><year>2016</year><publisher-name>Ministry of Health and Welfare and Korea Institute for Health and Social Affairs</publisher-name><publisher-loc>Sejong, Korea</publisher-loc></element-citation></ref><ref id="bib0003"><label>3</label><element-citation publication-type="journal" id="sbref0003"><person-group person-group-type="author"><name><surname>Millet</surname><given-names>JK</given-names></name><name><surname>Whittaker</surname><given-names>GR</given-names></name></person-group><article-title>Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein</article-title><source>Proc Natl Acad Sci USA</source><volume>111</volume><year>2014</year><fpage>15214</fpage><lpage>15219</lpage><pub-id pub-id-type="pmid">25288733</pub-id></element-citation></ref><ref id="bib0004"><label>4</label><element-citation publication-type="journal" id="sbref0004"><person-group person-group-type="author"><name><surname>Otto</surname><given-names>HH</given-names></name><name><surname>Schirmeister</surname><given-names>T</given-names></name></person-group><article-title>Cysteine proteases and their inhibitors</article-title><source>Chem Rev</source><volume>97</volume><year>1997</year><fpage>133</fpage><lpage>172</lpage><pub-id pub-id-type="pmid">11848867</pub-id></element-citation></ref><ref id="bib0005"><label>5</label><element-citation publication-type="journal" id="sbref0005"><person-group person-group-type="author"><name><surname>Park</surname><given-names>J-Y</given-names></name><name><surname>Kim</surname><given-names>JH</given-names></name><name><surname>Kim</surname><given-names>YM</given-names></name><name><surname>Jeong</surname><given-names>HJ</given-names></name><name><surname>Kim</surname><given-names>DW</given-names></name><name><surname>Park</surname><given-names>KH</given-names></name></person-group><article-title>Tanshinones as selective and slow-binding inhibitors for SARS-CoV cysteine proteases</article-title><source>Bioorg Med Chem</source><volume>20</volume><year>2012</year><fpage>5928</fpage><lpage>5935</lpage><pub-id pub-id-type="pmid">22884354</pub-id></element-citation></ref></ref-list><sec id="sec0002" sec-type="supplementary-material"><label>Appendix</label><title>Supplementary materials</title><p id="para0010a"><supplementary-material content-type="local-data" id="ecom0001"><media xlink:href="mmc1.docx"><alt-text>Image, application 1</alt-text></media></supplementary-material></p></sec><ack id="ack0001"><title>Acknowledgement</title><p>We thank the core facility of Laboratory Animal Research at the Convergence Medicine Research Center, Asan Medical Center.</p><sec id="sec0011a"><title>Funding</title><p id="para0011">This study was supported by the Korean Health Technology R&D Project through the Korean Health Industry Development Institute (KHIDI), funded by the <funding-source id="gs0001">Ministry of Health & Welfare, Republic of Korea</funding-source> [grant no. HI16C0272] and the Asan Institute for Life Sciences [2016-462].</p></sec><sec id="sec0011b"><title>Competing Interests</title><p id="para0012">None</p></sec><sec id="sec0011c"><title>Ethical Approval</title><p id="para0013">The study protocol was approved by the Institutional Review Board of Asan Medical Center (IRB 2016-0569).</p></sec></ack><fn-group><fn id="sec0001" fn-type="supplementary-material"><p id="para0002a">Supplementary material associated with this article can be found, in the online version, at doi:<ext-link ext-link-type="uri" xlink:href="https://doi.org/10.1016/j.ijantimicag.2018.05.003" id="interref0001">10.1016/j.ijantimicag.2018.05.003</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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