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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CL<sup>Pro</sup> inhibitors</title><author><name sortKey="Kumar, Vathan" sort="Kumar, Vathan" uniqKey="Kumar V" first="Vathan" last="Kumar">Vathan Kumar</name><affiliation><nlm:aff id="aff1">Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Shin, Jin Soo" sort="Shin, Jin Soo" uniqKey="Shin J" first="Jin Soo" last="Shin">Jin Soo Shin</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Shie, Jiun Jie" sort="Shie, Jiun Jie" uniqKey="Shie J" first="Jiun-Jie" last="Shie">Jiun-Jie Shie</name><affiliation><nlm:aff id="aff3">Institute of Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Ku, Keun Bon" sort="Ku, Keun Bon" uniqKey="Ku K" first="Keun Bon" last="Ku">Keun Bon Ku</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Chonsaeng" sort="Kim, Chonsaeng" uniqKey="Kim C" first="Chonsaeng" last="Kim">Chonsaeng Kim</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Go, Yun Young" sort="Go, Yun Young" uniqKey="Go Y" first="Yun Young" last="Go">Yun Young Go</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Huang, Kai Fa" sort="Huang, Kai Fa" uniqKey="Huang K" first="Kai-Fa" last="Huang">Kai-Fa Huang</name><affiliation><nlm:aff id="aff1">Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Kim, Meehyein" sort="Kim, Meehyein" uniqKey="Kim M" first="Meehyein" last="Kim">Meehyein Kim</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Liang, Po Huang" sort="Liang, Po Huang" uniqKey="Liang P" first="Po-Huang" last="Liang">Po-Huang Liang</name><affiliation><nlm:aff id="aff1">Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28216367</idno><idno type="pmc">7113684</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113684</idno><idno type="RBID">PMC:7113684</idno><idno type="doi">10.1016/j.antiviral.2017.02.007</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000A62</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A62</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CL<sup>Pro</sup> inhibitors</title><author><name sortKey="Kumar, Vathan" sort="Kumar, Vathan" uniqKey="Kumar V" first="Vathan" last="Kumar">Vathan Kumar</name><affiliation><nlm:aff id="aff1">Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Shin, Jin Soo" sort="Shin, Jin Soo" uniqKey="Shin J" first="Jin Soo" last="Shin">Jin Soo Shin</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Shie, Jiun Jie" sort="Shie, Jiun Jie" uniqKey="Shie J" first="Jiun-Jie" last="Shie">Jiun-Jie Shie</name><affiliation><nlm:aff id="aff3">Institute of Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Ku, Keun Bon" sort="Ku, Keun Bon" uniqKey="Ku K" first="Keun Bon" last="Ku">Keun Bon Ku</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Chonsaeng" sort="Kim, Chonsaeng" uniqKey="Kim C" first="Chonsaeng" last="Kim">Chonsaeng Kim</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Go, Yun Young" sort="Go, Yun Young" uniqKey="Go Y" first="Yun Young" last="Go">Yun Young Go</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Huang, Kai Fa" sort="Huang, Kai Fa" uniqKey="Huang K" first="Kai-Fa" last="Huang">Kai-Fa Huang</name><affiliation><nlm:aff id="aff1">Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation></author><author><name sortKey="Kim, Meehyein" sort="Kim, Meehyein" uniqKey="Kim M" first="Meehyein" last="Kim">Meehyein Kim</name><affiliation><nlm:aff id="aff2">Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Liang, Po Huang" sort="Liang, Po Huang" uniqKey="Liang P" first="Po-Huang" last="Liang">Po-Huang Liang</name><affiliation><nlm:aff id="aff1">Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan</nlm:aff></affiliation><affiliation><nlm:aff id="aff4">Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan</nlm:aff></affiliation></author></analytic><series><title level="j">Antiviral Research</title><idno type="ISSN">0166-3542</idno><idno type="eISSN">1872-9096</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3C<sup>pro</sup>), 3C-like protease (3CL<sup>pro</sup>) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3C<sup>pro</sup> (<bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold>) inhibited 3CL<sup>pro</sup> of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC<sub>50</sub> values ranging from 1.7 to 4.7 μM and from 0.2 to 0.7 μM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC<sub>50</sub> values ranging from 0.6 to 1.4 μM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and β-CoVs from human and feline origin. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Antiviral Res</journal-id><journal-id journal-id-type="iso-abbrev">Antiviral Res</journal-id><journal-title-group><journal-title>Antiviral Research</journal-title></journal-title-group><issn pub-type="ppub">0166-3542</issn><issn pub-type="epub">1872-9096</issn><publisher><publisher-name>Elsevier B.V.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28216367</article-id><article-id pub-id-type="pmc">7113684</article-id><article-id pub-id-type="publisher-id">S0166-3542(16)30508-3</article-id><article-id pub-id-type="doi">10.1016/j.antiviral.2017.02.007</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CL<sup>Pro</sup> inhibitors</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Kumar</surname><given-names>Vathan</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Shin</surname><given-names>Jin Soo</given-names></name><xref rid="aff2" ref-type="aff">b</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Shie</surname><given-names>Jiun-Jie</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Ku</surname><given-names>Keun Bon</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Kim</surname><given-names>Chonsaeng</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Go</surname><given-names>Yun Young</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author" id="au7"><name><surname>Huang</surname><given-names>Kai-Fa</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author" id="au8"><name><surname>Kim</surname><given-names>Meehyein</given-names></name><email>mkim@krict.re.kr</email><xref rid="aff2" ref-type="aff">b</xref><xref rid="cor2" ref-type="corresp">∗∗</xref></contrib><contrib contrib-type="author" id="au9"><name><surname>Liang</surname><given-names>Po-Huang</given-names></name><email>phliang@gate.sinica.edu.tw</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff4" ref-type="aff">d</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan</aff><aff id="aff2"><label>b</label>Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea</aff><aff id="aff3"><label>c</label>Institute of Chemistry, Academia Sinica, Taipei, Taiwan</aff><aff id="aff4"><label>d</label>Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author. Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. <email>phliang@gate.sinica.edu.tw</email></corresp><corresp id="cor2"><label>∗∗</label>Corresponding author. Center for Virus Research and Testing, Korea Research Institute of Chemical Technology, 141 Gajeongro, Yuseong, Daejeon 34114, Republic of Korea. <email>mkim@krict.re.kr</email></corresp><fn id="fn1"><label>1</label><p id="ntpara0010">These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>17</day><month>2</month><year>2017</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>5</month><year>2017</year></pub-date><pub-date pub-type="epub"><day>17</day><month>2</month><year>2017</year></pub-date><volume>141</volume><fpage>101</fpage><lpage>106</lpage><history><date date-type="received"><day>13</day><month>9</month><year>2016</year></date><date date-type="rev-recd"><day>10</day><month>2</month><year>2017</year></date><date date-type="accepted"><day>14</day><month>2</month><year>2017</year></date></history><permissions><copyright-statement>© 2017 Elsevier B.V. All rights reserved.</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>Elsevier B.V.</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><abstract id="abs0010"><p>Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3C<sup>pro</sup>), 3C-like protease (3CL<sup>pro</sup>) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3C<sup>pro</sup> (<bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold>) inhibited 3CL<sup>pro</sup> of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC<sub>50</sub> values ranging from 1.7 to 4.7 μM and from 0.2 to 0.7 μM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC<sub>50</sub> values ranging from 0.6 to 1.4 μM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and β-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against emerging CoVs and picornaviruses.</p></abstract><abstract abstract-type="graphical" id="abs0015"><title>Graphical abstract</title><p><fig id="undfig1" position="anchor"><alt-text id="alttext0010">Image 1</alt-text><graphic xlink:href="fx1_lrg"></graphic></fig></p></abstract><abstract abstract-type="author-highlights" id="abs0020"><title>Highlights</title><p><list list-type="simple" id="ulist0010"><list-item id="u0010"><label>•</label><p id="p0010">Aldehyde-containing peptidomimetics were identified to be potent inhibitors against MERS-CoV 3CL<sup>pro</sup>.</p></list-item><list-item id="u0015"><label>•</label><p id="p0015">The active inhibitor showed sub-μM EC<sub>50</sub> in killing MERS-CoV.</p></list-item><list-item id="u0020"><label>•</label><p id="p0020">Compounds were also effective against other α and β-CoVs of both human and feline origin.</p></list-item><list-item id="u0025"><label>•</label><p id="p0025">We identified broad-spectrum antiviral agents effective against both coronaviruses and picornaviruses.</p></list-item></list></p></abstract><kwd-group id="kwrds0010"><title>Keywords</title><kwd>MERS-CoV</kwd><kwd>SARS-CoV</kwd><kwd>3C-like protease</kwd><kwd>Peptidomimetic inhibitor</kwd><kwd>Coronavirus</kwd><kwd>Picornavirus</kwd></kwd-group></article-meta></front><body><sec id="sec1"><label>1</label><title>Introduction</title><p id="p0030">Coronaviruses (CoVs) affecting upper respiratory tract were first identified in humans in mid-1960 (<xref rid="bib34" ref-type="bibr">Tyrrell and Bynoe, 1965</xref>). In late 2002, there was emergence of a life threatening CoV of atypical pneumonia, named severe acute respiratory syndrome CoV (SARS-CoV). SARS-CoV belongs to the family <italic>Coronaviridae</italic>, and is an enveloped, positive-stranded RNA virus with ∼30,000 nucleotides (<xref rid="bib26" ref-type="bibr">Rota et al., 2003</xref>). Its genome encodes two polyproteins, pp1a (∼490 kDa) and pp1ab (∼790 kDa) which are processed by 3C-like protease (3CL<sup>pro</sup>) and papain-like protease (PL<sup>pro</sup>) to generate non-structural proteins essential for viral replication (<xref rid="bib30" ref-type="bibr">Thiel et al., 2001</xref>, <xref rid="bib31" ref-type="bibr">Thiel et al., 2003</xref>). Due to its vital role in replication, 3CL<sup>pro</sup> has been regarded as a validated drug target. Many inhibitors of SARS-CoV 3CL<sup>pro</sup> were discovered by high throughput screening and structure-based rational design as summarized in the review articles (<xref rid="bib10" ref-type="bibr">Hilgenfeld and Peiris, 2013</xref>, <xref rid="bib16" ref-type="bibr">Kumar et al., 2013</xref>, <xref rid="bib18" ref-type="bibr">Kuo and Liang, 2015</xref>, <xref rid="bib24" ref-type="bibr">Pillaiyar et al., 2016</xref>, <xref rid="bib25" ref-type="bibr">Ramajayam et al., 2011</xref>, <xref rid="bib33" ref-type="bibr">Tong, 2009</xref>, <xref rid="bib38" ref-type="bibr">Zhao et al., 2013</xref>). After SARS-CoV infection subsided, Middle East respiratory syndrome CoV (MERS-CoV), has emerged in Saudi Arabia in 2012 and spread worldwide, killing 36% of the reported 1826 patients (<ext-link ext-link-type="uri" xlink:href="http://www.who.int/mediacentre/factsheets/mers-cov/en/" id="intref0010">http://www.who.int/mediacentre/factsheets/mers-cov/en/</ext-link>). Due to the similar maturation pathway, MERS-CoV 3CL<sup>pro</sup> is also regarded as a target for developing antiviral drugs (<xref rid="bib32" ref-type="bibr">Tomar et al., 2015</xref>). Though tremendous efforts have been made to develop inhibitors, therapeutic interventions for such continuous CoV outbreaks are yet to reach market (<xref rid="bib4" ref-type="bibr">Barnard and Kumaki, 2011</xref>, <xref rid="bib14" ref-type="bibr">Kilianski and Baker, 2014</xref>).</p><p id="p0035">These CoVs' 3CL<sup>pro</sup> are functionally similar to the 3C<sup>pro</sup> in picornaviruses and both adopt chymotrypsin fold (<xref rid="bib3" ref-type="bibr">Anand et al., 2003</xref>). However, 3CL<sup>pro</sup> is a dimer with Cys-His dyad, whereas 3C<sup>pro</sup> is a monomer with Cys-His-Glu triad (<xref rid="bib11" ref-type="bibr">Hsu et al., 2005</xref>, <xref rid="bib21" ref-type="bibr">Lee et al., 2009</xref>, <xref rid="bib36" ref-type="bibr">Yang et al., 2003</xref>). Picornaviruses are small, non-enveloped RNA virus with genome size of 7500–8000 nucleotides. Based on their genetic organization, the family is composed of 31 genera including <italic>Enterovirus</italic> (enterovirus and rhinovirus), <italic>Aphthovirus</italic> (foot-and-mouth disease virus), <italic>Cardiovirus</italic> (encephalomyocarditis virus), <italic>Hepatovirus</italic> (hepatitis A virus) and others (<ext-link ext-link-type="uri" xlink:href="http://www.picornaviridae.com/" id="intref0015">http://www.picornaviridae.com/</ext-link>). As 3C<sup>pro</sup> is produced in all genera of <italic>Picornaviridae</italic> virus family, its inhibitors showed broad-spectrum, potent antiviral activity against rhinovirus, coxsackievirus and enterovirus (<xref rid="bib13" ref-type="bibr">Jetsadawisut et al., 2016</xref>, <xref rid="bib15" ref-type="bibr">Kim et al., 2015</xref>, <xref rid="bib28" ref-type="bibr">St John et al., 2015</xref>). Though 3C<sup>pro</sup> and 3CL<sup>pro</sup> share similar structures at their active sites, subtle differences often discriminate inhibitors. AG7088, an established 3C<sup>pro</sup> inhibitor, was inactive against SARS-CoV 3CL<sup>pro</sup> prior to the modifications (<xref rid="bib9" ref-type="bibr">Ghosh et al., 2005</xref>, <xref rid="bib27" ref-type="bibr">Shie et al., 2005</xref>, <xref rid="bib29" ref-type="bibr">Thanigaimalai et al., 2013</xref>, <xref rid="bib37" ref-type="bibr">Yang et al., 2006</xref>). Unlike AG7088 which contains α, β-unsaturated ester for forming covalent bond with the active-site Cys, our previously reported potent peptidomimetic inhibitors of 3C<sup>pro</sup> from enterovirus 71 (EV71) contains aldehyde as electrophilic warhead (<xref rid="bib20" ref-type="bibr">Kuo et al., 2008</xref>). In this work, we screened those EV71 3C<sup>pro</sup> inhibitors against MERS-CoV 3CL<sup>pro</sup> and further evaluated the hits by cell-based assays using live MERS-CoV. Our best compounds <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold> inhibited MERS-CoV 3CL<sup>pro</sup> with IC<sub>50</sub> values ranging from 1.7 to 4.7 μM and also suppressed viral replication with EC<sub>50</sub> values between 0.6 and 1.4 μM. These derivatives represent some of few cell-based assay-confirmed anti-MERS-CoV agents and also showed broad-spectrum activity against both α- and β-types of CoVs as described herein.</p></sec><sec id="sec2"><label>2</label><title>Materials and methods</title><sec id="sec2.1"><label>2.1</label><title>Synthesis of compounds</title><p id="p0040">Compounds reported here were synthesized using previously reported procedures with some modifications (<xref rid="bib20" ref-type="bibr">Kuo et al., 2008</xref>). Test compounds and gemcitabine hydrochloride (GEM; Sigma-Aldrich, St. Louis, MO) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at 50 μM concentration.</p></sec><sec id="sec2.2"><label>2.2</label><title>Viruses and cells</title><p id="p0045">Patient-derived isolate MERS-CoV (MERS-CoV/KOR/KNIH/002_05_2015; GenBank accession No. KT029139) was provided by the Korea Center for Disease Control and Prevention. Huh-7 and Vero cells (Cat. No. CCL-81) were obtained from Prof. D.-E. Kim at Konkuk University (Seoul, Republic of Korea) and American Type Culture Collection (ATCC, Manassas, VA), respectively. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) at 37 °C and 5% CO<sub>2</sub>. To minimize adaptive mutation probability of MERS-CoV to another species during passage, MERS-CoV was amplified by infection of a human cell line, Huh-7 cells. The infectious viral titers from culture supernatants at day 2 post-infection (p.i.) were measured by a plaque assay using Vero cells according to other reports (<xref rid="bib5" ref-type="bibr">Chan et al., 2013</xref>, <xref rid="bib6" ref-type="bibr">de Wilde et al., 2013</xref>). MERS-CoV was maintained under biosafety level 3 conditions in Korea Research Institute of Chemical Technology (KRICT).</p><p id="p0050">Human CoV strains, 229E (Cat. No. VR-740) and OC43 (Cat. No. VR-1558) were purchased from ATCC. They were amplified by infecting human fetal lung fibroblast MRC-5 cells (ATCC, Cat. No. CCL-171). Feline infectious peritonitis coronavirus (FIPV) strain (Cat. No. VR-990) and its host cell line Crandall feline kidney (CRFK) (Cat. No. 10094) were obtained from ATCC and Korean Cell Line Bank (Seoul, Republic of Korea), respectively.</p></sec><sec id="sec2.3"><label>2.3</label><title>Expression and purification of SARS- and MERS-CoV 3CL<sup>pro</sup></title><p id="p0055">The expression and purification of SARS-CoV 3CL<sup>pro</sup> followed our reported procedure (<xref rid="bib19" ref-type="bibr">Kuo et al., 2004</xref>). For expression of MERS-CoV 3CL<sup>pro</sup> (<xref rid="bib17" ref-type="bibr">Kumar et al., 2016</xref>), the Factor Xa cleavage site (IEGR) and the 3CL<sup>pro</sup> (accession <ext-link ext-link-type="uri" xlink:href="http://KJ361502.1" id="interref0010">KJ361502.1</ext-link>, Ser3248–Gln3553) DNA sequence was synthesized and cloned into the pET32 expression vector by Mission Biotech. Company (Taiwan) and was transformed into <italic>E. coli</italic> BL21 (DE3). A 10 ml overnight culture of a single transformant was used to inoculate 1L of fresh LB medium containing 100 μg/ml ampicillin. The cells were grown at 37 °C to A<sub>600</sub> = 0.8 and induced with 0.4 mM isopropyl-β-thiogalactopyranoside (IPTG) for 22 h at 16 °C. The cells were harvested by centrifugation at 7000 × <italic>g</italic> for 15 min and the pellet was suspended in lysis buffer (12 mM Tris-HCl, 120 mM NaCl, 0.1 mM EDTA, and 5 mM DTT, pH 7.5). A French-press instrument (Constant Cell Disruption System) was used to disrupt the cells at 20,000 psi and centrifuged at 20,000 × <italic>g</italic> for 1 h to discard the debris. The cell-free extract was loaded onto Ni-NTA column which was equilibrated with lysis buffer containing 5 mM imidazole. After exhaustive washing with lysis buffer, the imidazole concentration of the washing buffer was increased to 30 mM. The protein eluted by lysis buffer containing 300 mM imidazole was dialyzed against lysis buffer to remove imidazole and then Factor Xa was added to a final concentration of 1% (w/w) and incubated at 16 °C for 24 h to remove the His-tag. Subsequently, the processed MERS-CoV 3CL<sup>pro</sup> was passed through a Ni-NTA column for purification. The protein concentration was determined by the protein assay kit (BioRad, USA) and BSA was used as standard.</p></sec><sec id="sec2.4"><label>2.4</label><title>Measurement of IC<sub>50</sub></title><p id="p0060">A fluorometric assay by using the fluorogenic peptide, Dabcyl-KTSAVLQSGFRKME-Edans as previously described (<xref rid="bib19" ref-type="bibr">Kuo et al., 2004</xref>) was used to determine the inhibition constants of compounds. The enhanced fluorescence due to the cleavage of this substrate catalyzed by the 3CL<sup>pro</sup> was monitored at 538 nm with excitation at 355 nm. The IC<sub>50</sub> value of individual sample was measured in a reaction mixture containing 50 nM SARS-CoV 3CL<sup>pro</sup> or 0.3 μM MERS-CoV 3CL<sup>pro</sup> and 10 μM of the fluorogenic substrate in 20 mM Bis-Tris (pH 7.0).</p></sec><sec id="sec2.5"><label>2.5</label><title>Cytopathic effect inhibition assay</title><p id="p0065">Huh-7 cells were seeded in 96-well plates (2 × 10<sup>4</sup> cells per well). On the next day, cells were infected with MERS-CoV at a multiplicity of infection (MOI) of 0.1 in DMEM without FBS for 1 h. After washing with PBS, mock-infected or virus-infected cells were treated with 3-fold serial dilutions of test compounds or GEM used as a positive control. At day 2 p.i., cell lysate was harvested for measuring cell viability using the CellTiter 96<sup>®</sup> AQ<sub>ueous</sub> One Solution Cell Proliferation Assay according to the manufacturer's instructions (Promega, Madison, WI). The 50% cytotoxic concentration (CC<sub>50</sub>) and 50% effective concentration (EC<sub>50</sub>) values were calculated using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). Antiviral assay for other CoVs, including 229E, OC43 and FIPV strains, were performed as mentioned above by using different cell lines. MRC-5 cells were used for culturing human CoVs, 229E and OC43, while CRFK cells for feline CoV, FIPV.</p></sec><sec id="sec2.6"><label>2.6</label><title>Western blot analysis</title><p id="p0070">Huh-7 cells seeded in 6-well plates (3 × 10<sup>5</sup> cells per well) were infected with MERS-CoV at an MOI of 0.02 for 1 h. After washing with PBS, cells were treated with 0.1, 1 and 10 μM of compounds <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold>. In parallel, 0.02% DMSO was treated as a compound vehicle. On day 1 p.i., 30 μg cell lysates suspended in sample loading buffer (Biosesang, Gyeonggi-do, Republic of Korea) were subjected to 10% SDS-PAGE and electro-transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). MERS-CoV NP was detected using a primary antibody specific for viral nucleocapsid protein (NP) (Cat. 100211-RP02; Sino Biological Inc., Beijing, China), followed by a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Thermo Scientific, Waltham, MA). The cellular β-actin protein, a loading control, was detected with an anti-β-actin-specific primary antibody (Cat. No. A1987; Sigma-Aldrich) and the HRP-conjugated goat anti-mouse secondary antibody (Thermo Scientific). After addition of a chemiluminescent HRP substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL), images were obtained using a LAS-4000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan).</p></sec><sec id="sec2.7"><label>2.7</label><title>Plaque inhibition assay</title><p id="p0075">Huh-7 cells were inoculated in 6-well plates at a density of 1 × 10<sup>6</sup> cells per well for 1 day. Culture supernatants treated with 1 μM compounds were harvested at day 1 p.i. They were serially diluted 10-fold in DMEM from 10<sup>−1</sup> to 10<sup>−3</sup> and 1 ml of each sample was used to infect Vero cells for 1 h. After washing with PBS to remove unabsorbed virus, DMEM containing 0.5% agarose (overlay medium) was added. On day 3 p.i., plaques were visualized with 50 μg/ml neutral red (Sigma-Aldrich).</p></sec></sec><sec id="sec3"><label>3</label><title>Results and discussion</title><sec id="sec3.1"><label>3.1</label><title>Inhibition of MERS-CoV 3CL<sup>pro</sup> and viral infection by <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold></title><p id="p0080">Peptidomimetic compounds were synthesized according to our reported method (<xref rid="bib20" ref-type="bibr">Kuo et al., 2008</xref>). Preliminary screening of these peptidomimetic compounds against MERS 3CL<sup>pro</sup> were done at 50 μM. Compounds inhibiting more than half of the protease activity under such condition were selected for further IC<sub>50</sub> measurements. Using enzymatic assay, the compounds <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold> showed IC<sub>50</sub> of 2.4, 4.7 and 1.7 μM against purified 3CL<sup>pro</sup> of MERS-CoV, respectively (<xref rid="tbl1" ref-type="table">Table 1</xref>). These compounds also inhibited SARS 3CL<sup>pro</sup> at lower IC<sub>50</sub> values of 0.7, 0.5 and 0.2 μM, respectively.<table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>Enzymatic and cell-based antiviral assays of selected compounds.</p></caption><alt-text id="alttext0035">Table 1</alt-text><table frame="hsides" rules="groups"><thead><tr><th rowspan="2">Compd</th><th rowspan="2">Ar</th><th rowspan="2">R</th><th colspan="2">IC<sub>50</sub> (μM)<hr></hr></th><th rowspan="2">CC<sub>50</sub> (μM)<xref rid="tbl1fna" ref-type="table-fn">a</xref></th><th rowspan="2">EC<sub>50</sub> (μM) MERS-CoV</th><th rowspan="2">S.I.<xref rid="tbl1fnb" ref-type="table-fn">b</xref></th></tr><tr><th>MERS-CoV 3CL<sup>pro</sup></th><th>SARS –CoV 3CL<sup>pro</sup></th></tr></thead><tbody><tr><td><bold>4a</bold></td><td>3,4-(OCH<sub>2</sub>O)C<sub>6</sub>H<sub>3</sub></td><td>—COOCH<sub>3</sub></td><td>>25</td><td>>25</td><td>ND<xref rid="tbl1fnc" ref-type="table-fn">c</xref></td><td>ND</td><td>ND</td></tr><tr><td><bold>5a</bold></td><td>3,4-(OCH<sub>2</sub>O)C<sub>6</sub>H<sub>3</sub></td><td>—CH<sub>2</sub>OH</td><td>>25</td><td>>25</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td><bold>6a</bold></td><td>3,4-(OCH<sub>2</sub>O)C<sub>6</sub>H<sub>3</sub></td><td>—CHO</td><td>>25</td><td>>25</td><td>>100</td><td>>100</td><td>ND</td></tr><tr><td><bold>6b</bold></td><td>3-BrC<sub>6</sub>H<sub>4</sub></td><td>—CHO</td><td>2.4 ± 0.3</td><td>0.7 ± 0.2</td><td>>100</td><td>1.4 ± 0.0</td><td>>71.4</td></tr><tr><td><bold>6c</bold></td><td>4-Me<sub>2</sub>NC<sub>6</sub>H<sub>4</sub></td><td>—CHO</td><td>4.7 ± 0.6</td><td>0.5 ± 0.1</td><td>>100</td><td>1.2 ± 0.6</td><td>>83.3</td></tr><tr><td><bold>6d</bold></td><td>4-Cl,2-FC<sub>6</sub>H<sub>3</sub></td><td>—CHO</td><td>1.7 ± 0.3</td><td>0.2 ± 0.07</td><td>58.6 ± 1.2</td><td>0.6 ± 0.0</td><td>97.9</td></tr><tr><td>GEM<xref rid="tbl1fnd" ref-type="table-fn">d</xref></td><td>–</td><td>–</td><td>ND</td><td>ND</td><td>>100</td><td>8.3 ± 0.9</td><td>>12.1</td></tr></tbody></table><graphic xlink:href="fx2_lrg"></graphic><table-wrap-foot><fn id="tbl1fna"><label>a</label><p id="ntpara0015">50% cytotoxic concentration in MDCK cells.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fnb"><label>b</label><p id="ntpara0020">Selectivity index.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fnc"><label>c</label><p id="ntpara0025">Not determined.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl1fnd"><label>d</label><p id="ntpara0030">Gemcitabine hydrochloride.</p></fn></table-wrap-foot></table-wrap></p><p id="p0085">To evaluate the ability of these compounds to block viral replication, we performed cytopathic inhibition assay using MERS-CoV-infected Huh-7 cells. As shown in <xref rid="fig1" ref-type="fig">Fig. 1</xref>, compounds <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold> efficiently suppressed viral replication with EC<sub>50</sub> of 1.4, 1.2 and 0.6 μM, respectively (<xref rid="tbl1" ref-type="table">Table 1</xref>). Though it is usual to see EC<sub>50</sub> higher than IC<sub>50</sub>, due to the presence of membrane barrier, we observed EC<sub>50</sub> to be smaller than IC<sub>50</sub>. This could be due to the higher concentration (300 nM) used for <italic>in-vitro</italic> enzymatic assay because it is a weakly associated dimer (<xref rid="bib32" ref-type="bibr">Tomar et al., 2015</xref>). In fact, they inhibited 50 nM SARS 3CL<sup>pro</sup>, a tight dimer, in the submicromolar range (<xref rid="tbl1" ref-type="table">Table 1</xref>). These compounds had CC<sub>50</sub> larger than 100 μM for <bold>6b</bold> and <bold>6c</bold> or 58.6 μM for <bold>6d</bold> against uninfected cells, resulting in selectivity index (S.I.) values larger than 71.4. As expected, compound <bold>6a</bold> which was inactive in the enzyme assay did not suppress viral replication. GEM, used as a positive control according to a previous report (<xref rid="bib7" ref-type="bibr">Dyall et al., 2014</xref>), was less potent in inhibiting MERS-CoV infection with an EC<sub>50</sub> value of 8.3 μM. It also showed marginal toxicity and thus decreased viability of mock cells by 20% or more at the concentrations above 3.7 μM (<xref rid="tbl1" ref-type="table">Table 1</xref> and <xref rid="fig1" ref-type="fig">Fig. 1</xref>).<fig id="fig1"><label>Fig. 1</label><caption><p><bold>Antiviral activity of 6b, 6c and 6d against MERS-CoV in Huh-7 cells.</bold> Huh-7 cells in 96-well plates were mock-infected or infected with MERS-CoV at an MOI of 0.1 for 1 h at 37 °C. After washing with PBS, cells were treated with 3-fold serial dilutions of test compounds (<bold>6a</bold>, <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold>) or a control compound (GEM). On day 2 p.i., cell lysates were harvested for measuring cell viability. The data represent the means ± standard deviations from three independent experiments.</p></caption><alt-text id="alttext0015">Fig. 1</alt-text><graphic xlink:href="gr1_lrg"></graphic></fig></p></sec><sec id="sec3.2"><label>3.2</label><title>Suppression of viral protein production and infectious MERS-CoV generation</title><p id="p0090">To confirm that the observed antiviral activity of compounds <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold> reflects inhibition of MERS-CoV infection, both viral protein and progeny production was measured after treatment of virus-infected cells with these compounds. Western blot analysis showed that viral NP was decreased by these inhibitors in a dose-dependent manner (<xref rid="fig2" ref-type="fig">Fig. 2</xref>). It is noteworthy that no viral NP detectable in the presence of 10 μM 3CL<sup>pro</sup> inhibitors.<fig id="fig2"><label>Fig. 2</label><caption><p><bold>Inhibition of MERS-CoV NP production by 6b, 6c and 6d in a dose-dependent manner.</bold> Huh-7 cells in 6-well plates were infected with MERS-CoV at an MOI of 0.02 for 1 h at 37 °C.The virus-infected cells were treated with increasing concentrations (0.1, 1 and 10 μM) of each compound. Co-treatment of interferon-alpha 2A (IFN; 50 ng/ml) and ribavirin (RBV; 100 μM) was used as a positive control. On day 1 p.i., cells were harvested and loaded to 10% SDS-PAGE (30 μg per well). Immunoblotting was performed using rabbit anti-NP antibody and HRP-conjugated goat anti-rabbit secondary antibody. β-Actin was used as a loading control.</p></caption><alt-text id="alttext0020">Fig. 2</alt-text><graphic xlink:href="gr2_lrg"></graphic></fig></p><p id="p0095">We further compared the number of infectious MERS-CoV particles in the culture supernatants, both in the presence and absence of 3CL<sup>pro</sup> inhibitors. The plaque assay showed that the viral titer in the absence of compound was 4.4 × 10<sup>5</sup> plaque forming units (pfu)/ml, but reduced to 1.7 × 10<sup>4</sup>, 2.9 × 10<sup>4</sup>, and 1.2 × 10<sup>4</sup> pfu/ml by 1 μM of compounds <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold>, respectively (<xref rid="fig3" ref-type="fig">Fig. 3</xref>). Taken together, the results suggest that the compounds originally selected as EV71 3C<sup>pro</sup> blockers efficiently inhibited MERS-CoV 3CL<sup>pro</sup> activity and suppressed viral protein production as well as viral progeny generation. The data also indicate that the inhibitors can penetrate virus-infected cell membrane to reach the active site of 3CL<sup>pro</sup>.<fig id="fig3"><label>Fig. 3</label><caption><p><bold>Downregulation of MERS-CoV progeny generation by 3CL</bold><sup><bold>pro</bold></sup><bold>inhibitors, 6b, 6c and 6d.</bold> MERS-CoV-infected Huh-7 cells in 6-well plates were treated with 1 μM <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold> for 1 day. Culture supernatants were harvested and serially diluted by 10-fold in DMEM (10<sup>−1</sup> to 10<sup>−3</sup>). Fresh Vero cells in 6-well plates were infected with the diluted cell culture inoculum for 1 h. And the number of infectious viral particles was counted by addition of the overlay medium for 3 days and by neutral red staining.</p></caption><alt-text id="alttext0025">Fig. 3</alt-text><graphic xlink:href="gr3_lrg"></graphic></fig></p></sec><sec id="sec3.3"><label>3.3</label><title>In silico molecular docking of <bold>6d</bold> against MERS-CoV 3CL<sup>pro</sup></title><p id="p0100">To rationalize potent inhibition, we docked <bold>6d</bold> into the active site of MERS-CoV 3CL<sup>pro</sup>. The initial pose of the complex was generated based on the X-ray structure (PDB code: <ext-link ext-link-type="uri" xlink:href="pdb:4RSP" id="intref0025">4RSP</ext-link>) of the 3CL<sup>pro</sup> in complex with a covalent inhibitor (<xref rid="bib32" ref-type="bibr">Tomar et al., 2015</xref>) and those of the human rhinovirus 3C<sup>pro</sup> bound with irreversible inhibitors (<xref rid="bib22" ref-type="bibr">Matthews et al., 1999</xref>, <xref rid="bib35" ref-type="bibr">Webber et al., 1998</xref>). Then, the simulation was done by Discovery Studio (Accelrys Inc., San Diego, CA). A Cys at subsite S1 of 3CL<sup>pro</sup> acts as a nucleophile to cleave substrates by attacking carbonyl carbon of the amide bond between the conserved Gln at P1 and the small amino acids such as Ser, Ala or Gly at P1' (<xref rid="bib8" ref-type="bibr">Fan et al., 2004</xref>, <xref rid="bib23" ref-type="bibr">Needle et al., 2015</xref>). Our modelling shows that the γ-sulfur of Cys148 forms a covalent bond with the <bold>6d</bold> aldehyde carbon and the resulting oxyanion is stabilized by His41 (<xref rid="fig4" ref-type="fig">Fig. 4</xref>). A cyclic lactam moiety with <italic>cis</italic>-amide geometry on binding to 3C<sup>pro</sup> was proposed to mimic the P1 Gln of peptide substrates (<xref rid="bib22" ref-type="bibr">Matthews et al., 1999</xref>). Based on our docking results, the P1 lactam moiety of <bold>6d</bold> binds to S1 subsite of 3CL<sup>pro</sup> by forming H-bonds with His166 and Glu169. The P2 phenylalanine moiety prefers to occupy S2 subsite. The cinnamoyl group of <bold>6d</bold> occupies S3 and may be extended to S4. The amide group between phenylalanine and cinnamoyl group further forms H-bonds with Gln192 and Glu169. We also docked <bold>6d</bold> based on the apo-form MERS-CoV 3CL<sup>pro</sup> structure (PDB code: <ext-link ext-link-type="uri" xlink:href="pdb:5c3n" id="intref0030">5c3n</ext-link>) (<xref rid="bib12" ref-type="bibr">Ho et al., 2015</xref>). Since the free-form and ligand-bound structures showed no significant difference in the active site, the binding modes of <bold>6d</bold> in these two structures are indeed very similar (data not shown).<fig id="fig4"><label>Fig. 4</label><caption><p><bold>Docking of inhibitor 6d with MERS-CoV 3CL</bold><sup><bold>pro</bold></sup>. Cys148 of the protease makes a covalent bond with the carbonyl carbon of the inhibitor aldehyde, forming a stable tetrahedral species (the inset), and the resulting oxyanion being stabilized by His 41. The protease is shown in a charge-potential surface. The putative substrate-binding subsites S1′, S1, S2, S3 and S4 are indicated. Moreover, the possible hydrogen bonds of <bold>6d</bold> to the protease are further drawn with dashed lines.</p></caption><alt-text id="alttext0030">Fig. 4</alt-text><graphic xlink:href="gr4_lrg"></graphic></fig></p><p id="p0105">From our experimental as well as modelling results, substituents on cinnamoyl groups of these peptidomimetic inhibitors seem to be critical for the activity. Substituent <italic>p-</italic>chloro in <bold>6d</bold> makes halogen bonding with His194, leading to better potency. Compound <bold>6b</bold> and <bold>6c</bold> with slightly bulkier <italic>m</italic>-bromo and <italic>p</italic>-dimethylamino moiety, respectively, displayed approximately 2-fold drop in IC<sub>50</sub>. Compound <bold>6a</bold> with the bulkiest 3,4-methylenedioxy substituent failed to inhibit the 3CL<sup>pro</sup> even at 25 μM. Compounds <bold>4a</bold> and <bold>5a</bold> that lacked aldehyde warhead failed to inhibit 3CL<sup>pro</sup>, emphasizing the importance of reactive aldehyde electrophile.</p></sec><sec id="sec3.4"><label>3.4</label><title>Broad spectrum activity against human and feline CoVs</title><p id="p0110">To investigate for broad spectrum activity, three compounds were tested against human and feline CoVs. The result showed both α-CoVs, human 229E strain and FIPV, and β-CoV (human OC43 strain) were sensitive to the compounds with EC<sub>50</sub> of 1.1–17.7 μM (<xref rid="tbl2" ref-type="table">Table 2</xref>), suggesting potent and broad-spectrum antiviral activities of these 3CL<sup>pro</sup> inhibitors. The CC<sub>50</sub> values measured using MRC-5 and CRFK cells, used to cultivate human and feline CoVs, respectively, were found to be more than 100 μM. Therefore, the S.I. values were ranged above 5.7 as shown in parentheses.<table-wrap position="float" id="tbl2"><label>Table 2</label><caption><p>Antiviral activity of <bold>6b</bold>, <bold>6c</bold>, and <bold>6d</bold> against 229E, OC43 and FIP.</p></caption><alt-text id="alttext0040">Table 2</alt-text><table frame="hsides" rules="groups"><thead><tr><th rowspan="2">Compd</th><th rowspan="2">CC<sub>50</sub><xref rid="tbl2fna" ref-type="table-fn">a</xref> (μM)</th><th colspan="3">EC<sub>50</sub> (μM)<break></break>(S.I)<hr></hr></th></tr><tr><th>229E<xref rid="tbl2fnb" ref-type="table-fn">b</xref></th><th>OC43<xref rid="tbl2fnc" ref-type="table-fn">c</xref></th><th>FIPV<xref rid="tbl2fnd" ref-type="table-fn">d</xref></th></tr></thead><tbody><tr><td><bold>6b</bold></td><td>>100</td><td>4.3 ± 0.1<break></break>(>25.0)</td><td>13.5 ± 0.8<break></break>(>7.3)</td><td>2.5 ± 1.1<break></break>(>40.0)</td></tr><tr><td><bold>6c</bold></td><td>>100</td><td>4.2 ± 0.3<break></break>(>23.8)</td><td>16.8 ± 0.3<break></break>(>6.0)</td><td>1.9 ± 0.2<break></break>(>52.6)</td></tr><tr><td><bold>6d</bold></td><td>>100</td><td>2.0 ± 0.2<break></break>(>50.0)</td><td>17.7 ± 1.6<break></break>(>5.7)</td><td>1.1 ± 0.3<break></break>(>90.9)</td></tr></tbody></table><table-wrap-foot><fn id="tbl2fna"><label>a</label><p id="ntpara0035">50% cytotoxic concentration in MRC-5 cells and in CRFK cells.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl2fnb"><label>b</label><p id="ntpara0040">Human alpha coronavirus.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl2fnc"><label>c</label><p id="ntpara0045">Human beta coronavirus.</p></fn></table-wrap-foot><table-wrap-foot><fn id="tbl2fnd"><label>d</label><p id="ntpara0050">Feline infectious peritonitis alpha coronavirus.</p></fn></table-wrap-foot></table-wrap></p><p id="p0115">Peptide and peptidomimetic aldehyde inhibitors against 3CL<sup>pro</sup> have been reported (<xref rid="bib2" ref-type="bibr">Akaji et al., 2008</xref>, <xref rid="bib1" ref-type="bibr">Akaji et al., 2011</xref>, <xref rid="bib39" ref-type="bibr">Zhu et al., 2011</xref>) but not tested on live CoVs. However, a potent SARS-CoV 3CL<sup>pro</sup> peptidomimetic aldehyde inhibitor, TG-0205221, has been shown to block SARS-CoV and human CoV 229E replications (<xref rid="bib37" ref-type="bibr">Yang et al., 2006</xref>). As shown in this study, we have identified potent and membrane-permeable MERS-CoV inhibitors <bold>6b</bold>, <bold>6c</bold> and <bold>6d</bold> using live MERS-CoV virus with EC<sub>50</sub> of 0.6–1.2 μM and S.I. over 71.4. These compounds with IC<sub>50</sub> < 0.5 μM against 3C<sup>pro</sup> (<xref rid="bib20" ref-type="bibr">Kuo et al., 2008</xref>) inhibited 3CL<sup>pro</sup> of MERS-CoV with IC<sub>50</sub> of 1.7–4.7 μM and SARS-CoV with IC<sub>50</sub> of 0.2–0.7 μM. Moreover, we found these inhibitors were active against other viruses, including α- and β-CoVs with EC<sub>50</sub> of 1.1–17.7 μM, but were less potent (higher EC<sub>50</sub>) in killing human β-CoV OC43. Although not as potent as inhibiting picornavirus EV71 with the EC<sub>50</sub> of 18 and 7 nM for <bold>6c</bold> and <bold>6d</bold>, respectively (<xref rid="bib20" ref-type="bibr">Kuo et al., 2008</xref>), they are the most potent inhibitors of live MERS-CoV identified so far. Their inhibitory activities against picornaviruses and CoVs make these compounds broad-spectrum antiviral agents. With the escalating cost of drug discovery, development of an antiviral agent with broad-spectrum activities might help in overcoming financial hurdles. More compounds are being synthesized for lead optimization. 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Res.</source><volume>92</volume><year>2011</year><fpage>204</fpage><lpage>212</lpage><pub-id pub-id-type="pmid">21854807</pub-id></element-citation></ref></ref-list><ack id="ack0010"><title>Acknowledgements</title><p>We appreciate Dr. Sung Soon Kim at Korea Centers for Disease Control and Prevention (KCDC) for providing MERS-CoV. This work was supported by Academia Sinica, Taiwan and the grants from <funding-source id="gs1">KRICT</funding-source> (KK1603) and <funding-source id="gs2">KCDC</funding-source> (2015-ER4808-00), Republic of Korea.</p></ack></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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