Individual and common inhibitors of coronavirus and picornavirus main proteases
Identifieur interne : 000877 ( Pmc/Corpus ); précédent : 000876; suivant : 000878Individual and common inhibitors of coronavirus and picornavirus main proteases
Auteurs : Chih-Jung Kuo ; Hun-Ge Liu ; Yueh-Kuei Lo ; Churl-Min Seong ; Kee-In Lee ; Young-Sik Jung ; Po-Huang LiangSource :
- Febs Letters [ 0014-5793 ] ; 2009.
Abstract
Picornaviruses (PV) and coronaviruses (CoV) are positive‐stranded RNA viruses which infect millions of people worldwide each year, resulting in a wide range of clinical outcomes. As reported in this study, using high throughput screening against ∼6800 small molecules, we have identified several novel inhibitors of SARS‐CoV 3CLpro with IC50 of low μM. Interestingly, one of them equally inhibited both 3Cpro and 3CLpro from PV and CoV, respectively. Using computer modeling, the structural features of these compounds as individual and common protease inhibitors were elucidated to enhance our knowledge for developing anti‐viral agents against PV and CoV.
Url:
DOI: 10.1016/j.febslet.2008.12.059
PubMed: 19166843
PubMed Central: 7094298
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PMC:7094298Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Individual and common inhibitors of coronavirus and picornavirus main proteases</title><author><name sortKey="Kuo, Chih Jung" sort="Kuo, Chih Jung" uniqKey="Kuo C" first="Chih-Jung" last="Kuo">Chih-Jung Kuo</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff2"></nlm:aff></affiliation></author><author><name sortKey="Liu, Hun Ge" sort="Liu, Hun Ge" uniqKey="Liu H" first="Hun-Ge" last="Liu">Hun-Ge Liu</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation></author><author><name sortKey="Lo, Yueh Kuei" sort="Lo, Yueh Kuei" uniqKey="Lo Y" first="Yueh-Kuei" last="Lo">Yueh-Kuei Lo</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation></author><author><name sortKey="Seong, Churl Min" sort="Seong, Churl Min" uniqKey="Seong C" first="Churl-Min" last="Seong">Churl-Min Seong</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff3"></nlm:aff></affiliation></author><author><name sortKey="Lee, Kee In" sort="Lee, Kee In" uniqKey="Lee K" first="Kee-In" last="Lee">Kee-In Lee</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff3"></nlm:aff></affiliation></author><author><name sortKey="Jung, Young Sik" sort="Jung, Young Sik" uniqKey="Jung Y" first="Young-Sik" last="Jung">Young-Sik Jung</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff3"></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="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff2"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19166843</idno><idno type="pmc">7094298</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094298</idno><idno type="RBID">PMC:7094298</idno><idno type="doi">10.1016/j.febslet.2008.12.059</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">000877</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000877</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Individual and common inhibitors of coronavirus and picornavirus main proteases</title><author><name sortKey="Kuo, Chih Jung" sort="Kuo, Chih Jung" uniqKey="Kuo C" first="Chih-Jung" last="Kuo">Chih-Jung Kuo</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff2"></nlm:aff></affiliation></author><author><name sortKey="Liu, Hun Ge" sort="Liu, Hun Ge" uniqKey="Liu H" first="Hun-Ge" last="Liu">Hun-Ge Liu</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation></author><author><name sortKey="Lo, Yueh Kuei" sort="Lo, Yueh Kuei" uniqKey="Lo Y" first="Yueh-Kuei" last="Lo">Yueh-Kuei Lo</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation></author><author><name sortKey="Seong, Churl Min" sort="Seong, Churl Min" uniqKey="Seong C" first="Churl-Min" last="Seong">Churl-Min Seong</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff3"></nlm:aff></affiliation></author><author><name sortKey="Lee, Kee In" sort="Lee, Kee In" uniqKey="Lee K" first="Kee-In" last="Lee">Kee-In Lee</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff3"></nlm:aff></affiliation></author><author><name sortKey="Jung, Young Sik" sort="Jung, Young Sik" uniqKey="Jung Y" first="Young-Sik" last="Jung">Young-Sik Jung</name><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff3"></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="feb2s0014579309000167-aff-aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="feb2s0014579309000167-aff-aff2"></nlm:aff></affiliation></author></analytic><series><title level="j">Febs Letters</title><idno type="ISSN">0014-5793</idno><idno type="eISSN">1873-3468</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Picornaviruses (PV) and coronaviruses (CoV) are positive‐stranded RNA viruses which infect millions of people worldwide each year, resulting in a wide range of clinical outcomes. As reported in this study, using high throughput screening against ∼6800 small molecules, we have identified several novel inhibitors of SARS‐CoV 3CL<sup>pro</sup> with IC<sub>50</sub> of low μM. Interestingly, one of them equally inhibited both 3C<sup>pro</sup> and 3CL<sup>pro</sup> from PV and CoV, respectively. Using computer modeling, the structural features of these compounds as individual and common protease inhibitors were elucidated to enhance our knowledge for developing anti‐viral agents against PV and CoV.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="brief-report"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">FEBS Lett</journal-id><journal-id journal-id-type="iso-abbrev">FEBS Lett</journal-id><journal-id journal-id-type="doi">10.1002/(ISSN)1873-3468</journal-id><journal-id journal-id-type="publisher-id">FEB2</journal-id><journal-title-group><journal-title>Febs Letters</journal-title></journal-title-group><issn pub-type="ppub">0014-5793</issn><issn pub-type="epub">1873-3468</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19166843</article-id><article-id pub-id-type="pmc">7094298</article-id><article-id pub-id-type="doi">10.1016/j.febslet.2008.12.059</article-id><article-id pub-id-type="publisher-id">FEB2S0014579309000167</article-id><article-categories><subj-group subj-group-type="overline"><subject>Short Communication</subject></subj-group><subj-group subj-group-type="heading"><subject>Short Communications</subject></subj-group></article-categories><title-group><article-title>Individual and common inhibitors of coronavirus and picornavirus main proteases</article-title></title-group><contrib-group><contrib id="feb2s0014579309000167-cr-0001" contrib-type="author"><name><surname>Kuo</surname><given-names>Chih-Jung</given-names></name><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff1"><sup>1</sup></xref><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff2"><sup>2</sup></xref></contrib><contrib id="feb2s0014579309000167-cr-0002" contrib-type="author"><name><surname>Liu</surname><given-names>Hun-Ge</given-names></name><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff1"><sup>1</sup></xref></contrib><contrib id="feb2s0014579309000167-cr-0003" contrib-type="author"><name><surname>Lo</surname><given-names>Yueh-Kuei</given-names></name><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff1"><sup>1</sup></xref></contrib><contrib id="feb2s0014579309000167-cr-0004" contrib-type="author"><name><surname>Seong</surname><given-names>Churl-Min</given-names></name><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff3"><sup>3</sup></xref></contrib><contrib id="feb2s0014579309000167-cr-0005" contrib-type="author"><name><surname>Lee</surname><given-names>Kee-In</given-names></name><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff3"><sup>3</sup></xref></contrib><contrib id="feb2s0014579309000167-cr-0006" contrib-type="author" corresp="yes"><name><surname>Jung</surname><given-names>Young-Sik</given-names></name><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff3"><sup>3</sup></xref><address><email>ysjung@krict.re.kr</email></address></contrib><contrib id="feb2s0014579309000167-cr-0007" contrib-type="author" corresp="yes"><name><surname>Liang</surname><given-names>Po-Huang</given-names></name><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff1"><sup>1</sup></xref><xref ref-type="aff" rid="feb2s0014579309000167-aff-aff2"><sup>2</sup></xref><address><email>phliang@gate.sinica.edu.tw</email></address></contrib></contrib-group><aff id="feb2s0014579309000167-aff-aff1"><label><sup>1</sup></label>Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, ROC</aff><aff id="feb2s0014579309000167-aff-aff2"><label><sup>2</sup></label>Taiwan International Graduate Program, Academia Sinica, Taipei 11529, Taiwan</aff><aff id="feb2s0014579309000167-aff-aff3"><label><sup>3</sup></label>Korea Research Institute of Chemical Technology, Daejeon 305-606, Republic of Korea</aff><author-notes><corresp id="correspondenceTo"><label>*</label>Corresponding authors. Address: Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, ROC. Fax: +886 2 2788 9759.</corresp></author-notes><pub-date pub-type="ppub"><day>04</day><month>2</month><year>2009</year></pub-date><pub-date pub-type="epub"><day>21</day><month>1</month><year>2009</year></pub-date><volume>583</volume><issue>3</issue><issue-id pub-id-type="doi">10.1002/feb2.2009.583.issue-3</issue-id><fpage>549</fpage><lpage>555</lpage><history><date date-type="received"><day>05</day><month>11</month><year>2008</year></date><date date-type="rev-recd"><day>17</day><month>12</month><year>2008</year></date><date date-type="accepted"><day>23</day><month>12</month><year>2008</year></date></history><permissions><pmc-comment> © 2015 Federation of European Biochemical Societies </pmc-comment>
<copyright-statement content-type="article-copyright">FEBS Letters 583 (2009) 1873-3468 © 2015 Federation of European Biochemical Societies</copyright-statement><license><license-p>This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="file:FEB2-583-549.pdf"></self-uri><abstract><p>Picornaviruses (PV) and coronaviruses (CoV) are positive‐stranded RNA viruses which infect millions of people worldwide each year, resulting in a wide range of clinical outcomes. As reported in this study, using high throughput screening against ∼6800 small molecules, we have identified several novel inhibitors of SARS‐CoV 3CL<sup>pro</sup> with IC<sub>50</sub> of low μM. Interestingly, one of them equally inhibited both 3C<sup>pro</sup> and 3CL<sup>pro</sup> from PV and CoV, respectively. Using computer modeling, the structural features of these compounds as individual and common protease inhibitors were elucidated to enhance our knowledge for developing anti‐viral agents against PV and CoV.</p></abstract><kwd-group><kwd id="feb2s0014579309000167-kwd-0001">Coronavirus</kwd><kwd id="feb2s0014579309000167-kwd-0002">Picornavirus</kwd><kwd id="feb2s0014579309000167-kwd-0003">3C protease</kwd><kwd id="feb2s0014579309000167-kwd-0004">Fluorescence assay</kwd><kwd id="feb2s0014579309000167-kwd-0005">High throughput screening</kwd><kwd id="feb2s0014579309000167-kwd-0006">Computer modeling</kwd></kwd-group><counts><fig-count count="4"></fig-count><table-count count="2"></table-count><ref-count count="21"></ref-count><page-count count="7"></page-count><word-count count="3418"></word-count></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>February 04, 2009</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.0 mode:remove_FC converted:15.04.2020</meta-value></custom-meta></custom-meta-group></article-meta><notes><p content-type="self-citation"><mixed-citation publication-type="journal" id="feb2s0014579309000167-cit-0000"><string-name><surname>Kuo</surname><given-names>Chih-Jung</given-names></string-name>,<string-name><surname>Liu</surname><given-names>Hun-Ge</given-names></string-name>,<string-name><surname>Lo</surname><given-names>Yueh-Kuei</given-names></string-name>,<string-name><surname>Seong</surname><given-names>Churl-Min</given-names></string-name>,<string-name><surname>Lee</surname><given-names>Kee-In</given-names></string-name>,<string-name><surname>Jung</surname><given-names>Young-Sik</given-names></string-name> and <string-name><surname>Liang</surname><given-names>Po-Huang</given-names></string-name>(<year>2009</year>), <article-title>Individual and common inhibitors of coronavirus and picornavirus main proteases</article-title>, <source xml:lang="en">FEBS Letters</source>, <volume>583</volume>, doi: 10.1016/j.febslet.2008.12.059</mixed-citation></p></notes></front><body id="feb2s0014579309000167-body-0001"><sec id="feb2s0014579309000167-sec-1"><label>1</label><title>Introduction</title><p>Picornaviruses (PV) are small nonenveloped RNA viruses with a single strand of genomic RNA of 7500–8000 nucleotides <xref rid="feb2s0014579309000167-bib-bib1" ref-type="ref">[1]</xref>. The members of PV include rhinoviruses (RV), enteroviruses (EV), coxsackieviruses (CV), polioviruses, echoviruses, encephalomyocarditis viruses, meningitis virus, foot and mouth viruses, hepatitis A virus, and so on. Among them, RV is the major cause of the common cold, whereas EV and CV infection can cause hand, foot, and mouth diseases in human and animals. In severe cases, EV can damage the central nervous systems leading to viral meningitis, encephalitis, and severe myocarditis, as well as fatal pulmonary edema [<xref rid="feb2s0014579309000167-bib-bib2" ref-type="ref">2</xref>, <xref rid="feb2s0014579309000167-bib-bib3" ref-type="ref">3</xref>, <xref rid="feb2s0014579309000167-bib-bib4" ref-type="ref">4</xref>, <xref rid="feb2s0014579309000167-bib-bib5" ref-type="ref">5</xref>]. CV strain B is a major human pathogen that causes meningitis and mycocarditis leading to heart failure in young adults and congestive heart failure <xref rid="feb2s0014579309000167-bib-bib6" ref-type="ref">[6]</xref>. In these PV, a chymotrypsin‐like protease (named 3C<sup>pro</sup>) is required to process polyproteins into mature proteins for viral replication, which represents a promising anti‐viral drug target <xref rid="feb2s0014579309000167-bib-bib7" ref-type="ref">[7]</xref>.</p><p>On the other hand, coronaviruses (CoV) are the positive‐stranded RNA viruses with larger genome of 27–32 kb, which typically cause respiratory and enteric diseases, pneumonia, exacerbation of asthma, neurological symptoms, and myocarditis in humans and domestic animals. An outbreak of severe acute respiratory syndrome (SARS), caused by a novel human CoV, was spread from China to 29 countries in 2003, infecting a total of ∼8000 people and killing ∼800 patients <xref rid="feb2s0014579309000167-bib-bib8" ref-type="ref">[8]</xref>. SARS‐CoV contains a 3C‐like protease (3CL<sup>pro</sup>) analogous to the 3C<sup>pro</sup> of PV, responsible for processing two overlapping polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Other members of human CoV including CoV‐229E, CoV‐OC43, CoV‐HKU1, and CoV‐NL63 also require a 3CL<sup>pro</sup> in the maturation of viral proteins.</p><p>Several inhibitors have been developed to inhibit the 3C<sup>pro</sup> of RV and EV [<xref rid="feb2s0014579309000167-bib-bib9" ref-type="ref">9</xref>, <xref rid="feb2s0014579309000167-bib-bib10" ref-type="ref">10</xref>, <xref rid="feb2s0014579309000167-bib-bib11" ref-type="ref">11</xref>, <xref rid="feb2s0014579309000167-bib-bib12" ref-type="ref">12</xref>] and 3CL<sup>pro</sup> of SARS‐CoV [<xref rid="feb2s0014579309000167-bib-bib13" ref-type="ref">13</xref>, <xref rid="feb2s0014579309000167-bib-bib14" ref-type="ref">14</xref>, <xref rid="feb2s0014579309000167-bib-bib15" ref-type="ref">15</xref>]. However, their inhibitors can not be mutually used without modification. For example, AG7088, a potent inhibitor of RV and other picornaviral 3C<sup>pro</sup><xref rid="feb2s0014579309000167-bib-bib16" ref-type="ref">[16]</xref>, failed to inhibit SARS‐CoV 3CL<sup>pro</sup><xref rid="feb2s0014579309000167-bib-bib17" ref-type="ref">[17]</xref>. Unlike the 3CL<sup>pro</sup>, which is dimeric and in which each subunit is composed of three domains, the 3C<sup>pro</sup> is a monomer with only the two catalytic domains. The structure‐based sequence alignment (<xref rid="feb2s0014579309000167-fig1" ref-type="fig">Fig. 1</xref>) shows some sequence differences, which may alter inhibitor specificity. In this study, we performed high throughput screening using a library of ∼6800 compounds to find five novel inhibitors of the SARS‐CoV 3CL<sup>pro</sup>, 4 of which also inhibited another human CoV‐229E 3CL<sup>pro</sup>, but did not inhibit the 3C<sup>pro</sup> from RV14, CVB3, and EV71. But, one compound was found to almost equally inhibit these 3CL<sup>pro</sup> and 3C<sup>pro</sup>. From computer modeling, we rationalized the binding discrepancy of the inhibitors against these proteases. The information is useful to further develop more potent individual or common inhibitors of 3C<sup>pro</sup> and 3CL<sup>pro</sup> of PV and CoV for anti‐viral drug discovery.</p><fig fig-type="Figure" xml:lang="en" id="feb2s0014579309000167-fig1" orientation="portrait" position="float"><label>Figure 1</label><caption><p>A structure‐based sequence alignment of SARS‐CoV 3CL<sup>pro</sup>, CoV‐229E 3CL<sup>pro</sup>, CVB3 3C<sup>pro</sup>, EV71 3C<sup>pro</sup>, and RV14 3C<sup>pro</sup>. The domains according to 3CL<sup>pro</sup> are shown above the sequence and the secondary elements according to the 3C<sup>pro</sup> structure are shown below it. Arrows indicate the essential catalytic amino acids His and Cys for 3CL<sup>pro</sup> and 3C<sup>pro</sup>, and Glu (only for 3C<sup>pro</sup>).</p></caption><graphic id="nlm-graphic-1" xlink:href="FEB2-583-549-g001"><alt-text>figure image</alt-text></graphic></fig></sec><sec id="feb2s0014579309000167-sec-2"><label>2</label><title>Methods</title><sec id="feb2s0014579309000167-sec-2.1"><label>2.1</label><title>Expression and purification of the proteases</title><p>Two types of proteases including 3CL<sup>pro</sup> from SARS‐CoV and CoV‐229E and 3C<sup>pro</sup> from CVB3, EV71, and RV14 were used to assay the inhibitors in this study. The SARS‐CoV 3CL<sup>pro</sup> and EV71 3C<sup>pro</sup> were prepared as reported previously [<xref rid="feb2s0014579309000167-bib-bib12" ref-type="ref">12</xref>, <xref rid="feb2s0014579309000167-bib-bib18" ref-type="ref">18</xref>]. For expressing CVB3, RV14, and CoV‐229E proteases, the genes were cloned from viral cDNAs by using polymerase chain reaction (PCR) as reported elsewhere.</p></sec><sec id="feb2s0014579309000167-sec-2.2"><label>2.2</label><title>Primary screening</title><p>For screening, 0.05 μM SARS 3CL<sup>pro</sup>, 6 μM fluorogenic substrate Dabcyl‐KTSAVLQSGFRKME‐Edans, and 50 μM of approximately 6800 compounds provided by Korea Chemical Bank (Daejeon, Korea) were used. Enhanced fluorescence of the reactions in the buffer of 20 mM Bis‐Tris at pH 7.0 was monitored at 538 nm with excitation at 355 nm using a fluorescence plate reader. The compounds which inhibited more than 50% of the protease activity at 50 μM were selected for the next assay run at 10 μM.</p></sec><sec id="feb2s0014579309000167-sec-2.3"><label>2.3</label><title>IC<sub>50</sub> determination</title><p>The five hits that inhibited SARS‐CoV 3CL<sup>pro</sup> at 10 μM were also evaluated against CoV‐229E 3CL<sup>pro</sup>, EV71 3C<sup>pro</sup>, CVB3 3C<sup>pro</sup>, and RV14 3C<sup>pro</sup>. In the assay solution, the activities of these proteases (0.5 μM) with 10 μM fluorogenic substrate in the buffers of 10 mM MES at pH 6.5 and 6.0 (the optimal pH for EV71 and RV14 proteases, respectively) and 10 mM HEPES at pH 7.5 (for CoV‐229E and CVB3 proteases) were measured in the presence of various concentrations of the inhibitors to obtain the IC<sub>50</sub> values.</p></sec><sec id="feb2s0014579309000167-sec-2.4"><label>2.4</label><title>Computer modeling of the inhibitors binding with the proteases</title><p>For the modeling analysis, we used the crystal structure of SARS 3CL<sup>pro</sup> in complex with a peptide inhibitor (PDB code 1UK4) <xref rid="feb2s0014579309000167-bib-bib19" ref-type="ref">[19]</xref>, the structures of CoV‐229E 3CL<sup>pro</sup> and CVB3 3C<sup>pro</sup> solved by us, and the structural model of EV71 3C<sup>pro</sup> constructed from the structure of RV 3C<sup>pro</sup> (PDB code 1CQQ) <xref rid="feb2s0014579309000167-bib-bib20" ref-type="ref">[20]</xref>. Docking process was performed using an automated ligand‐docking subprogram of the Discovery Studio Modeling 1.2 SBD (Accelrys Inc., San Diego, CA), with a set of parameters chosen to control the precise operation of the genetic algorithm. Docking runs were carried out using standard default settings “grid resolution” of 5 Å, “site opening” of 12 Å, and “binding site” selected for defining the active site cavity.</p></sec></sec><sec id="feb2s0014579309000167-sec-3"><label>3</label><title>Results</title><sec id="feb2s0014579309000167-sec-3.1"><label>3.1</label><title>Screening of the protease inhibitors</title><p>We first screened against a library of ∼6800 compounds for inhibiting SARS‐CoV 3CL<sup>pro</sup>. From the primary screening, there were 66 compounds which showed more than 50% inhibition of the enzyme activity at 50 μM. We further tested their inhibitory activities at 10 μM and five of them (21155, 22723, 27548, 43146, and 48511) showed IC<sub>50</sub> values smaller than 10 μM. According to their dose–response curves as shown in <xref rid="feb2s0014579309000167-fig2" ref-type="fig">Fig. 2A–E</xref>, the five hits 21155, 22723, 27548, 43146, and 48511 displayed IC<sub>50</sub> values of 7.2 ± 0.7, 10.6 ± 1.3, 7.0 ± 0.8, 3.3 ± 0.2, and 8.1 ± 0.9 μM, respectively, against the SARS 3CL<sup>pro</sup>. Similar inhibition results were observed for 3CL<sup>pro</sup> of CoV‐229E (data summarized in <xref rid="feb2s0014579309000167-tbl1" ref-type="table">Table 1</xref>), but not for 3C<sup>pro</sup>. However, 43146 inhibited both 3C<sup>pro</sup> and 3CL<sup>pro</sup> with IC<sub>50</sub> values of 10.3 ± 1.1 μM, 5.4 ± 0.2 μM, 3.3 ± 0.3, and 5.2 ± 0.6 μM, respectively, against CoV‐229E 3CL<sup>pro</sup>, CVB3 3C<sup>pro</sup>, EV71 3C<sup>pro</sup> and RV14 3C<sup>pro</sup> (<xref rid="feb2s0014579309000167-fig3" ref-type="fig">Fig. 3A–D</xref>and summarized in <xref rid="feb2s0014579309000167-tbl1" ref-type="table">Table 1</xref>). This compound contains a dihydropyrazole ring with three substituents, two phenyl groups and a lengthy N‐butyl‐benzimidazolylamino‐toluene.</p><fig fig-type="Figure" xml:lang="en" id="feb2s0014579309000167-fig2" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Dose–response curves for the five hits against SARS‐CoV 3CL<sup>pro</sup> from the screening. IC<sub>50</sub> values were determined from the curves using equation 1. These were (A) 7.2 ± 0.7 μM (21155), (B) 10.6 ± 1.3 μM (22723), (C) 7.0 ± 0.8 μM (27548), (D) 3.3 ± 0.2 μM (48511), and (E) 8.1 ± 0.9 μM (43146). The structures and activities of these inhibitors are summarized in <xref rid="feb2s0014579309000167-tbl1" ref-type="table">Table 1</xref>.</p></caption><graphic id="nlm-graphic-3" xlink:href="FEB2-583-549-g002"><alt-text>figure image</alt-text></graphic></fig><fig fig-type="Figure" xml:lang="en" id="feb2s0014579309000167-fig3" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Dose–response curves for 43146 against 229E 3CL<sup>pro</sup>, CVB3 3C<sup>pro</sup>, EV71 3C<sup>pro</sup> and RV14 3C<sup>pro</sup>. IC<sub>50</sub> values were determined from the curves using equation 1. These were (A) 10.3 ± 1.1 μM (229E 3CL<sup>pro</sup>), (B) 5.4 ± 0.2 μM (CVB3 3C<sup>pro</sup>), (C) 3.3 ± 0.3 μM (EV71 3C<sup>pro</sup>), and (D) 5.2 ± 0.6 μM (RV14 3C<sup>pro</sup>).</p></caption><graphic id="nlm-graphic-5" xlink:href="FEB2-583-549-g003"><alt-text>figure image</alt-text></graphic></fig><table-wrap id="feb2s0014579309000167-tbl1" xml:lang="en" orientation="portrait" position="float"><label>Table Table 1</label><caption><p>Summary of IC<sub>50</sub> values (μM) of the five hits with SARS‐CoV 3CL<sup>pro</sup>, and other 3C(L) proteases</p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><thead><tr style="border-bottom:solid 1px #000000" valign="top"><th valign="top" rowspan="1" colspan="1">Compound ID</th><th valign="top" rowspan="1" colspan="1">Structure</th><th valign="top" rowspan="1" colspan="1">SARS 3CL</th><th valign="top" rowspan="1" colspan="1">229E 3CL</th><th valign="top" rowspan="1" colspan="1">CVB3 3C</th><th valign="top" rowspan="1" colspan="1">EV71 3C</th><th valign="top" rowspan="1" colspan="1">RV14 3C</th></tr></thead><tbody><tr valign="top"><td valign="top" rowspan="1" colspan="1">21155</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e001.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">7.2 ± 0.7</td><td valign="top" rowspan="1" colspan="1">5.6 ± 1.0</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td></tr><tr valign="top"><td valign="top" rowspan="1" colspan="1">22723</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e002.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">10.6 ± 1.3</td><td valign="top" rowspan="1" colspan="1">12.4 ± 0.8</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td></tr><tr valign="top"><td valign="top" rowspan="1" colspan="1">27548</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e003.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">7.0 ± 0.8</td><td valign="top" rowspan="1" colspan="1">6.6 ± 0.3</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td></tr><tr valign="top"><td valign="top" rowspan="1" colspan="1">48511</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e004.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">3.3 ± 0.2</td><td valign="top" rowspan="1" colspan="1">1.8 ± 0.7</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td><td valign="top" rowspan="1" colspan="1">>50</td></tr><tr valign="top"><td valign="top" rowspan="1" colspan="1">43146</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e005.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">8.1 ± 0.9</td><td valign="top" rowspan="1" colspan="1">10.3 ± 1.1</td><td valign="top" rowspan="1" colspan="1">5.4 ± 0.2</td><td valign="top" rowspan="1" colspan="1">3.3 ± 0.3</td><td valign="top" rowspan="1" colspan="1">5.2 ± 0.6</td></tr></tbody></table></table-wrap></sec><sec id="feb2s0014579309000167-sec-3.2"><label>3.2</label><title>Inhibition potencies of the 43146 analogues</title><p>Since 43146 inhibited 3CL<sup>pro</sup> and 3C<sup>pro</sup>, its analogues including 45240, 68638, 55688, and 55585 obtained from another compound library were evaluated. As shown in <xref rid="feb2s0014579309000167-tbl2" ref-type="table">Table 2</xref>, all of them showed good potencies against the five proteases. The most potent compound was 45240, and its IC<sub>50</sub> values in inhibiting the 3C(L) proteases were measured to be 2.5 ± 0.2 μM (SARS‐CoV 3CL<sup>pro</sup>), 2.6 ± 0.4 μM (CoV‐229E 3CL<sup>pro</sup>), 1.2 ± 0.3 μM (CVB3 3C<sup>pro</sup>), 0.5 ± 0.1 μM (EV71 3C<sup>pro</sup>), and 1.7 ± 0.1 μM (RV14 3C<sup>pro</sup>) (<xref rid="feb2s0014579309000167-tbl2" ref-type="table">Table 2</xref>). This compound contains four rings, three phenyl groups and one imidazole, surrounding a central dihydropyrazole ring, without the lengthy side chain as seen in 43146. Compound 68638 with benzylcyclohexane ring fused with the dihydropyrazole ring and acetyl and iodobenzyl groups attached to the central ring showed less inhibition against both 3C<sup>pro</sup> and 3CL<sup>pro</sup> (<xref rid="feb2s0014579309000167-tbl2" ref-type="table">Table 2</xref>). The other two compounds, 55688 and 55585, with shorter side chains attached to the benzimidazolyl group showed similar inhibitory activities as compared to 43146 (<xref rid="feb2s0014579309000167-tbl2" ref-type="table">Table 2</xref>).</p><table-wrap id="feb2s0014579309000167-tbl2" xml:lang="en" orientation="portrait" position="float"><label>Table Table 2</label><caption><p>IC<sub>50</sub> values (μM) of compound 43146 analogs with SARS‐CoV 3CL<sup>pro</sup>, and other 3C(L) proteases</p></caption><table frame="hsides" rules="groups"><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><col align="left" span="1"></col><thead><tr style="border-bottom:solid 1px #000000" valign="top"><th valign="top" rowspan="1" colspan="1">Compound ID</th><th valign="top" rowspan="1" colspan="1">Structure</th><th valign="top" rowspan="1" colspan="1">SARS 3CL</th><th valign="top" rowspan="1" colspan="1">229E 3CL</th><th valign="top" rowspan="1" colspan="1">CVB3 3C</th><th valign="top" rowspan="1" colspan="1">EV71 3C</th><th valign="top" rowspan="1" colspan="1">RV14 3C</th></tr></thead><tbody><tr valign="top"><td valign="top" rowspan="1" colspan="1">45240</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e006.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">2.5 ± 0.2</td><td valign="top" rowspan="1" colspan="1">2.6 ± 0.4</td><td valign="top" rowspan="1" colspan="1">1.2 ± 0.3</td><td valign="top" rowspan="1" colspan="1">0.5 ± 0.1</td><td valign="top" rowspan="1" colspan="1">1.7 ± 0.1</td></tr><tr valign="top"><td valign="top" rowspan="1" colspan="1">68638</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e007.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">9.8 ± 0.8</td><td valign="top" rowspan="1" colspan="1">12.4 ± 0.8</td><td valign="top" rowspan="1" colspan="1">7.0 ± 0.8</td><td valign="top" rowspan="1" colspan="1">10.6 ± 1.3</td><td valign="top" rowspan="1" colspan="1">5.3 ± 1.1</td></tr><tr valign="top"><td valign="top" rowspan="1" colspan="1">55688</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e008.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">8.0 ± 0.5</td><td valign="top" rowspan="1" colspan="1">9.6 ± 0.3</td><td valign="top" rowspan="1" colspan="1">6.1 ± 0.5</td><td valign="top" rowspan="1" colspan="1">8.5 ± 0.6</td><td valign="top" rowspan="1" colspan="1">7.7 ± 1.0</td></tr><tr valign="top"><td valign="top" rowspan="1" colspan="1">55585</td><td valign="top" rowspan="1" colspan="1"><inline-graphic xlink:href="FEB2-583-549-e009.jpg" xlink:title="equation image"></inline-graphic></td><td valign="top" rowspan="1" colspan="1">8.4 ± 0.2</td><td valign="top" rowspan="1" colspan="1">10.2 ± 0.7</td><td valign="top" rowspan="1" colspan="1">6.5 ± 0.6</td><td valign="top" rowspan="1" colspan="1">4.7 ± 0.2</td><td valign="top" rowspan="1" colspan="1">6.4 ± 0.3</td></tr></tbody></table></table-wrap></sec><sec id="feb2s0014579309000167-sec-3.3"><label>3.3</label><title>Computer modeling of 21155, 22723, 27548, and 48511 binding to the proteases</title><p>These inhibitors are competitive inhibitors with respect to the substrate (data not shown), indicating they bind in the active site. To rationalize the binding discrepancy of these inhibitors against these proteases, their binding modes with SARS‐CoV 3CL<sup>pro</sup> and four other proteases were modeled and some of them are shown in <xref rid="feb2s0014579309000167-fig4" ref-type="fig">Fig. 4</xref>. The first four inhibitors of SARS‐CoV 3CL<sup>pro</sup> are more rigid because the thiazolopyridine in 21155, the dichlorobenzoquinolinone in 22723, the isoindoledione in 27548, and the oxazole in 48511 adopt planar structures and the three substituents of the oxazole ring in 48511 are fixed in a conformer, due to the 1,2‐steric interaction between the acetate group and the N‐aryl imino group as well as the biaryl interaction between the phenyl and oxazole to prohibit their free rotation. All these compounds can be considered as two rigid aromatic moieties connected by a small linker. Based on the computer modeling, each of these aromatic moieties is bound to S1 or S2 site of SARS protease by forming H‐bonds and hydrophobic interactions (<xref rid="feb2s0014579309000167-fig4" ref-type="fig">Fig. 4A–D</xref>). As shown in the computer modeling, Glu166 side chain of SARS 3CL<sup>pro</sup> forms H‐bonds with these four inhibitors. However, the corresponding amino acid residue in 3C<sup>pro</sup> is Gly164, which lacks the side chain to form H‐bond with any of these compounds (also see <xref rid="feb2s0014579309000167-fig4" ref-type="fig">Fig. 4</xref>F), leading to loss of inhibition. In addition, the 3C<sup>pro</sup> have more open but shallow S2 site (due to its partial blockage by Leu127) than 3CL<sup>pro</sup> according to the crystal structures of RV 3C<sup>pro</sup><xref rid="feb2s0014579309000167-bib-bib20" ref-type="ref">[20]</xref> and CVB3 3C<sup>pro</sup> (Lee et al., unpublished results) compared to 3CL<sup>pro</sup> (also see <xref rid="feb2s0014579309000167-fig4" ref-type="fig">Fig. 4</xref>F). Thus, 3C<sup>pro</sup> can not hold these compounds tightly.</p><fig fig-type="Figure" xml:lang="en" id="feb2s0014579309000167-fig4" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Computer modeling of the binding modes of the inhibitors in the active site of the SARS 3CL<sup>pro</sup>. The more rigid moieties of the inhibitors 21155, 22723, 27548, 48511 are probably bound to S1 and S2 sites as shown in (A), (B), (C), and (D), respectively. (E) 43146 binds SARS‐CoV 3CL<sup>pro</sup> differently with the biphenyl 4,5‐dihydro‐1H‐pyrazole moiety anchored at the S1’ and S2 sites and the rest of the molecule at the S3 and the following sites. The hydrogen bond interactions were represented by yellow dotted lines.</p></caption><graphic id="nlm-graphic-7" xlink:href="FEB2-583-549-g004"><alt-text>figure image</alt-text></graphic></fig></sec><sec id="feb2s0014579309000167-sec-3.4"><label>3.4</label><title>Binding modes of 43146 and its analogues to the proteases</title><p>In contrast, the compound 43146 is more flexible, because the dihydropyrazole is not planar, and the phenyl group is linked to the sp<sup>3</sup>‐hybridized carbon of the dihydropyrazole ring, so it is free for rotation. Different from the binding modes of the other 4 inhibitors, the diphenyl 4,5‐dihydro‐1H‐pyrazole moiety of 43146 fits well at the S1′ and S2 sites in the SARS 3CL<sup>pro</sup> (<xref rid="feb2s0014579309000167-fig4" ref-type="fig">Fig. 4</xref>E) with the rest of the molecule at the S3 site and beyond. With this binding mode, the compound was predicted to also bind well in the 3C<sup>pro</sup>, consistent with the inhibition data. In fact, RV 3C<sup>pro</sup> prefers a phenyl group at the S2 site as evidenced by its strong inhibition by AG7088 which has a P2‐fluorophenylalanine. Thus, it could be rationalized by computer modeling that only 43146 among the five hits can inhibit the three 3C<sup>pro</sup> in addition to the 3CL<sup>pro</sup>.</p><p>The analogues of 43146, including 45240, 68638, 55688, and 55585, bind in the 3C<sup>pro</sup> and 3CL<sup>pro</sup> active sites with similar modes to that of 43146 (data not shown). Compared to 43146, 55688 and 55585 only have minor structural difference with shorter alkyl groups attached to the benzimidazole ring, so that they showed similar inhibition against the proteases. The fused ring system and the phenyl group in 68638 may also span from S1′ to S2 sites in both kinds of proteases, yielding similar inhibition. However, 45240 showed a significantly better inhibition against the 3C<sup>pro</sup> than 43146. Apparently, the lengthy side chain attached to the phenyl group in the compound did not provide additional interaction with the protease, consistent with the binding mode shown in <xref rid="feb2s0014579309000167-fig4" ref-type="fig">Fig. 4</xref>E. However, the additional interaction is provided by the pyridine ring bound near the more open S1′ site in 3C<sup>pro</sup>.</p></sec></sec><sec id="feb2s0014579309000167-sec-4"><label>4</label><title>Discussion</title><p>AG7088 is the best inhibitor identified so far for 3C<sup>pro</sup>, which not only inhibits the 3C<sup>pro</sup> from RV, but also those from CV and EV <xref rid="feb2s0014579309000167-bib-bib16" ref-type="ref">[16]</xref>. However, it did not inhibit 3CL<sup>pro</sup> from SARS‐CoV <xref rid="feb2s0014579309000167-bib-bib17" ref-type="ref">[17]</xref>. This may be partially due to the blockage of its P1‐lactam ring by the relatively larger Glu166 side chain and also the S2 site of 3CL<sup>pro</sup> is narrower although it is deeper. Therefore, when the P2‐phenylalanine is changed to non‐planar leucine or cyclohexane without changing the P1‐lactam, they became good inhibitors of SARS 3CL<sup>pro</sup><xref rid="feb2s0014579309000167-bib-bib21" ref-type="ref">[21]</xref>. Unlike AG7088, which is a ketomethyl isostere of a tripeptide‐conjugated ester, compound 43146 is not peptide‐like. From the random screening as shown in the study, we have found a starting point toward the development of non‐peptide multiple‐function inhibitors against CoV and PV. With further modification of these individual and common inhibitors of the viral proteases, we hope to find solution for the possible reoccurrence of SARS and other diseases caused by the viruses with the 3C<sup>pro</sup> and 3CL<sup>pro</sup>.</p></sec></body><back><ack id="feb2s0014579309000167-sec-0000"><title>Acknowledgements</title><p>This work was supported by a grant from Academia Sinica to PHL, and we thank Korea Chemical Bank for providing their chemical library with which this work was conducted.</p></ack><ref-list content-type="cited-references"><title>References</title><ref id="feb2s0014579309000167-bib-bib1"><label>1</label><mixed-citation publication-type="miscellaneous" id="feb2s0014579309000167-cit-bib1">Melnick, J.L. (1996) Enterovirus: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In Virology, 3rd ed., Lippincott-Raven, Philadephia, PA.
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