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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Inhibitor Recognition Specificity of MERS-CoV Papain-like
Protease May Differ from That of SARS-CoV</title><author><name sortKey="Lee, Hyun" sort="Lee, Hyun" uniqKey="Lee H" first="Hyun" last="Lee">Hyun Lee</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Lei, Hao" sort="Lei, Hao" uniqKey="Lei H" first="Hao" last="Lei">Hao Lei</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Santarsiero, Bernard X0a D" sort="Santarsiero, Bernard X0a D" uniqKey="Santarsiero B" first="Bernard X0a D." last="Santarsiero">Bernard X0a D. Santarsiero</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Gatuz, Joseph X0a L" sort="Gatuz, Joseph X0a L" uniqKey="Gatuz J" first="Joseph X0a L." last="Gatuz">Joseph X0a L. Gatuz</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Cao, Shuyi" sort="Cao, Shuyi" uniqKey="Cao S" first="Shuyi" last="Cao">Shuyi Cao</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Rice, Amy J" sort="Rice, Amy J" uniqKey="Rice A" first="Amy J." last="Rice">Amy J. Rice</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Patel, Kavankumar" sort="Patel, Kavankumar" uniqKey="Patel K" first="Kavankumar" last="Patel">Kavankumar Patel</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Szypulinski, Michael Z" sort="Szypulinski, Michael Z" uniqKey="Szypulinski M" first="Michael Z." last="Szypulinski">Michael Z. Szypulinski</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Ojeda, Isabel" sort="Ojeda, Isabel" uniqKey="Ojeda I" first="Isabel" last="Ojeda">Isabel Ojeda</name></author><author><name sortKey="Ghosh, Arun K" sort="Ghosh, Arun K" uniqKey="Ghosh A" first="Arun K." last="Ghosh">Arun K. Ghosh</name><affiliation><nlm:aff id="aff2">Departments of Chemistry and Medicinal Chemistry,<institution>Purdue University</institution>, 560 Oval Drive, West Lafayette, Indiana 47907,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Johnson, Michael E" sort="Johnson, Michael E" uniqKey="Johnson M" first="Michael E." last="Johnson">Michael E. Johnson</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25746232</idno><idno type="pmc">4845099</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845099</idno><idno type="RBID">PMC:4845099</idno><idno type="doi">10.1021/cb500917m</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000123</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000123</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Inhibitor Recognition Specificity of MERS-CoV Papain-like
Protease May Differ from That of SARS-CoV</title><author><name sortKey="Lee, Hyun" sort="Lee, Hyun" uniqKey="Lee H" first="Hyun" last="Lee">Hyun Lee</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Lei, Hao" sort="Lei, Hao" uniqKey="Lei H" first="Hao" last="Lei">Hao Lei</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Santarsiero, Bernard X0a D" sort="Santarsiero, Bernard X0a D" uniqKey="Santarsiero B" first="Bernard X0a D." last="Santarsiero">Bernard X0a D. Santarsiero</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Gatuz, Joseph X0a L" sort="Gatuz, Joseph X0a L" uniqKey="Gatuz J" first="Joseph X0a L." last="Gatuz">Joseph X0a L. Gatuz</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Cao, Shuyi" sort="Cao, Shuyi" uniqKey="Cao S" first="Shuyi" last="Cao">Shuyi Cao</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Rice, Amy J" sort="Rice, Amy J" uniqKey="Rice A" first="Amy J." last="Rice">Amy J. Rice</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Patel, Kavankumar" sort="Patel, Kavankumar" uniqKey="Patel K" first="Kavankumar" last="Patel">Kavankumar Patel</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Szypulinski, Michael Z" sort="Szypulinski, Michael Z" uniqKey="Szypulinski M" first="Michael Z." last="Szypulinski">Michael Z. Szypulinski</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Ojeda, Isabel" sort="Ojeda, Isabel" uniqKey="Ojeda I" first="Isabel" last="Ojeda">Isabel Ojeda</name></author><author><name sortKey="Ghosh, Arun K" sort="Ghosh, Arun K" uniqKey="Ghosh A" first="Arun K." last="Ghosh">Arun K. Ghosh</name><affiliation><nlm:aff id="aff2">Departments of Chemistry and Medicinal Chemistry,<institution>Purdue University</institution>, 560 Oval Drive, West Lafayette, Indiana 47907,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Johnson, Michael E" sort="Johnson, Michael E" uniqKey="Johnson M" first="Michael E." last="Johnson">Michael E. Johnson</name><affiliation><nlm:aff id="aff1">Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></nlm:aff></affiliation></author></analytic><series><title level="j">ACS Chemical Biology</title><idno type="ISSN">1554-8929</idno><idno type="eISSN">1554-8937</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="cb500917m_0008" id="ab-tgr1"></graphic></p><p>The Middle East Respiratory Syndrome
coronavirus (MERS-CoV) papain-like
protease (PLpro) blocking loop 2 (BL2) structure differs significantly
from that of SARS-CoV PLpro, where it has been proven to play a crucial
role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead
inhibitors were tested against MERS-CoV PLpro, none of which were
effective against MERS-CoV PLpro. Structure and sequence alignments
revealed that two residues, Y269 and Q270, responsible for inhibitor
binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV
PLpro, making critical binding interactions difficult to form for
similar types of inhibitors. High-throughput screening (HTS) of 25 000
compounds against both PLpro enzymes identified a small fragment-like
noncovalent dual inhibitor. Mode of inhibition studies by enzyme kinetics
and competition surface plasmon resonance (SPR) analyses suggested
that this compound acts as a competitive inhibitor with an IC<sub>50</sub> of 6 μM against MERS-CoV PLpro, indicating that it
binds to the active site, whereas it acts as an allosteric inhibitor
against SARS-CoV PLpro with an IC<sub>50</sub> of 11 μM. These
results raised the possibility that inhibitor recognition specificity
of MERS-CoV PLpro may differ from that of SARS-CoV PLpro. In addition,
inhibitory activity of this compound was selective for SARS-CoV and
MERS-CoV PLpro enzymes over two human homologues, the ubiquitin C-terminal
hydrolases 1 and 3 (hUCH-L1 and hUCH-L3).</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Anderson, L J" uniqKey="Anderson L">L. J. Anderson</name></author><author><name sortKey="Baric, R S" uniqKey="Baric R">R. S. Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, J F" uniqKey="Chan J">J. F. Chan</name></author><author><name sortKey="Li, K S" uniqKey="Li K">K. S. Li</name></author><author><name sortKey="To, K K" uniqKey="To K">K. K. To</name></author><author><name sortKey="Cheng, V C" uniqKey="Cheng V">V. C. Cheng</name></author><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name></author><author><name sortKey="Yuen, K Y" uniqKey="Yuen K">K. Y. Yuen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zaki, A M" uniqKey="Zaki A">A. M. Zaki</name></author><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S. van Boheemen</name></author><author><name sortKey="Bestebroer, T M" uniqKey="Bestebroer T">T. M. Bestebroer</name></author><author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A. D. Osterhaus</name></author><author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R. A. Fouchier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De X0a Groot, R J" uniqKey="De X0a Groot R">R. J. de
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<front><journal-meta><journal-id journal-id-type="nlm-ta">ACS Chem Biol</journal-id><journal-id journal-id-type="iso-abbrev">ACS Chem. Biol</journal-id><journal-id journal-id-type="publisher-id">cb</journal-id><journal-id journal-id-type="coden">acbcct</journal-id><journal-title-group><journal-title>ACS Chemical Biology</journal-title></journal-title-group><issn pub-type="ppub">1554-8929</issn><issn pub-type="epub">1554-8937</issn><publisher><publisher-name>American Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25746232</article-id><article-id pub-id-type="pmc">4845099</article-id><article-id pub-id-type="doi">10.1021/cb500917m</article-id><article-categories><subj-group><subject>Articles</subject></subj-group></article-categories><title-group><article-title>Inhibitor Recognition Specificity of MERS-CoV Papain-like
Protease May Differ from That of SARS-CoV</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Lee</surname><given-names>Hyun</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="notes-1" ref-type="notes">§</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Lei</surname><given-names>Hao</given-names></name><xref rid="aff1" ref-type="aff">†</xref><xref rid="notes-1" ref-type="notes">§</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Santarsiero</surname><given-names>Bernard
D.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>Gatuz</surname><given-names>Joseph
L.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath5"><name><surname>Cao</surname><given-names>Shuyi</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath6"><name><surname>Rice</surname><given-names>Amy J.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath7"><name><surname>Patel</surname><given-names>Kavankumar</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath8"><name><surname>Szypulinski</surname><given-names>Michael Z.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath9"><name><surname>Ojeda</surname><given-names>Isabel</given-names></name></contrib><contrib contrib-type="author" id="ath10"><name><surname>Ghosh</surname><given-names>Arun K.</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath11"><name><surname>Johnson</surname><given-names>Michael E.</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref></contrib><aff id="aff1"><label>†</label>Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy,<institution>University of Illinois at Chicago</institution>, 900 S. Ashland, Chicago, Illinois 60607,<country>United States</country></aff><aff id="aff2"><label>‡</label>Departments of Chemistry and Medicinal Chemistry,<institution>Purdue University</institution>, 560 Oval Drive, West Lafayette, Indiana 47907,<country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>mjohnson@uic.edu</email>.</corresp></author-notes><pub-date pub-type="epub"><day>06</day><month>03</month><year>2015</year></pub-date><pub-date pub-type="ppub"><day>19</day><month>06</month><year>2015</year></pub-date><volume>10</volume><issue>6</issue><fpage>1456</fpage><lpage>1465</lpage><history><date date-type="received"><day>12</day><month>11</month><year>2014</year></date><date date-type="accepted"><day>06</day><month>03</month><year>2015</year></date></history><permissions><copyright-statement>Copyright © 2015 American Chemical
Society</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>American Chemical
Society</copyright-holder><license license-type="open-access"><license-p>This article is made available via the PMC Open Access Subset 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 the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="cb500917m_0008" id="ab-tgr1"></graphic></p><p>The Middle East Respiratory Syndrome
coronavirus (MERS-CoV) papain-like
protease (PLpro) blocking loop 2 (BL2) structure differs significantly
from that of SARS-CoV PLpro, where it has been proven to play a crucial
role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead
inhibitors were tested against MERS-CoV PLpro, none of which were
effective against MERS-CoV PLpro. Structure and sequence alignments
revealed that two residues, Y269 and Q270, responsible for inhibitor
binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV
PLpro, making critical binding interactions difficult to form for
similar types of inhibitors. High-throughput screening (HTS) of 25 000
compounds against both PLpro enzymes identified a small fragment-like
noncovalent dual inhibitor. Mode of inhibition studies by enzyme kinetics
and competition surface plasmon resonance (SPR) analyses suggested
that this compound acts as a competitive inhibitor with an IC<sub>50</sub> of 6 μM against MERS-CoV PLpro, indicating that it
binds to the active site, whereas it acts as an allosteric inhibitor
against SARS-CoV PLpro with an IC<sub>50</sub> of 11 μM. These
results raised the possibility that inhibitor recognition specificity
of MERS-CoV PLpro may differ from that of SARS-CoV PLpro. In addition,
inhibitory activity of this compound was selective for SARS-CoV and
MERS-CoV PLpro enzymes over two human homologues, the ubiquitin C-terminal
hydrolases 1 and 3 (hUCH-L1 and hUCH-L3).</p></abstract><funding-group><funding-statement><funding-source>National Institutes of Health, United States</funding-source></funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>cb500917m</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>cb500917m</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta><notes id="notes-d1e21-autogenerated"><fn-group><fn fn-type="" id="d30e208"><p>This article is made available for a limited time sponsored by ACS under the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/freetoread/index.html">ACS Free to Read License</ext-link>, which permits copying and redistribution of the article for non-commercial scholarly purposes.</p></fn></fn-group></notes></front><body><p id="sec1">Middle East
Respiratory Syndrome
coronavirus (MERS-CoV), previously called human coronavirus-Erasmus
Medical Center (HCoV-EMC), was first reported in Saudi Arabia in 2012
and spread to 20 different countries,<sup><xref ref-type="bibr" rid="ref1">1</xref>−<xref ref-type="bibr" rid="ref4">4</xref></sup> resulting in 853 infections with 301 deaths
as of October 2, 2014.<sup><xref ref-type="bibr" rid="ref5">5</xref></sup> The unusually high
case-fatality rate (CFR) of MERS-CoV infections (∼35%) is alarming
as it far exceeds that of all other known human coronaviruses, including
the human severe acute respiratory syndrome coronavirus (SARS-CoV).
SARS-CoV caused a fatal global outbreak in 2003, resulting in 800
deaths (∼10% CFR).<sup><xref ref-type="bibr" rid="ref6">6</xref></sup> There are over
20 known coronaviruses (CoV), six of which are identified as human
coronaviruses (HCoV; <xref rid="notes-4" ref-type="notes">Supplementary Figure S1</xref>). Coronaviruses are classified into four genera (α, β,
γ, and δ), and each genus can be divided into lineage
subgroups. Of the six HCoVs, two (NL63 and 229E) belong to genus α,
and the remaining four (HKU1, OC43, SARS-CoV, and MERS-CoV) belong
to genus β. Within the betacoronavirus genus, SARS-CoV is classified
as lineage group B, while MERS-CoV is categorized into lineage group
C based on their genomes. Two bat CoVs from lineage group C, BtCoV-HKU4
and BtCoV-HKU5, are the most closely related to the MERS-CoV.<sup><xref ref-type="bibr" rid="ref2">2</xref>,<xref ref-type="bibr" rid="ref7">7</xref>−<xref ref-type="bibr" rid="ref9">9</xref></sup> MERS-CoV and SARS-CoV are highly pathogenic, with
evidence of person-to-person transmission via either household or
hospital contacts.<sup><xref ref-type="bibr" rid="ref10">10</xref>,<xref ref-type="bibr" rid="ref11">11</xref></sup> MERS-CoV and SARS-CoV use different
receptors, dipeptidyl peptidase 4 (DPP4 or CD26) and angiotensin-converting
enzyme 2 (ACE2), respectively,<sup><xref ref-type="bibr" rid="ref12">12</xref>,<xref ref-type="bibr" rid="ref13">13</xref></sup> and the epidemiology
of MERS-CoV is still being investigated. Both MERS-CoV and SARS-CoV
exhibit as a severe respiratory infection, while MERS-CoV exhibits
an additional unique symptom of renal failure.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> Even though the MERS-CoV transmission rate is slower than that of
SARS-CoV, the number of MERS-CoV infections continues to grow.<sup><xref ref-type="bibr" rid="ref11">11</xref>,<xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref15">15</xref></sup> Due to the recent emergence of
this new coronavirus and the potential of SARS-CoV retransmission
from zoonotic reservoirs to humans,<sup><xref ref-type="bibr" rid="ref16">16</xref>−<xref ref-type="bibr" rid="ref18">18</xref></sup> the possibility of another
deadly pandemic has been seriously raised. However, there is still
no effective therapeutic available against either coronavirus. Therefore,
developing treatments against both coronaviruses is important.</p><p>Both MERS-CoV and SARS-CoV are single-stranded positive-sense RNA
viruses with approximately 30 kb genome sizes. Each of their genes
encodes two polyproteins called pp1a and pp1b (Figure <xref rid="fig1" ref-type="fig">1</xref>A) that are processed by two proteases, a 3-C-like protease
(3CLpro) and a papain-like protease (PLpro). Many coronaviruses contain
two PLpro enzymes (PLP1 and PLP2), but MERS-CoV and SARS-CoV have
only one PLpro enzyme.<sup><xref ref-type="bibr" rid="ref19">19</xref>,<xref ref-type="bibr" rid="ref20">20</xref></sup> PLpro enzymes are part of a large
nonstructural protein 3 (nsp3) that contains four other domains, a
ubiquitin-like fold (UB1), an ADP-ribose-1d-phosphatase (ADRP) domain,
a SARS-unique domain (SUD), and a transmembrane (TM) domain (Figure <xref rid="fig1" ref-type="fig">1</xref>A). PLpro is responsible for cleavage of the first
three positions of its polyprotein, while 3CLpro cleaves the remaining
11 locations, releasing a total of 16 nonstructural proteins (nsp)
in both MERS-CoV and SARS-CoV. Sequence motifs recognized by MERS-CoV
PLpro (MERS-PLpro) and SARS-CoV PLpro (SARS-PLpro) are (L/I)XGG↓(A/D)X
and LXGG↓(A/K)X, respectively (Figure <xref rid="fig1" ref-type="fig">1</xref>B). Unlike 3CLpro, SARS-PLpro has been shown to be a multifunctional
protein involved in de-ISGylation, deubiquitination, and viral evasion
of the innate immune response in addition to viral peptide cleavage
as a protease.<sup><xref ref-type="bibr" rid="ref16">16</xref>,<xref ref-type="bibr" rid="ref21">21</xref></sup> Researchers have discovered that
the MERS-PLpro also exhibits deubiquitination and de-ISGylation functions,
blocking the interferon regulatory factor 3 (IRF3) pathway.<sup><xref ref-type="bibr" rid="ref22">22</xref>,<xref ref-type="bibr" rid="ref23">23</xref></sup> Both 3CLpro and PLpro are known to be essential for viral replication,
making them attractive targets in antiviral drug discovery.<sup><xref ref-type="bibr" rid="ref20">20</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup> In this work we investigated four known SARS-PLpro lead inhibitors
against MERS-PLpro. In addition, high-throughput screening (HTS) of
a 25 000-compound antimicrobial focused library against both
MERS-CoV and SARS-PLpro enzymes identified a low molecular weight
compound that showed activity against both PLpro enzymes via two different
modes of inhibition.</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Schematics of SARS-CoV and MERS-CoV polyproteins. (A)
Cleavage
positions of PLpro (pink) and 3CLpro (cyan) are shown by different
colored arrows in their polyproteins. (B) Cleavage site comparison
between SARS and MERS PLpro enzymes. Sequence motifs recognized by
SARS-CoV PLpro (SARS-PLpro) and MERS-CoV PLpro (MERS-PLpro) are LXGG↓(A/K)X
and (L/I)XGG↓(A/D)X, respectively.</p></caption><graphic xlink:href="cb500917m_0001" id="gr1" position="float"></graphic></fig><sec id="sec2"><title>Results and Discussion</title><sec id="sec2.1"><title>SARS-PLpro Lead Inhibitors Do Not Inhibit
MERS-PLpro</title><p>We and others have previously identified and developed
a series of
noncovalent SARS-PLpro inhibitors using high-throughput screening
(HTS) and structure-based drug design that can be classified into
two distinct scaffolds.<sup><xref ref-type="bibr" rid="ref25">25</xref>−<xref ref-type="bibr" rid="ref28">28</xref></sup> Four inhibitor structures from these two scaffolds
are shown in Figure <xref rid="fig2" ref-type="fig">2</xref>A, with <bold>I-1</bold> and <bold>I-2</bold> representing scaffold 1 and <bold>I-3</bold> and <bold>I-4</bold> representing scaffold 2. Inhibitors <bold>I-2</bold> and <bold>I-3</bold> exhibited excellent inhibitory activities with
IC<sub>50</sub> values of 0.34 μM and 0.6 μM against SARS-PLpro,
with SARS antiviral activities of 2 μM and 15 μM, respectively.<sup><xref ref-type="bibr" rid="ref25">25</xref>,<xref ref-type="bibr" rid="ref28">28</xref></sup> The four SARS-PLpro lead inhibitors (<bold>I-1</bold>–<bold>I-4</bold>) were tested against MERS-PLpro to determine whether these
two PLpro enzymes behave similarly or not. Surprisingly, none of them
showed any inhibitory activity against MERS-PLpro. This result led
us to further analyze what determined the interaction between SARS-PLpro
and its inhibitors.</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>SARS-PLpro lead inhibitors and structures. (A) Structures
of four
SARS-PLpro lead inhibitors (<bold>I-1</bold>–<bold>I-4</bold>).<sup><xref ref-type="bibr" rid="ref25">25</xref>,<xref ref-type="bibr" rid="ref26">26</xref></sup> (B) X-ray crystal structure of inhibitor <bold>I-2</bold> bound to SARS-PLpro (PDB: 3E9S). The amide group of inhibitor <bold>I-2</bold> forms two hydrogen bonds with D165 and Q270 in the BL2
loop. (C) X-ray crystal structure of inhibitor <bold>I-3</bold> bound
to SARS-PLpro (PDB: 3MJ5). The amide group of inhibitor <bold>I-3</bold> forms a hydrogen
bond with Q270 in the BL2 loop. The aromatic ring of Y269 forms a
hydrophobic interaction with the naphthyl rings of both <bold>I-2</bold> and <bold>I-3</bold>. The three catalytic site residues are shown
in green. (D) Overlay of the SARS-PLpro blocking loop 2 (BL2) and
the corresponding loop of MERS-PLpro.</p></caption><graphic xlink:href="cb500917m_0002" id="gr2" position="float"></graphic></fig><p>There are seven SARS-PLpro crystal structures available to
date,
four of which are complexes with an inhibitor and two are substrate-bound.<sup><xref ref-type="bibr" rid="ref21">21</xref>,<xref ref-type="bibr" rid="ref25">25</xref>−<xref ref-type="bibr" rid="ref28">28</xref></sup> In the case of MERS-PLpro, one apo and two substrate-bound complexes
have been recently published.<sup><xref ref-type="bibr" rid="ref29">29</xref>,<xref ref-type="bibr" rid="ref30">30</xref></sup> We also determined
the X-ray crystal structure of unbound MERS-PLpro (PDB code: 4RNA) at 1.8 Å (<xref rid="notes-4" ref-type="notes">Supporting Information Table S1</xref>) in addition
to our previously released apo structure at lower resolution (PDB
code: 4PT5).
Both SARS-PLpro and MERS-PLpro contain two blocking loops named BL1
and BL2 that could be structurally important. The two corresponding
loops of human ubiquitin-specific protease 14 (USP14) have been proven
to be crucial in blocking accessibility to the active site.<sup><xref ref-type="bibr" rid="ref31">31</xref></sup> Indeed, two SARS-PLpro complex crystal structures,
with lead inhibitors from each scaffold (<bold>I-2</bold> or <bold>I-3</bold>) revealed that inhibitors bind not to the catalytic site
of the PLpro enzyme but to the BL2 loop, blocking the entrance of
the active site. This appears to prevent substrate access to the catalytic
site, inhibiting PLpro enzyme activity. The amide group of inhibitor <bold>I-2</bold> forms two hydrogen bonds with D165 and Q270 (Figure <xref rid="fig2" ref-type="fig">2</xref>B). The amide group of inhibitor <bold>I-3</bold> also forms a hydrogen bond with Q270 in the BL2 loop (Figure <xref rid="fig2" ref-type="fig">2</xref>C). The aromatic ring of Y269 forms a hydrophobic
interaction with the naphthyl ring of both <bold>I-2</bold> and <bold>I-3</bold>. Therefore, residues Q270 and Y269 form common key interactions
in both scaffold 1 and 2 lead inhibitors of SARS-CoV PLpro. However,
neither Q270 nor Y269 residues exist in MERS-PLpro (Figure <xref rid="fig2" ref-type="fig">2</xref>D). In MERS-PLpro, A275 exists in place of Q270
of SARS-PLpro, eliminating potential hydrogen bonding with inhibitors.
Additionally, the second key interaction is also impossible because
T274 of the MERS-PLpro does not have the aromatic ring of Y269. Apparently
due to the lack of these two key residues, none of the SARS-PLpro
lead inhibitors had any inhibitory efficacy against MERS-PLpro.</p><p>In order to further analyze the interaction between SARS-PLpro
and its inhibitors, we aligned all four available SARS-PLpro–inhibitor
complexes, one SARS-PLpro in complex with a ubiquitin aldehyde substrate,
one MERS-PLpro complex with a ubiquitin substrate, and the apo structures
of both SARS-PLpro and MERS-PLpro (Figure <xref rid="fig3" ref-type="fig">3</xref>).<sup><xref ref-type="bibr" rid="ref21">21</xref>,<xref ref-type="bibr" rid="ref25">25</xref>−<xref ref-type="bibr" rid="ref28">28</xref>,<xref ref-type="bibr" rid="ref30">30</xref></sup> In addition, all residues involved in inhibitor binding by SARS-PLpro
and their corresponding residues in MERS-PLpro are compared to three
bat coronaviruses because of the similarities of MERS-CoV with bat
coronaviruses BtCoV-HKU4 and BtCoV-HKU5. The residues compared include
the active site, catalytic triad, and BL2 loop residues (Figure <xref rid="fig3" ref-type="fig">3</xref>E). All eight structures aligned well with each
other, except for several distinct locations, including the zinc-binding
motif and BL2 regions. The zinc atoms were shifted similar distances
and locations in all five SARS-PLpro complex structures as compared
to the apo structure, as noted with a red arrow pointed to the right
in the bottom insert in Figure <xref rid="fig3" ref-type="fig">3</xref>A. The equivalent
zinc atom in MERS-PLpro was located in a different position and was
shifted in a different direction from those in SARS-PLpro (dark brown
arrow in Figure <xref rid="fig3" ref-type="fig">3</xref>A). Residues involved in the
SARS-PLpro inhibitor binding are shown in Figure <xref rid="fig3" ref-type="fig">3</xref>B, along with inhibitor <bold>I-3</bold>, in order to illustrate
the orientation of the inhibitor, inhibitor-interacting SARS-PLpro
residues, and the flexible BL2 loop. Figure <xref rid="fig3" ref-type="fig">3</xref>C and D show the binding locations of inhibitors and ubiquitin substrates
and the flexible BL2 loop in exactly the same orientation. The SARS-PLpro
flexible BL2 loop blocks the entrance of the tunnel to the active
site when it is unbound and becomes well-ordered upon binding of either
an inhibitor or a substrate (Figure <xref rid="fig3" ref-type="fig">3</xref>C and
D) through conformational changes.<sup><xref ref-type="bibr" rid="ref21">21</xref>,<xref ref-type="bibr" rid="ref26">26</xref>−<xref ref-type="bibr" rid="ref28">28</xref>,<xref ref-type="bibr" rid="ref32">32</xref>,<xref ref-type="bibr" rid="ref33">33</xref></sup> For MERS-PLpro, the flexible BL2 loop is positioned much further
from its active site than that of SARS-PLpro when it is unbound. Upon
substrate binding to MERS-PLpro, its BL2 loop moves to an orientation
similar to that of the substrate bound SARS-PLpro loop, as noted with
red and dark brown arrows in Figure <xref rid="fig3" ref-type="fig">3</xref>D.<sup><xref ref-type="bibr" rid="ref30">30</xref></sup> Both BL1 and BL2 loops of USP14 appear to play
a regulatory role in its deubiquitinating activity,<sup><xref ref-type="bibr" rid="ref31">31</xref></sup> while mainly BL2 seems to serve this role in SARS-PLpro.
The question of whether the BL2 loop of MERS-PLpro plays a regulatory
role in its deubiquitinating activity remains to be answered.</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Structure comparison
of SARS-PLpro complexes and MERS-PLpro. (A)
Overlay of five SARS-PLpro complex structures with an inhibitor or
a substrate, apo SARS-PLpro, apo MERS-PLpro, and MERS-CoV-PLpro complex
with a ubiquitin. The PDB codes of aligned structures are Apo MERS-CoV-PLpro
(PDB: 4RNA),
MERS-CoV-PLpro complex with a ubiquitin (PDB: 4RF1), Apo SARS-CoV-PLpro
(PDB: 2FE8),
SARS-CoV-PLpro inhibitor complex with inhibitor <bold>I-2</bold> (PDB: 3E9S), SARS-CoV-PLpro
complex with inhibitor <bold>I-3</bold> (PDB: 3MJ5), SARS-CoV-PLpro
complex with inhibitor <bold>3k</bold> (PDB: 4OVZ), SARS-CoV-PLpro
complex with inhibitor <bold>3j</bold> (PDB: 4OW0), and SARS-CoV-PLpro
complex with ubiquitin aldehyde substrate (PDB: 4MM3). (B) Expanded overlaid
structures of BL2 and surrounding residues involved with inhibitor
binding. (C) Different orientation of Figure <xref rid="fig4" ref-type="fig">4</xref>B, showing catalytic residues and relative BL2 orientations. (D)
Expanded overlaid structures of BL2 loops and a ubiquitin aldehyde
(blue) and ubiquitin (orange) substrates for SARS-PLpro and MERS-PLpro,
respectively. Ubiquitin is hidden, and only part of the each substrate
is shown in this figure due to space constraints. (E) The active site,
catalytic triad (CT), and two blocking loop (BL1 and BL2) residues
of MERS-PLpro and their corresponding aligned residues in the active
sites of SARS-PLpro and three bat coronaviral PLpro enzymes.</p></caption><graphic xlink:href="cb500917m_0003" id="gr3" position="float"></graphic></fig></sec><sec id="sec2.2"><title>Prescreen Assay Optimization
for High-Throughput Screening</title><p>Prescreen assay optimization
prior to a large scale HTS is crucial
to achieving a high quality outcome. Essential factors for consideration
include the enzyme and substrate concentrations, additives (detergent
and reducing agent), the DMSO tolerance, and enzyme stability. The
most important factor is the substrate concentration, and it is recommended
to use a substrate concentration near or slightly lower than the Michaelis
constant (<italic>K</italic><sub>M</sub>) value in order to select
for both competitive and noncompetitive inhibitors.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> The <italic>K</italic><sub>M</sub> was determined with
the ubiquitin-derived peptide substrate, RLRGG-AMC, for both SARS-PLpro
and MERS-PLpro enzymes side by side for comparison. The substrate <italic>K</italic><sub>M</sub> value of MERS-PLpro was ∼2-fold larger
at 142 μM and 75.9 μM for MERS-PLpro and SARS-CoV, respectively
(<xref rid="notes-4" ref-type="notes">Supporting Information Table S2</xref>). Therefore,
a higher substrate concentration was used for MERS-PLpro than for
the SARS-PLpro HTS screen. The <italic>k</italic><sub>cat</sub> (turnover
number) value of SARS-CoV was ∼25-fold larger than that of
MERS-PLpro, which made the catalytic efficiency (<italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>M</sub>) of SARS-PLpro for
this substrate ∼45-fold higher than that of MERS-PLpro. Accordingly,
a 20-fold higher MERS-PLpro enzyme concentration was necessary to
yield an enzyme activity signal similar to that of SARS-PLpro. DMSO
tolerance, reducing agent effect, two additives (BSA and Triton X-100)
effect, and enzyme stability at RT were determined in addition to
optimal substrate and enzyme concentrations for HTS.</p></sec><sec id="sec2.3"><title>High-Throughput
Screening (HTS) and Hit Validation</title><p>To search for MERS-PLpro
inhibitors, HTS was performed with a 25 000-compound
Life Chemicals antimicrobial/antiviral focused library against both
SARS-PLpro and MERS-PLpro. The overall screening and hit validation
process are described in Figure <xref rid="fig4" ref-type="fig">4</xref>A. The primary
HTS screen against SARS-PLpro was performed in duplicate, generating
average Z′-factors of 0.64 ± 0.08. The MERS-PLpro primary
screen was done in a single pass with Z′-factors of 0.65 ±
0.11. HTS hits with over 50% inhibition at 50 μM compound concentration
were cherry picked and reanalyzed by a continuous kinetic assay to
filter out false positives. The enzyme omission assay with exactly
the same assay conditions, but without the PLpro enzyme, was performed
to remove fluorescence signal interfering compounds. Confirmed hits
were repurchased, and their inhibitory activities (IC<sub>50</sub> values) were determined from full inhibition curves.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>HTS results from Life
Chemicals antimicrobial/antiviral focused
library and hit validation. (A) Schematic of HTS with 25 000
Life Chemicals compounds and hit validation process. (B) Bar graphs
of IC<sub>50</sub> values and the dissociation equilibrium constants
(<italic>K</italic><sub>D</sub>) of six hit compounds determined by
fluorescence-based enzymatic assay and Surface Plasmon Resonance (SPR),
respectively. All data were normalized for immobilization levels of
target proteins and reference. Bars that reach the top of the graph
represent either IC<sub>50</sub> or <italic>K</italic><sub>D</sub> values of over 200 μM (no inhibition or no binding).</p></caption><graphic xlink:href="cb500917m_0004" id="gr4" position="float"></graphic></fig><p>Of ∼25 000 compounds,
four (compounds <bold>1</bold>–<bold>4</bold> in Figure <xref rid="fig4" ref-type="fig">4</xref>B) and three
(compounds <bold>4</bold>–<bold>6</bold> in Figure <xref rid="fig4" ref-type="fig">4</xref>B) exhibited inhibitory activity with IC<sub>50</sub> values below 50 μM for SARS-PLpro and MERS-PLpro, respectively.
Surface plasmon resonance (SPR) was used as a secondary orthogonal
binding assay to eliminate false positives from the primary hits since
our primary screen was done by a fluorescence-based enzymatic assay.
Binding affinity of each hit compound can be determined by measuring
the dissociation equilibrium constant (<italic>K</italic><sub>D</sub>) using SPR, and IC<sub>50</sub> and <italic>K</italic><sub>D</sub> values for the six hit compounds are compared in Figure <xref rid="fig4" ref-type="fig">4</xref>B. Compound <bold>2</bold> from the four SARS-PLpro
hits did not bind to the enzyme, indicating that it is a false positive,
while the remaining three were confirmed to be binders with <italic>K</italic><sub>D</sub> values below 50 μM. The binding affinities
of these validated hits varied from 26.3 μM to 39.9 μM,
and their corresponding IC<sub>50</sub> values varied from 10.9–31.4
μM. The IC<sub>50</sub> and <italic>K</italic><sub>D</sub> values
of <bold>3</bold> are 31.4 μM and 39.9 μM, respectively,
which are similar. The <italic>K</italic><sub>D</sub> value of <bold>4</bold> (26.3 μM) is approximately 2.4-fold greater than its
IC<sub>50</sub> value (10.9 μM). Of the three MERS-PLpro hits,
only <bold>4</bold> showed specific binding to the enzyme, with 18.4
μM binding affinity, while <bold>5</bold> and <bold>6</bold> were false positives. Compound <bold>5</bold> did not bind to MERS-PLpro
at all, whereas compound <bold>6</bold> bound nonspecifically. These
three distinct binding patterns determined by SPR, specific, no binding,
and nonspecific interactions, are shown in <xref rid="notes-4" ref-type="notes">Supporting
Information Figure S3A–C</xref>. Interestingly, compound <bold>4</bold> showed similar strength of inhibitory activities and binding
affinity against both SARS-PLpro and MERS-PLpro enzymes.</p></sec><sec id="sec2.4"><title>Mechanism of
Inhibition</title><p>There have been two types of
noncovalent small molecule inhibitor scaffolds against SARS-PLpro
previously discovered by our research group. Mechanism of inhibition
studies revealed that these compounds are mixed-type inhibitors with
α values greater than 1, indicating that they bind to an allosteric
site other than its catalytic site but behave as if they are competitive
inhibitors.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> As noted above, these lead
inhibitors bind to the flexible BL2 region and induce conformational
changes to block substrate access to the catalytic site of the enzyme.<sup><xref ref-type="bibr" rid="ref26">26</xref>,<xref ref-type="bibr" rid="ref28">28</xref></sup> There have been no MERS-PLpro inhibitors published to date; we have
identified one compound (<bold>4</bold>) that inhibits both SARS-PLpro
and MERS-PLpro. Our mode of inhibition studies with compound <bold>4</bold> were done with a series of increasing substrate concentrations
and enzyme-compound complexes (Figure <xref rid="fig5" ref-type="fig">5</xref>A and
B). The kinetic data were fit to four different enzyme inhibition
models (competitive, noncompetitive, uncompetitive, and mixed-type)
using the Sigmaplot Enzyme Kinetics Module. The best fit equation
was selected based on Akaike Information Criterion-corrected (AICc)
values.<sup><xref ref-type="bibr" rid="ref36">36</xref></sup> The equation with the lowest
AICc value corresponds to the best fit, and a minimum of a two AICc
unit difference from the next lowest is required to be considered
statistically significant. Interestingly, compound <bold>4</bold> exhibited
mixed-type inhibition for SARS-PLpro but competitive inhibition for
MERS-PLpro for the same substrate. The second best fit equation of
compound <bold>4</bold> with SARS-PLpro was noncompetitive inhibition
with 7.1 AICc values lower than the best fit (mixed-type) inhibition,
which was also 38.6 AICc values lower than competitive inhibition,
clearly indicating that compound <bold>4</bold> is an allosteric inhibitor
of SARS-PLpro. For MERS-PLpro, the AICc value of competitive inhibition
was 19.4 units lower than the next lowest, noncompetitive inhibition.
Because of the large AICc value differences from the next best fit
equations for both SARS and MERS-PLpro, it is clear that the same
compound acts as an allosteric inhibitor for SARS-PLpro and acts as
a competitive inhibitor for MERS-PLpro. Therefore, this compound inhibits
the two PLpro enzymes via two different inhibitory mechanisms even
though the inhibitory activities are similar, with IC<sub>50</sub> values of 10.9 μM (SARS-PLpro) and 6.2 μM (MERS-PLpro).
The <italic>K</italic><sub><italic>i</italic></sub> values of compound <bold>4</bold> against SARS-PLpro and MERS-PLpro are 11.5 μM and
7.6 μM, respectively (Figure <xref rid="fig5" ref-type="fig">5</xref>C).</p><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Mechanism of
inhibition. Dixon plots of compound <bold>4</bold> against SARS-PLpro
(A) and MERS-PLpro (B). (C) Summary table of
kinetic mode of inhibition of compound <bold>4</bold>. Mechanism of
enzyme inhibition of compound <bold>4</bold> was determined to be
a mixed inhibition for SARS-PLpro and a competitive inhibition for
MERS-PLpro. Determined <italic>K</italic><sub><italic>i</italic></sub> values of compound <bold>4</bold> were 11.5 μM and 7.5 μM
for SARS-PLpro and MERS-PLpro, respectively. (D) IC<sub>50</sub> value
comparison of four SARS-PLpro lead inhibitors in combination with
the newly identified compound <bold>4</bold> to determine if they
inhibit synergistically. (E) Bar graphs of the dissociation equilibrium
constants (<italic>K</italic><sub>D</sub>) of compound <bold>4</bold> in the absence (solid bars) and in the presence (striped bars) of
substrate determined by Surface Plasmon Resonance (SPR).</p></caption><graphic xlink:href="cb500917m_0005" id="gr5" position="float"></graphic></fig><p>Compound <bold>4</bold> appears to interact with
MERS-PLpro by
binding to the catalytic site since it is a competitive inhibitor
with respect to the substrate. However, we were uncertain where <bold>4</bold> might bind to SARS-PLpro since it is an allosteric inhibitor.
Our first hypothesis was that it may bind to the BL2 loop where two
other SARS-PLpro lead inhibitors (<bold>I-2</bold> and <bold>I-3</bold>) bound. We thus evaluated inhibition by <bold>4</bold> in the presence
of each of the four SARS-PLpro lead inhibitors (<bold>I-1</bold>–<bold>I-4</bold>) in order to see if it has an additive effect (Figure <xref rid="fig5" ref-type="fig">5</xref>D). If <bold>4</bold> binds to the same location
as <bold>I-2</bold> and <bold>I-3</bold>, their inhibitory activities
(IC<sub>50</sub> values) would not be improved by the addition of
compound <bold>4</bold>. But their IC<sub>50</sub> values should be
enhanced if compound <bold>4</bold> binds elsewhere. Inhibitory activities
of the four lead inhibitors alone varied from 0.23 μM to 2.26
μM, and their IC<sub>50</sub> values were enhanced up to almost
7-fold (0.034–0.67 μM) in the presence of <bold>4</bold> at a concentration of 10 μM, slightly lower than its IC<sub>50</sub> value. This led us to conclude that <bold>4</bold> binds
to an allosteric site of the SARS-PLpro other than the BL2 loop. In
addition to enzymatic mode of inhibition analysis, competition SPR
studies of compound <bold>4</bold> with SARS-PLpro and MERS-PLpro
were each performed in the presence and in the absence of the substrate.
Binding affinity of compound <bold>4</bold> to SARS-PLpro was the
same regardless of substrate presence, whereas that of compound <bold>4</bold> to MERS-PLpro was 4.5-fold weaker in the presence of the
substrate than <bold>4</bold> alone. These results indicate that the
substrate is competing with <bold>4</bold> for the same binding site
in MERS-PLpro (Figure <xref rid="fig5" ref-type="fig">5</xref>E). Therefore, both
the enzymatic mechanism of inhibition and SPR studies support compound <bold>4</bold> being an allosteric inhibitor for SARS-PLpro, while <bold>4</bold> is a competitive inhibitor for MERS-PLpro. In order to further
clarify this, cocrystallizations of both SARS-PLpro and MERS-PLpro
with compound <bold>4</bold> are currently in process.</p></sec><sec id="sec2.5"><title>Inhibitor Selectivity</title><p>A concern of potential nonspecificity
was raised due to the fact that our newly identified dual inhibitor
is a small fragment-like compound and also exhibited inhibitory activity
against SARS-CoV 3CLpro with an IC<sub>50</sub> value of 13.9 μM.<sup><xref ref-type="bibr" rid="ref37">37</xref></sup> X-ray crystallography and mode of inhibition
studies showed that <bold>4</bold> binds to the dimer interface of
the SARS-CoV 3CLpro, inhibiting its enzyme activity by breaking the
dimer since SARS-CoV 3CLpro is a functional dimer (unpublished data).
Therefore, compound <bold>4</bold> acts as an allosteric inhibitor
against both SARS-CoV proteases (3CLpro and PLpro), whereas it acts
as a competitive inhibitor against MERS-PLpro. We further investigated
the specificity of <bold>4</bold> and a lead inhibitor <bold>I-3</bold> (control) against two human cysteine proteases also called human
ubiquitin C-terminal hydrolases (hUCH-L1 and hUCH-L3) and two unrelated
enzymes (Hepatitis C Virus NS3 serine protease and <italic>Bacillus
anthracis</italic> dihydroorotase). The hUCH-L1 is one of the human
homologues most closely related to PLpro, which makes it an excellent
control to test the selectivity of a newly identified inhibitor. Structural
alignment of these two human homologues revealed that their catalytic
triads are very similar (Figure <xref rid="fig6" ref-type="fig">6</xref>A). Compound <bold>4</bold> was selective for SARS-PLpro and MERS-PLpro proteases over
the two human cysteine proteases and both unrelated enzymes (Figure <xref rid="fig6" ref-type="fig">6</xref>B).</p><fig id="fig6" position="float"><label>Figure 6</label><caption><p>Selectivity of compound <bold>4</bold>. (A) Structural
alignment
of MERS-PLpro with two human deubiquitinating enzymes. The aligned
catalytic triads of two human ubiquitin C-terminal hydrolases, hUCH-L1
(green, PDB: 2ETL)<sup><xref ref-type="bibr" rid="ref38">38</xref></sup> and hUCH-L3 (orange, PDB: 1UCH),<sup><xref ref-type="bibr" rid="ref39">39</xref></sup> are shown with that of MERS-PLpro (tan, PDB: 4RNA) in the expanded
box. (B) Selectivity of the confirmed hit compound <bold>4</bold>.
In addition to two human cysteine proteases (hUCH-L1 and hUCH-L3),
two unrelated enzymes, Hepatitis C Virus NS3 serine protease (NS3)
and <italic>Bacillus anthracis</italic> dihydroorotase (PyrC), were
also tested along with both PLpro enzymes.</p></caption><graphic xlink:href="cb500917m_0006" id="gr6" position="float"></graphic></fig></sec><sec id="sec2.6"><title>Active-Site Comparison and Oxyanion Hole Stabilization</title><p>The
cysteine/serine protease mechanism involves tetrahedral intermediate
formation, following nucleophilic attack by the cysteine/serine side
chain. Generally, there is an oxyanion close to the catalytic site,
which interacts with a negatively charged tetrahedral intermediate
to stabilize it. Ratia et al. demonstrated that the SARS-PLpro W107
located below the catalytic cysteine plays this vital role in forming
a hydrogen bond (H-bond) with an intermediate as an H-bond donor in
the active site by showing that the SARS-PLpro W107A mutant completely
lost catalytic activity.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> However, in
the MERS-PLpro active site, the equivalent position is occupied by
L106, which is not capable of being an H-bond donor (Figure <xref rid="fig7" ref-type="fig">7</xref>A). Lei et al. recently demonstrated that the L106W
mutation resulted in catalytic activity enhancement of MERS-PLpro,<sup><xref ref-type="bibr" rid="ref29">29</xref></sup> indicating that the MERS-PLpro oxyanion hole
may not be complete in comparison to that of SARS-PLpro. Interestingly,
the leucine residue at this position is highly conserved in three
bat coronaviruses (BtCoV-HKU4, BtCoV-HKU5, and BtCoV-133) that belong
to the same lineage group C as MERS-CoV (Figure <xref rid="fig7" ref-type="fig">7</xref>B). On the other hand, two human coronavirus (NL63 and 229E) PLpro
enzymes have a residue (Q or T) that can be an H-bond donor similar
to SARS-PLpro. Therefore, another residue must play this intermediate-stabilizing
function in MERS-PLpro. Asparagine (N110) in SARS-PLpro is highly
conserved among various coronavirus PLpro enzymes, and Ratia et al.
suggested that this residue could be another residue contributing
to the oxyanion hole stabilization in addition to W107.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> From the structural alignment of the active
site, we noted that the N109 of MERS-PLpro overlaps with N110 of SARS-PLpro.
This suggested that N109, located above the catalytic cysteine, might
be the residue that plays this critical role in MERS-PLpro. We hypothesized
two potential mechanisms: First, the side chain amine group of N109
could form an H-bond with the intermediate’s oxyanion as an
H-bond donor (Figure <xref rid="fig7" ref-type="fig">7</xref>C). Alternatively, the
carbonyl group of N109 could bind to a water molecule, followed by
the water forming another H-bond with the negatively charged intermediate
(Figure <xref rid="fig7" ref-type="fig">7</xref>D). The positions of N109 in these
two scenarios could differ. We generated two MERS-PLpro mutants, N109A
and N109D, to investigate these two hypotheses. Enzyme activity of
the N109A mutant was completely abolished, while the N109D mutant
exhibited only ∼13.8% of the wild-type MERS-PLpro activity
(Figure <xref rid="fig7" ref-type="fig">7</xref>E). This indicates that the N109 residue
is indeed crucial for stabilizing the intermediate for the enzyme
to perform its catalytic function. If N109 stabilized the intermediate
via the second hypothesis, the side chain of N109D could still form
an H-bond with a water molecule through the carbonyl group of aspartic
acid, rescuing the MERS-PLpro enzyme activity. However, the N109D
mutant also showed very low enzyme activity as compared to the wild-type,
suggesting that the second hypothesis is not likely to be the main
stabilization mechanism. This result suggests that N109 is a critical
residue for intermediate stabilization, most likely through an H-bond
formation with the side chain amine group of N109.</p><fig id="fig7" position="float"><label>Figure 7</label><caption><p>Active site analysis
of the MERS-PLpro. (A) Active site alignment
of MERS-PLpro (tan) and SARS-PLpro (cyan). The three catalytic triad
residues (C111, H278, and D293) of MERS-PLpro are aligned with the
SARS-PLpro catalytic triad (C112, H273 and D287). (B) Sequence alignment
of important residues near the catalytic triad between various CoV.
Residue numbers are shown for MERS-PLpro. (C) Potential mechanism
1 for oxyanion hole stabilization via N109. Active site and substrate
residues are shown in green and pink, respectively. (D) Potential
mechanism 2 for oxyanion hole stabilization via N109. (E) Enzyme activity
comparison between wild-type and two mutant MERS-PLpro enzymes.</p></caption><graphic xlink:href="cb500917m_0007" id="gr7" position="float"></graphic></fig><p>In addition to containing a crucial
residue that stabilizes the
oxyanion, the small loop (residues 101–108) next to the active
site in SARS-PLpro is important for its catalytic activity via controlling
active site access. The hydrogen bond between D109 from this loop
and W94 restrains the loop conformation, preventing it from moving
to block active site access.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> These two
residues (D108 and W93 in MERS-PLpro) are conserved in MERS-PLpro,
playing the same role as that of SARS-PLpro (Figure <xref rid="fig7" ref-type="fig">7</xref>B).</p></sec></sec><sec id="sec3"><title>Conclusion</title><p>SARS-CoV and MERS-CoV
cause contagious and highly virulent infectious
diseases in humans, threatening public health.<sup><xref ref-type="bibr" rid="ref10">10</xref>,<xref ref-type="bibr" rid="ref27">27</xref></sup> Both coronaviruses apparently originated from animal reservoirs
such as bats or camels but surprisingly have rapidly evolved to human-to-human
transmission, although limited cases have been reported for MERS-CoV.<sup><xref ref-type="bibr" rid="ref11">11</xref></sup> SARS-CoV has been contained by public health
measures since 2003, but MERS-CoV has spread into 12 different countries
so far, and the numbers of infections continue to rise. There is currently
no specific treatment or vaccine available.</p><p>In this study, we
determined that none of the tested SARS-PLpro
lead inhibitors were effective against MERS-PLpro. Thorough structural
comparison between these two PLpro enzymes using all available structures
revealed crucial structural differences, providing insights for developing
inhibitors against PLpro. The overall MERS-PLpro structure is similar
to that of SARS-PLpro including the N-terminal Ubl-domain. However,
the flexible BL2 loop of MERS-PLpro differs significantly from that
of SARS-PLpro, which raised the possibility of differing roles in
inhibitor binding. This may explain the observation that all of the
tested SARS-PLpro lead inhibitors were ineffective against MERS-PLpro.
It was surprising to discover that SARS-PLpro has a deubiquitinating
function,<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> and now it has been shown that
MERS-PLpro also exhibits the same function.<sup><xref ref-type="bibr" rid="ref22">22</xref>,<xref ref-type="bibr" rid="ref23">23</xref></sup> We determined the catalytic activity of MERS-PLpro in direct comparison
with that of SARS-PLpro. The catalytic efficiency (<italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>M</sub>) of SARS-PLpro was
∼45-fold higher (8.2 × 10<sup>5</sup> M<sup>–1</sup> s<sup>–1</sup>) than that of MERS-PLpro (1.9 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>). Although the
deubiquitinating activity of MERS-PLpro is lower than SARS-PLpro,
it is still much more active than two closely related human homologues
of PLpro, herpes-associated ubiquitin-specific protease (HAUSP) and
ubiquitin-specific protease 14 (USP14), which exhibit catalytic efficiencies
of 2.2 × 10<sup>3</sup> M<sup>–1</sup> s<sup>–1</sup> and 107 M<sup>–1</sup> s<sup>–1</sup>, respectively.<sup><xref ref-type="bibr" rid="ref40">40</xref></sup></p><p>We performed HTS of 25 000 compounds
against both PLpro
enzymes, and identified a dual noncovalent inhibitor that was active
against both PLpro enzymes. Interestingly, this inhibitor was determined
to be a competitive inhibitor against MERS-PLpro, whereas it was an
allosteric inhibitor against SARS-PLpro. These results suggest that
inhibitor recognition specificity of MERS-PLpro may differ from that
of SARS-PLpro even though the overall structures of the whole protein
and the catalytic sites are very similar. The most probable contributing
factor for inhibitor selectivity of these two PLpro enzymes could
be attributed to the structural differences of the BL2 loop.</p></sec><sec id="sec4"><title>Materials and Methods</title><p>Details
about cloning, expression, and purification; crystallization,
confirmation assay, and IC<sub>50</sub> value determination by dose
response curve; and reversibility of inhibition are provided in the <xref rid="notes-4" ref-type="notes">SI Materials and Methods</xref>.</p><sec id="sec4.1"><title>Primary High-Throughput
Screening</title><p>The 25 000-compound
Life Chemicals library was screened against the two PLpro cysteine
proteases from SARS-CoV and MERS-CoV. All assays against SARS-PLpro
were done in duplicate, and those against MERS-PLpro were done in
a single pass in black 384-well plates (Matrix Technologies). The
SARS-PLpro enzyme (20 nM final concentration) was prepared in an assay
buffer (50 mM HEPES, pH 7.5, 0.01% Triton X-100 (v/v), 0.1 mg mL<sup>–1</sup> BSA, and 2 mM GSH). The MERS-PLpro enzyme (400 nM
final concentration) was prepared in the same assay buffer with 5
mM DTT in place of 2 mM GSH. A total of 30 μL of enzyme solution
was dispensed into wells, and then 200 nL of 10 mM compounds (50 μM
final concentrations) was added and incubated for 5 min. Enzyme reactions
were initiated with 10 μL of substrate Z-Arg-Leu-Arg-Gly-Gly-AMC
(Bachem Bioscience; 50 μM and 75 μM for SARS- and MERS-PLpro,
respectively) dissolved in the assay buffer and incubated for 6 min,
followed by adding 10 μL of 10% SDS (w/v) as a stop solution.
Fluorescence intensity was monitored at 360 nm (excitation) and 450
nm (emission).</p></sec><sec id="sec4.2"><title>Determination of Dissociation Equilibrium
Constant (<italic>K</italic><sub>D</sub>) by SPR</title><p>Compound
solutions with a
series of increasing concentrations (0–200 μM at 1.5-fold
dilution) were applied to all four channels at a 30 μL/min flow
rate. Sensorgrams were analyzed using the Biacore T200 evaluation
software 2.0, and response units were measured during the equilibration
phase at each concentration. Each PLpro enzyme was immobilized on
a CM5 sensor chip using standard amine-coupling with running buffer
HBS-P (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P-20, pH 7.4) using
a Biacore T200 instrument. The MERS-PLpro enzyme was immobilized to
flow channels 2 and 3, and immobilization levels of flow channels
2 and 3 were ∼16 900 RU and ∼16 700 RU,
respectively. SARS-PLpro was immobilized to flow channel 4 at the
immobilization level of ∼14 600 RU to be compared with
MERS-PLpro. Data were referenced with blank (enthanolamine) RU values.
SigmaPlot 12.0 was used to fit the data to a single rectangular hyperbolic
curve to determine <italic>K</italic><sub>D</sub> values. The hyperbolic, <italic>y</italic> = <italic>y</italic><sub>max</sub>·<italic>x</italic>/(<italic>K</italic><sub>D</sub> + <italic>x</italic>), was used
to plot response units and corresponding concentration, where <italic>y</italic> is the response, <italic>y</italic><sub>max</sub> is the
maximum response, and <italic>x</italic> is the compound concentration.</p></sec><sec id="sec4.3"><title>Mechanism of Inhibition</title><p>Enzyme activities of both MERS-PLpro
and SARS-PLpro were monitored in the same way as the primary screen
with varying concentrations of inhibitors and substrates (0–300
μM). The concentration of compounds was varied from 0 to at
least 10× the IC<sub>50</sub> value of each compound. The data
were fit to four equations (shown in <xref rid="notes-4" ref-type="notes">SI Materials
and Methods</xref>) using SigmaPlot Enzyme Kinetics Module 1.3 in
order to determine the best fit inhibition mechanism and kinetic parameters
for each compound.</p></sec><sec id="sec4.4"><title>Inhibitor Selectivity Assay</title><p>To test
for selectivity,
two human ubiquitin C-terminal hydrolases (UCH-L1 and UCH-L3) and
two unrelated enzymes (Hepatitis C Virus NS3 serine protease and <italic>B. anthracis</italic> dihydroorotase) were tested with the top hit
compound from HTS and a lead SARS-PLpro inhibitor (<bold>I-1</bold>) using a fluorometric assay. The fluorogenic substrate used in this
study was ubiquitin-AMC (Boston Biochem). All assays were performed
in 384-well black plates (Corning) in a total volume of 24 μL
of the assay buffer containing 50 mM HEPES (pH 7.5), 5 mM DTT, 0.1
mg mL<sup>–1</sup> BSA, and 0.01% Triton X-100 (v/v) in triplicate.
A series of compound concentrations (0 to 200 μM final concentration
at 2-fold serial dilution) in 100% DMSO was prepared in a 384-well
plate. Then 3× compound solutions were prepared in the assay
buffer prior to assays. A total of 8 μL of each enzyme solution
was distributed into wells, and 8 μL of varying concentrations
of compounds was added and incubated for 10 min. The enzyme reaction
was initiated by adding 8 μL of the substrate (50 μM final
concentration), and fluorescence intensity was continuously monitored
at excitation/emission wavelengths of 350 nm/460 nm for 10 min.</p></sec><sec id="sec4.5"><title>X-ray Data Collection, Processing, and Structure Solvation</title><p>Data were collected at the LS-CAT end station 21-ID-F at the Advanced
Photon Source, Argonne National Laboratory, using a wavelength of
λ = 0.97872 Å, and the crystal at 100 K under a dry liquid
nitrogen stream. Data were recorded by a MAR CCD 225 mm detector with
an oscillation angle of 1.0° using a total of 190 frames. Data
were processed and scaled by XDS.<sup><xref ref-type="bibr" rid="ref41">41</xref></sup> The
crystal space group belonged to C2, containing one monomer in the
asymmetric unit. The Matthews coefficient (<italic>V</italic><sub>M</sub>) was calculated as 2.5, and solvent content was estimated
to be 50%. Molecular replacement was carried out using Phaser<sup><xref ref-type="bibr" rid="ref42">42</xref></sup> from the CCP4 package. The SARS-PLpro crystal
structure (2FE8)<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> was used as a search
model. The zinc binding domain in the initial model was truncated
and manually rebuilt by Coot.<sup><xref ref-type="bibr" rid="ref43">43</xref></sup> Structural
refinement was conducted using Refmac5.5.<sup><xref ref-type="bibr" rid="ref44">44</xref></sup></p></sec></sec></body><back><notes id="notes-4" notes-type="si"><title>Supporting Information Available</title><p>Additional Materials and Methods,
Tables S1 and S2, Figures S1–S3, and NMR Spectra and MS analysis.
This material is available free of charge via the Internet.</p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="cb500917m_si_001.pdf"><caption><p>cb500917m_si_001.pdf</p></caption></media></supplementary-material></sec><notes notes-type="accession-codes" id="notes-2"><title>Accession Codes</title><p>4PT5 (unbound
MERS-PLpro at 2.5 Å resolution), 4RNA (unbound MERS-PLpro at
1.8 Å resolution)</p></notes><notes notes-type="" id="notes-1"><title>Author Contributions</title><p><sup>§</sup> Equal contributors.</p></notes><notes notes-type="" id="notes-5"><title>Author Contributions</title><p>H. Lee performed
all experiments with the assistance of J.L.G., K.P., M.Z.S., and I.O.
H. Lei and B.D.S. solved the MERS-PLpro structure. S.C. performed
computational studies. A.K.G. synthesized current SARS-PLpro lead
compounds. H. Lee, H. Lei, and M.E.J. designed the experiments, and
H. Lee, H. Lei, S.C., A.J.R., and M.E.J. wrote the manuscript.</p></notes><notes notes-type="COI-statement" id="NOTES-d391e1224-autogenerated"><p>The authors declare
no
competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>This work was supported in part by National Institutes of
Health Grants R56 AI089535. We thank K. Ratia for performing HTS and
primary screening data analysis and J. Ren for computational assistance.
A.J.R. was supported during a portion of this work by NIDCR T32-DE018381,
UIC College of Dentistry, MOST program. This work used the Extreme
Science and Engineering Discovery Environment (XSEDE), which is supported
by National Science Foundation, grant number OCI-1053575. Use of the
APS, an Office of Science User Facility operated for the U.S. Department
of Energy (DOE) Office of Science by Argonne National Laboratory,
was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.
We thank ChemAxon for a free academic license of their cheminformatics
suite including JChem and JChem for excel for HTS data analysis.</p></ack><glossary id="dl1"><def-list><title>Abbreviations</title><def-item><term>MERS-CoV</term><def><p>Middle East Respiratory Syndrome
coronavirus</p></def></def-item><def-item><term>HCoV-EMC</term><def><p>human coronavirus-Erasmus Medical Center</p></def></def-item><def-item><term>SARS-CoV</term><def><p>Severe Acute Respiratory Syndrome</p></def></def-item><def-item><term>3CLpro</term><def><p>3C-like protease</p></def></def-item><def-item><term>PLpro</term><def><p>papain-like protease</p></def></def-item><def-item><term>nsp</term><def><p>nonstructural protein</p></def></def-item><def-item><term>CFR</term><def><p>case-fatality rate</p></def></def-item><def-item><term>BL2</term><def><p>blocking loop 2</p></def></def-item><def-item><term>HTS</term><def><p>high-throughput
screening</p></def></def-item><def-item><term>SPR</term><def><p>surface
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