Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro)
Identifieur interne : 000D85 ( Pmc/Corpus ); précédent : 000D84; suivant : 000D86Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro)
Auteurs : Sakshi Tomar ; Melanie L. Johnston ; Sarah E. St. John ; Heather L. Osswald ; Prasanth R. Nyalapatla ; Lake N. Paul ; Arun K. Ghosh ; Mark R. Denison ; Andrew D. MesecarSource :
- The Journal of Biological Chemistry [ 0021-9258 ] ; 2015.
Abstract
Url:
DOI: 10.1074/jbc.M115.651463
PubMed: 26055715
PubMed Central: 4528106
Links to Exploration step
PMC:4528106Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Ligand-induced Dimerization of Middle East Respiratory Syndrome
(MERS) Coronavirus nsp5 Protease (3CL<sup>pro</sup>
)</title>
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<author><name sortKey="Johnston, Melanie L" sort="Johnston, Melanie L" uniqKey="Johnston M" first="Melanie L." last="Johnston">Melanie L. Johnston</name>
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<author><name sortKey="St John, Sarah E" sort="St John, Sarah E" uniqKey="St John S" first="Sarah E." last="St. John">Sarah E. St. John</name>
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<author><name sortKey="Osswald, Heather L" sort="Osswald, Heather L" uniqKey="Osswald H" first="Heather L." last="Osswald">Heather L. Osswald</name>
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<author><name sortKey="Nyalapatla, Prasanth R" sort="Nyalapatla, Prasanth R" uniqKey="Nyalapatla P" first="Prasanth R." last="Nyalapatla">Prasanth R. Nyalapatla</name>
<affiliation><nlm:aff id="aff2">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
</affiliation>
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<author><name sortKey="Paul, Lake N" sort="Paul, Lake N" uniqKey="Paul L" first="Lake N." last="Paul">Lake N. Paul</name>
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<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">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
</affiliation>
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<author><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R." last="Denison">Mark R. Denison</name>
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</affiliation>
<affiliation><nlm:aff id="aff2">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
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<idno type="doi">10.1074/jbc.M115.651463</idno>
<date when="2015">2015</date>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Ligand-induced Dimerization of Middle East Respiratory Syndrome
(MERS) Coronavirus nsp5 Protease (3CL<sup>pro</sup>
)</title>
<author><name sortKey="Tomar, Sakshi" sort="Tomar, Sakshi" uniqKey="Tomar S" first="Sakshi" last="Tomar">Sakshi Tomar</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
</author>
<author><name sortKey="Johnston, Melanie L" sort="Johnston, Melanie L" uniqKey="Johnston M" first="Melanie L." last="Johnston">Melanie L. Johnston</name>
<affiliation><nlm:aff id="aff2">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
</affiliation>
</author>
<author><name sortKey="St John, Sarah E" sort="St John, Sarah E" uniqKey="St John S" first="Sarah E." last="St. John">Sarah E. St. John</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
</author>
<author><name sortKey="Osswald, Heather L" sort="Osswald, Heather L" uniqKey="Osswald H" first="Heather L." last="Osswald">Heather L. Osswald</name>
<affiliation><nlm:aff id="aff2">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Nyalapatla, Prasanth R" sort="Nyalapatla, Prasanth R" uniqKey="Nyalapatla P" first="Prasanth R." last="Nyalapatla">Prasanth R. Nyalapatla</name>
<affiliation><nlm:aff id="aff2">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Paul, Lake N" sort="Paul, Lake N" uniqKey="Paul L" first="Lake N." last="Paul">Lake N. Paul</name>
<affiliation><nlm:aff id="aff3"></nlm:aff>
</affiliation>
</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">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R." last="Denison">Mark R. Denison</name>
<affiliation><nlm:aff id="aff4"></nlm:aff>
</affiliation>
</author>
<author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Chemistry, Purdue University, West Lafayette, Indiana 47907,</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">The Journal of Biological Chemistry</title>
<idno type="ISSN">0021-9258</idno>
<idno type="eISSN">1083-351X</idno>
<imprint><date when="2015">2015</date>
</imprint>
</series>
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<front><div type="abstract" xml:lang="en"><p><bold>Background:</bold>
3CL<sup>pro</sup>
protease is required for coronaviral
polyprotein processing and is only active as a dimer.</p>
<p><bold>Results:</bold>
MERS-CoV 3CL<sup>pro</sup>
is a weakly associated dimer
requiring ligand binding for dimer formation.</p>
<p><bold>Conclusion:</bold>
Ligand-induced dimerization is a key mechanism for
regulating the enzymatic activity of MERS-CoV 3CL<sup>pro</sup>
during
polyprotein processing.</p>
<p><bold>Significance:</bold>
Activation via ligand-induced dimerization may add
complexity for the development of MERS-CoV 3CL<sup>pro</sup>
inhibitors as
antivirals.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id>
<journal-id journal-id-type="iso-abbrev">J. Biol. Chem</journal-id>
<journal-id journal-id-type="publisher-id">J. Biol. Chem</journal-id>
<journal-id journal-id-type="hwp">jbc</journal-id>
<journal-id journal-id-type="pmc">jbc</journal-id>
<journal-id journal-id-type="publisher-id">JBC</journal-id>
<journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title>
</journal-title-group>
<issn pub-type="ppub">0021-9258</issn>
<issn pub-type="epub">1083-351X</issn>
<publisher><publisher-name>American Society for Biochemistry and Molecular
Biology</publisher-name>
<publisher-loc>11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110,
U.S.A.</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">26055715</article-id>
<article-id pub-id-type="pmc">4528106</article-id>
<article-id pub-id-type="publisher-id">M115.651463</article-id>
<article-id pub-id-type="doi">10.1074/jbc.M115.651463</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Protein Structure and Folding</subject>
</subj-group>
</article-categories>
<title-group><article-title>Ligand-induced Dimerization of Middle East Respiratory Syndrome
(MERS) Coronavirus nsp5 Protease (3CL<sup>pro</sup>
)</article-title>
<subtitle>IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS<xref ref-type="fn" rid="FN1">*</xref>
</subtitle>
<alt-title alt-title-type="short">Ligand-induced Dimerization Regulates MERS-CoV
3CL<sup>pro</sup>
</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Tomar</surname>
<given-names>Sakshi</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
<xref ref-type="author-notes" rid="FN2"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Johnston</surname>
<given-names>Melanie L.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>St. John</surname>
<given-names>Sarah E.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Osswald</surname>
<given-names>Heather L.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Nyalapatla</surname>
<given-names>Prasanth R.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Paul</surname>
<given-names>Lake N.</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>¶</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ghosh</surname>
<given-names>Arun K.</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Denison</surname>
<given-names>Mark R.</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>‖</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Mesecar</surname>
<given-names>Andrew D.</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
<xref ref-type="corresp" rid="cor1"><sup>2</sup>
</xref>
</contrib>
<aff id="aff1">From the Departments of<label>‡</label>
Biological Sciences and</aff>
<aff id="aff2"><label>§</label>
Chemistry, Purdue University, West Lafayette, Indiana 47907,</aff>
<aff id="aff3">the<label>¶</label>
Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, and</aff>
<aff id="aff4">the<label>‖</label>
Departments of Pediatrics and Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>2</label>
To whom correspondence should be addressed:
<addr-line>Dept. of Biological Sciences, Purdue University, 915 W. State
St., West Lafayette, IN 47907.</addr-line>
Tel.:
<phone>765-494-1924</phone>
; E-mail:
<email>amesecar@purdue.edu</email>
.</corresp>
<fn fn-type="supported-by" id="FN2"><label>1</label>
<p>Supported by a grant from the Purdue Research Foundation.</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><day>7</day>
<month>8</month>
<year>2015</year>
</pub-date>
<pub-date pub-type="epub"><day>8</day>
<month>6</month>
<year>2015</year>
</pub-date>
<volume>290</volume>
<issue>32</issue>
<fpage>19403</fpage>
<lpage>19422</lpage>
<history><date date-type="received"><day>11</day>
<month>3</month>
<year>2015</year>
</date>
<date date-type="rev-recd"><day>3</day>
<month>6</month>
<year>2015</year>
</date>
</history>
<permissions><copyright-statement>© 2015 by The American Society for Biochemistry and
Molecular Biology, Inc.</copyright-statement>
<copyright-year>2015</copyright-year>
<license><license-p>This article is made available via the PMC Open Access Subset for
unrestricted 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 COVID-19 pandemic or until permissions are revoked in
writing. Upon expiration of these permissions, PMC is granted a perpetual
license to make this article available via PMC and Europe PMC, consistent
with existing copyright protections.</license-p>
</license>
</permissions>
<self-uri xlink:title="pdf" xlink:type="simple" xlink:href="zbc03215019403.pdf"></self-uri>
<abstract abstract-type="teaser"><p><bold>Background:</bold>
3CL<sup>pro</sup>
protease is required for coronaviral
polyprotein processing and is only active as a dimer.</p>
<p><bold>Results:</bold>
MERS-CoV 3CL<sup>pro</sup>
is a weakly associated dimer
requiring ligand binding for dimer formation.</p>
<p><bold>Conclusion:</bold>
Ligand-induced dimerization is a key mechanism for
regulating the enzymatic activity of MERS-CoV 3CL<sup>pro</sup>
during
polyprotein processing.</p>
<p><bold>Significance:</bold>
Activation via ligand-induced dimerization may add
complexity for the development of MERS-CoV 3CL<sup>pro</sup>
inhibitors as
antivirals.</p>
</abstract>
<abstract><p>All coronaviruses, including the recently emerged Middle East respiratory
syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the
proteolytic activity of the nsp5 protease (also known as 3C-like protease,
3CL<sup>pro</sup>
) during virus replication, making it a high value target
for the development of anti-coronavirus therapeutics. Kinetic studies indicate
that in contrast to 3CL<sup>pro</sup>
from other β-CoV 2c members,
including HKU4 and HKU5, MERS-CoV 3CL<sup>pro</sup>
is less efficient at
processing a peptide substrate due to MERS-CoV 3CL<sup>pro</sup>
being a weakly
associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL<sup>pro</sup>
enzymes
are tightly associated dimers. Analytical ultracentrifugation studies support
that MERS-CoV 3CL<sup>pro</sup>
is a weakly associated dimer
(<italic>K<sub>d</sub>
</italic>
∼52 μ<sc>m</sc>
) with a
slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL<sup>pro</sup>
were
synthesized and utilized in analytical ultracentrifugation experiments and
demonstrate that MERS-CoV 3CL<sup>pro</sup>
undergoes significant ligand-induced
dimerization. Kinetic studies also revealed that designed reversible inhibitors
act as activators at a low compound concentration as a result of induced
dimerization. Primary sequence comparisons and x-ray structural analyses of two
MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal
remarkable structural similarity of the dimer interface with 3CL<sup>pro</sup>
from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial
differences in the dimerization ability suggest that long range interactions by
the nonconserved amino acids distant from the dimer interface may control
MERS-CoV 3CL<sup>pro</sup>
dimerization. Activation of MERS-CoV
3CL<sup>pro</sup>
through ligand-induced dimerization appears to be unique
within the genogroup 2c and may potentially increase the complexity in the
development of MERS-CoV 3CL<sup>pro</sup>
inhibitors as antiviral agents.</p>
</abstract>
<kwd-group><kwd>analytical ultracentrifugation</kwd>
<kwd>enzyme inactivation</kwd>
<kwd>enzyme inhibitor</kwd>
<kwd>enzyme kinetics</kwd>
<kwd>viral protease</kwd>
<kwd>X-ray crystallography</kwd>
<kwd>β-CoV</kwd>
<kwd>MERS-CoV 3CLpro</kwd>
<kwd>ligand-induced dimerization</kwd>
<kwd>monomer-dimer equilibrium</kwd>
</kwd-group>
<funding-group><award-group><funding-source id="CS100">National Institutes of Health</funding-source>
<award-id rid="CS100">AI08508</award-id>
<award-id rid="CS100">AI026603</award-id>
<award-id rid="CS100">P30 CA023168</award-id>
</award-group>
</funding-group>
</article-meta>
</front>
<body><sec sec-type="intro"><title>Introduction</title>
<p>Coronaviruses (CoVs)<xref ref-type="fn" rid="FN3"><sup>3</sup>
</xref>
are enveloped,
positive-strand RNA viruses that infect a variety of vertebrates, including bats,
livestock, pets, poultry, and humans (<xref rid="B1" ref-type="bibr">1</xref>
<xref ref-type="bibr" rid="B2">–</xref>
<xref rid="B3" ref-type="bibr">3</xref>
). Although human CoVs cause respiratory illnesses of mild to moderate
severity (<xref rid="B4" ref-type="bibr">4</xref>
<xref ref-type="bibr" rid="B5">–</xref>
<xref rid="B9" ref-type="bibr">9</xref>
), two recently emerged
CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East
respiratory syndrome coronavirus (MERS-CoV), have demonstrated their potential to
become a serious threat to public health. MERS-CoV emerged late in 2012, and unlike
its predecessor SARS-CoV, MERS-CoV continues to exhibit up to a 35% fatality rate
(<xref rid="B10" ref-type="bibr">10</xref>
<xref ref-type="bibr" rid="B11">–</xref>
<xref rid="B12" ref-type="bibr">12</xref>
).</p>
<p>Based on the sequence analysis of seven genes of the replicase domain, MERS-CoV has
been classified as a β-CoV genogroup 2c member, along with closely related
bat coronaviruses HKU5 (<italic>Pipistrellus</italic>
bat) and HKU4
(<italic>Tylonycteris</italic>
bat) (<xref rid="B13" ref-type="bibr">13</xref>
,
<xref rid="B14" ref-type="bibr">14</xref>
). Increasing evidence suggests that
bats may serve as zoonotic reservoirs for MERS-CoV (<xref rid="B15" ref-type="bibr">15</xref>
, <xref rid="B16" ref-type="bibr">16</xref>
). Evidence presented by
recent studies also supports the local zoonotic transmission of MERS-CoV from
dromedary camels to humans (<xref rid="B17" ref-type="bibr">17</xref>
, <xref rid="B18" ref-type="bibr">18</xref>
). Alarmingly, human-to-human transmission
during close contact, especially in elderly or patients with underlying health
conditions, has also been reported for MERS-CoV (<xref rid="B19" ref-type="bibr">19</xref>
<xref ref-type="bibr" rid="B20">–</xref>
<xref rid="B22" ref-type="bibr">22</xref>
). In the wake of the recent upsurge in the
laboratory-confirmed cases of MERS-CoV, including two recently identified cases in
the United States (<xref rid="B23" ref-type="bibr">23</xref>
), there is an urgent
need to study and characterize the properties of important drug targets of MERS-CoV
for the development of effective therapeutics.</p>
<p>Coronaviruses express a >800-kDa replicase polyprotein, which is processed by
viral 3CL<sup>pro</sup>
protease (or nsp5) at 11 distinct cleavage sites to yield
intermediate and mature nonstructural proteins (nsp) responsible for many aspects of
virus replication (<xref rid="B3" ref-type="bibr">3</xref>
, <xref rid="B24" ref-type="bibr">24</xref>
<xref ref-type="bibr" rid="B25">–</xref>
<xref rid="B26" ref-type="bibr">26</xref>
). Because of its indispensable role in the
virus life cycle, 3CL<sup>pro</sup>
is an important target for therapeutic
intervention against coronavirus infections (<xref rid="B27" ref-type="bibr">27</xref>
<xref ref-type="bibr" rid="B28">–</xref>
<xref rid="B33" ref-type="bibr">33</xref>
).</p>
<p>A number of kinetic, biophysical, and x-ray structural studies have demonstrated that
SARS-CoV 3CL<sup>pro</sup>
is only active <italic>in vitro</italic>
as a tightly
associated dimer with a dimer dissociation constant (<italic>K<sub>d</sub>
</italic>
)
in the low nanomolar range (<xref rid="B34" ref-type="bibr">34</xref>
<xref ref-type="bibr" rid="B35">–</xref>
<xref rid="B38" ref-type="bibr">38</xref>
). The addition or deletion of amino acids, <italic>e.g.</italic>
His<sub>6</sub>
affinity tags, at either the N or C terminus drastically reduces
the enzymatic rate and decreases the ability of SARS-CoV 3CL<sup>pro</sup>
to
dimerize (<xref rid="B37" ref-type="bibr">37</xref>
). Although cellular evidence for
the auto-cleavage mechanism (<italic>cis versus trans</italic>
) of 3CL<sup>pro</sup>
is lacking, models for how 3CL<sup>pro</sup>
cleaves itself from the polyprotein to
form the mature dimer have been proposed based on <italic>in vitro</italic>
studies
using purified 3CL<sup>pro</sup>
(<xref rid="B34" ref-type="bibr">34</xref>
, <xref rid="B39" ref-type="bibr">39</xref>
, <xref rid="B40" ref-type="bibr">40</xref>
).
A current model posits that two inactive 3CL<sup>pro</sup>
molecules within two
separate polyproteins recognize each other and form an immature dimer capable of
cleaving the nsp4↓nsp5 and nsp5↓nsp6 sites in
<italic>trans</italic>
, followed by formation of an active and mature dimer that can
then rapidly process other cleavage sites and multiple polyproteins. It has also
been proposed that substrate-induced dimerization regulates the enzymatic activity
of SARS-CoV 3CL<sup>pro</sup>
during virus replication; however, no experimental
evidence of this has ever been demonstrated in infected cells (<xref rid="B40" ref-type="bibr">40</xref>
). Although our knowledge of SARS-CoV 3CL<sup>pro</sup>
is extensive, the dimerization properties of 3CL<sup>pro</sup>
from MERS-CoV and
other coronaviruses, as well as the factors regulating their enzymatic activity,
remain largely unknown.</p>
<p>To understand the properties of MERS-CoV 3CL<sup>pro</sup>
, we conducted a series of
kinetic, biophysical and x-ray structural studies. Here, we report a detailed
kinetic and biophysical analysis of MERS-CoV 3CL<sup>pro</sup>
activity and
dimerization. These kinetic and biophysical studies provide evidence for a weakly
associated MERS-CoV 3CL<sup>pro</sup>
dimer. In addition, we utilized our previous
knowledge on the design of potent SARS-CoV 3CL<sup>pro</sup>
peptidic inhibitors to
design a series of inhibitors of MERS-CoV 3CL<sup>pro</sup>
that exhibit low
micromolar potency. We demonstrate that MERS-CoV 3CL<sup>pro</sup>
requires the
binding of a ligand for dimer formation, indicating that ligand-induced dimerization
is likely a key mechanism in the regulation of MERS-CoV 3CL<sup>pro</sup>
activity
during virus infection.</p>
</sec>
<sec sec-type="methods"><title>Experimental Procedures</title>
<sec><title></title>
<sec><title></title>
<sec><title>Construct Design and Expression of MERS-CoV 3CL<sup>pro</sup>
</title>
<p>The gene encoding 3CL<sup>pro</sup>
protease of MERS-CoV (amino acid
residues 3248–3553 in the replicase polyprotein,
GenBank<sup>TM</sup>
accession number <ext-link ext-link-type="gen" xlink:href="AHC74086.1">AHC74086.1</ext-link>
) was codon-optimized
for optimal expression in <italic>E. coli</italic>
(BioBasic Inc). The
gene was subcloned into pET-11a expression vector with an N-terminal
His<sub>6</sub>
tag followed by the nsp4↓nsp5 auto-cleavage
site using the forward primer
5′-ATATACATATGCACCACCACCACCACCACAGCGGTGTTCTGCAGTCTGGTC-3′
and the reverse primer
5′-GACGGATCCTTACTGCATCACAACACCCATGATCTGC-3′. The
construct was verified by DNA sequencing at the Purdue University
Genomics Core Facility. This construct results in the expression of
MERS-CoV 3CL<sup>pro</sup>
without any N- or C-terminal extensions.
MERS-CoV 3CL<sup>pro</sup>
was expressed through auto-induction in
<italic>Escherichia coli</italic>
BL21-DE3 cells in the presence of
100 μg/ml carbenicillin as described previously (<xref rid="B41" ref-type="bibr">41</xref>
). Cells were harvested by centrifugation
at 5000 × <italic>g</italic>
for 20 min at 4 °C, and the
pellets were stored at −80 °C until further use.</p>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Purification</title>
<p>Frozen pellets from 4 liters of bacterial cell culture were thawed on ice
and resuspended in 250 ml of Buffer A (20 m<sc>m</sc>
Tris, pH 7.5, 0.05
m<sc>m</sc>
EDTA, 10% glycerol, and 5 m<sc>m</sc>
β-mercaptoethanol (BME)), containing 500 μg of lysozyme
and a small amount of DNase. Cells were then lysed using a single pass
through a French press at 1200 p.s.i., and cell debris was removed from
the cleared lysate by centrifuging at 29,000 ×
<italic>g</italic>
for 30 min. Solid ammonium sulfate was added to
the cleared lysate to a final concentration of 1 <sc>m</sc>
through
gradual mixing on ice.</p>
</sec>
<sec><title>Hydrophobic Interaction Chromatography</title>
<p>The cleared lysate, mixed with ammonium sulfate, was loaded at a flow
rate of 3 ml/min onto a 60-ml phenyl-Sepharose 6 fast-flow high-sub
column (Amersham Biosciences) equilibrated with Buffer B (50 m<sc>m</sc>
Tris, pH 7.5, 1 <sc>m</sc>
ammonium sulfate, 0.05 m<sc>m</sc>
EDTA, 10%
glycerol, and 5 m<sc>m</sc>
BME). The column was then washed with
5× column volume (300 ml) of Buffer B at a flow rate of 4
ml/min. Protein was eluted using a 5× column volume (300 ml)
linear gradient to 100% Buffer A. Fractions (12 ml) were collected, and
those containing MERS-CoV 3CL<sup>pro</sup>
, as judged through SDS-PAGE
analysis and specific activity measurements, were pooled (120 ml) and
exchanged into 2 liters of Buffer A via overnight dialysis in a 10,000
molecular weight cutoff SnakeSkin® dialysis tubing (Thermo
Scientific).</p>
</sec>
<sec><title>DEAE Anion-exchange Chromatography</title>
<p>Dialyzed sample from the previous step was loaded at a flow rate of 3
ml/min onto a 120- ml DEAE anion-exchange column (Amersham Biosciences)
equilibrated with Buffer A. The column was then washed with 2×
column volume (240 ml) of Buffer A at a flow rate of 4 ml/min. A linear
gradient (total volume 480 ml) to 40% Buffer C (50 m<sc>m</sc>
Tris, pH
7.5, 1 <sc>m</sc>
NaCl, 0.05 m<sc>m</sc>
EDTA, 10% glycerol, and 5
m<sc>m</sc>
BME) was used to elute the protein. Fractions (6 ml)
were collected, and those containing MERS-CoV 3CL<sup>pro</sup>
were
pooled (66 ml) and dialyzed for 4 h in 4 liters of Buffer D (20
m<sc>m</sc>
MES, pH 5.5, 0.05 m<sc>m</sc>
EDTA, 10% glycerol, and 5
m<sc>m</sc>
BME).</p>
</sec>
<sec><title>Mono S Cation-exchange Chromatography</title>
<p>Following dialysis, the pH of the sample was manually adjusted to 5.5
using 1 <sc>m</sc>
solution of MES, pH 5.5, and any precipitated protein
was removed by filtering through a 0.22-μm pore size Millex-GP
filter (Millipore). The filtered sample was then loaded at a flow rate
of 2 ml/min onto an 8-ml Mono S 10/100 column (Amersham Biosciences)
equilibrated in Buffer D. The column was then washed with 5×
column volume (40 ml) of Buffer D at a flow rate of 2 ml/min. Protein
was eluted using a 25× column volume (200 ml) and a linear
gradient to 50% Buffer E (50 m<sc>m</sc>
MES, pH 5.5, 1 <sc>m</sc>
NaCl,
0.05 m<sc>m</sc>
EDTA, 10% glycerol, and 5 m<sc>m</sc>
BME). Fractions
(2 ml) were collected, and those containing MERS-CoV 3CL<sup>pro</sup>
were pooled (22 ml) and concentrated to ∼5 mg/ml.</p>
</sec>
<sec><title>Gel Filtration Chromatography</title>
<p>As the final purification step, the concentrated protein sample was
loaded onto the preparation grade Superdex 75 26/60 gel filtration
column (Amersham Biosciences) equilibrated with Buffer F (25 m<sc>m</sc>
HEPES, pH 7.5, 10% glycerol, 2.5 m<sc>m</sc>
dithiothreitol (DTT)).
Protein was eluted isocratically at a flow rate of 1 ml/min with Buffer
F. Fractions (2 ml) containing MERS-CoV 3CL<sup>pro</sup>
were pooled
(total volume of 34 ml) and concentrated to ∼5 mg/ml. For final
storage of the purified MERS-CoV 3CL<sup>pro</sup>
enzyme,
300-μl protein aliquots were placed into 1-ml screw-cap vials,
flash-frozen under liquid nitrogen, and then stored at −80
°C until further use.</p>
</sec>
<sec><title>Purification of SARS-CoV, HKU4-CoV, and HKU5-CoV
3CL<sup>pro</sup>
</title>
<p>SARS-CoV 3CL<sup>pro</sup>
and HKU5-CoV 3CL<sup>pro</sup>
with authentic
N and C termini were expressed and purified as described previously
(<xref rid="B37" ref-type="bibr">37</xref>
, <xref rid="B42" ref-type="bibr">42</xref>
). HKU4-CoV 3CL<sup>pro</sup>
was purified
utilizing a modified protocol from Ref. <xref rid="B42" ref-type="bibr">42</xref>
. Final protein yield was calculated based on the
measurement of total activity units (μ<sc>m</sc>
product/min),
specific activity (units/mg), and milligrams of protein obtained
(Bio-Rad protein assay) after each chromatographic step.</p>
</sec>
<sec><title>Synthesis of Compounds <bold>1</bold>
–<bold>11</bold>
</title>
<p>The peptidomimetic compounds with Michael acceptor groups (compounds
<bold>1</bold>
-<bold>9</bold>
, <xref rid="T3" ref-type="table">Table
3</xref>
) were synthesized via very similar methods to those
published previously (<xref rid="B30" ref-type="bibr">30</xref>
, <xref rid="B43" ref-type="bibr">43</xref>
). Synthesis of noncovalent
peptidomimetic compounds <bold>10</bold>
and <bold>11</bold>
(<xref rid="T3" ref-type="table">Table 3</xref>
) has been described
previously (<xref rid="B33" ref-type="bibr">33</xref>
).</p>
</sec>
<sec><title>Fluorescence-based Kinetic Assays</title>
<p>The enzymatic activity of 3CL<sup>pro</sup>
was measured using the
following custom-synthesized peptide:
HilyteFluor<sup>TM</sup>
-488-ESATLQSGLRKAK-(QXL<sup>TM</sup>
-520)-NH<sub>2</sub>
(AnaSpec, Inc.). The HilyteFluor<sup>TM</sup>
-488 fluorescence group was
internally quenched by QXL<sup>TM</sup>
-520 dye. This substrate works as
a generic peptide substrate for 3CL<sup>pro</sup>
enzymes and was
designed based on the nsp4↓nsp5 cleavage sequence for many
coronavirus 3CL<sup>pro</sup>
enzymes. The rate of enzymatic activity
was determined at 25 °C by following the increase in
fluorescence (λ<sub>excitation</sub>
= 485 nm,
λ<sub>emission</sub>
= 528 nm, bandwidths = 20 nm) of Hilyte
Fluor<sup>TM</sup>
-488 upon peptide hydrolysis by the enzyme as a
function of time. Assays were conducted in black, half-area, 96-well
plates (Corning Glass) in assay buffer (50 m<sc>m</sc>
HEPES, pH 7.5,
0.1 mg/ml BSA, 0.01% Triton X-100, and 2 m<sc>m</sc>
DTT) using a final
reaction volume of 100 μl. The resulting florescence was
monitored using a BioTek Synergy H1 plate reader. The rate of the
reaction in arbitrary fluorescence units/s (AFU/s) was determined by
measuring the initial slope of the progress curves, which were then
converted to units of micromolars of product produced per min
(μ<sc>m</sc>
/min) using experimentally determined values of
fluorescence “extinction coefficient” as described
previously (<xref rid="B37" ref-type="bibr">37</xref>
). All reactions
were carried out in triplicate.</p>
</sec>
<sec><title>Determination of Enzymatic Efficiency</title>
<p>The apparent enzymatic efficiency for each of the 3CL<sup>pro</sup>
enzymes was determined by measuring the rate of enzymatic activity as a
function of varying substrate concentration in 100-μl reactions.
Reactions were initiated by the addition of enzyme to the wells of an
assay plate containing varying concentrations of substrate. The final
substrate concentrations varied over a range from 0 to 2
μ<sc>m</sc>
. The final enzyme concentrations for each
3CL<sup>pro</sup>
studied were as follows: MERS-CoV
3CL<sup>pro</sup>
at 1 μ<sc>m</sc>
, SARS-CoV
3CL<sup>pro</sup>
at 100 n<sc>m</sc>
, HKU5-CoV 3CL<sup>pro</sup>
at
250 n<sc>m</sc>
, and HKU4-CoV 3CL<sup>pro</sup>
at 200 n<sc>m</sc>
.
Because 3CL<sup>pro</sup>
enzymes cannot be saturated with this
substrate at a substrate concentration that would still allow accurate
fluorescent measurements without the inner filter effect, only the
apparent <italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
</italic>
values can be determined from the slope of the line that results from a
plot of the enzymatic activity (<italic>y</italic>
axis), normalized for
the total enzyme concentration, against the substrate concentration
(<italic>x</italic>
axis).</p>
</sec>
<sec><title>Influence of Dimerization on the Activity of 3CL<sup>pro</sup>
Enzymes</title>
<p>The dependence of the enzymatic activity on the total enzyme
concentration was determined using the FRET-based assay described above.
The final enzyme concentrations were varied over a concentration range
from 2 μ<sc>m</sc>
to 100 n<sc>m</sc>
for MERS-CoV
3CL<sup>pro</sup>
, 500 to 10 n<sc>m</sc>
for SARS-CoV
3CL<sup>pro</sup>
, 250 to 0.6 n<sc>m</sc>
for HKU5-CoV
3CL<sup>pro</sup>
, and 200 to 10 n<sc>m</sc>
for HKU4-CoV
3CL<sup>pro</sup>
. Reactions were initiated by the addition of
substrate, at a final concentration of 2 μ<sc>m</sc>
, to the
assay plates containing varying enzyme concentrations in the assay
buffer. Initial rates were determined from the initial slopes of the
progress curves at each enzyme concentration.</p>
<p>The rates of the 3CL<sup>pro</sup>
-catalyzed reactions measured over a
range of enzyme concentrations can be fit to either <xref ref-type="disp-formula" rid="FD1">Equation 1</xref>
or <xref ref-type="disp-formula" rid="FD2">2</xref>
to determine the values
of the dissociation constant for the monomer-dimer equilibrium as well
as the turnover numbers. Nonlinear regression and the program TableCurve
2D version 4.0 were used to fit the data to either <xref ref-type="disp-formula" rid="FD1">Equation 1</xref>
or <xref ref-type="disp-formula" rid="FD2">2</xref>
below (<xref rid="B44" ref-type="bibr">44</xref>
). <disp-formula id="FD1"><graphic xlink:href="zbc03215-2168-m01.jpg" mimetype="image" position="float" orientation="portrait"></graphic>
</disp-formula>
In <xref ref-type="disp-formula" rid="FD1">Equation 1</xref>
,
<italic>V</italic>
<sub>max</sub>
is the rate of the enzymatic
activity calculated at each enzyme concentration
(<italic>C<sub>T</sub>
</italic>
); <italic>K<sub>d</sub>
</italic>
is
the monomer-dimer equilibrium dissociation constant, and
<italic>k</italic>
<sub>cat,</sub>
<italic><sub>M</sub>
</italic>
and <italic>k</italic>
<sub>cat,</sub>
<italic><sub>D</sub>
</italic>
are the turnover numbers for the monomer
and the dimer, respectively. <disp-formula id="FD2"><graphic xlink:href="zbc03215-2168-m02.jpg" mimetype="image" position="float" orientation="portrait"></graphic>
</disp-formula>
In <xref ref-type="disp-formula" rid="FD2">Equation 2</xref>
,
<italic>V</italic>
<sub>max</sub>
, <italic>C<sub>T</sub>
</italic>
,
and <italic>K<sub>d</sub>
</italic>
have been described previously, and
<italic>k</italic>
<sub>cat</sub>
is the turnover number for the
dimer only.</p>
</sec>
<sec><title>Inhibition Assays</title>
<p>To determine the percent inhibition for compounds
<bold>1–9</bold>
, the total concentration of the substrate
was fixed at 1.0 μ<sc>m</sc>
, and the enzymes were fixed at 250
n<sc>m</sc>
for SARS-CoV 3CL<sup>pro</sup>
, HKU5-CoV
3CL<sup>pro</sup>
, HKU4-CoV 3CL<sup>pro</sup>
, and at 500
n<sc>m</sc>
for MERS-CoV 3CL<sup>pro</sup>
. DMSO stocks
(100×) of the compounds were diluted a hundred-fold to a final
concentration of 50 μ<sc>m</sc>
in 80 μl of the enzyme
solution and incubated for 20 min. After 20 min, the enzymatic activity
was measured as initial slope of the progress curve, obtained by
initiating the reaction with 20 μl of 5 μ<sc>m</sc>
substrate. % inhibition was calculated using <xref ref-type="disp-formula" rid="FD3">Equation 3</xref>
. <disp-formula id="FD3"><graphic xlink:href="zbc03215-2168-m03.jpg" mimetype="image" position="float" orientation="portrait"></graphic>
</disp-formula>
In <xref ref-type="disp-formula" rid="FD3">Equation 3</xref>
,
rate<sub>sample</sub>
is the initial slope of the progress curve in
AFU/s measured in the presence of the compound; rate<sub>pos</sub>
is
the initial slope measured in the absence of any compound, and
rate<sub>neg</sub>
is the baseline substrate hydrolysis calculated
in the absence of enzyme. All the reactions were carried out in
triplicate and contained a final DMSO concentration of 1%. For compounds
displaying more than 50% inhibition, a more extensive characterization
of the inactivation kinetics was performed through progress curve
analysis. To the reaction well, 20 μl of 5 μ<sc>m</sc>
substrate was added to a final concentration of 1 μ<sc>m</sc>
,
and the total inhibitor concentration
[<italic>I</italic>
]<sub>total</sub>
was varied from 0 to 50
μ<sc>m</sc>
. The reaction was initiated with the addition of
80 μl of MERS-CoV 3CL<sup>pro</sup>
to a final concentration of
500 n<sc>m</sc>
. Fluorescence intensity was then measured over time as
AFU<italic><sub>t</sub>
</italic>
for a period of 70 min. <xref ref-type="disp-formula" rid="FD4">Equation 4</xref>
describes the
resulting time course of reaction. <disp-formula id="FD4"><graphic xlink:href="zbc03215-2168-m04.jpg" mimetype="image" position="float" orientation="portrait"></graphic>
</disp-formula>
In <xref ref-type="disp-formula" rid="FD4">Equation 4</xref>
,
<italic>v<sub>i</sub>
</italic>
is the initial velocity of the
reaction; <italic>k</italic>
<sub>obs</sub>
is the observed first-order
rate constant for the reaction in the absence and presence of inhibitor;
<italic>t</italic>
is the time in minutes;
[<italic>P</italic>
]<italic><sub>t</sub>
</italic>
is the
concentration of product produced at time <italic>t</italic>
, and
[<italic>P</italic>
]<italic><sub>i</sub>
</italic>
is the initial
product concentration, which is zero. Product concentrations were
calculated from the values of AFU<italic><sub>t</sub>
</italic>
, using
the experimentally determined fluorescence extinction coefficient. The
resulting values of [<italic>P</italic>
]<italic><sub>t</sub>
</italic>
were then plotted against time <italic>t,</italic>
and the data were fit
to <xref ref-type="disp-formula" rid="FD4">Equation 4</xref>
with
[<italic>P</italic>
]<italic><sub>i</sub>
</italic>
= 0 using the
nonlinear regression program TableCurve 2D to derive the fitted
parameters <italic>v<sub>i</sub>
</italic>
and
<italic>k</italic>
<sub>obs</sub>
and their associated errors
Δ<italic>v<sub>i</sub>
</italic>
and
Δ<italic>k</italic>
<sub>obs</sub>
.</p>
<p>Values for each <italic>k</italic>
<sub>obs</sub>
were then plotted
against [<italic>I</italic>
]<sub>total</sub>
and the data were fit to
<xref ref-type="disp-formula" rid="FD5">Equation 5</xref>
.
<disp-formula id="FD5"><graphic xlink:href="zbc03215-2168-m05.jpg" mimetype="image" position="float" orientation="portrait"></graphic>
</disp-formula>
In <xref ref-type="disp-formula" rid="FD5">Equation 5</xref>
,
<italic>k</italic>
<sub>inact</sub>
defines the maximum rate of
inactivation at infinite inhibitor concentration, and
<italic>K<sub>I</sub>
</italic>
defines the concentration of
inhibitor that yields a rate of inactivation equal to
½<italic>k</italic>
<sub>inact</sub>
. The half-life of
inactivation at infinite inhibitor concentration, which is a measure of
inactivation efficiency, is defined as
<italic>t</italic>
<sub>½</sub>
<sup>∞</sup>
=
0.693/<italic>k</italic>
<sub>inact</sub>
.</p>
</sec>
<sec><title>AUC Analysis</title>
<p>To determine the oligomeric state of MERS-CoV 3CL<sup>pro</sup>
,
sedimentation velocity experiments were performed at 20 °C on
the Beckman-Coulter XLA ultracentrifuge using varying concentrations of
MERS-CoV 3CL<sup>pro</sup>
(4–23 μ<sc>m</sc>
) in 25
m<sc>m</sc>
HEPES, pH 7.5, 50 m<sc>m</sc>
NaCl, and 1 m<sc>m</sc>
tris(2-carboxyethyl)phosphine at 50,000 rpm. To characterize the effect
of the ligand on the monomer-dimer equilibrium of MERS-CoV
3CL<sup>pro</sup>
, sedimentation velocity experiments were conducted
on the Beckman-Coulter XLI instrument using different stoichiometric
ratios of MERS-CoV 3CL<sup>pro</sup>
with compounds <bold>6</bold>
and
<bold>10</bold>
. Samples were prepared by mixing 25
μ<sc>m</sc>
MERS-CoV 3CL<sup>pro</sup>
with 25, 50, and 100
μ<sc>m</sc>
compound <bold>6</bold>
or <bold>10</bold>
and
incubating the mixture overnight at 4 °C before performing the
experiments. Absorbance optics (280 nm) and interference optics were
utilized for protein detection. Solvent density, viscosity, and partial
specific volumes were calculated using SEDNTERP. SEDPHAT was used to fit
the data to the monomer-dimer self-association model to estimate the
sedimentation coefficients (<italic>s</italic>
), apparent molecular
weights, and <italic>K<sub>d</sub>
</italic>
and
<italic>k</italic>
<sub>off</sub>
values from size distribution
analysis. To obtain exact molecular weights, sedimentation equilibrium
experiments were performed at concentrations of 3 and 17
μ<sc>m</sc>
MERS-CoV 3CL<sup>pro</sup>
. The experiments were
done at 20 °C utilizing a two-channel centerpiece and run at
multiple speeds (8100, 13,800 and 24,000 rpm) in a AN-60 Ti rotor.</p>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Activation and Inhibition by a Noncovalent
Inhibitor</title>
<p>The rates of the MERS-CoV 3CL<sup>pro</sup>
-catalyzed reactions were
determined at final enzyme concentrations of 0.5, 1.0, and 2.0
μ<sc>m</sc>
and in the absence and presence of varying
concentrations (0.1–60 μ<sc>m</sc>
) of compound
<bold>10</bold>
. The substrate concentration was fixed at 2.0
μ<sc>m</sc>
. DMSO stocks (100×) of compound
<bold>10</bold>
were diluted a hundred-fold in 80 μl of
enzyme solution and incubated for 10 min. At the same time, a
zero-inhibitor control reaction was set up by mixing DMSO to a final
concentration of 1% into 80 μl of enzyme solution. After 10 min,
the rate of the enzymatic activity was measured as the initial slope of
the progress curve, obtained by initiating the reaction with 20
μl of 10 μ<sc>m</sc>
substrate. <xref ref-type="disp-formula" rid="FD6">Equation 6</xref>
was utilized to
calculate the percent activity. <disp-formula id="FD6"><graphic xlink:href="zbc03215-2168-m06.jpg" mimetype="image" position="float" orientation="portrait"></graphic>
</disp-formula>
The rate<sub>sample</sub>
,
rate<sub>pos</sub>
, and rate<sub>neg</sub>
are as described above
for <xref ref-type="disp-formula" rid="FD3">Equation 3</xref>
.</p>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Crystallization, X-ray Data Collection,
and Structure Determination</title>
<p>Purified MERS-CoV 3CL<sup>pro</sup>
was concentrated to 1.6 mg/ml in 25
m<sc>m</sc>
HEPES, pH 7.5, and 2.5 m<sc>m</sc>
DTT. Inhibitor
complexes of MERS-CoV 3CL<sup>pro</sup>
with compounds <bold>6</bold>
and <bold>11</bold>
were formed by incubating MERS-CoV 3CL<sup>pro</sup>
with the compounds in a 1:3 stoichiometric ratio at 4 °C
overnight. After iterative rounds of optimization of the crystallization
conditions based on the initial hits obtained from high throughput
screening of Qiagen Nextel Screens, crystals of MERS-CoV
3CL<sup>pro</sup>
inhibitor complexes suitable for x-ray diffraction
were grown by the hanging-drop, vapor diffusion method at 20 °C
in 0.2 <sc>m</sc>
sodium acetate, 0.1 <sc>m</sc>
BisTris, pH 7.0, and
20% PEG-3350 for the MERS-CoV 3CL<sup>pro</sup>
and <bold>6</bold>
complex, and 0.2 <sc>m</sc>
ammonium acetate, 0.1 <sc>m</sc>
BisTris, pH
5.5, 12% PEG-3350 for the MERS-CoV 3CL<sup>pro</sup>
and <bold>11</bold>
complex. For x-ray data collection, crystals were flash-cooled in liquid
nitrogen after dragging the crystals through a cryo-solution that
contained the crystallization solution supplemented with 15%
2-methyl-2,4-pentanediol.</p>
<p>X-ray diffraction data were collected for MERS-CoV 3CL<sup>pro</sup>
and
<bold>6</bold>
and MERS-CoV 3CL<sup>pro</sup>
and <bold>11</bold>
complexes at the Lilly Research Laboratories Collaborative Access Team
(LRL-CAT) Sector 31 and the Life Sciences Collaborative Access Team
(LS-CAT) Sector 21 at the Advanced Photon Source, Argonne National
Laboratory, respectively. Data were processed and scaled using Mosflm
version 7.0.5 (<xref rid="B45" ref-type="bibr">45</xref>
) and HKL2000
version 706 (<xref rid="B46" ref-type="bibr">46</xref>
). The method of
molecular replacement was used to obtain initial phases using the
program PHASER-MR in Phenix suite version 1.8.4 (<xref rid="B47" ref-type="bibr">47</xref>
). For MERS-CoV 3CL<sup>pro</sup>
and
<bold>6</bold>
complex, the x-ray structure of SARS-CoV
3CL<sup>pro</sup>
(PDB code <ext-link ext-link-type="pdb" xlink:href="3V3M">3V3M</ext-link>
) was used as a phasing model
(<xref rid="B32" ref-type="bibr">32</xref>
). The final MERS-CoV
3CL<sup>pro</sup>
and <bold>6</bold>
complex structure was then used
to calculate the initial phases for the MERS-CoV 3CL<sup>pro</sup>
and
<bold>11</bold>
complex model. Automated model building using
Autobuild in Phenix was initially used to build a preliminary model of
the MERS-CoV 3CL<sup>pro</sup>
and <bold>6</bold>
inhibitor complex.
Each structure was then refined using iterative cycles of refinement
using Phenix Refine coupled to manual model building using COOT (<xref rid="B48" ref-type="bibr">48</xref>
) based on
<italic>F<sub>o</sub>
</italic>
−
<italic>F<sub>c</sub>
</italic>
and 2<italic>F<sub>o</sub>
</italic>
− <italic>F<sub>c</sub>
</italic>
maps. Coordinates and molecular
library files for inhibitor molecules were built using the program eLBOW
in the Phenix suite. Water molecules were added to peaks in residual
(<italic>F<sub>o</sub>
</italic>
−
<italic>F<sub>c</sub>
</italic>
) density maps that were greater than
3σ using the “Find Water” function in COOT.
MolProbity was used to assess structural quality of the final model
(<xref rid="B49" ref-type="bibr">49</xref>
). The measured structure
factor amplitudes and the atomic coordinates for the final structures
were deposited in the Protein Data Bank with accession codes <ext-link ext-link-type="pdb" xlink:href="4RSP">4RSP</ext-link>
(MERS-CoV
3CL<sup>pro</sup>
and <bold>6</bold>
complex) and <ext-link ext-link-type="pdb" xlink:href="4YLU">4YLU</ext-link>
(MERS-CoV
3CL<sup>pro</sup>
and <bold>11</bold>
complex), respectively.
Structural superposition was performed using the method of least squares
fitting of C-α atoms in COOT. PyMOL was used to generate figures
of all the structures (<xref rid="B50" ref-type="bibr">50</xref>
).</p>
</sec>
</sec>
</sec>
</sec>
<sec sec-type="results"><title>Results</title>
<sec><title></title>
<sec><title></title>
<sec><title>Production of MERS-CoV 3CL<sup>pro</sup>
with Authentic N and C
Termini</title>
<p>Insertion of the nsp4↓nsp5 cleavage site between the N-terminal
His<sub>6</sub>
tag and the coding region for MERS-CoV
3CL<sup>pro</sup>
results in autoprocessing of the His tag and
overexpression of MERS-CoV 3CL<sup>pro</sup>
without any N-terminal
extension in <italic>E. coli</italic>
BL21-DE3 cells. MERS-CoV
3CL<sup>pro</sup>
was purified to high purity and an overall yield
of 10% using four sequential chromatographic steps. A summary of the
percent enzyme yield, total activity units, and the fold-purification
after each chromatographic step is summarized in <xref rid="T1" ref-type="table">Table 1</xref>
. Approximately 12 mg of highly pure MERS-CoV
3CL<sup>pro</sup>
can be obtained per liter of bacterial cell
culture.</p>
<table-wrap id="T1" orientation="portrait" position="float"><label>TABLE 1</label>
<caption><p><bold>Purification summary of MERS-CoV 3CL<sup>pro</sup>
per
liter of <italic>E.coli</italic>
BL21-DE3 cells</bold>
</p>
</caption>
<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="1" colspan="1">Sample</th>
<th align="center" rowspan="1" colspan="1">Protein</th>
<th align="center" rowspan="1" colspan="1">Total activity
units</th>
<th align="center" rowspan="1" colspan="1">Specific
activity</th>
<th align="center" rowspan="1" colspan="1">Fold
purification</th>
<th align="center" rowspan="1" colspan="1">% yield</th>
</tr>
</thead>
<tbody valign="top"><tr><td rowspan="1" colspan="1"></td>
<td align="center" rowspan="1" colspan="1"><italic>mg</italic>
</td>
<td rowspan="1" colspan="1"></td>
<td align="center" rowspan="1" colspan="1"><italic>units/mg</italic>
</td>
<td rowspan="1" colspan="1"></td>
<td rowspan="1" colspan="1"></td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Lysate</td>
<td align="left" rowspan="1" colspan="1">1102</td>
<td align="left" rowspan="1" colspan="1">1168</td>
<td align="left" rowspan="1" colspan="1">1</td>
<td align="left" rowspan="1" colspan="1">1</td>
<td align="left" rowspan="1" colspan="1">100</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Phenyl-Sepharose</td>
<td align="left" rowspan="1" colspan="1">219</td>
<td align="left" rowspan="1" colspan="1">185</td>
<td align="left" rowspan="1" colspan="1">1</td>
<td align="left" rowspan="1" colspan="1">1</td>
<td align="left" rowspan="1" colspan="1">16</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">DEAE</td>
<td align="left" rowspan="1" colspan="1">22</td>
<td align="left" rowspan="1" colspan="1">189</td>
<td align="left" rowspan="1" colspan="1">8</td>
<td align="left" rowspan="1" colspan="1">8</td>
<td align="left" rowspan="1" colspan="1">16</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Mono S</td>
<td align="left" rowspan="1" colspan="1">15</td>
<td align="left" rowspan="1" colspan="1">142</td>
<td align="left" rowspan="1" colspan="1">9</td>
<td align="left" rowspan="1" colspan="1">9</td>
<td align="left" rowspan="1" colspan="1">12</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">Superdex 75</td>
<td align="left" rowspan="1" colspan="1">12</td>
<td align="left" rowspan="1" colspan="1">114</td>
<td align="left" rowspan="1" colspan="1">10</td>
<td align="left" rowspan="1" colspan="1">10</td>
<td align="left" rowspan="1" colspan="1">10</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>To verify the production of the enzyme with correct N and C termini, the
molecular mass of purified MERS-CoV 3CL<sup>pro</sup>
was determined by
MALDI to be 33.4 kDa, which is close to the theoretical molecular mass
of 33.3 kDa for the authentic/mature MERS-CoV 3CL<sup>pro</sup>
monomer.
Western blot analysis of purified MERS-CoV 3CL<sup>pro</sup>
using an
anti-His<sub>6</sub>
antibody also confirmed the absence of the N
terminus His<sub>6</sub>
tag associated with the expression plasmid
(data not shown). These results demonstrate that the N-terminal
His<sub>6</sub>
tag is auto-catalytically removed by MERS-CoV
3CL<sup>pro</sup>
during its expression in <italic>E. coli</italic>
,
indicating MERS-CoV 3CL<sup>pro</sup>
is enzymatically active when
expressed in <italic>E. coli</italic>
.</p>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Hydrolyzes a Fluorescent Peptide Substrate
with Lower Efficiency than Other 3CL<sup>pro</sup>
Enzymes</title>
<p>A FRET-based peptide substrate was used to measure the enzymatic activity
of MERS-CoV 3CL<sup>pro</sup>
as a function of substrate concentration
over a substrate concentration range from 0 to 2.0 μ<sc>m</sc>
(<xref ref-type="fig" rid="F1">Fig. 1</xref>
<italic>A</italic>
). We
observed that MERS-CoV 3CL<sup>pro</sup>
cannot be saturated by the
substrate over this concentration range, which is typical for other
coronavirus 3CL<sup>pro</sup>
enzymes because the
<italic>K<sub>m</sub>
</italic>
values for peptide substrates
approach 1 m<sc>m</sc>
(<xref rid="B51" ref-type="bibr">51</xref>
<xref ref-type="bibr" rid="B52">–</xref>
<xref rid="B54" ref-type="bibr">54</xref>
). Therefore, the slope of the kinetic
response of MERS-CoV 3CL<sup>pro</sup>
to increasing substrate
concentration was determined to derive an apparent
(<italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
</italic>
)
value, which is a measure of enzymatic efficiency. We also determined
and compared the apparent
(<italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
</italic>
)
values for 3CL<sup>pro</sup>
enzymes from SARS-CoV, HKU5-CoV, and
HKU4-CoV under similar experimental conditions (<xref ref-type="fig" rid="F1">Fig. 1</xref>
<italic>B</italic>
). MERS-CoV
3CL<sup>pro</sup>
is able to hydrolyze the peptide substrate;
however, the enzymatic efficiency of MERS-CoV 3CL<sup>pro</sup>
(<italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
</italic>
=
3.1 ± 0.03 × 10<sup>−2</sup>
μ<sc>m</sc>
<sup>−1</sup>
min<sup>−1</sup>
)
is noticeably lower than other 3CL<sup>pro</sup>
enzymes tested.
Specifically, MERS-CoV 3CL<sup>pro</sup>
was 5-fold less efficient at
processing the peptide substrate when compared with SARS-CoV
3CL<sup>pro</sup>
. Even among the β-CoVs from the same 2c
genogroup (MERS, HKU5, and HKU4), MERS-CoV 3CL<sup>pro</sup>
was the
least efficient enzyme.</p>
<fig id="F1" orientation="portrait" position="float"><label>FIGURE 1.</label>
<caption><p><bold>Comparison of enzymatic efficiencies
(<italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
</italic>
)
of 3CL<sup>pro</sup>
enzymes from different CoVs.</bold>
<italic>A,</italic>
rates for the enzymatic activity, normalized
to the total enzyme concentration, are plotted as a function of
varying substrate concentrations. Total concentration of each
enzyme in the final reaction is as follows: MERS-CoV
3CL<sup>pro</sup>
at 1 μ<sc>m</sc>
; SARS-CoV
3CL<sup>pro</sup>
at 100 n<sc>m</sc>
; HKU5-CoV
3CL<sup>pro</sup>
at 250 n<sc>m;</sc>
and HKU4-CoV
3CL<sup>pro</sup>
at 200 n<sc>m</sc>
. Slope of the
<italic>line</italic>
represents the apparent value of
<italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
.
Error bars</italic>
represent the standard deviation for
triplicate data. <italic>B,</italic>
*, apparent value of
<italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
</italic>
for the nonsaturable substrate, calculated as the slope of the
linear plot from panel <italic>A</italic>
.</p>
</caption>
<graphic xlink:href="zbc0341521680001"></graphic>
</fig>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Is a Weakly Associated Dimer</title>
<p>Because a dimer has consistently been shown to be the catalytically
active form of all 3CL<sup>pro</sup>
enzymes studied to date, we tested
the hypothesis that the lower enzymatic efficiency of MERS-CoV
3CL<sup>pro</sup>
is a result of the reduction in its ability to
dimerize. Therefore, we determined the dependence of the enzymatic
activity of MERS-CoV 3CL<sup>pro</sup>
on the total enzyme concentration
and compared it with other 3CL<sup>pro</sup>
enzymes from HKU4, HKU5,
and SARS coronaviruses (<xref ref-type="fig" rid="F2">Fig.
2</xref>
).</p>
<fig id="F2" orientation="portrait" position="float"><label>FIGURE 2.</label>
<caption><p><bold>Dependence of the enzymatic activity of MERS-CoV, HKU4-CoV,
HKU5-CoV, and SARS-CoV 3CL<sup>pro</sup>
on the total enzyme
concentration.</bold>
<italic>A,</italic>
kinetic response of each CoV
3CL<sup>pro</sup>
to increasing enzyme concentration is
plotted along with the resulting fit of the data to <xref ref-type="disp-formula" rid="FD2">Equation 2</xref>
.
Resulting values for the apparent turnover number,
<italic>k</italic>
<sub>cat</sub>
, and the monomer-dimer
equilibrium constant, <italic>K<sub>d</sub>
</italic>
, are shown
in <xref rid="T2" ref-type="table">Table 2</xref>
. Final enzyme
concentrations varied over the concentration ranges of 2
μ<sc>m</sc>
to 100 n<sc>m</sc>
for MERS-CoV
3CL<sup>pro</sup>
, 500 to 10 n<sc>m</sc>
for SARS-CoV
3CL<sup>pro</sup>
, 250 to 0.6 n<sc>m</sc>
for HKU5-CoV
3CL<sup>pro</sup>
, and 200 to 10 n<sc>m</sc>
for HKU4-CoV
3CL<sup>pro</sup>
. Final substrate concentration was fixed
at 2 μ<sc>m</sc>
. Experiments were done in triplicate.
<italic>Error bars</italic>
represent the standard deviation
for triplicate data. <italic>Shaded box</italic>
represents the
data that are plotted in <italic>B. B,</italic>
enlarged view of
the fitted data at low total enzyme concentrations, marked in
<italic>shaded box</italic>
in <italic>A</italic>
,
illustrating the nonlinear dependence of enzymatic activity on
the total concentrations of 3CL<sup>pro</sup>
from SARS-CoV,
HKU5-CoV, and HKU4-CoV.</p>
</caption>
<graphic xlink:href="zbc0341521680002"></graphic>
</fig>
<p>It is immediately apparent from the data plotted in <xref ref-type="fig" rid="F2">Fig. 2</xref>
that the response of MERS-CoV
3CL<sup>pro</sup>
enzymatic activity to an increasing enzyme
concentration is nonlinear. The strong curvature suggests that a dimer
is either the most active form or the only active form of MERS-CoV
3CL<sup>pro</sup>
. To determine the mechanism of dimerization, the
data in <xref ref-type="fig" rid="F2">Fig. 2</xref>
were first fit to
<xref ref-type="disp-formula" rid="FD1">Equation 1</xref>
(see
“Experimental Procedures”), which describes a model
where both the monomer and the dimer are active. A fit of the data to
<xref ref-type="disp-formula" rid="FD1">Equation 1</xref>
yielded a
negative turnover value for the monomer
(<italic>k</italic>
<sub>cat,</sub>
<italic><sub>M</sub>
</italic>
), suggesting the monomer is inactive and
that the dimer is the only active form of the enzyme. Therefore, the
data were fit to <xref ref-type="disp-formula" rid="FD2">Equation
2</xref>
(see “Experimental Procedures”), which
considers only the dimer as the active form of the enzyme. The kinetic
data for all four 3CL<sup>pro</sup>
enzymes, MERS-CoV, HKU4-CoV,
HKU5-CoV, and SARS-CoV, fit well to this model, and the resulting values
for the monomer-dimer equilibrium dissociation constant,
<italic>K<sub>d</sub>
</italic>
, and apparent turnover number,
<italic>k</italic>
<sub>cat</sub>
, for each enzyme are provided in
<xref rid="T2" ref-type="table">Table 2</xref>
.</p>
<table-wrap id="T2" orientation="portrait" position="float"><label>TABLE 2</label>
<caption><p><bold>Comparison of the apparent turnover number,
<italic>k</italic>
<sub>cat</sub>
, and the monomer-dimer
dissociation constant, <italic>K<sub>d</sub>
</italic>
, for
3CL<sup>pro</sup>
from different CoVs</bold>
</p>
</caption>
<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="2" colspan="1">3CL<sup>pro</sup>
</th>
<th align="center" colspan="2" rowspan="1">Nonlinear fitting
of kinetic data<xref ref-type="table-fn" rid="TF2-1"><italic><sup>a</sup>
</italic>
</xref>
<hr></hr>
</th>
</tr>
<tr><th align="center" rowspan="1" colspan="1"><italic>k</italic>
<sub>cat</sub>
<xref ref-type="table-fn" rid="TF2-2"><italic><sup>b</sup>
</italic>
</xref>
</th>
<th align="center" rowspan="1" colspan="1"><italic>K<sub>d</sub>
</italic>
</th>
</tr>
</thead>
<tbody valign="top"><tr><td rowspan="1" colspan="1"></td>
<td align="center" rowspan="1" colspan="1"><italic>min</italic>
<sup>−<italic>1</italic>
</sup>
</td>
<td align="center" rowspan="1" colspan="1">μ<italic><sc>m</sc>
</italic>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">MERS-CoV</td>
<td align="left" rowspan="1" colspan="1">0.2 ±
0.02</td>
<td align="left" rowspan="1" colspan="1">7.8 ±
1.3</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">SARS-CoV</td>
<td align="left" rowspan="1" colspan="1">0.47 ±
0.03</td>
<td align="left" rowspan="1" colspan="1">0.06 ±
0.01</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">HKU5-CoV</td>
<td align="left" rowspan="1" colspan="1">0.53 ±
0.02</td>
<td align="left" rowspan="1" colspan="1">0.06 ±
0.01</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">HKU4-CoV</td>
<td align="left" rowspan="1" colspan="1">0.84 ±
0.07</td>
<td align="left" rowspan="1" colspan="1">0.1 ±
0.03</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="TF2-1"><p><italic><sup>a</sup>
</italic>
Values were determined through
nonlinear fitting of the kinetic data to <xref ref-type="disp-formula" rid="FD2">Equation 2</xref>
.</p>
</fn>
<fn id="TF2-2"><p><italic><sup>b</sup>
k</italic>
<sub>cat</sub>
represents the
apparent turnover number.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>The lower <italic>k</italic>
<sub>cat</sub>
value for MERS-CoV
3CL<sup>pro</sup>
, when compared with other coronavirus
3CL<sup>pro</sup>
enzymes, indicates a moderate reduction
(2–4-fold) in its ability to turn over the substrate, which is
consistent with the observed lower apparent
(<italic>k</italic>
<sub>cat</sub>
/<italic>K<sub>m</sub>
</italic>
)
value. In contrast, there is a substantial reduction in the ability of
MERS-CoV 3CL<sup>pro</sup>
to dimerize compared with the other
3CL<sup>pro</sup>
enzymes. Based on the
<italic>K<sub>d</sub>
</italic>
values, the capacity of MERS-CoV
3CL<sup>pro</sup>
to dimerize is ∼78–130-fold weaker
than the other enzymes (<xref rid="T2" ref-type="table">Table 2</xref>
).
These results indicate that the MERS-CoV 3CL<sup>pro</sup>
dimer is much
more weakly associated than the other coronavirus 3CL<sup>pro</sup>
enzymes studied, and these results raise questions as to the structural
and mechanistic differences among the 3CL<sup>pro</sup>
enzymes that
ultimately regulate protease activity during coronavirus
replication.</p>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Inhibition by Designed Peptidomimetic
Compounds</title>
<p>In an effort to develop potent inhibitors of MERS-CoV 3CL<sup>pro</sup>
,
we designed and synthesized nine peptidomimetic compounds containing a
Michael acceptor group, <italic>i.e.</italic>
an
α,β-unsaturated carbonyl, capable of irreversibly
reacting with the active site cysteine of MERS-CoV 3CL<sup>pro</sup>
(<xref rid="T3" ref-type="table">Table 3</xref>
). These compounds
were designed and synthesized based on our understanding and knowledge
of the interactions of similar inhibitor molecules with SARS-CoV
3CL<sup>pro</sup>
(<xref rid="B30" ref-type="bibr">30</xref>
, <xref rid="B31" ref-type="bibr">31</xref>
). At a concentration of 50
μ<sc>m</sc>
, compounds <bold>6–9</bold>
displayed
more than 50% inhibition of MERS-CoV 3CL<sup>pro</sup>
and were further
evaluated for their ability to inactivate the enzyme in a time- and
concentration-dependent manner (<xref ref-type="fig" rid="F3">Fig.
3</xref>
). Data from the kinetic progress curve for compound
<bold>6</bold>
(<xref ref-type="fig" rid="F3">Fig. 3</xref>
), as
well as for compounds <bold>7</bold>
-<bold>9</bold>
(data not shown),
were fit to the appropriate equations (see under “Experimental
Procedures”) to obtain the kinetic parameters,
<italic>k</italic>
<sub>inact</sub>
,
<italic>t</italic>
<sub>½</sub>
<sup>∞</sup>
, and
<italic>K<sub>I</sub>
</italic>
, and the resulting values are
provided in <xref rid="T3" ref-type="table">Table 3</xref>
.</p>
<table-wrap id="T3" orientation="portrait" position="float"><label>TABLE 3</label>
<caption><p><bold>Chemical structures and inhibitory activity of compounds 1
to 11 against MERS-CoV 3CL<sup>pro</sup>
</bold>
</p>
<p>The Michael acceptor group for compound 1 is shaded to highlight
this group for all the compounds. The stereochemistry at the
benzyl stereocenter of compound 5 is a 1:1 mixture of
enantiomers (racemic); therefore, the compound was tested as a
mixture of diastereomers.</p>
</caption>
<graphic xlink:href="zbc034152168t003"></graphic>
<table-wrap-foot><fn><p>* % inhibition was measured as the % loss in enzymatic
activity after 20 min of incubation of 500 n<sc>m</sc>
MERS-CoV 3CL<sup>pro</sup>
with 50 μ<sc>m</sc>
of
the compound.</p>
</fn>
<fn><p><italic><sup>a</sup>
</italic>
As compounds 1–5 showed
<50% inhibition of MERS-CoV 3CL<sup>pro</sup>
,
values of <italic>k</italic>
<sub>inact</sub>
,
<italic>t</italic>
<sub>1/2</sub>
<sup>∞</sup>
and
<italic>K<sub>I</sub>
</italic>
were not determined
(nd) for these compounds.</p>
</fn>
<fn><p><italic><sup>b</sup>
k</italic>
<sub>inact</sub>
is
×10<sup>−3</sup>
s<sup>−1</sup>
.</p>
</fn>
<fn><p><italic><sup>c</sup>
t</italic>
<sub>1/2</sub>
<sup>∞</sup>
is
× 10<sup>3</sup>
s.</p>
</fn>
<fn><p><italic><sup>d</sup>
K<sub>I</sub>
</italic>
is in
μ<sc>m</sc>
.</p>
</fn>
<fn><p><italic><sup>e</sup>
</italic>
IC<sub>50</sub>
values for
compounds 10 and 11 were calculated from a dose- response
curve determined after 10 min of incubation of 1
μ<sc>m</sc>
MERS-CoV 3CL<sup>pro</sup>
with
varying concentrations of compounds. IC<sub>50</sub>
is in
μ<sc>m</sc>
.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<fig id="F3" orientation="portrait" position="float"><label>FIGURE 3.</label>
<caption><p><bold>Progress curves for the MERS-CoV
3CL<sup>pro</sup>
-catalyzed reaction in the presence of
compound 6.</bold>
Time-dependent hydrolysis of 1
μ<sc>m</sc>
substrate catalyzed by 500 n<sc>m</sc>
MERS-CoV 3CL<sup>pro</sup>
was measured over a time period of 70
min and at fixed variable concentrations of compound
<bold>6</bold>
ranging from 0 to 50 μ<sc>m</sc>
.
Values for the inactivation kinetic parameters
<italic>k</italic>
<sub>inact</sub>
,
<italic>t</italic>
<sub>½</sub>
<sup>∞</sup>
,
and <italic>K<sub>I</sub>
</italic>
were calculated by fitting
the progress curve data to <xref ref-type="disp-formula" rid="FD4">Equations 4</xref>
and <xref ref-type="disp-formula" rid="FD5">5</xref>
. Chemical
structure of compound <bold>6</bold>
is shown in the
<italic>inset</italic>
.</p>
</caption>
<graphic xlink:href="zbc0341521680003"></graphic>
</fig>
<p>We identified four compounds, <bold>6–9</bold>
, as micromolar
inhibitors of MERS-CoV 3CL<sup>pro</sup>
with
<italic>K<sub>I</sub>
</italic>
values less than 10
μ<sc>m</sc>
(<xref rid="T3" ref-type="table">Table
3</xref>
). Analysis of structure-activity relationships of these
compounds suggests that the <italic>S</italic>
<sub>2</sub>
subsite
pocket of MERS-CoV 3CL<sup>pro</sup>
is small and can only accommodate a
smaller <italic>P</italic>
<sub>2</sub>
-isobutyl substituent (compounds
<bold>6</bold>
-<bold>9</bold>
) but not bigger substituents such as
<italic>P</italic>
<sub>2</sub>
-benzyl or
<italic>P</italic>
<sub>2</sub>
-isobutylenyl (compounds
<bold>1</bold>
-<bold>5</bold>
). It was also observed that replacing
the <italic>P</italic>
<sub>4</sub>
-ethoxy (compound <bold>6</bold>
) with
<italic>P</italic>
<sub>4</sub>
-isopropoxy (compounds <bold>7</bold>
and <bold>8</bold>
) had no effect on the inhibitory activity of the
compounds. Finally, these compounds provide an excellent chemical
scaffold to study the molecular details of interactions of
substrate-like compounds with the enzyme and to develop more potent
inhibitors of MERS-CoV 3CL<sup>pro</sup>
for therapeutic
intervention.</p>
<p>To evaluate broad spectrum specificity of these compounds, we also
calculated % inhibition of SARS-CoV 3CL<sup>pro</sup>
, HKU5-CoV
3CL<sup>pro</sup>
, and HKU4-CoV 3CL<sup>pro</sup>
after 20 min of
incubation in the presence of 50 μ<sc>m</sc>
compounds
<bold>6–9</bold>
. Except for compound <bold>9</bold>
, which
inhibited SARS-CoV 3CL<sup>pro</sup>
by 76%, we observed 100% inhibition
of all other enzymes in the presence of compounds
<bold>6</bold>
-<bold>9</bold>
. Furthermore, we performed progress
curve analysis of HKU5-CoV 3CL<sup>pro</sup>
and HKU4-CoV
3CL<sup>pro</sup>
in the presence of varying concentrations of
compounds <bold>6–9</bold>
. The <italic>K<sub>I</sub>
</italic>
values of compounds <bold>6–9</bold>
for HKU5-CoV
3CL<sup>pro</sup>
are 0.49 ± 0.16, 0.60 ± 0.21, 1.30
± 0.53, and 0.47 ± 0.06 μ<sc>m</sc>
,
respectively. The <italic>K<sub>I</sub>
</italic>
values of compounds
<bold>6–9</bold>
for HKU4-CoV 3CL<sup>pro</sup>
are 0.39
± 0.14, 0.50 ± 0.17, 0.85 ± 0.33, and 0.64
± 0.25 μ<sc>m</sc>
, respectively. These data suggest
that peptidomimetic compounds <bold>6–9</bold>
have the
potential to be developed as coronavirus 3CL<sup>pro</sup>
inhibitors
with broad spectrum specificity.</p>
</sec>
<sec><title>Weak Association of the MERS-CoV 3CL<sup>pro</sup>
Dimer Is Supported
by AUC Studies</title>
<p>To further explore the mechanism of MERS-CoV 3CL<sup>pro</sup>
dimerization, we performed analytical ultracentrifugation sedimentation
velocity (AUC-SV) studies at varying concentrations of MERS-CoV
3CL<sup>pro</sup>
(<xref ref-type="fig" rid="F4">Fig.
4</xref>
<italic>A</italic>
). Unlike enzyme kinetics, AUC allows
determination of the monomer-dimer equilibrium constant
(<italic>K<sub>d</sub>
</italic>
) in the absence of substrate.
MERS-CoV 3CL<sup>pro</sup>
displayed a continuous size distribution at
different protein concentrations. Two distinct peaks corresponding to
monomer (2.9 S) and dimer (3.9 S) species are observed, with the dimer
peak becoming more pronounced at higher enzyme concentrations (<xref ref-type="fig" rid="F4">Fig. 4</xref>
<italic>A</italic>
). We fit the
AUC data to a monomer-dimer equilibrium model to determine the values
for <italic>K<sub>d</sub>
</italic>
and <italic>k</italic>
<sub>off</sub>
,
where <italic>K<sub>d</sub>
</italic>
is the equilibrium dissociation
constant for a monomer from the dimer, and
<italic>k</italic>
<sub>off</sub>
is the rate constant for
dissociation of the monomer from the dimer. The resulting best fit value
for <italic>K<sub>d</sub>
</italic>
is 52 ± 5 μ<sc>m</sc>
and that for <italic>k</italic>
<sub>off</sub>
is 10<sup>−4</sup>
s<sup>−1</sup>
. The <italic>K<sub>d</sub>
</italic>
value of
52 μ<sc>m</sc>
for MERS 3CL<sup>pro</sup>
is dramatically
different from SARS-CoV 3CL<sup>pro</sup>
, which has reported
<italic>K<sub>d</sub>
</italic>
values ranging from low nanomolar
up to 10 μ<sc>m</sc>
depending on the enzyme construct used and
the experimental conditions and methods utilized to determine the
dissociation constant (<xref rid="B37" ref-type="bibr">37</xref>
). The
dimer affinity of MERS-CoV 3CL<sup>pro</sup>
is substantially weaker
than that for SARS-CoV 3CL<sup>pro</sup>
, when comparing the same enzyme
construct, <italic>i.e.</italic>
the enzyme without any N- or C-terminal
modifications. The AUC-SV calculated <italic>K<sub>d</sub>
</italic>
value for MERS-CoV 3CL<sup>pro</sup>
is ∼150,000 times higher
than the value of 0.35 n<sc>m</sc>
determined for SARS-CoV
3CL<sup>pro</sup>
(<xref rid="B34" ref-type="bibr">34</xref>
).</p>
<fig id="F4" orientation="portrait" position="float"><label>FIGURE 4.</label>
<caption><p><bold>AUC-SV analyses of ligand-induced dimerization of MERS-CoV
3CL<sup>pro</sup>
.</bold>
<italic>A,</italic>
sedimentation coefficient distribution for
varying concentrations of MERS-CoV 3CL<sup>pro</sup>
(4.1 to 23
μ<sc>m</sc>
) with sedimentation coefficient values
of 2.9S and 3.9S for the monomer and the dimer, respectively.
The best fit value for AUC-SV-calculated
<italic>K<sub>d</sub>
</italic>
is 52 ± 5
μ<sc>m</sc>
. <italic>B,</italic>
sedimentation
coefficient distribution of MERS-CoV 3CL<sup>pro</sup>
(25
μ<sc>m</sc>
) in the presence of different
stoichiometric ratios of compound <bold>6</bold>
(25, 50, and
100 μ<sc>m</sc>
). <italic>C,</italic>
sedimentation
coefficient distribution of MERS-CoV 3CL<sup>pro</sup>
(25
μ<sc>m</sc>
) in the presence of different
stoichiometric ratios of compound <bold>10</bold>
(25, 50, and
100 μ<sc>m</sc>
). A significant shift in the 2.9S peak
(monomer) to a 4.1S peak (dimer) is detected upon addition of
increasing concentrations of compounds <bold>6</bold>
and
<bold>10.</bold>
</p>
</caption>
<graphic xlink:href="zbc0341521680004"></graphic>
</fig>
<p>The AUC results (<xref ref-type="fig" rid="F4">Fig.
4</xref>
<italic>A</italic>
) show that the monomer peak at
∼2.9S does not gradually shift peak position toward the dimer
peak at ∼3.9S with increasing concentrations of MERS-CoV
3CL<sup>pro</sup>
; rather, the two peaks change in area, which is
indicative of very slow monomer-dimer exchange rate
(<italic>k</italic>
<sub>off</sub>
∼10<sup>−4</sup>
s<sup>−1</sup>
) and the formation of hydrodynamically stable
monomer and dimer species (<xref rid="B55" ref-type="bibr">55</xref>
).
This <italic>k</italic>
<sub>off</sub>
value is 1000 times slower than
the <italic>k</italic>
<sub>off</sub>
value (10<sup>−1</sup>
s<sup>−1</sup>
) reported for SARS-CoV 3CL<sup>pro</sup>
indicating that the SARS-CoV enzyme has a significantly more rapid
monomer-dimer exchange rate (<xref rid="B56" ref-type="bibr">56</xref>
).
These observations support a model whereby the MERS-CoV
3CL<sup>pro</sup>
dimer is weakly associated, suggesting the enzyme
exists mainly as a monomer in solution.</p>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Undergoes Extensive Ligand-induced
Dimerization</title>
<p>The weak association of MERS-CoV 3CL<sup>pro</sup>
monomers engenders the
following questions. “Are higher levels of expression of
3CL<sup>pro</sup>
in MERS-CoV-infected cells necessary to allow
formation of active dimer?” “Are other mechanisms such
as substrate- or ligand-induced dimerizations involved in activating
3CL<sup>pro</sup>
?” To explore the latter question of
ligand-induced dimerization of MERS-CoV 3CL<sup>pro</sup>
, we performed
AUC experiments in the presence of compound <bold>6</bold>
, which acts
as a substrate mimetic and mechanism-based inhibitor, also known as a
suicide substrate. Peptidomimetic compounds such as compound
<bold>6</bold>
, which contains a Michael acceptor group, interact
and react with the active site cysteine of cysteine proteases to
covalently modify them. We utilized compound <bold>6</bold>
to form a
covalent MERS-CoV 3CL<sup>pro</sup>
and inhibitor <bold>6</bold>
complex
that is stable over long periods of time, making it amenable to analysis
by AUC-SV experiments. In contrast, incubation of a normal peptide
substrate with the enzyme would lead to immediate hydrolysis of the
substrate and dissociation of the products from the enzyme, confounding
AUC experiments and subsequent data analysis.</p>
<p>MERS-CoV 3CL<sup>pro</sup>
was incubated with varying concentrations of
compound <bold>6</bold>
in stoichiometric ratios of 1:1, 1:2, and 1:4.
The modified enzyme was then subjected to AUC studies to determine the
influence of compound <bold>6</bold>
on the monomer-dimer equilibrium
(<xref ref-type="fig" rid="F4">Fig. 4</xref>
<italic>B</italic>
). A
significant shift in the area under 2.9S peak (monomer) to 4.1S peak
(dimer) is detected upon addition of increasing concentrations of
compound <bold>6</bold>
. We obtained similar results when AUC studies
were performed utilizing a complex of MERS-CoV 3CL<sup>pro</sup>
with a
noncovalent peptidomimetic inhibitor (compound <bold>10</bold>
, <xref ref-type="fig" rid="F4">Figs. 4</xref>
<italic>C</italic>
). The
transition of MERS-CoV 3CL<sup>pro</sup>
from monomer to dimer in the
presence of compounds <bold>6</bold>
and <bold>10</bold>
suggests that
the enzyme undergoes extensive dimerization upon substrate binding.</p>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Is Activated by Ligand-induced
Dimerization</title>
<p>The observed ligand-induced dimerization of MERS-CoV 3CL<sup>pro</sup>
,
as demonstrated through AUC studies, prompted us to investigate whether
or not the enzymatic activity of MERS-CoV 3CL<sup>pro</sup>
could be
increased at low concentrations of a compound via ligand-induced
dimerization. To do so, we chose to use a noncovalent peptidomimetic
compound (compound <bold>10</bold>
, <xref ref-type="fig" rid="F5">Fig.
5</xref>
<italic>A</italic>
) that we previously identified as an
inhibitor of SARS-CoV 3CL<sup>pro</sup>
. Because of the time-dependent,
irreversible nature of the reaction between compound <bold>6</bold>
and
MERS-CoV 3CL<sup>pro</sup>
, use of compound <bold>6</bold>
was not ideal
for these kinetic studies as it would further complicate kinetic data
analysis.</p>
<fig id="F5" orientation="portrait" position="float"><label>FIGURE 5.</label>
<caption><p><bold>Activation of MERS-CoV 3CL<sup>pro</sup>
via ligand-induced
dimerization.</bold>
<italic>A,</italic>
enzymatic activity of 0.5, 1.0, and 2.0
μ<sc>m</sc>
MERS-CoV 3CL<sup>pro</sup>
was measured
in the absence and presence of varying concentrations of
compound <bold>10.</bold>
Substrate concentration was fixed at
2.0 μ<sc>m</sc>
. % activity, normalized to zero
inhibitor enzymatic activity, was plotted as a function of
increasing inhibitor concentrations. <italic>Error bars</italic>
represent the standard deviation for triplicate data. Increase
in enzymatic activity (highlighted in <italic>cyan-shaded
box</italic>
) is observed in the presence of low
concentrations of compound <bold>10</bold>
. Inhibition of
enzymatic activity is observed at higher inhibitor
concentrations (highlighted in <italic>yellow-shaded
box</italic>
). <italic>B,</italic>
kinetic model describing
the equilibrium between different species of MERS-CoV
3CL<sup>pro</sup>
that are formed in the absence
(<italic>blue box</italic>
) and presence (<italic>green
box</italic>
) of a ligand is shown. Based on the
AUC-calculated <italic>K<sub>d</sub>
</italic>
value of ∼
52 μ<sc>m</sc>
, MERS-CoV 3CL<sup>pro</sup>
primarily
exists as a monomer in solution in the absence of a ligand. Upon
ligand binding (inhibitor <italic>I</italic>
in our case) to the
monomer, the monomer-dimer equilibrium shifts toward dimer
formation. Next, under lower inhibitor concentrations
(<italic>cyan-shaded box</italic>
), substrate
(<italic>S</italic>
) binds in the second active site and
catalysis takes place. However, under higher inhibitor
concentrations (<italic>yellow-shaded box</italic>
), inhibitor
directly competes with the substrate for the second active site,
and inhibition of the enzymatic activity is observed.</p>
</caption>
<graphic xlink:href="zbc0341521680005"></graphic>
</fig>
<p>The kinetic response of MERS-CoV 3CL<sup>pro</sup>
to increasing
concentrations of compound <bold>10</bold>
was first measured at a
single enzyme concentration of 1.0 μ<sc>m</sc>
(<xref ref-type="fig" rid="F5">Fig. 5</xref>
<italic>A</italic>
).
Interestingly, an increase in the activity of MERS-CoV
3CL<sup>pro</sup>
, as high as 195%, was observed in the presence of low
inhibitor concentrations (0.1 to 20 μ<sc>m</sc>
). Inhibition of
enzymatic activity was observed only at higher inhibitor concentrations
(40 μ<sc>m</sc>
or greater). These results suggest that at low
concentrations, compound <bold>10</bold>
binds to a monomer and induces
the formation of a dimer. The resulting dimer then has one free active
site that is capable of processing the substrate. At higher
concentrations of inhibitor, the substrate and inhibitor directly
compete for the free active site.</p>
<p>The model of activation and inhibition suggested by the data at 1
μ<sc>m</sc>
enzyme would predict that at higher enzyme
concentrations less activation by a compound would be observed at lower
inhibitor concentrations, and the inhibition of activity would be
detected at lower inhibitor concentrations because the equilibrium would
be pushed toward dimer formation. In contrast, lower enzyme
concentrations would result in higher activation by compounds, and
inhibition by the compound would occur at significantly higher compound
concentrations. Therefore, we further measured the activity of MERS-CoV
3CL<sup>pro</sup>
at two additional enzyme concentrations (0.5 and
2.0 μ<sc>m</sc>
) in the presence of varying concentrations of
compound <bold>10</bold>
. Remarkably, we observed that the activation
effect was most pronounced at the lowest MERS-CoV 3CL<sup>pro</sup>
concentration tested (0.5 μ<sc>m</sc>
), and the effect decreased
as the enzyme concentration was increased (1.0 and 2.0
μ<sc>m</sc>
) (<xref ref-type="fig" rid="F5">Fig.
5</xref>
<italic>A</italic>
). Moreover, inhibition by compound
<bold>10</bold>
occurred at lower compound concentrations when
higher concentrations of enzyme were used. These observations further
support a model whereby enzyme activation can occur through
ligand-induced dimerization.</p>
<p>The activation and inhibition of MERS-CoV 3CL<sup>pro</sup>
by compound
<bold>10</bold>
can be explained by a simple kinetic model depicted
in <xref ref-type="fig" rid="F5">Fig. 5</xref>
<italic>B</italic>
. The
MERS-CoV 3CL<sup>pro</sup>
monomer exists in equilibrium with the dimer,
and their relative concentrations depend on the total enzyme
concentration. In the absence of substrate or compound, the
<italic>K<sub>d</sub>
</italic>
value is 52 μ<sc>m</sc>
,
and the equilibrium is represented by the <italic>gray spheres</italic>
(<italic>blue box</italic>
) in <xref ref-type="fig" rid="F5">Fig.
5</xref>
<italic>B</italic>
. The monomer is unable to hydrolyze the
substrate and is therefore inactive. Binding of inhibitor (<xref ref-type="fig" rid="F5">Fig. 5</xref>
<italic>B, green
triangle</italic>
) to the monomer results in monomer to dimer switch
leading to the formation of a dimer that contains inhibitor bound in one
of the active sites. Once the dimer is formed, the substrate binds in
the second active site and catalysis takes place. Under high inhibitor
concentrations, however, the inhibitor molecule directly competes with
substrate for the free dimer active site, and inhibition of the
enzymatic activity is observed as a result.</p>
<p>We would also expect to observe induced dimerization and activation in
the presence of the substrate. Indeed, the monomer-dimer kinetic studies
performed in <xref ref-type="fig" rid="F2">Fig. 2</xref>
were performed
at a fixed concentration of substrate at 2 μ<sc>m</sc>
. In this
experiment, the <italic>K<sub>d</sub>
</italic>
value for the MERS-CoV
3CL<sup>pro</sup>
dimer was determined to be 7.8
μ<sc>m</sc>
, which is lower than the
<italic>K<sub>d</sub>
</italic>
value determined in the absence of
substrate using AUC, thereby supporting substrate-induced dimerization.
Given the high <italic>K<sub>m</sub>
</italic>
value of 3CL<sup>pro</sup>
for the peptide substrate (<xref rid="B51" ref-type="bibr">51</xref>
<xref ref-type="bibr" rid="B52">–</xref>
<xref rid="B54" ref-type="bibr">54</xref>
), even higher substrate
concentrations would be required to observe substrate activation in a
plot of catalytic activity <italic>versus</italic>
substrate
concentration. However, we are limited to use our FRET-based substrate
only at low concentrations due to a significant inner filter effect at
higher concentrations of substrate. Therefore, a compound that both
mimics substrate and has higher binding affinity can act as a useful
surrogate for the substrate, allowing the observation of ligand-induced
dimerization and activation even at low substrate concentrations.</p>
</sec>
<sec><title>X-ray Structure of MERS-CoV 3CL<sup>pro</sup>
in Complex with
Compound <bold>6</bold>
</title>
<p>To gain atomic level detail and molecular insight into the mechanism for
substrate-induced dimerization of MERS-CoV 3CL<sup>pro</sup>
, we
attempted to crystallize and determine the x-ray structures of the
unliganded MERS-CoV 3CL<sup>pro</sup>
monomer and the MERS-CoV
3CL<sup>pro</sup>
covalently modified with compound <bold>6</bold>
.
Unfortunately, we were unable to crystallize the unliganded MERS-CoV
3CL<sup>pro</sup>
monomer after multiple attempts, but we were able
to crystallize and determine the x-ray structure of MERS-CoV
3CL<sup>pro</sup>
in complex with compound <bold>6</bold>
to a
resolution of 1.6 Å. The statistics for x-ray data collection,
processing, and refinement are summarized in <xref rid="T4" ref-type="table">Table 4</xref>
. The MERS-CoV 3CL<sup>pro</sup>
and
<bold>6</bold>
complex crystallized as a biologically relevant,
symmetrical dimer in space group <italic>C</italic>
2 with one monomer in
the asymmetric unit. Electron density for the entire protein was clearly
visible and strong electron density (<italic>F<sub>o</sub>
</italic>
− <italic>F<sub>c</sub>
</italic>
>4σ) was
present for compound <bold>6</bold>
within the active site (<xref ref-type="fig" rid="F6">Fig. 6</xref>
<italic>A</italic>
).</p>
<table-wrap id="T4" orientation="portrait" position="float"><label>TABLE 4</label>
<caption><p><bold>X-ray data collection and refinement statistics</bold>
</p>
</caption>
<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th rowspan="1" colspan="1"></th>
<th align="center" rowspan="1" colspan="1">MERS-CoV
3CL<sup>pro</sup>
·6</th>
<th align="center" rowspan="1" colspan="1">MERS-CoV
3CL<sup>pro</sup>
·11</th>
</tr>
</thead>
<tbody valign="top"><tr><td align="left" rowspan="1" colspan="1">Beamline</td>
<td align="left" rowspan="1" colspan="1">LRL-CAT sector 31
ID-D</td>
<td align="left" rowspan="1" colspan="1">LS-CAT sector 21
ID-G</td>
</tr>
<tr><td align="left" colspan="3" rowspan="1"><bold>Data
collection</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Wavelength
(Å)</td>
<td align="left" rowspan="1" colspan="1">0.9793</td>
<td align="left" rowspan="1" colspan="1">0.9786</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Resolution range
(Å)</td>
<td align="left" rowspan="1" colspan="1">19.35–1.62
(1.68–1.62)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">50.00–2.10
(2.14–2.10)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Protein monomers in
asymmetric unit</td>
<td align="left" rowspan="1" colspan="1">1</td>
<td align="left" rowspan="1" colspan="1">4</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Space group</td>
<td align="left" rowspan="1" colspan="1"><italic>C</italic>
2</td>
<td align="left" rowspan="1" colspan="1"><italic>P</italic>
2<sub>1</sub>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Unit cell
dimensions</td>
<td rowspan="1" colspan="1"></td>
<td rowspan="1" colspan="1"></td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> <italic>a,
b, c</italic>
(Å)</td>
<td align="left" rowspan="1" colspan="1">106.49, 57.31,
48.88</td>
<td align="left" rowspan="1" colspan="1">63.44, 114.93,
92.34</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> α,
β, γ (°)</td>
<td align="left" rowspan="1" colspan="1">90, 112.78, 90</td>
<td align="left" rowspan="1" colspan="1">90, 90.89, 90</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Total no. of
reflections</td>
<td align="left" rowspan="1" colspan="1">63,855</td>
<td align="left" rowspan="1" colspan="1">816,216</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> No. of unique
reflections</td>
<td align="left" rowspan="1" colspan="1">32,851</td>
<td align="left" rowspan="1" colspan="1">76,865</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Multiplicity</td>
<td align="left" rowspan="1" colspan="1">1.9 (1.9)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">2.2 (2.2)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Completeness
(%)</td>
<td align="left" rowspan="1" colspan="1">95.0 (93.8)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">96.8 (93.8)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Mean
<italic>I</italic>
/σ<italic>I</italic>
</td>
<td align="left" rowspan="1" colspan="1">5.2 (1.3)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">11.17 (1.83)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic>
<sub>merge</sub>
(%))<xref ref-type="table-fn" rid="TF4-2"><italic><sup>b</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">8.3 (67.2)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">8.8 (58.6)<xref ref-type="table-fn" rid="TF4-1"><italic><sup>a</sup>
</italic>
</xref>
</td>
</tr>
<tr><td colspan="3" rowspan="1"><hr></hr>
</td>
</tr>
<tr><td align="left" colspan="3" rowspan="1"><bold>Refinement</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Resolution range
(Å)</td>
<td align="left" rowspan="1" colspan="1">19.35–1.62</td>
<td align="left" rowspan="1" colspan="1">42.59–2.10</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> No. of reflections
in working set</td>
<td align="left" rowspan="1" colspan="1">30824</td>
<td align="left" rowspan="1" colspan="1">76623</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> No. of reflections
in test set</td>
<td align="left" rowspan="1" colspan="1">2026</td>
<td align="left" rowspan="1" colspan="1">2019</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic>
<sub>work</sub>
(%)<xref ref-type="table-fn" rid="TF4-3"><italic><sup>c</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">17.8</td>
<td align="left" rowspan="1" colspan="1">15.91</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic>
<sub>free</sub>
(%)<xref ref-type="table-fn" rid="TF4-3"><italic><sup>c</sup>
</italic>
</xref>
</td>
<td align="left" rowspan="1" colspan="1">21.7</td>
<td align="left" rowspan="1" colspan="1">21.51</td>
</tr>
<tr><td align="left" colspan="2" rowspan="1"> No. of non-hydrogen
atoms</td>
<td rowspan="1" colspan="1"></td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Protein/water</td>
<td align="left" rowspan="1" colspan="1">2380/208</td>
<td align="left" rowspan="1" colspan="1">9383/995</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> r.m.s.d.,<xref ref-type="table-fn" rid="TF4-4"><italic><sup>d</sup>
</italic>
</xref>
bond lengths
(Å)</td>
<td align="left" rowspan="1" colspan="1">0.007</td>
<td align="left" rowspan="1" colspan="1">0.013</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> r.m.s.d., bond
angles (°)</td>
<td align="left" rowspan="1" colspan="1">1.09</td>
<td align="left" rowspan="1" colspan="1">1.35</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Ramachandran
favored (%)</td>
<td align="left" rowspan="1" colspan="1">99</td>
<td align="left" rowspan="1" colspan="1">98</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Ramachandran
outliers (%)</td>
<td align="left" rowspan="1" colspan="1">0</td>
<td align="left" rowspan="1" colspan="1">0</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Molprobity clash
score</td>
<td align="left" rowspan="1" colspan="1">3.3</td>
<td align="left" rowspan="1" colspan="1">1.94</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Average
<italic>B</italic>
-factor
(Å<sup>2</sup>
)</td>
<td align="left" rowspan="1" colspan="1">20.4</td>
<td align="left" rowspan="1" colspan="1">33.1</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Protein</td>
<td align="left" rowspan="1" colspan="1">19.8</td>
<td align="left" rowspan="1" colspan="1">32.5</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Ligands</td>
<td align="left" rowspan="1" colspan="1">16.6</td>
<td align="left" rowspan="1" colspan="1">41.1</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Solvent</td>
<td align="left" rowspan="1" colspan="1">27.7</td>
<td align="left" rowspan="1" colspan="1">37.9</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="TF4-1"><p><italic><sup>a</sup>
</italic>
Values in parentheses are for
highest resolution shell.</p>
</fn>
<fn id="TF4-2"><p><italic><sup>b</sup>
R</italic>
<sub>merge</sub>
=
Σ<italic><sub>h</sub>
</italic>
Σ<italic><sub>i</sub>
</italic>
|<italic>I<sub>i</sub>
</italic>
(<italic>h</italic>
)
−
〈<italic>I</italic>
(<italic>h</italic>
)〉|/Σ<italic><sub>h</sub>
</italic>
Σ<italic><sub>i</sub>
I<sub>i</sub>
</italic>
(<italic>h</italic>
),
where <italic>I<sub>i</sub>
</italic>
(<italic>h</italic>
) is
the <italic>i</italic>
th measurement and
〈<italic>I</italic>
(<italic>h</italic>
)〉
is the weighted mean of all measurements of
<italic>I</italic>
(<italic>h</italic>
).</p>
</fn>
<fn id="TF4-3"><p><italic><sup>c</sup>
R</italic>
<sub>work</sub>
and
<italic>R</italic>
<sub>free</sub>
=
<italic>h</italic>
(|<italic>F</italic>
(<italic>h</italic>
)<italic><sub>o</sub>
</italic>
|
− |
<italic>F</italic>
(<italic>h</italic>
)<italic><sub>c</sub>
</italic>
|)/<italic>h</italic>
|
<italic>F</italic>
(<italic>h</italic>
)<italic><sub>o</sub>
</italic>
|
for reflections in the working and test sets,
respectively.</p>
</fn>
<fn id="TF4-4"><p><italic><sup>d</sup>
</italic>
r.m.s.d. is root mean square
deviation.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<fig id="F6" orientation="portrait" position="float"><label>FIGURE 6.</label>
<caption><p><bold>X-ray crystal structure of MERS-CoV 3CL<sup>pro</sup>
in
complex with inhibitors.</bold>
<italic>A,</italic>
solvent-accessible surface
(<italic>gray-shaded surface</italic>
) of MERS-CoV
3CL<sup>pro</sup>
and compound <bold>6</bold>
complex.
Compound <bold>6</bold>
is displayed in <italic>ball and
stick</italic>
model with atoms colored as follows: carbons
(<italic>orange</italic>
), nitrogens
(<italic>blue</italic>
), and oxygens (<italic>red</italic>
).
Electron density associated with compound <bold>6</bold>
is
shown as an <italic>F<sub>o</sub>
</italic>
−
<italic>F<sub>c</sub>
</italic>
electron density
difference map contoured to 3σ (<italic>green
mesh</italic>
). Substrate binding pockets
<italic>S</italic>
<sub>4</sub>
-<italic>S</italic>
′<sub>1</sub>
are labeled, where <italic>asterisk</italic>
indicates the
electrophilic carbon of compound <bold>6</bold>
that forms a
C–S covalent bond with the active site cysteine Cys-148.
<italic>B,</italic>
MERS-CoV 3CL<sup>pro</sup>
and compound
<bold>6</bold>
complex with the MERS-CoV 3CL<sup>pro</sup>
backbone represented as a <italic>ribbon</italic>
model and
relevant amino acids that interact with compound <bold>6</bold>
represented as <italic>ball and sticks</italic>
. MERS-CoV
3CL<sup>pro</sup>
carbon atoms are colored
<italic>blue</italic>
, and compound <bold>6</bold>
carbon
atoms are colored <italic>orange</italic>
. Nitrogen atoms are
colored <italic>blue</italic>
, and oxygen atoms are colored
<italic>red</italic>
. Catalytic residues Cys-148 and His-41
are also shown. Hydrogen bonds are depicted as <italic>red
dashed lines. C,</italic>
sequence logos showing amino acid
conservation for the 11 polyprotein cleavage sites of different
3CL<sup>pro</sup>
enzymes (MERS-CoV, HKU5-CoV, HKU4-CoV, and
SARS-CoV), generated using the WebLogo server (<xref rid="B63" ref-type="bibr">63</xref>
). Residues
<italic>P</italic>
<sub>2</sub>
-<italic>P</italic>
′<sub>1</sub>
are shown. <italic>Height</italic>
of each letter corresponds to
the amino acid conservation at that position.
<italic>D,</italic>
solvent-accessible surface
(<italic>gray-shaded surface</italic>
) of MERS-CoV
3CL<sup>pro</sup>
and compound <bold>11</bold>
complex.
Compound <bold>11</bold>
is displayed in <italic>ball and
stick</italic>
model. Electron density associated with
compound <bold>11</bold>
is shown as a
2<italic>F<sub>o</sub>
</italic>
−
<italic>F<sub>c</sub>
</italic>
electron density
difference map contoured to 1.5σ (<italic>green
mesh</italic>
). Functional groups of compound
<bold>11</bold>
with their corresponding binding pockets are
highlighted in <italic>yellow, green,</italic>
and <italic>blue
ellipses</italic>
. Chemical structure of compound
<bold>11</bold>
is shown in the <italic>inset. E,</italic>
interactions between MERS-CoV 3CL<sup>pro</sup>
and compound
<bold>11</bold>
are illustrated. Catalytic residues Cys-148
and His-41 are also shown. Hydrogen bonds are depicted as
<italic>red dashed lines</italic>
.</p>
</caption>
<graphic xlink:href="zbc0341521680006"></graphic>
</fig>
</sec>
<sec><title>MERS-CoV 3CL<sup>pro</sup>
Has a Smaller S<sub>2</sub>
Pocket than
SARS-CoV 3CL<sup>pro</sup>
</title>
<p>The active site of MERS-CoV 3CL<sup>pro</sup>
bound with compound
<bold>6</bold>
is shown in <xref ref-type="fig" rid="F6">Fig.
6</xref>
, <italic>A</italic>
and <italic>B</italic>
. Compound
<bold>6</bold>
is covalently bound to the active site cysteine
(Cys-148) via a 1.8 Å bond between the γ-sulfur and the
electrophilic β-carbon of the Michael acceptor. The
<italic>P</italic>
′<sub>1</sub>
-ethyl ester carbonyl, which
mimics the carbonyl of the scissile bond in a substrate, forms a
hydrogen bond with the backbone NH of Gly-146 that forms part of the
oxyanion hole (<xref ref-type="fig" rid="F6">Fig.
6</xref>
<italic>B</italic>
). Within the
<italic>S</italic>
<sub>1</sub>
subsite, the
<italic>P</italic>
<sub>1</sub>
-lactam carbonyl, which is a surrogate
for the amide of <italic>P</italic>
<sub>1</sub>
-glutamine of substrates,
participates in a hydrogen bonding interaction with the imidazole ring
of His-166, and the <italic>P</italic>
<sub>1</sub>
-lactam NH forms a
hydrogen bond with the carboxylate oxygen of Glu-169. The
<italic>P</italic>
<sub>2</sub>
-backbone amide NH forms a hydrogen
bond with the side chain carbonyl of Gln-192 (<xref ref-type="fig" rid="F6">Fig. 6</xref>
<italic>B</italic>
). The
<italic>P</italic>
<sub>2</sub>
-leucine side chain atoms of the
inhibitor make hydrophobic contacts with the side chains of Met-168 and
Leu-49 that line the <italic>S</italic>
<sub>2</sub>
subsite pocket.
Moreover, compared with the equivalent residue Thr-25 in SARS-CoV
3CL<sup>pro</sup>
, Met-25 in the <italic>S</italic>
<sub>2</sub>
pocket of MERS-CoV 3CL<sup>pro</sup>
is expected to reduce the size of
the hydrophobic pocket, which is supported by our observed SAR described
above.</p>
<p>The smaller size of the <italic>S</italic>
<sub>2</sub>
pocket in MERS-CoV
3CL<sup>pro</sup>
is also consistent with the preference for a
smaller leucine residue at the <italic>P</italic>
<sub>2</sub>
position
of cleavage sites instead of a bulkier phenylalanine or methionine
residue. Indeed, analysis of the preference for leucine or phenylalanine
at the <italic>P</italic>
<sub>2</sub>
position for the 11
3CL<sup>pro</sup>
cleavage sites within the polyprotein of MERS-CoV
shows that none of the 11 cleavage sites contain a phenylalanine residue
at this position (<xref ref-type="fig" rid="F6">Fig.
6</xref>
<italic>C</italic>
). Leucine is the predominantly favored
residue at this position followed by methionine. Analysis of the
cleavage sites from SARS-CoV, HKU4-CoV, and HKU5-CoV shows that none of
the 11 cleavage sites from group 2c members (MERS-CoV, HKU4-CoV, and
HKU5-CoV) contain a phenylalanine residue at the
<italic>P</italic>
<sub>2</sub>
position; however, the SARS-CoV
nsp5↓nsp6 cleavage site contains a phenylalanine residue at this
position.</p>
<p>Other interactions are also observed to play a significant role in
stabilizing the MERS-CoV 3CL<sup>pro</sup>
-compound <bold>6</bold>
complex. The <italic>P</italic>
<sub>3</sub>
-carbonyl and
<italic>P</italic>
<sub>3</sub>
-NH participate in hydrogen bonding
interactions with the backbone NH and carbonyl of Glu-169. The
<italic>P</italic>
<sub>4</sub>
-serine side chain is within hydrogen
bonding distance of the side chain carboxamide of Gln-195 and the
backbone carbonyl of Lys-191.</p>
</sec>
<sec><title>X-ray Structure of MERS-CoV 3CL<sup>pro</sup>
in Complex with a
Noncovalent Inhibitor</title>
<p>We were also able to obtain diffraction quality crystals of MERS-CoV
3CL<sup>pro</sup>
in complex with compound <bold>11</bold>
, which
has an almost identical chemical structure as that of compound
<bold>10</bold>
(<xref ref-type="fig" rid="F6">Fig.
6</xref>
<italic>D</italic>
). We previously showed that compounds
similar to <bold>10</bold>
and <bold>11</bold>
act as potent noncovalent
inhibitors of 3CL<sup>pro</sup>
from SARS-CoV (<xref rid="B33" ref-type="bibr">33</xref>
). The x-ray structure of compound
<bold>11</bold>
bound to MERS-CoV 3CL<sup>pro</sup>
was determined
to a resolution of 2.1 Å and the x-ray data collection,
processing, and refinement statistics are summarized in <xref rid="T4" ref-type="table">Table 4</xref>
. The MERS-CoV
3CL<sup>pro</sup>
and <bold>11</bold>
complex crystallized in space
group <italic>P</italic>
2<sub>1</sub>
with two biologically relevant
dimers in the asymmetric unit. The overall root mean square deviation
between the C-α atoms of the four chains was less than 1
Å, with the highest C-α root mean square deviation of
0.719 Å between chains C and D. Strong electron density
(<italic>F<sub>o</sub>
</italic>
−
<italic>F<sub>c</sub>
</italic>
>4σ) was present for
compound <bold>11</bold>
within all the four active sites of the two
dimers (<xref ref-type="fig" rid="F6">Fig.
6</xref>
<italic>D</italic>
).</p>
<p>The binding orientation for compound <bold>11</bold>
in the active site
of MERS-CoV 3CL<sup>pro</sup>
is similar to the binding orientation of
related compounds in the active site of SARS-CoV 3CL<sup>pro</sup>
(PDB
code <ext-link ext-link-type="pdb" xlink:href="4MDS">4MDS</ext-link>
).
The benzotriazole group binds in the <italic>S</italic>
<sub>1</sub>
subsite; phenyl propionamidyl occupies the
S′<sub>1</sub>
<italic>-S</italic>
<sub>2</sub>
subsite, and
the thiophene group binds in the <italic>S</italic>
<sub>2</sub>
subsite.
Compound <bold>11</bold>
also forms two direct and one water-mediated
hydrogen bond interactions with amino acids in the MERS-CoV
3CL<sup>pro</sup>
active site (<xref ref-type="fig" rid="F6">Fig.
6</xref>
<italic>E</italic>
). The <italic>N</italic>
3 of the
benzotriazole ring forms a hydrogen bond with the side chain
ϵ-nitrogen of conserved His-166, and the central acetamide
oxygen forms a hydrogen bond with the backbone NH of conserved Glu-169.
The NH of the phenyl propionamidyl group interacts with backbone
carbonyl oxygen of the catalytic His-41 residue through a water-mediated
hydrogen bond, and the imidazole ring of His-41 engages with the phenyl
ring of phenyl propionamidyl group through T-shaped π stacking.
The phenyl ring also form hydrophobic contacts with Leu-49.</p>
</sec>
<sec><title>Interactions at the 3CL<sup>pro</sup>
Dimer Interface</title>
<p>Analysis of the MERS-CoV 3CL<sup>pro</sup>
and <bold>6</bold>
and
MERS-CoV 3CL<sup>pro</sup>
and <bold>11</bold>
crystal structures
reveals key differences between the dimer interface of MERS-CoV and
SARS-CoV 3CL<sup>pro</sup>
(PDB code <ext-link ext-link-type="pdb" xlink:href="2ALV">2ALV</ext-link>
) (<xref ref-type="fig" rid="F7">Fig. 7</xref>
) (<xref rid="B30" ref-type="bibr">30</xref>
). Two
arginine residues, Arg-4 and Arg-298 (<xref ref-type="fig" rid="F7">Fig.
7</xref>
, <italic>A–C</italic>
), form some of the key
interactions at the dimer interface of SARS-CoV 3CL<sup>pro</sup>
, and
mutation of either of these amino acids results in a drastic loss of
dimerization in SARS-CoV 3CL<sup>pro</sup>
(<xref rid="B36" ref-type="bibr">36</xref>
, <xref rid="B38" ref-type="bibr">38</xref>
). Interestingly, these two arginine residues (Arg-4 and
Arg-298) are substituted in MERS-CoV 3CL<sup>pro</sup>
by two
hydrophobic residues (Val-4 and Met-298) that are unable to participate
in the formation of hydrogen bonds or salt bridges. Therefore, we
initially thought that the loss of these key interactions might simply
explain the >100,000-fold weaker dimerization observed for
MERS-CoV 3CL<sup>pro</sup>
compared with SARS-CoV 3CL<sup>pro</sup>
.
Surprisingly, however, structural analysis of the dimer interface from
the available x-ray structure of HKU4-CoV 3CL<sup>pro</sup>
(PDB code
<ext-link ext-link-type="pdb" xlink:href="2YNB">2YNB</ext-link>
;
<xref ref-type="fig" rid="F7">Fig. 7</xref>
, <italic>B</italic>
and
<italic>C</italic>
), and primary sequence alignment of
3CL<sup>pro</sup>
from MERS-CoV, HKU5-CoV, HKU4-CoV and SARS-CoV
(<xref ref-type="fig" rid="F8">Fig. 8</xref>
) revealed that Val-4
and Met-298 are conserved between all the β-CoV 2c members
studied here. Substantial differences between the ability of MERS-CoV
3CL<sup>pro</sup>
and HKU4/HKU5-CoV 3CL<sup>pro</sup>
to dimerize,
despite their high sequence identity, led us to the hypothesis that
nonconserved residues between MERS-CoV and other β-CoV 2c
members that are remote from the dimer interface may play a significant
role in dimer formation.</p>
<fig id="F7" orientation="portrait" position="float"><label>FIGURE 7.</label>
<caption><p><bold>Comparison of x-ray crystal structures of 3CL<sup>pro</sup>
dimers from MERS-CoV, HKU4-CoV, and SARS-CoV.</bold>
<italic>A,</italic>
superposition of dimers of MERS-CoV
3CL<sup>pro</sup>
(<italic>pink color</italic>
), HKU4-CoV
3CL<sup>pro</sup>
(<italic>yellow color</italic>
, PDB code
<ext-link ext-link-type="pdb" xlink:href="2YNB">2YNB</ext-link>
), and SARS-CoV 3CL<sup>pro</sup>
(<italic>blue color</italic>
, PDB code <ext-link ext-link-type="pdb" xlink:href="2ALV">2ALV</ext-link>
). For
SARS-CoV 3CL<sup>pro</sup>
, residues Arg-4 and Ser-123 from
monomer A, and residues Gln-127, Lys-137, Glu-290, and Met-298
from monomer B are represented as <italic>spheres. B,</italic>
for SARS-CoV 3CL<sup>pro</sup>
, interactions between the side
chain of Arg-4 from monomer A and Gln-127, Glu-290, and Lys-137
residues from monomer B are shown. The corresponding residues in
MERS-CoV 3CL<sup>pro</sup>
and HKU4-CoV 3CL<sup>pro</sup>
are
Val-4 in monomer A and Glu-290 in monomer B, which do not
interact at the dimer interface. <italic>C,</italic>
for
SARS-CoV 3CL<sup>pro</sup>
, Ser-123 from monomer A engages in
hydrogen bonding with Arg-298 from monomer B across the dimer
interface. The corresponding residue in monomer B of MERS-CoV
3CL<sup>pro</sup>
and HKU4-CoV 3CL<sup>pro</sup>
is Met-298,
which does not participate in any interaction with Thr-126 from
monomer A across the dimer interface.</p>
</caption>
<graphic xlink:href="zbc0341521680007"></graphic>
</fig>
<fig id="F8" orientation="portrait" position="float"><label>FIGURE 8.</label>
<caption><p><bold>Sequence alignment of 3CL<sup>pro</sup>
enzymes from
MERS-CoV, HKU5-CoV, HKU4-CoV, and SARS-CoV.</bold>
Programs
MultAlin (<xref rid="B64" ref-type="bibr">64</xref>
) and ESPript
(<xref rid="B65" ref-type="bibr">65</xref>
) were used for
the sequence alignment and visualization. Secondary structural
elements of MERS-CoV 3CL<sup>pro</sup>
are represented as
<italic>spirals</italic>
for α-helix,
<italic>arrows</italic>
for β-strands, η for
3<sub>10</sub>
helix, and <italic>T</italic>
for
β-turns. Residues Val-4 and Met-298 in MERS-CoV,
HKU5-CoV, HKU4-CoV 3CL<sup>pro</sup>
, and Arg-4 and Arg-298 in
SARS-CoV are shown in a <italic>green box</italic>
; catalytic
residues His-41 and Cys-148 are highlighted in a <italic>purple
box</italic>
. The nonconserved residues of MERS-CoV
3CL<sup>pro</sup>
are marked with <italic>pink
arrows</italic>
. % identity with MERS-CoV 3CL<sup>pro</sup>
is shown.</p>
</caption>
<graphic xlink:href="zbc0341521680008"></graphic>
</fig>
</sec>
<sec><title>Analysis of Nonconserved Residues of MERS-CoV
3CL<sup>pro</sup>
</title>
<p>Analysis of our current crystal structures does not reveal a clear
mechanism for the monomer to dimer switch of MERS-CoV 3CL<sup>pro</sup>
upon ligand binding. Therefore, we attempted to identify the
nonconserved residues in MERS-CoV 3CL<sup>pro</sup>
that might affect
enzymatic activity due to their proximity to key residues involved in
substrate binding and/or dimer formation.</p>
<p>Based on a sequence alignment, MERS-CoV 3CL<sup>pro</sup>
contains
∼24 nonconserved amino acids (<italic>pink arrows</italic>
in
<xref ref-type="fig" rid="F8">Fig. 8</xref>
). Upon analyzing the
position of these amino acids in the crystal structure, we observed that
a remarkable number of these amino acids are present in the loop
regions. <xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>A</italic>
illustrates the nonconserved residues present in the loop regions as
<italic>gray</italic>
(monomer A) and <italic>pink</italic>
(monomer
B) spheres. Interestingly, we also observed that there are hot spots in
the protein structure where most of these amino acids are clustered.
These hot spots include the N-terminal region, the active site region,
the inter-domain loop (loop between the catalytic fold and domain III),
and the domain III. In MERS-CoV 3CL<sup>pro</sup>
, nonconserved amino
acid His-8, which forms van der Waals contacts with Lys-155 of the same
monomer and Thr-128 of the other monomer, is present at the end of the
N-terminal finger (<xref ref-type="fig" rid="F9">Fig. 9</xref>
,
<italic>B</italic>
and <italic>C</italic>
), whereas amino acids
Asp-12 and Ala-15 are part of the N-terminal helix (<xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>B</italic>
). Additionally, amino acids
Thr-128, Lys-155, and Ser-158 are present within 6 Å of the
N-terminal region (<xref ref-type="fig" rid="F9">Fig.
9</xref>
<italic>B</italic>
). Substitution to these amino acids in
MERS-CoV 3CL<sup>pro</sup>
might have changed the protein dynamics in a
way that only ligand binding populates the monomer conformation, which
is more amenable to dimer formation.</p>
<fig id="F9" orientation="portrait" position="float"><label>FIGURE 9.</label>
<caption><p><bold>Analysis of the nonconserved amino acids of MERS-CoV
3CL<sup>pro</sup>
.</bold>
<italic>A,</italic>
representation of MERS-CoV 3CL<sup>pro</sup>
dimer with monomers A and B colored in <italic>orange</italic>
and <italic>yellow</italic>
, respectively. Nonconserved residues
that are present in the loop regions are shown as
<italic>spheres</italic>
in <italic>gray</italic>
and
<italic>pink</italic>
for monomers A and B, respectively.
Other nonconserved residues are represented as
<italic>spheres</italic>
with the corresponding chain color.
Domains I–III and the inter-domain loop are labeled.
Catalytic residues His-41 and Cys-148 are shown as <italic>green
spheres</italic>
. Inhibitor molecule is shown in both active
sites in <italic>blue sticks. B–G,</italic>
residues of
monomer B are shown (<italic>yellow</italic>
and
<italic>pink</italic>
), unless otherwise labeled.
<italic>B,</italic>
clustering of some of the nonconserved
amino acids, His-8, Asp-12, Ala-15, Thr-128, Lys-155, and
Ser-158, near the N-terminal region is shown. N-terminal helices
for both monomers are labeled. <italic>C,</italic>
His-8 from
the N-terminal region forms van der Waals contacts with Lys-155
of the same monomer and Thr-128 of the other monomer in the
dimer. <italic>D,</italic>
nonconserved residue Met-61 forms
hydrophobic contacts with the Met-43 residue, which is in close
proximity to the catalytic residue His-41. <italic>E,</italic>
loop containing the nonconserved residue Ala-171 forms the
<italic>S</italic>
<sub>1</sub>
pocket along with residues
His-166 and His-175. <italic>F,</italic>
Val-132 forms
hydrophobic contacts with a residue within the same domain
(Ala-114), as well as Glu-290 from domain III.
<italic>G,</italic>
nonconserved residue Tyr-137 makes
hydrophobic contacts with Tyr-185; Tyr-185 along with two other
nonconserved residues Thr-183 and Met-189 are present on the
inter-domain loop.</p>
</caption>
<graphic xlink:href="zbc0341521680009"></graphic>
</fig>
<p>We also observe that some of the nonconserved residues in MERS-CoV
3CL<sup>pro</sup>
are located in proximity to the substrate-binding
site and might contribute toward ligand-induced dynamic changes
favorable for dimer formation. For example, nonconserved amino acid
Met-61 forms hydrophobic interactions with Met-43, which in turn is in
close proximity to the catalytic residue His-41 (<xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>D</italic>
). Residue Ala-171 is
present on a loop, and this loop, along with conserved residues His-166
and His-175, forms the <italic>S</italic>
<sub>1</sub>
subsite for
binding the <italic>P</italic>
<sub>1</sub>
amino acid of the substrate
(<xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>E</italic>
). In
addition to its influence on substrate binding, Ala-171 may also
contribute toward dimer formation upon substrate binding due to its
close proximity with Glu-169. This glutamate residue in SARS-CoV
3CL<sup>pro</sup>
(Glu-166) has been established as a key residue
linking the substrate-binding site to the dimer interface (<xref rid="B56" ref-type="bibr">56</xref>
). Val-132 forms hydrophobic
interaction with other nonconserved residue Ala-114 within domain II
(<xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>F</italic>
).
Additionally, Val-132 is present within van der Waals contact distance
of Glul-290 from extra-helical domain III (<xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>F</italic>
). It is noteworthy that Glu-290
forms a salt bridge with Arg-4 across the dimer interface in SARS-CoV
3CL<sup>pro</sup>
. However, this interaction is not formed in
MERS-CoV 3CL<sup>pro</sup>
due to the substitution of Arg-4 with Val-4.
Tyr-137 forms hydrophobic contacts with the conserved residue Tyr-185
(<xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>G</italic>
).</p>
<p>Besides amino acid Val-132 that connects domains II and III, residue
Tyr-185, along with two other nonconserved residues, Thr-183 and
Met-189, is present on the inter-domain loop that connects the catalytic
fold (domains I and II) with the extra-helical domain III (<xref ref-type="fig" rid="F9">Fig. 9</xref>
<italic>G</italic>
).
Flexibility within these residues might affect the orientation of domain
III required for dimer formation.</p>
</sec>
</sec>
</sec>
</sec>
<sec sec-type="discussion"><title>Discussion</title>
<sec><title></title>
<sec><title></title>
<sec><title>Model for Regulation of the Enzymatic Activity of MERS-CoV
3CL<sup>pro</sup>
during Polyprotein Processing</title>
<p>Enzymatic activity of coronavirus 3CL<sup>pro</sup>
is required for the
processing of viral polyproteins at 11 distinct cleavage sites, allowing
the release of nonstructural proteins that subsequently form a
replication complex for virus genome replication. Because of its
indispensable role in the virus life cycle, regulation of the enzymatic
activity of 3CL<sup>pro</sup>
is instrumental for efficient replication
of coronaviruses. Based on our experimental results, we propose a model
to explain the mechanism for regulating the enzymatic activity of
MERS-CoV 3CL<sup>pro</sup>
in the context of polyprotein processing
during virus infection (<xref ref-type="fig" rid="F10">Fig.
10</xref>
).</p>
<fig id="F10" orientation="portrait" position="float"><label>FIGURE 10.</label>
<caption><p><bold>Proposed model for polyprotein processing in MERS-CoV
regulated by ligand-induced dimerization of MERS-CoV
3CL<sup>pro</sup>
.</bold>
MERS-CoV 3CL<sup>pro</sup>
domains I and II are together represented as the
<italic>rectangular box</italic>
, and domain III is
represented as a <italic>cylinder</italic>
. The N and C termini
are labeled, and the <italic>yellow cylinder</italic>
labeled
<italic>S</italic>
represents a ligand that can be a peptide
inhibitor, peptide substrate, or 3CL<sup>pro</sup>
cleavage
sites in the polyprotein. Various steps required for the
auto-release of 3CL<sup>pro</sup>
from the polyprotein and
subsequent processing of the polyprotein cleavage sites are
described in the text. Suggested by our AUC and kinetic studies,
the <italic>shaded</italic>
region (<italic>steps 5</italic>
and
<italic>6</italic>
) highlights the additional steps MERS-CoV
3CL<sup>pro</sup>
would undertake during polyprotein
processing and have been described in the kinetic model depicted
in <xref ref-type="fig" rid="F5">Fig.
5</xref>
<italic>B</italic>
.</p>
</caption>
<graphic xlink:href="zbc0341521680010"></graphic>
</fig>
<p>A number of <italic>in vitro</italic>
studies performed on SARS-CoV
3CL<sup>pro</sup>
have established the mechanism for
3CL<sup>pro</sup>
auto-release from the polyprotein (<xref rid="B34" ref-type="bibr">34</xref>
, <xref rid="B39" ref-type="bibr">39</xref>
, <xref rid="B40" ref-type="bibr">40</xref>
). Based upon these
studies and our data on MERS-CoV 3CL<sup>pro</sup>
, we propose the
polyprotein processing model in <xref ref-type="fig" rid="F10">Fig.
10</xref>
. The steps proposed for auto-release of MERS-CoV
3CL<sup>pro</sup>
from the polyprotein (<italic>steps
1–4</italic>
, <xref ref-type="fig" rid="F10">Fig. 10</xref>
)
have been adapted from Chen <italic>et al.</italic>
(<xref rid="B39" ref-type="bibr">39</xref>
), where it is suggested that the
N-terminal auto-processing does not require the formation of a mature
3CL<sup>pro</sup>
dimer for SARS-CoV. Based on the differences
between the properties of SARS-CoV 3CL<sup>pro</sup>
and MERS-CoV
3CL<sup>pro</sup>
, as highlighted in our studies, we added two
additional steps (<italic>steps 5</italic>
and <italic>6</italic>
, <xref ref-type="fig" rid="F10">Fig. 10</xref>
) that MERS-CoV
3CL<sup>pro</sup>
may need to utilize for efficient polyprotein
processing. In <xref ref-type="fig" rid="F10">Fig. 10</xref>
,
<italic>step 1</italic>
, two immature MERS-CoV 3CL<sup>pro</sup>
monomers in the polyprotein approach each other and form an immature
dimer via interactions between domain III, which allows each of the
monomers to insert their N termini into the active site of the other
monomer. In <italic>step 2,</italic>
the N termini are cleaved, and the
dimer with uncleaved C termini adopts a conformation similar to the
mature dimer. Our observation of auto-cleavage of the N-terminal
His<sub>6</sub>
tag from MERS-CoV 3CL<sup>pro</sup>
during
expression in bacterial cells supports <italic>steps 1</italic>
and
<italic>2</italic>
, where formation of an immature dimer capable of
auto-processing the N terminus occurs. In <italic>step 3</italic>
, two
dimers with uncleaved C termini approach each other, followed by
insertion of the C terminus from one dimer into one of the active sites
of the other dimer. In <italic>step 4</italic>
, the C termini are
cleaved and mature dimer is released from the polyprotein.</p>
<p>For SARS-CoV, the 3CL<sup>pro</sup>
dimer formed in <italic>step
4</italic>
continues to process cleavage sites in the polyprotein,
effectively skipping <italic>steps 5</italic>
and <italic>6</italic>
(<italic>red arrow</italic>
in <xref ref-type="fig" rid="F10">Fig.
10</xref>
) because the dimer is tightly associated. However, the
high <italic>K<sub>d</sub>
</italic>
value of MERS-CoV 3CL<sup>pro</sup>
dimer suggests that the active and mature dimer may dissociate into
inactive, mature monomers in the absence of any ligand (<italic>step
5</italic>
). In order for polyprotein processing to proceed, another
step (<italic>step 6</italic>
) must occur. In <italic>step 6</italic>
, a
substrate S, <italic>e.g.</italic>
one of the 11 polyprotein cleavage
sites, would induce dimer formation and hence activate catalysis and
cleavage at the substrate recognition sites. Our AUC results and the
kinetic activation studies performed in the absence and presence of
inhibitors support <italic>steps 5</italic>
and <italic>6</italic>
where
the inactive but mature monomers require binding of a ligand to undergo
ligand-induced dimerization and formation of an active, mature dimer
that can then process the polyprotein cleavage sites.</p>
</sec>
<sec><title>Nonconserved Amino Acids of MERS-CoV 3CL<sup>pro</sup>
May Regulate
the Dimer Formation</title>
<p>Long range interactions have been reported to modulate dimerization and
activity of 3CL<sup>pro</sup>
enzymes. Barrila <italic>et al.</italic>
(<xref rid="B57" ref-type="bibr">57</xref>
) demonstrated that
mutation of a conserved amino acid Ser-147, which is distant from the
dimer interface, results in a total loss of dimerization and enzymatic
activity of SARS-CoV 3CL<sup>pro</sup>
. Although Ser-147 does not form
direct interactions at the dimer interface, disruption of the dimer upon
mutation stems from the fact that Ser-147 makes several interactions
with other residues involved in forming a hydrogen bonding network
within SARS-CoV 3CL<sup>pro</sup>
. Site-directed mutagenesis studies on
domain III of SARS-CoV 3CL<sup>pro</sup>
, where N214A and
S284A/T285A/I286A mutants were characterized, revealed that despite
being present on an entirely different domain, these residues affect
catalysis through a network of residues undergoing correlated motions
across the entire protease (<xref rid="B58" ref-type="bibr">58</xref>
,
<xref rid="B59" ref-type="bibr">59</xref>
). Utilizing
3CL<sup>pro</sup>
temperature-sensitive mutants of MHV, Stobart
<italic>et al.</italic>
(<xref rid="B60" ref-type="bibr">60</xref>
)
have also demonstrated that second-site mutation physically distant from
the temperature-sensitive mutation suppresses the temperature-sensitive
phenotype through long range interactions, thereby regulating
3CL<sup>pro</sup>
enzymatic activity during polyprotein processing
and virus replication.</p>
<p>Our studies also suggest that long range interactions among the
nonconserved residues can significantly alter the properties of MERS-CoV
3CL<sup>pro</sup>
. A detailed analysis of nonconserved residues of
MERS-CoV 3CL<sup>pro</sup>
among β-CoV 2c members identified hot
spots, including the N-terminal finger and helix, the active site
region, the inter-domain loop, and the domain III, where these residues
are clustered. Several studies done on SARS-CoV 3CL<sup>pro</sup>
have
demonstrated that amino acids from the N-terminal finger, the N-terminal
helix, and domain III significantly contribute toward dimer
formation.</p>
<p>In addition to the direct interactions at the dimer interface, correct
orientation between the catalytic fold and domain III is also crucial
for dimer formation. Wu <italic>et al.</italic>
(<xref rid="B61" ref-type="bibr">61</xref>
) showed that the most dramatic difference
between the crystal structures of monomer and the ligand-bound dimer of
the R298A mutant of SARS-CoV 3CL<sup>pro</sup>
was a 33°
rotation of domain III (<xref rid="B38" ref-type="bibr">38</xref>
). This
rotation results in a steric clash between domain III from two monomers
and would essentially block dimer formation. However, upon addition of a
ligand, domain III of the R298A mutant adopts the correct orientation
and results in the formation of a dimer structure. Similar to the
SARS-CoV 3CL<sup>pro</sup>
R298A mutant, ligand binding into the active
site of the MERS-CoV 3CL<sup>pro</sup>
monomer possibly stabilizes the
inter-domain loop conformation that maintains domain III in the correct
orientation for dimer formation. Most of the nonconserved residues
within domain III are present on the surface and also are distant from
the dimer interface. These residues may be involved in providing the
flexibility required for conformational changes during the monomer to
dimer switch.</p>
<p>We have identified several amino acids in MERS-CoV 3CL<sup>pro</sup>
that
may contribute to the dimer formation upon ligand binding. However,
single amino acid mutagenesis alone is unlikely to reveal significant
differences in the dimerization properties. As demonstrated by Myers
<italic>et al.</italic>
(<xref rid="B62" ref-type="bibr">62</xref>
)
for ornithine decarboxylase, the response of single amino acid to ligand
binding may be limited to only local conformational changes and may not
have significant contribution toward dimer stability. However, local
conformational changes in a network of residues may propagate larger
effects that stabilize dimer formation upon ligand binding. Analysis of
the nonconserved residues of MERS-CoV 3CL<sup>pro</sup>
discussed here
sets forth a framework to perform systematic single or multiple
mutagenesis studies to gain insights into the mechanism for
ligand-induced dimerization of the enzyme.</p>
</sec>
<sec><title>Development of 3CL<sup>pro</sup>
Inhibitors with Broad Spectrum
Specificity</title>
<p>Insights into the mechanistic and structural similarities as well as
differences between 3CL<sup>pro</sup>
enzymes from different coronavirus
subgroups are instrumental for the development of 3CL<sup>pro</sup>
inhibitors with broad spectrum specificity. To evaluate the broad
spectrum specificity of our peptidomimetic compounds, we determined
their inhibitory activity against 3CL<sup>pro</sup>
from MERS-CoV,
SARS-CoV, HKU5-CoV, and HKU4-CoV. Our inhibitory data and
<italic>K<sub>I</sub>
</italic>
values clearly show that
compounds <bold>6–9</bold>
inhibit all the 3CL<sup>pro</sup>
enzymes tested here. The x-ray structure of MERS-CoV 3CL<sup>pro</sup>
in complex with compound <bold>6</bold>
revealed that out of eight
direct hydrogen bonds formed between compound <bold>6</bold>
and
MERS-CoV 3CL<sup>pro</sup>
, four of these hydrogen bonds involve
interactions with conserved structural elements of the peptide backbone
of the enzyme. Furthermore, the amino acids that form hydrogen bonds
with compound <bold>6</bold>
through side chain interactions are
conserved in all the coronavirus 3CL<sup>pro</sup>
enzymes evaluated
here, as well as 3CL<sup>pro</sup>
enzymes from other
β-coronaviruses like MHV, OC43, and HKU1. These results suggest
that canonical structural features exist among the 3CL<sup>pro</sup>
enzymes that can be exploited for structure-based design of broad
spectrum inhibitors.</p>
<p>For the noncovalent inhibitor compound <bold>11</bold>
, the x-ray
structure reveals two direct hydrogen bonding interactions between the
compound and MERS-CoV 3CL<sup>pro</sup>
. One of the hydrogen bonds forms
with the side chain ϵ-nitrogen of conserved His-166, and the
second involves the backbone NH of conserved Glu-169. We speculate these
interactions remain conserved in other 3CL<sup>pro</sup>
enzymes as
well, because His-166 and Glu-169 amino acids are conserved in all
3CL<sup>pro</sup>
enzymes. In fact, the crystal structure of
SARS-CoV 3CL<sup>pro</sup>
in complex with an inhibitor similar to
compound <bold>11</bold>
(PDB code <ext-link ext-link-type="pdb" xlink:href="4MDS">4MDS</ext-link>
) reveals that the interactions of
the inhibitor with the amino acids His-166 and Glu-169 are
conserved.</p>
<p>The identification of 3CL<sup>pro</sup>
-inhibitor interactions utilizing
conserved elements of the protein structure, including the peptide
backbone and conserved side chains of active site residues, suggests
that the development of broad-spectrum inhibitors of coronavirus
3CL<sup>pro</sup>
is feasible.</p>
<p>Our studies here demonstrate the unique properties of MERS-CoV
3CL<sup>pro</sup>
among β-CoV 2c members, evident from the
requirement for a ligand to induce dimerization. Although the
peptidomimetic compounds containing a Michael acceptor group (for
example, compounds <bold>6–9</bold>
) induce dimer formation of
MERS-CoV 3CL<sup>pro</sup>
, the irreversible nature of their reaction
with the active site cysteine ensures complete inhibition of the enzyme
at stoichiometric ratios in a time-dependent manner. On the contrary,
noncovalent peptidomimetic compounds (for example, compounds
<bold>10</bold>
and <bold>11</bold>
) inhibit the enzymatic activity
of MERS-CoV 3CL<sup>pro</sup>
only at high compound concentrations.
Based on these observations, compounds that irreversibly modify the
3CL<sup>pro</sup>
active site may serve as better candidates for the
development of inhibitors for MERS-CoV 3CL<sup>pro</sup>
.</p>
</sec>
<sec><title>Potential Complexity in the Development of MERS-CoV 3CL<sup>pro</sup>
Inhibitors as Antiviral Agents</title>
<p>Induced dimerization of MERS-CoV 3CL<sup>pro</sup>
, as seen in the
presence of peptidomimetic inhibitors, has significant implications in
the development of antiviral agents targeting MERS-CoV
3CL<sup>pro</sup>
. As a consequence of enzyme activation, the
development of an effective antiviral agent may necessitate the
development of a compound that can inhibit the MERS-CoV
3CL<sup>pro</sup>
monomer and stabilize it without inducing
dimerization and/or inhibit the active sites of the dimer at low doses,
ensuring inactivation of both the active sites within the dimer. On the
contrary, it is also possible that the presence of an inhibitor could
enhance the activity of MERS-CoV 3CL<sup>pro</sup>
to an extent that
results in a complete loss of the temporal and spatial regulation of the
enzymatic activity, thereby disrupting viral genome replication.
Ramifications of ligand-induced dimerization and activation of MERS-CoV
3CL<sup>pro</sup>
, as seen in the presence of lower concentrations
of inhibitor, will need to be further explored in virus-infected
cells.</p>
</sec>
</sec>
</sec>
</sec>
<sec><title>Author Contributions</title>
<p>S. T. and A. D. M. conceived and coordinated the study and wrote the paper. S. T., M.
L. J. and S. E. S. J. designed, performed, and analyzed the experiments shown in
<xref ref-type="fig" rid="F1">Figs. 1</xref>
and <xref ref-type="fig" rid="F2">2</xref>
. S. T. and L. N. P. designed, performed, and analyzed the experiments
shown in <xref ref-type="fig" rid="F4">Fig. 4</xref>
. S. T. and M. L. J. designed,
performed, and analyzed the experiments shown in <xref ref-type="fig" rid="F5">Fig.
5</xref>
. S. T. and A. D. M. determined the crystal structures. H. L. O., P. R.
N., and A. K. G. synthesized compounds <bold>1–9</bold>
. S. E. S. J., A. K.
G., and M. R. D. provided substantial contributions to analysis and interpretation
of data.</p>
</sec>
</body>
<back><fn-group><fn fn-type="supported-by" id="FN1"><label>*</label>
<p>This work was supported, in whole or in part, by National Institutes of Health
Grants AI08508 (to A. D. M.) and AI026603 (to A. D. M. and M. R. D.). This work
was also supported by the Walther Cancer Foundation (to A. D. M.). All authors
reviewed the results and approved the final version of the manuscript. The
authors declare that they have no conflicts of interest with the contents of
this article.</p>
</fn>
<fn fn-type="other"><p>The atomic coordinates and structure factors (codes <ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=4RSP">4RSP</ext-link>
and <ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=4YLU">4YLU</ext-link>
) have been deposited in the Protein Data Bank (<ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/">http://wwpdb.org/</ext-link>
).</p>
</fn>
</fn-group>
<fn-group content-type="abbreviations"><fn id="FN3"><label>3</label>
<p>The abbreviations used are: <def-list><def-item><term id="G1">CoV</term>
<def><p>coronavirus</p>
</def>
</def-item>
<def-item><term id="G2">MERS</term>
<def><p>Middle East respiratory syndrome</p>
</def>
</def-item>
<def-item><term id="G3">SARS</term>
<def><p>severe acute respiratory syndrome</p>
</def>
</def-item>
<def-item><term id="G4">nsp</term>
<def><p>nonstructural protein</p>
</def>
</def-item>
<def-item><term id="G5">3CL<sup>pro</sup>
</term>
<def><p>3-chymotrypsin-like protease</p>
</def>
</def-item>
<def-item><term id="G6">AUC</term>
<def><p>analytical ultracentrifugation</p>
</def>
</def-item>
<def-item><term id="G7">SV</term>
<def><p>sedimentation velocity</p>
</def>
</def-item>
<def-item><term id="G8">BME</term>
<def><p>β-mercaptoethanol</p>
</def>
</def-item>
<def-item><term id="G9">BisTris</term>
<def><p>2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol</p>
</def>
</def-item>
<def-item><term id="G10">PDB</term>
<def><p>Protein Data Bank.</p>
</def>
</def-item>
</def-list>
</p>
</fn>
</fn-group>
<ack><title>Acknowledgments</title>
<p>Crystallization and DNA sequencing were partially supported by the Purdue Center for
Cancer Research Macromolecular Crystallography and DNA Sequencing Shared Resources,
which are partially supported by National Institutes of Health Grant P30 CA023168.
We acknowledge the LS-CAT and LRL-CAT beam line staff for their help in acquiring
x-ray data. Use of the Advanced Photon Source, an Office of Science User Facility
operated for the United States Department of Energy Office of Science by Argonne
National Laboratory, was supported by the United States Department of Energy under
Contract DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the
Michigan Economic Development Corp. and the Michigan Technology Tri-Corridor Grant
085P1000817. Use of the Lilly Research Laboratories Collaborative Access Team
(LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli
Lilly Co., which operates the facility. We thank Yahira Baez-Santos, Dia Beachboard
and Sergey Savinov for their helpful suggestions during manuscript preparation.</p>
</ack>
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