Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design
Identifieur interne : 000133 ( Pmc/Corpus ); précédent : 000132; suivant : 000134Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design
Auteurs : Shan-Meng Lin ; Shih-Chao Lin ; Jia-Ning Hsu ; Chung-Ke Chang ; Ching-Ming Chien ; Yong-Sheng Wang ; Hung-Yi Wu ; U-Ser Jeng ; Kylene Kehn-Hall ; Ming-Hon HouSource :
- Journal of Medicinal Chemistry [ 0022-2623 ] ; 2020.
Abstract
Structure-based stabilization of protein–protein interactions (PPIs) is a promising strategy for drug discovery. However, this approach has mainly focused on the stabilization of native PPIs, and non-native PPIs have received little consideration. Here, we identified a non-native interaction interface on the three-dimensional dimeric structure of the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV N-NTD). The interface formed a conserved hydrophobic cavity suitable for targeted drug screening. By considering the hydrophobic complementarity during the virtual screening step, we identified 5-benzyloxygramine as a new N protein PPI orthosteric stabilizer that exhibits both antiviral and N-NTD protein-stabilizing activities. X-ray crystallography and small-angle X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD dimers through simultaneous hydrophobic interactions with both partners, resulting in abnormal N protein oligomerization that was further confirmed in the cell. This unique approach based on the identification and stabilization of non-native PPIs of N protein could be applied toward drug discovery against CoV diseases.
Url:
DOI: 10.1021/acs.jmedchem.9b01913
PubMed: 32105468
PubMed Central: 7094172
Links to Exploration step
PMC:7094172Le document en format XML
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Structure-Based
Stabilization of Non-native Protein–Protein
Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug
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<author><name sortKey="Hsu, Jia Ning" sort="Hsu, Jia Ning" uniqKey="Hsu J" first="Jia-Ning" last="Hsu">Jia-Ning Hsu</name>
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<author><name sortKey="Wang, Yong Sheng" sort="Wang, Yong Sheng" uniqKey="Wang Y" first="Yong-Sheng" last="Wang">Yong-Sheng Wang</name>
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<author><name sortKey="Kehn Hall, Kylene" sort="Kehn Hall, Kylene" uniqKey="Kehn Hall K" first="Kylene" last="Kehn-Hall">Kylene Kehn-Hall</name>
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<series><title level="j">Journal of Medicinal Chemistry</title>
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<front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="jm9b01913_0005" id="ab-tgr1"></graphic>
</p>
<p>Structure-based
stabilization of protein–protein interactions
(PPIs) is a promising strategy for drug discovery. However, this approach
has mainly focused on the stabilization of native PPIs, and non-native
PPIs have received little consideration. Here, we identified a non-native
interaction interface on the three-dimensional dimeric structure of
the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV
N-NTD). The interface formed a conserved hydrophobic cavity suitable
for targeted drug screening. By considering the hydrophobic
complementarity during the virtual screening step, we identified 5-benzyloxygramine
as a new N protein PPI orthosteric stabilizer that exhibits both antiviral
and N-NTD protein-stabilizing activities. X-ray crystallography and
small-angle
X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD
dimers through simultaneous hydrophobic interactions with both partners,
resulting in abnormal N protein oligomerization that was further confirmed
in the cell. This unique approach based on the identification and
stabilization of non-native PPIs of N protein could be applied toward
drug discovery against CoV diseases.</p>
</div>
</front>
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</TEI>
<pmc article-type="research-article" xml:lang="EN"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Med Chem</journal-id>
<journal-id journal-id-type="iso-abbrev">J. Med. Chem</journal-id>
<journal-id journal-id-type="publisher-id">jm</journal-id>
<journal-id journal-id-type="coden">jmcmar</journal-id>
<journal-title-group><journal-title>Journal of Medicinal Chemistry</journal-title>
</journal-title-group>
<issn pub-type="ppub">0022-2623</issn>
<issn pub-type="epub">1520-4804</issn>
<publisher><publisher-name>American Chemical
Society</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">32105468</article-id>
<article-id pub-id-type="pmc">7094172</article-id>
<article-id pub-id-type="doi">10.1021/acs.jmedchem.9b01913</article-id>
<article-categories><subj-group><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Structure-Based
Stabilization of Non-native Protein–Protein
Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug
Design</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Lin</surname>
<given-names>Shan-Meng</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="notes-3" ref-type="notes">○</xref>
</contrib>
<contrib contrib-type="author" id="ath2"><name><surname>Lin</surname>
<given-names>Shih-Chao</given-names>
</name>
<xref rid="aff3" ref-type="aff">§</xref>
<xref rid="notes-3" ref-type="notes">○</xref>
</contrib>
<contrib contrib-type="author" id="ath3"><name><surname>Hsu</surname>
<given-names>Jia-Ning</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="notes-3" ref-type="notes">○</xref>
</contrib>
<contrib contrib-type="author" id="ath4"><name><surname>Chang</surname>
<given-names>Chung-ke</given-names>
</name>
<xref rid="aff5" ref-type="aff">⊥</xref>
<xref rid="notes-3" ref-type="notes">○</xref>
</contrib>
<contrib contrib-type="author" id="ath5"><name><surname>Chien</surname>
<given-names>Ching-Ming</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
</contrib>
<contrib contrib-type="author" id="ath6"><name><surname>Wang</surname>
<given-names>Yong-Sheng</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath7"><name><surname>Wu</surname>
<given-names>Hung-Yi</given-names>
</name>
<xref rid="aff4" ref-type="aff">∥</xref>
</contrib>
<contrib contrib-type="author" id="ath8"><name><surname>Jeng</surname>
<given-names>U-Ser</given-names>
</name>
<xref rid="aff6" ref-type="aff">#</xref>
<xref rid="aff7" ref-type="aff">∇</xref>
</contrib>
<contrib contrib-type="author" id="ath9"><name><surname>Kehn-Hall</surname>
<given-names>Kylene</given-names>
</name>
<xref rid="aff3" ref-type="aff">§</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" id="ath10"><name><surname>Hou</surname>
<given-names>Ming-Hon</given-names>
</name>
<xref rid="cor1" ref-type="other">*</xref>
<xref rid="aff1" ref-type="aff">†</xref>
<xref rid="aff2" ref-type="aff">‡</xref>
</contrib>
<aff id="aff1"><label>†</label>
Institute of Genomics and Bioinformatics,<institution>National Chung Hsing University</institution>
, Taichung 402,<country>Taiwan</country>
</aff>
<aff id="aff2"><label>‡</label>
Department of Life Sciences,<institution>National Chung Hsing University</institution>
, Taichung 402,<country>Taiwan</country>
</aff>
<aff id="aff3"><label>§</label>
National Center for Biodefense and Infectious Diseases, School of Systems Biology,<institution>George Mason University</institution>
, Manassas, Virginia 20110,<country>United States</country>
</aff>
<aff id="aff4"><label>∥</label>
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine,<institution>National Chung Hsing University</institution>
, Taichung 40227,<country>Taiwan</country>
</aff>
<aff id="aff5"><label>⊥</label>
<institution>Institute of Biomedical Sciences, Academia Sinica</institution>
, Taipei 115,<country>Taiwan</country>
</aff>
<aff id="aff6"><label>#</label>
<institution>National Synchrotron Radiation Research Center</institution>
, 101 Hsin-Ann Road, Hsinchu Science Park, Hsinchu 30076,<country>Taiwan</country>
</aff>
<aff id="aff7"><label>∇</label>
Department of Chemical Engineering,<institution>National Tsing Hua University</institution>
, Hsinchu 30013,<country>Taiwan</country>
</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>*</label>
Email: <email>mhho@nchu.edu.tw</email>
. Tel.: <phone>+886-4-2284-0338</phone>
. Fax: <fax>+886-4-2285-9329</fax>
(ext. 7011).</corresp>
</author-notes>
<pub-date pub-type="epub"><day>27</day>
<month>02</month>
<year>2020</year>
</pub-date>
<pub-date pub-type="ppub"><day>26</day>
<month>03</month>
<year>2020</year>
</pub-date>
<volume>63</volume>
<issue>6</issue>
<fpage>3131</fpage>
<lpage>3141</lpage>
<history><date date-type="received"><day>19</day>
<month>11</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2020 American Chemical Society</copyright-statement>
<copyright-year>2020</copyright-year>
<copyright-holder>American Chemical Society</copyright-holder>
<license license-type="open-access"><license-p>This article is made available via the PMC Open Access Subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p>
</license>
</permissions>
<abstract><p content-type="toc-graphic"><graphic xlink:href="jm9b01913_0005" id="ab-tgr1"></graphic>
</p>
<p>Structure-based
stabilization of protein–protein interactions
(PPIs) is a promising strategy for drug discovery. However, this approach
has mainly focused on the stabilization of native PPIs, and non-native
PPIs have received little consideration. Here, we identified a non-native
interaction interface on the three-dimensional dimeric structure of
the N-terminal domain of the MERS-CoV nucleocapsid protein (MERS-CoV
N-NTD). The interface formed a conserved hydrophobic cavity suitable
for targeted drug screening. By considering the hydrophobic
complementarity during the virtual screening step, we identified 5-benzyloxygramine
as a new N protein PPI orthosteric stabilizer that exhibits both antiviral
and N-NTD protein-stabilizing activities. X-ray crystallography and
small-angle
X-ray scattering showed that 5-benzyloxygramine stabilizes the N-NTD
dimers through simultaneous hydrophobic interactions with both partners,
resulting in abnormal N protein oligomerization that was further confirmed
in the cell. This unique approach based on the identification and
stabilization of non-native PPIs of N protein could be applied toward
drug discovery against CoV diseases.</p>
</abstract>
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<meta-value>jm9b01913</meta-value>
</custom-meta>
<custom-meta><meta-name>document-id-new-14</meta-name>
<meta-value>jm9b01913</meta-value>
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<meta-value></meta-value>
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</article-meta>
<notes id="notes-d1e21-autogenerated"><fn-group><fn fn-type="" id="d30e250"><p>This article is made available for a limited time sponsored by ACS under the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/freetoread/index.html">ACS Free to Read License</ext-link>
, which permits copying and redistribution of the article for non-commercial scholarly purposes.</p>
</fn>
</fn-group>
</notes>
</front>
<body><sec id="sec1"><title>Introduction</title>
<p>Small-molecule stabilization
of protein–protein interactions
(PPIs) is an extremely promising strategy in drug discovery. It can
be used to treat cancers and viral infections.<sup><xref ref-type="bibr" rid="ref1">1</xref>
−<xref ref-type="bibr" rid="ref3">3</xref>
</sup>
Stabilizing
PPIs with small molecules may be allosteric or direct (also called
orthosteric). This process alters the oligomerization equilibrium
of the protein and enables small molecules to modulate its physiological
function.<sup><xref ref-type="bibr" rid="ref4">4</xref>
−<xref ref-type="bibr" rid="ref7">7</xref>
</sup>
The anticancer drug paclitaxel, for example, allosterically enhances
microtubule structure assembly by binding to β-tubulin.<sup><xref ref-type="bibr" rid="ref8">8</xref>
,<xref ref-type="bibr" rid="ref9">9</xref>
</sup>
On the other hand, rapamycin, another anticancer agent, binds directly
to the interface between FKBP12 and mTOR and stabilizes the structure
of the complex.<sup><xref ref-type="bibr" rid="ref10">10</xref>
</sup>
The most well-characterized
PPIs suitable as targets for drug development form natively under
physiological conditions. However, non-native interactions, which
may form under extreme circumstances such as inside a crystal lattice,
are also potential drug targets. For example, nucleozin exerts its
antiviral activity by stabilizing the non-native PPI interface between
the two neighboring nucleoprotein trimers within the influenza virus,
which results in abnormal protein oligomerization and loss of viral
viability.<sup><xref ref-type="bibr" rid="ref11">11</xref>
</sup>
</p>
<p>Middle East respiratory
syndrome coronavirus (MERS-CoV) belongs
to the betacoronavirus (β-CoVs) family. It causes severe respiratory
distress with a high mortality rate in humans.<sup><xref ref-type="bibr" rid="ref12">12</xref>
−<xref ref-type="bibr" rid="ref14">14</xref>
</sup>
Recently, a
closely related novel coronavirus, coronavirus disease 2019 (COVID-19),
caused an outbreak of pneumonia in Wuhan, which further underscored
the risk of CoVs to the global public health.<sup><xref ref-type="bibr" rid="ref15">15</xref>
,<xref ref-type="bibr" rid="ref16">16</xref>
</sup>
However, there is no effective treatment for CoVs. Thus, there is
an urgent need to develop new antiviral agents against CoVs.<sup><xref ref-type="bibr" rid="ref14">14</xref>
,<xref ref-type="bibr" rid="ref17">17</xref>
</sup>
MERS-CoV packages its genome in a nucleocapsid (N) protein and forms
a ribonucleoprotein (RNP) complex. The RNP is essential for viral
transcription and assembly. Several studies suggested that the modulation
of CoV N protein oligomerization by small molecules is a feasible
antiviral drug development strategy.<sup><xref ref-type="bibr" rid="ref18">18</xref>
,<xref ref-type="bibr" rid="ref19">19</xref>
</sup>
The CoV N
protein is organized into the N-terminal domain (NTD) and the C-terminal
domain (CTD), with both domains participating in RNA binding.<sup><xref ref-type="bibr" rid="ref20">20</xref>
,<xref ref-type="bibr" rid="ref21">21</xref>
</sup>
All CoV N-NTD structures are folded in a monomeric conformation.
In contrast, the CoV N-CTDs are always dimeric and are responsible
for N protein oligomerization via protein–protein interactions.<sup><xref ref-type="bibr" rid="ref22">22</xref>
,<xref ref-type="bibr" rid="ref23">23</xref>
</sup>
</p>
<p>Here, we report the crystal structure of MERS-CoV N-NTD in
a non-native
dimeric configuration. We used the non-native dimer interface as the
target in virtual screening for an orthosteric stabilizer. To this
end, we considered the
binding scores and hydrophobic complementarity of the acquired poses,
and further selected the potential leads P1–P3 from Acros and
ZINC drug databases. Of these, only 5-benzyloxygramine (P3) had both
antiviral and stabilizing activities on the N protein. Small-angle
X-ray scattering (SAXS) and cell-based assays showed that P3 induces
abnormal full-length N protein oligomerization in vitro and at the
cell level. We also described the structure of MERS-CoV N-NTD complexed
with 5-benzyloxygramine and revealed its stabilizing mechanism. Our
findings provide insight into the development of a new therapeutic
approach based on stabilizing a non-native protein interaction interface.
It may lead to the discovery and development of new treatments for
various infectious diseases.</p>
</sec>
<sec id="sec2"><title>Results</title>
<sec id="sec2.1"><title>Structure of the N-Terminal
Domain of the MERS-CoV N Protein</title>
<p>We determined the crystal
structure of MERS-CoV N-NTD by molecular
replacement (MR) using the structure of HCoV-OC43 N-NTD (PDB ID: 4J3K) as the search model.<sup><xref ref-type="bibr" rid="ref24">24</xref>
</sup>
The final structure was refined to R-factor
and R-free values of 0.26 and 0.29, respectively, at a resolution
of 2.6 Å (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Table S1</ext-link>
). Each asymmetric
unit contained four N-NTD molecules assembled into two identical dimers
with an overall RMSD of 0.28 Å between the dimers (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Figure S1A,B</ext-link>
). The monomers shared a similar
structural core preceded by a flexible region (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Figure S1D</ext-link>
). The core consisted of a five-stranded antiparallel
β-sheet sandwiched between loops arranged in a right-handed,
fist-shaped structure conserved among the CoVs.<sup><xref ref-type="bibr" rid="ref25">25</xref>
</sup>
In our structure, however, the loop connecting strands
β2 and β3 protruding out of the core into other CoV N
proteins was absent. Unlike the reported structures that have a monomeric
conformation, our structure was atypically dimeric.</p>
<p><xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
B shows the details
of the interactions in the MERS-CoV N protein dimer. We named the
units monomer 1 and monomer 2 (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
A). According to the amino acid composition of the
binding site on monomer 2, we divided the dimeric interface into two
areas: one located on the N-terminus flexible region and the other
on the loop between β4 and β5 of the N protein. In the
first area, W43, N66, N68, Y102, and F135 of monomer 1 generated a
conserved hydrophobic pocket permitting the side chain of M38 of monomer
2 to enter this hole by a hydrophobic contact (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
C,D). H37 and N39 of monomer 2 were packed
against W43 and F135 of monomer 1, respectively, and contributed to
the hydrophobic interaction. The side chains of N39 of monomer 2 formed
one hydrogen bond with the N68 backbone in monomer 1 at a distance
of 2.6 Å. The second area was relatively more hydrophilic. The
main chain oxygens of G104, F135, and T137 of monomer 2 formed three
hydrogen bonds with the side chains of Q73 and T134 of monomer 1 at
distances of 3.8, 3.2, and 3.7 Å, respectively. The side chain
of N139 on monomer 2 formed a hydrogen bond with the main chain oxygen
of T137 on monomer 1 at a distance of 3.6 Å. The interactions
of the first and second areas comprise buried surface areas (BSA)
of 289 and 103 Å<sup>2</sup>
, respectively. The small surface
area buried at the interface accounts for ∼5 kcal mol<sup>–1</sup>
binding energy,<sup><xref ref-type="bibr" rid="ref26">26</xref>
</sup>
which translates to
a dissociation constant of ∼200 mM. Thus, the dimer described
here is unique in that it is non-native and relies on vector-fusion
residues (H37 and M38) to maintain its dimeric status. This property
may also explain why the present structure has an oligomeric status
different from previously reported structures for the CoV N protein.<sup><xref ref-type="bibr" rid="ref24">24</xref>
,<xref ref-type="bibr" rid="ref27">27</xref>
−<xref ref-type="bibr" rid="ref30">30</xref>
</sup>
We used cross-linking experiments to analyze the oligomeric capacity
of MERS N-NTD containing the vector-fusion residues in solution. MERS
N-NTD had a dimeric conformation in solution. Our structure indicated
that W43 played an essential role in forming the hydrophobic pocket
accommodating the vector-fusion residues and, therefore, mediated
the N-NTD dimer formation. The W43A mutation significantly reduced
the oligomeric tendency of N-NTD (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Figure S1C</ext-link>
). This further supports that the “exogenous residues”
encoded by the vector backbone mediated the formation of the non-native
dimer. We also superimposed the previously published structure of
MERS-CoV N-NTD (PDB ID: 4ud1)<sup><xref ref-type="bibr" rid="ref27">27</xref>
</sup>
containing a native
N-terminal flexible region with our dimer structure. The side chain
of N38 in the native structure could not interact with the hydrophobic
pocket as the former is hydrophilic and short (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Figure S1E</ext-link>
). Thus, it may be possible to utilize small compounds
to replace the vector-fusion residues and stabilize the PPI through
hydrophobic interactions.</p>
<fig id="fig1" position="float"><label>Figure 1</label>
<caption><p>Structure and sequence of MERS-CoV N-NTD. (A)
Overall structure
of the MERS-CoV N-NTD dimer containing monomers 1 and 2 is depicted
as a cartoon and colored yellow and green, respectively. Residues
involved in dimerization are shown as sticks and highlighted in (B).
(B) Interactions among MERS-CoV N-NTDs. Dimerization is mediated mainly
by vector-fusion residues interacting with the conserved hydrophobic
regions on the core structure (first area) along with the residues
surrounding it (second area). Interacting residues on monomers 1 and
2 are labeled in black and blue, respectively. Vector-fusion and conserved
hydrophobic regions are colored cyan and red, respectively. The color
of all other interacting residues is the same as that for each monomer
in (A). Polar contacts are indicated with red dashed lines. (C) Upper
panel: close-up of the interacting region of vector-fusion residues.
The surface was colored according to the hydrophobicity level at the
protein surface. Vector-fusion residues (black) are shown as sticks
to emphasize the hydrophobic pocket. Lower panel: 2D diagrams of the
interaction between the hydrophobic pocket and the vector-fusion residues.
The latter are labeled in black. The hydrophobic contacts are indicated
with black dashed lines. (D) Sequence alignment of various CoV N proteins
in the N-terminal region. Red letters indicate strictly conserved
residues. Cyan indicates conservative substitution sites. Hydrophobic
regions involved in unusual dimerization are indicated by black triangles.</p>
</caption>
<graphic xlink:href="jm9b01913_0003" id="gr1" position="float"></graphic>
</fig>
</sec>
<sec id="sec2.2"><title>Direct Targeting of the Non-native Dimer
Interface for Antiviral
Screening</title>
<p>We performed a structure-based virtual screening
by targeting W43 in the hydrophobic pocket of the N-NTD dimeric interface.
H37 and M38 were removed from the template to identify compounds that
could replace the vector-fusion residues and, therefore, contribute
to the stabilizing effect (<xref rid="fig2" ref-type="fig">Figures <xref rid="fig2" ref-type="fig">2</xref>
</xref>
A and <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">S2A,B</ext-link>
). We chose the
highest-scoring hits, listed in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Table S2</ext-link>
, based on shape complementarity, the presence of aromatic moieties,
and the ability to stack onto W43 of N-NTD. Because the formation
of the non-native dimers was primarily mediated by hydrophobic interactions
in our structure (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
C,D), we next considered the hydrophobic complementarity between
the acquired ligands and N-NTD in the form of the lipophilic match
surface (<italic>S</italic>
<sub>L/L</sub>
).<sup><xref ref-type="bibr" rid="ref31">31</xref>
</sup>
We also took into account the ability of the drug to permeate cells
by aiming for lower topological polar surface areas (TPSA). Based
on the above criteria, three compounds were finally chosen for further
study. Benzyl-2-(hydroxymethyl)-1-indolinecarboxylate (P1) and 5-benzyloxygramine
(P3) had higher <italic>S</italic>
<sub>L/L</sub>
and docking scores
and lower TPSA. The clinical drug etodolac (P2) had a comparable <italic>S</italic>
<sub>L/L</sub>
but a lower docking score. It too was selected
as a candidate.
However, only P3 induced a comparatively larger blue shift in the
intrinsic N-NTD fluorescence spectrum, indicating that the microenvironment
surrounding the tryptophans of the protein increased in rigidity and
hydrophobicity in the presence of P3.<sup><xref ref-type="bibr" rid="ref32">32</xref>
</sup>
The result also indicated that P3 bound more tightly to the N protein
than P1 or P2 by interacting with the W43 pocket (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
B). Fluorescent thermal stability
assays disclosed that the N-NTD denaturation melting temperature had
increased from 42 to 45 °C when P3 was added. The sigmoidal melting
curve for MERS-CoV N-NTD changed in the presence of P3. The delay
in protein denaturation suggests that P3 stabilized the MERS-CoV N-NTD
dimer structure (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
C). We then measured the cytotoxic concentration (CC<sub>50</sub>
) and effective concentration (EC<sub>50</sub>
) for each compound
using Vero E6 cells infected with MERS-CoV. <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref>
</xref>
shows that P3 had a favorable therapeutic
index among the lead compounds tested in this study. Therefore, P3
is an excellent candidate inhibitor against MERS-CoV.</p>
<fig id="fig2" position="float"><label>Figure 2</label>
<caption><p>Compound P3 was a potent
stabilizer of the MERS-CoV N protein.
(A) Schematic depicting the rationale used in designing the allosteric
stabilizer of this study. An orthosteric stabilizer may be used to
bind to the non-native interaction interface of the N protein and
stabilize the abnormal interaction between proteins. (B) Conformation
and (C) stability analyses were performed based on the FL spectra
of NTD (1 μM) incubated with P1–P3 (10 μM) for
1 h with a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150 mM
NaCl.</p>
</caption>
<graphic xlink:href="jm9b01913_0002" id="gr2" position="float"></graphic>
</fig>
<table-wrap id="tbl1" position="float"><label>Table 1</label>
<caption><title>CC<sub>50</sub>
and
EC<sub>50</sub>
and Therapeutic Indexes of Lead Compounds</title>
</caption>
<table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col>
<col align="char" char="."></col>
<col align="left"></col>
<col align="left"></col>
</colgroup>
<thead><tr><th colspan="4" align="center">quantal dose–response relationship (μM)<hr></hr>
</th>
</tr>
<tr><th style="border:none;" align="center">compound</th>
<th style="border:none;" align="center" char=".">CC<sub>50</sub>
<xref rid="t1fn1" ref-type="table-fn">a</xref>
</th>
<th style="border:none;" align="center">EC<sub>50</sub>
<xref rid="t1fn2" ref-type="table-fn">b</xref>
</th>
<th style="border:none;" align="center">TI<xref rid="t1fn3" ref-type="table-fn">c</xref>
(CC<sub>50</sub>
/EC<sub>50</sub>
)</th>
</tr>
</thead>
<tbody><tr><td style="border:none;" align="left">P1</td>
<td style="border:none;" align="char" char=".">459.69</td>
<td style="border:none;" align="left">>100</td>
<td style="border:none;" align="left">NA<xref rid="t1fn4" ref-type="table-fn">d</xref>
</td>
</tr>
<tr><td style="border:none;" align="left">P2</td>
<td style="border:none;" align="char" char=".">569.77</td>
<td style="border:none;" align="left">>100</td>
<td style="border:none;" align="left">NA<xref rid="t1fn4" ref-type="table-fn">d</xref>
</td>
</tr>
<tr><td style="border:none;" align="left">P3</td>
<td style="border:none;" align="char" char=".">805.32</td>
<td style="border:none;" align="left">32.1</td>
<td style="border:none;" align="left">25.1</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t1fn1"><label>a</label>
<p>CC<sub>50</sub>
: Half maximal toxicity
concentration.</p>
</fn>
<fn id="t1fn2"><label>b</label>
<p>EC<sub>50</sub>
: Half maximum effective
concentration.</p>
</fn>
<fn id="t1fn3"><label>c</label>
<p>TI: Therapeutic
index.</p>
</fn>
<fn id="t1fn4"><label>d</label>
<p>NA: Nonavailable.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="sec2.3"><title>Structural Model of P3-Induced
MERS-CoV N Protein Aggregation</title>
<p>We used SAXS to assess the
effects of P3 on the full-length MERS-CoV
N protein structure. The fitted distance distribution function of
the protein with and without P3 are shown in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
A. P3 increased the maximum dimension (<italic>D</italic>
<sub>max</sub>
) and radius of gyration (<italic>R</italic>
<sub>g</sub>
) of the protein from 207 to 230 Å and from 58 to
65 Å, respectively. Thus, the size of the MERS-CoV N protein
in solution was altered upon binding to P3.</p>
<fig id="fig3" position="float"><label>Figure 3</label>
<caption><p>P3-induced abnormal aggregation
on the full-length MERS-CoV N protein.
(A–E) SAXS analysis of the full-length MERS-CoV N protein.
(A) Normalized results from GNOM showing pairwise distance distribution <italic>P</italic>
(<italic>r</italic>
) and maximum distance. The radius of
gyration fitted to 207 and 230 Å for the N protein and the N-P3
complex, respectively. “<italic>r</italic>
” represents
pairwise distances. (B, C) Scattering profiles of the N protein (B)
and the N-P3 complex (C) and normalization fitting with GNOM (dashed
lines). (D, E) Representative models of the N protein (D) and the
N-P3 complex (E) generated by CRYSOL simulations of the SAXS data.
Only α carbons are shown. NTD (yellow), CTD (green), and disorder
region (cyan). (F, G) Conformation (F) and stability (G) analyses
based on FL spectra of the MERS-CoV N protein (1 μM) incubated
with P3 (10 μM) for 1 h in a buffer consisting of 50 mM Tris-HCl,
150 mM NaCl (pH 8.3). (H) Schematic of the P3 inhibition mechanism.
Left panel: in the absence of RNA, N proteins organize as a dimeric
building block contributed by N-CTD dimerization. Middle panel: P3
promoted the dimerization of N-NTDs from different building blocks,
by which the distance between CTD cuboids was shortened and N protein
aggregation occurred. Right panel: octameric conformation of building
blocks buried in the RNA-binding surface of N-CTDs. It hindered the
formation of filamentous ribonucleocapsids.</p>
</caption>
<graphic xlink:href="jm9b01913_0004" id="gr3" position="float"></graphic>
</fig>
<p>The presence of multiple intrinsically disordered regions in the
N protein precluded the determination of its structure by X-ray crystallography.
Instead, we used rigid body modeling of the SAXS data with the N-terminal
domain (NTD; solved in this study) and the C-terminal domains (CTD,
PDB ID: 6G13).<sup><xref ref-type="bibr" rid="ref23">23</xref>
</sup>
In this way, we obtained structural
models for the free N protein and its complex with P3 (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
B,C). Excellent fits were obtained.
Representative structural models for the full-length protein without
and with P3 are shown in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
D,E, respectively. The free N protein formed a tetramer
through CTD with the NTD freely hanging in solution (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
D). The conformation of the
solution was consistent with structures previously reported for other
CoV N proteins.<sup><xref ref-type="bibr" rid="ref33">33</xref>
</sup>
The N-P3 complex formed
a compact hexadecamer with a sunburst configuration (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
E). The CTDs formed a central
ring and non-native NTD dimers formed “spikes” protruding
from the ring. Consistent with ligand-induced aggregation, we observed
a “blue shift” in the fluorescence spectrum of the full-length
MERS-CoV N protein in the presence of P3 (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
F). The addition of P3 also delayed N protein
thermal denaturation and changed the shape of the denaturation curve,
further suggesting that large protein aggregates formed in the presence
of P3 (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
G).
The structure explains how N-NTD dimerization decreased MERS-CoV viability.
The N protein packages the viral genome into an RNP complex. Several
models for N-CTD dimer assembly have been proposed for the formation
of filamentous RNPs.<sup><xref ref-type="bibr" rid="ref33">33</xref>
</sup>
All of the proposed
interfaces between N-CTD dimers occurred on the side-faces of the
CTD cuboid perpendicular to the proposed RNA-binding surface (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
H). Combinatorial
use of any region on the side-faces of the CTD dimer cuboid may facilitate
manipulation of the RNP length and curvature without obstructing the
RNA-binding surface.<sup><xref ref-type="bibr" rid="ref28">28</xref>
,<xref ref-type="bibr" rid="ref34">34</xref>
</sup>
However, the SAXS results indicated
that N-CTD aggregation occurred on the β-sheet floor of the
CTD cuboid. For this reason, the RNA-binding surface of the CTD is
occluded by the neighboring CTD on the ring and by the non-native
NTD dimer making direct contact with the CTD (<xref rid="fig3" ref-type="fig">Figures <xref rid="fig3" ref-type="fig">3</xref>
</xref>
H and <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">S3</ext-link>
). In
addition, the CTD cuboids in the aggregation naturally form a topologically
closed octamer, leaving no open ends for further addition of CTD cuboids
to form a long filamentous RNP. Both the loss of the RNA-binding surface
and the inability to incorporate further N protein molecules beyond
an octamer may inhibit the formation of the RNP. Therefore, P3 may
inhibit MERS-CoV RNP formation by inducing N protein aggregation.</p>
</sec>
<sec id="sec2.4"><title>P3 Inhibits MERS-CoV by Inducing N Protein Aggregation</title>
<p>We
demonstrated that P3 had the best characteristics as a therapeutic
candidate. To determine the anti-MERS-CoV activity of P3 in the cell,
the effects of P3 incubation on extracellular viral titers and intracellular
viral RNA levels were assessed by plaque assays on Vero E6 cells (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
A) and by RT-qPCR
(<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
B), respectively.
At 50 μM, P3 marginally affected the viral titer after 48 h
but suppressed viral RNA replication by 40%. At 100 μM, P3 halted
both viral production and replication after 48 h. This result proved
the capacity of P3 as an antiviral compound. We then examined MERS-CoV
N protein distribution and expression in the infected cells with or
without P3 treatment to confirm our SAXS findings. Immunofluorescence
microscopy (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
C) showed condensation of the intracellular N protein fluorescence
signal in infected Vero E6 cells treated with 50 μM P3. Thus,
P3 may induce intracellular N protein aggregation. At 100 μM,
P3 suppressed N protein expression in most cells. However, a few presented
with intense N protein signals. P3 may have restrained the MERS-CoV
N proteins inside the infected cells that promoted the formation of
new virions that could not be released. In this way, the adjacent
cells could not be infected with MERS-CoV. The data, therefore, suggest
that P3 may inhibit MERS-CoV by inducing abnormal aggregation of the
N protein inside the cell. This finding is consistent with the results
of our structure-based assays.</p>
<fig id="fig4" position="float"><label>Figure 4</label>
<caption><p>Compound P3 was a potential inhibitor
against MERS-CoV. (A, B)
Viral titers (A) and RNA (B) of MERS-CoV measured by plaque assay
and RT-qPCR, respectively, decreased after P3 treatment for 48 h.
Relative RNA levels were determined by comparing MERS alone at each
time point. GAPDH RNA was the internal control. All values are presented
as mean ± SE (standard error of mean). One-way Anova was used
for statistics (*<italic>p</italic>
< 0.05, **<italic>p</italic>
< 0.01, ***<italic>p</italic>
< 0.001). (C) MERS-CoV nucleocapsid
protein decreased after 48 h P3 treatment. Nucleocapsid protein expressions
(red) were examined under a confocal microscope at ×680. Nuclei
were stained blue with DAPI.</p>
</caption>
<graphic xlink:href="jm9b01913_0001" id="gr4" position="float"></graphic>
</fig>
</sec>
<sec id="sec2.5"><title>Crystal Structure of MERS-CoV N-NTD Complexed with Potent Compounds</title>
<p>We attempted to obtain crystals of MERS-CoV N-NTD in complex with
compounds P1, P2, and P3 by cocrystallization or ligand-soaking. With
the exception of P2, the complex structures of N-NTD with P1 and P3
were solved at resolutions of 3.09 and 2.77 Å, respectively (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Table S1</ext-link>
). The overall structures of the complexes
resembled that of apo-MERS-CoV. Both complexes revealed well-defined
unbiased densities in the dimer interface and permitted detailed analysis
of the interactions between the compounds and MERS-CoV N-NTD (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
). The interactions
between the N protein and each compound were calculated with the Discovery
Studio Client (v19.1.0.18287). Most interactions were hydrophobic
contacts, which were consistent with our selection rationale. In the
P1 complex, N68, F135, and D143 on monomer 1 and V41, G106, P107,
and T137 on monomer 2 packed against P1 to create a dimer (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
A). In addition,
two nonbonding interactions were detected between P1 and the monomers.
There was a π-anion interaction between the benzene ring of
the P1 indoline moiety and D143 of monomer 1. There was also a π-donor
hydrogen bond between the other P1 benzene ring and the T137 side
chain of monomer 2 (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
B). Relative to P1, P3 bound more deeply into the dimer interface
and interacted with a larger number of residues on both N-NTD monomers.
The amino acid composition of this binding region was W43, N66, N68,
S69, T70, N73, and F135 on monomer 1 and V41, G104, T105, G106, A109,
and T137 on monomer 2. These residues along with P3 generated a massive
hydrophobic driving force allowing the proteins and ligands to pack
against each other and stabilize the dimeric conformation of the N
protein (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
C).
Several nonbonding interactions were also observed at the P3-binding
site. These included the interaction between the P3 benzene ring and
N68 of monomer 1 and A109 of monomer 2 via π-lone pair and π-alkyl
interactions. The dimethylaminomethyl moiety of P3 was a major source
of nonbonding interactions. Three π-cation interactions formed
between this moiety and the aromatic groups of W43 and F135 in monomer
1. This moiety also formed a π-lone pair interaction with N66
and a π-sigma interaction with W43 of monomer 1 (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
D). The structural analyses
explain the comparatively stronger binding of P3 to N-NTD (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
B) and corroborated
the thermal stabilization effects (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
C) and antiviral activities (<xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref>
</xref>
) of the compounds.</p>
<fig id="fig5" position="float"><label>Figure 5</label>
<caption><p>Structures
of MERS-CoV N-NTD complexed with potent compounds. The
structures were solved using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.<sup><xref ref-type="bibr" rid="ref24">24</xref>
</sup>
Left panel: (Upper) structural superimposition
of the MERS-CoV N-NTD:P1 complex (monomers 1 and 2 are in purple and
pink, respectively) and the MERS-CoV N-NTD:P3 complex (monomers 1
and 2 are in brown and green, respectively) with compounds depicted
as stick structures. (Lower) Interactions involving vector-fusion
residues in the non-native dimer of the apoprotein shown for comparison
with (A) and (B). Color is the same as in <xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
A. Right panel: detailed interactions among
MERS-CoV N-NTD and P1 (A, B) and P3 (C, D). Different Fo–Fc
maps were contoured at ∼2.5 σ. (A) Detailed stereoview
of interactions at the P1-binding site. The color of each monomer
is the same as in the left panel. Residues constructing the P1-binding
pocket are labeled and showed as sticks. (B) Schematic of P1 bound
to MERS-CoV N-NTD. Hydrophobic contacts between P1 and each monomer
are displayed as dashed lines. Nonbonding interactions are indicated
by cyan arrows. (C) Detailed stereoview of interactions at the P3-binding
site. The color of each monomer is the same as in the left panel.
Residues belonging to the P3-binding pocket are labeled and shown
as sticks. (D) Schematic of P3 bound to MERS-CoV N-NTD. Hydrophobic
contacts between P3 and each monomer are displayed as dashed lines.
Nonbonding interactions are indicated by red arrows.</p>
</caption>
<graphic xlink:href="jm9b01913_0006" id="gr5" position="float"></graphic>
</fig>
</sec>
</sec>
<sec id="sec3"><title>Discussion and Conclusions</title>
<p>The use of small molecules
to stabilize PPI interfaces has been
shown to be a viable approach for the development of new therapeutics.
Most compounds created by structure-based design to manipulate the
PPI depend on detailed knowledge of the native interacting interfaces.<sup><xref ref-type="bibr" rid="ref35">35</xref>
−<xref ref-type="bibr" rid="ref37">37</xref>
</sup>
In contrast, most of the compounds with therapeutic potential that
stabilize non-native PPI interfaces were discovered by chance alone.<sup><xref ref-type="bibr" rid="ref38">38</xref>
</sup>
The anti-influenza compound nucleozin was initially
discovered using a chemical genetics approach, and its ability to
stabilize non-native nucleoprotein oligomers was elucidated much later.<sup><xref ref-type="bibr" rid="ref39">39</xref>
,<xref ref-type="bibr" rid="ref40">40</xref>
</sup>
Swinholide A, a cytotoxic marine macrolide, has been known to disrupt
the actin cytoskeleton and act as an anticancer agent, but it took
ten years to discover that it stabilized G-actin as a non-native homodimer
complex.<sup><xref ref-type="bibr" rid="ref41">41</xref>
−<xref ref-type="bibr" rid="ref43">43</xref>
</sup>
However, these examples did underscore the importance
of hydrophobicity as a crucial factor stabilizing protein–protein
and protein–ligand associations.<sup><xref ref-type="bibr" rid="ref44">44</xref>
</sup>
Here, we demonstrated the possibility of using the hydrophobic interactions
on non-native interfaces as targets for virtual screening. Combined
with suitable selection criteria focusing on both shape and hydrophobic
complementarities between the ligand and the receptor, small-molecule
compounds that stabilize non-native PPIs may be identified in a rational
manner. Using the above approach, we successfully identified a compound
P3 that affected the biochemical activity of our target protein and
showed efficacy against our target pathogen MERS-CoV (<xref rid="fig2" ref-type="fig">Figures <xref rid="fig2" ref-type="fig">2</xref>
</xref>
and <xref rid="fig4" ref-type="fig">4</xref>
). P3-mediated non-native MERS-CoV N-NTD dimerization induced abnormal
N protein aggregation by influencing the oligomeric behavior of N-CTD
and eventually halting its function in RNP formation. To the best
of our knowledge, this structure-based strategy for targeting non-native
interfaces has never been proposed for therapeutic design. Thus, non-native
interaction interfaces in proteins may comprise a new drug development
target class. For β-coronaviruses such as MERS-CoV, the amino
acids comprising the non-native interaction interface on N-NTD are
relatively conserved. The hydrophobic pocket surrounding W43 and F135
on monomer 1 is shared among other β-coronaviruses.<sup><xref ref-type="bibr" rid="ref24">24</xref>
,<xref ref-type="bibr" rid="ref28">28</xref>
</sup>
The interacting surface on monomer 2, which includes G104, T105,
G106, and A109, is also highly conserved (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Figure S4</ext-link>
). This conservation may provide certain advantages when
developing compounds with broad-spectrum activity against a target
pathogen family, including COVID-19. When we tested P3 on mouse hepatitis
virus (MHV), we observed a reduction in viral titer, indicating that
P3 may also inhibit MHV replication (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Figure S5</ext-link>
). On the other hand, targeting non-native interaction interfaces
is not trivial. As interface formation is induced by an external agent,
computations aimed at predicting native PPI structures may not be
able to identify potential non-native interfaces. Nevertheless, several
stratagems may assist in the identification of potential non-native
interaction interfaces. One strategy is to search for target protein
structures with crystal packing contacts known to be biologically
irrelevant. Another approach is to identify weakly interacting sites
through NMR chemical shift perturbation and hydrogen-deuterium exchange
MS.<sup><xref ref-type="bibr" rid="ref45">45</xref>
</sup>
Once these potential non-native interaction
interfaces are identified, standard screening and then functional
characterization may be conducted for small compounds that bind to
the interface.</p>
<p>To summarize, we demonstrated that non-native
interaction interfaces
might form when proteins abnormally oligomerize. Using the MERS-CoV
N protein as an example, we showed that non-native interfaces might
serve as targets for small-molecule, structure-based screening. We
also proposed several stratagems to rationally identify potential
non-native interface targets for screening. We believe that we have
discovered and tested an alternative drug discovery paradigm that
could help expand the repertoire of lead compounds against various
pathogens.</p>
</sec>
<sec id="sec4"><title>Experimental Section</title>
<sec id="sec4.1"><title>Chemicals</title>
<p>The
compounds P1, P2, and P3 were purchased
from Maybridge Chemical Company, TCI Chemicals, and Sigma-Aldrich
Corporation, respectively. The reagents used in this study were purchased
from Sigma Chemical Co. (St. Louis, MO). The purity of all compounds
is higher than 95% and was used without further purification.</p>
</sec>
<sec id="sec4.2"><title>Cloning,
Protein Expression, and Purification</title>
<p>The MERS-CoV
N proteins were prepared according to previously described methods.<sup><xref ref-type="bibr" rid="ref46">46</xref>
</sup>
In brief, the cDNA fragments of MERS-CoV N proteins
were cloned into a pET-28a expression vector (Merck, Darmstadt, Germany)
containing a histidine tag-encoding sequence. Vectors encoding the
single mutants N39A, N39G, and W43A were generated using the <italic>QuikChange</italic>
site-directed mutagenesis protocol with the primers
listed in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Table S3</ext-link>
. The vectors were transformed
into <italic>Escherichia coli</italic>
BL21 (DE3) pLysS
cells. The cells were grown to an optical density range of 0.6–0.8
at 600 nm at 37 °C and protein expression induced with 1.0 mM
isopropyl β-<sc>d</sc>
-1-thiogalactopyranoside (IPTG), followed
by incubation at 10 °C for 24 h. The cells were harvested by
centrifugation (6000<italic>g</italic>
, 12 min, 4 °C) and resuspended
in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, and
1 mM PMSF; pH 7.5). The cells were lysed by sonication and centrifuged
(10000<italic>g</italic>
, 40 min, 4 °C) to remove debris. The
supernatant was purified by injection into a Ni–NTA column
(Merck, Darmstadt, Germany) and eluted with buffer containing imidazole
at a gradient range of 15–300 mM. Pure protein fractions were
collected, dialyzed with low-salt buffer, concentrated, and quantified
by the Bradford method (BioShop Canada Inc., Burlington, ON, Canada).</p>
</sec>
<sec id="sec4.3"><title>Crystallization and Data Collection</title>
<p>MERS-CoV N-NTD
crystals were grown as previously described:<sup><xref ref-type="bibr" rid="ref46">46</xref>
</sup>
MERS-CoV N-NTD was crystallized at room temperature (∼25
°C) by the sitting-drop vapor-diffusion method. A protein solution
(2 μL; 10 mg mL<sup>–1</sup>
) was mixed with an equal
volume of crystallization solution consisting of 75 mM ammonium sulfate,
2 mM NaBr, and 29% PEG 3350 (Sigma-Aldrich Corp., St. Louis, MO) and
equilibrated against a 300 μL solution. MERS-CoV N-NTD:P3 co-crystals
were obtained using a crystallization solution containing 2 mM P3.
MERS-CoV N-NTD crystals in complex with P1 were obtained by soaking
native MERS-CoV N-NTD crystals for 90 s at room temperature in a crystallization
solution containing 2 mM P1. Diffraction datasets for MERS-CoV N-NTD
alone and in complex with P1 were collected at beamline 13B1 of the
Taiwan Light Source (TLS) of the National Synchrotron Research Center
(NSRRC; Hsinchu City, Taiwan). Diffraction of the MERS-CoV N-NTD:P3
complex was performed at the beamline SP44XU of SPring-8 (Hyogo, Japan).</p>
</sec>
<sec id="sec4.4"><title>Structural Determination and Refinement</title>
<p>Diffraction
data were processed and scaled with HKL-2000 software. The structures
were solved by molecular replacement (MR) in Phenix<sup><xref ref-type="bibr" rid="ref47">47</xref>
</sup>
using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.<sup><xref ref-type="bibr" rid="ref24">24</xref>
</sup>
The initial models were rebuilt and refined by Coot<sup><xref ref-type="bibr" rid="ref48">48</xref>
</sup>
and Phenix. Structures were visualized using
PyMOL (The PyMOL Molecular Graphics System, version 2.3.0).<sup><xref ref-type="bibr" rid="ref49">49</xref>
</sup>
</p>
</sec>
<sec id="sec4.5"><title>Chemical Cross-Link Assay</title>
<p>Protein
solutions containing
40 μM of wild-type or mutated MERS-CoV N-NTD were incubated
with glutaraldehyde at a final concentration of 1% v/v. The reaction
was conducted at room temperature for 10 min and quenched with the
addition of 1 M Tris-HCl (pH 7.5). The samples were then stored on
ice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).</p>
</sec>
<sec id="sec4.6"><title>Discovery of Orthosteric PPI Stabilizers
for MERS N Protein</title>
<p>To screen for compounds that induce hydrophobic
PPI between MERS
N-NTDs, a model of dimeric MERS-CoV N-NTD without the H37 and M38
residues was used in virtual drug screening. The Sigma-Aldrich, Acros
Organics, and ZINC drug databases were screened with LIBDOCK molecular
docking software to obtain compounds acting on the N protein. The
N protein binding pocket was represented by a set of spheres. Each
compound in the database was docked in a pocket comprising W43. The
hydrophobic complementarity between ligands and receptors was calculated
with PLATINUM.<sup><xref ref-type="bibr" rid="ref31">31</xref>
</sup>
Compounds with higher
docking scores are listed in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">Table S2</ext-link>
.</p>
</sec>
<sec id="sec4.7"><title>Fluorescence Measurements</title>
<p>Fluorescence assays were
performed in a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150
mM NaCl. One micromolar N protein was incubated either with the control
buffer or each compound (10 μM) at 4 °C for 1 h. Tryptophan
fluorescence was acquired with a Jasco FP-8300 fluorescence spectrometer
(JASCO International Co. Ltd., Tokyo, Japan) at an excitation wavelength
of 280 nm and an emission wavelength range of 300–400 nm.</p>
</sec>
<sec id="sec4.8"><title>Thermostability Measurements</title>
<p>Thermostability assays
were conducted in a buffer consisting of 50 mM Tris-HCl (pH 7.5) and
150 mM NaCl and with a JASCO FP-8300 fluorescence spectrometer (JASCO
International Co. Ltd., Tokyo, Japan). One micromolar N protein was
incubated either with the control buffer or each compound (10 μM)
at 4 °C for 2 h. UV absorbance vs temperature profiles were acquired
by ramping the temperature from 4–95 °C at a 1 °C
min<sup>–1</sup>
and recording the absorbance at 280 nm every
0.5 min.</p>
</sec>
<sec id="sec4.9"><title>Determining CC<sub>50</sub>
and EC<sub>50</sub>
of Hit Compounds</title>
<p>Vero E6 cells were infected with MERS-CoV with M.O.I = 0.1 and
treated with lead compounds for 48 h. Cell viability was determined
by the neutral red uptake assay. CC<sub>50</sub>
and EC<sub>50</sub>
were determined by % cell viability. CC<sub>50</sub>
was determined
for cells treated with drugs only. EC<sub>50</sub>
was determined
for MERS-infected cells after drug treatments.</p>
</sec>
<sec id="sec4.10"><title>Small-Angle
X-ray Scattering (SAXS) Experiments</title>
<p>SAXS
experiments were performed at the BL23A SAXS beamline at the TLS of
NSRRC, using a monochromatic X-ray beam (λ = 0.828 Å),
with an integrated HPLC system of an Agilent-Bio SEC-3 300 Å
column (Agilent Technologies, Inc. Santa Clara, CA). Protein samples
(44 μM MERS-CoV N and MERS-CoV N:P3 complex prepared by incubating
the 44 μM native protein with 440 μM P3) were prepared
in a buffer consisting of 50 mM Tris-HCl (pH 8.5) and 150 mM NaCl
on ice for 1 h. Then, a 100 μL aliquot was injected into the
column at a flow rate of 0.02 mL min<sup>–1</sup>
. After passing
through the column, the sample solution was directed
into a quartz capillary (2 mm dia.) for subsequent buffer and sample
SAXS measurement at 288 K. The sample-to-detector distance of 2.5
m used covered a scattering vector <italic>q</italic>
range of 0.01–0.20
Å<sup>–1</sup>
. Here, <italic>q</italic>
is defined as <italic>q</italic>
= (4π/λ) sin θ, with the
scattering angle 2θ. Thirty-six frames were collected for each
sample elution with an X-ray frame exposure time of 30 s. Frames of
good data overlapping (namely, of low radiation damage effects) were
merged for improved data statistics and analyzed to determine initial <italic>R</italic>
<sub>g</sub>
using PRIMUS (version 3.1). The <italic>P</italic>
(<italic>r</italic>
) distance distribution and <italic>D</italic>
<sub>max</sub>
were calculated from the experimental scattering curve
using GNOM (version 4.1). An ensemble optimization method (EOM) analysis
was performed through the EMBL Hamburg web interface.<sup><xref ref-type="bibr" rid="ref50">50</xref>
</sup>
Modeling of the rigid body crystal structure was calculated
and generated using CRYSOL (ATSAS Program Suite v. 2.8.2).<sup><xref ref-type="bibr" rid="ref51">51</xref>
</sup>
The crystal structures of MERS-CoV NTD (PDB
ID: 4UD1)<sup><xref ref-type="bibr" rid="ref27">27</xref>
</sup>
and MERS-CoV NTD:P3 (solved in this study) and
the CTD domain of MERS-CoV N protein (PDB ID: 6G13)<sup><xref ref-type="bibr" rid="ref23">23</xref>
</sup>
were used as rigid bodies in EOM analysis. With the EOM analysis, 1000 models were generated
in the beginning as a structural pool. Selected from the SAXS profiles
of the structural pool was an ensemble of models that could fit the
experimental scattering curve with their linear combination. Tetrameric
MERS-CoV NP conformations and 16-mer MERS-CoV:P3 conformations were
selected because their ensemble generated curves fit best to the experimental
SAXS results.</p>
</sec>
<sec id="sec4.11"><title>Viral Infection</title>
<p>Vero E6 cells (ATCC
No: CRL-1586) were
seeded onto culture plates with complete Dulbecco’s modified
Eagle’s medium (DMEM) and incubated overnight prior to infection.
MERS-CoV (HCoV-EMC/2012) at a multiplicity of infection (M.O.I.) of
0.1 was added to the cells and incubated at 37 °C for 1 h, followed
by washing thrice with phosphate-buffered saline (PBS) to remove the
unattached virus. Fresh complete culture medium was then added to
the plates.</p>
</sec>
<sec id="sec4.12"><title>Plaque Assay</title>
<p>Vero E6 cells were
seeded in 12-well plates
and incubated overnight before the assays. Samples containing MERS-CoV
were serially diluted 10× with MEM, added to the wells, and incubated
for 1 h with agitation every 15 min. After incubation, the inocula
were removed and washed with PBS. An overlay medium comprising 2×
MEM and 1.5% (w/v) agarose (1:1) was added to the wells followed by
incubation at 37 °C and 5% CO<sub>2</sub>
for 3 days. The plates
were fixed with 10% (v/v) formalin containing 0.2% (w/v) crystal violet,
and the plaques were counted.</p>
</sec>
<sec id="sec4.13"><title>RT-qPCR</title>
<p>Total
RNA of infected Vero E6 cells was extracted
with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. Reverse transcription and PCR
amplification were performed with an iTaq Universal One-Step RT-qPCR
Kit (Bio-Rad Laboratories, Hercules, CA). Real-time PCR was performed
in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster
City, CA). The primer pairs used to amplify the viral RNA were as
follows: GAPDH-F: 5′-GAAGGTGAAGGTCGGAGTC-3′; GAPDH-R:
5′-GAAGATGGTGATGGGATTTC-3′;<sup><xref ref-type="bibr" rid="ref53">53</xref>
</sup>
MERS-CoV-F: 5′-CCACTACTCCCATTTCGTCAG-3′; MERS-CoV-R:
5′-CAGTATGTGTAGTGCGCATATAAGCA-3′.<sup><xref ref-type="bibr" rid="ref54">54</xref>
</sup>
The MERS RNA levels were normalized to that of GAPDH and
compared between MERS-CoV groups at 24 h.p.i. and at 48 h.p.i.</p>
</sec>
<sec id="sec4.14"><title>Immunofluorescence
Assay</title>
<p>Vero E6 cells were seeded
in eight-well chamber slides and incubated overnight prior to infection
with MERS-CoV at M.O.I. = 0.1. The cells were fixed with 4% (v/v)
paraformaldehyde for 20 min at 4 °C, followed by permeabilization
in 0.1% (v/v) Triton X-100 for 10 min. Then, 7.5% (v/v) BSA was used
as a blocking buffer for 30 min at 37 °C. Anti-MERS-CoV N primary
antibody (1:500 dilution; Sino Biological Inc., Beijing, China) was
used to stain the virus. The cells were incubated overnight, washed
thrice with PBS, and incubated with Alex Fluor 568 anti-rabbit secondary
antibody (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA)
for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole
(DAPI) was added during the PBS wash. MERS nucleocapsid expression
was examined under a confocal microscope (LSM-700; Carl Zeiss AG,
Oberkochen, Germany).</p>
</sec>
</sec>
</body>
<back><notes id="notes-1" notes-type="si"><title>Supporting Information Available</title>
<p>The Supporting Information
is available free of charge at <ext-link ext-link-type="uri" xlink:href="https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01913?goto=supporting-info">https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01913</ext-link>
.<list id="silist" list-type="simple"><list-item><p>Dimeric conformation of
MERS-CoV N-NTD (Figure S1);
LibDock pose of dimeric MERS-CoV N-NTD with potent compounds (Figure
S2); P3-induced aggregation of the MERS-CoV N protein sequesters the
RNA-binding region on the N-CTD dimer (Figure S3); potential target
sites for broad-spectrum antiviral compound development (Figure S4);
antiviral activity of P3 against MHV (mouse hepatitis virus) (Figure
S5); crystallographic data collection and refinement statistics (Table
S1); pW43 docking pose with chemical structures, docking scores, and
biochemical properties of 17 potential hits (Table S2); primers used
for mutagenesis in this study (Table S3); molecular formula strings
(<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_001.pdf">PDF</ext-link>
)</p>
</list-item>
<list-item><p>Molecular
Formula Strings_JMC_revision (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01913/suppl_file/jm9b01913_si_002.csv">CSV</ext-link>
)</p>
</list-item>
</list>
</p>
</notes>
<sec sec-type="supplementary-material"><title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="sifile1"><media xlink:href="jm9b01913_si_001.pdf"><caption><p>jm9b01913_si_001.pdf</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="sifile2"><media xlink:href="jm9b01913_si_002.csv"><caption><p>jm9b01913_si_002.csv</p>
</caption>
</media>
</supplementary-material>
</sec>
<notes notes-type="accession-codes" id="notes-2"><title>Accession Codes</title>
<p>For PDB codes 6KL2 (native), 6KL5 (N-P1 complex),
and 6KL6 (N-P3
complex), authors will release the atomic coordinates and experimental
data upon article publication.</p>
</notes>
<notes notes-type="" id="notes-3"><title>Author Contributions</title>
<p><sup>○</sup>
S.-M.L.,
S.-C.L., J.-N.H., and C.-k.C. contributed equally to this
work.</p>
</notes>
<notes notes-type="" id="notes-4"><title>Author Contributions</title>
<p>Jia N. Hsu,
Shih C. Lin, Yong S. Wang, and Ming H. Hou designed research; Jia
N. Hsu, Shih C. Lin, Shan M. Lin, Ching M. Chien, and Yong S. Wang
performed research; Hung Y. Wu, U S. Jeng, K. Kehn-Hall, and Ming
H. Hou contributed new reagents or analytic tools; Shih C. Lin, Shan
M. Lin, Ching M. Chien, and Ming H. Hou analyzed the data; Shih C.
Lin, Shan M. Lin, Chung K. Chang, Ching M. Chien, and Ming H. Hou
wrote the paper.</p>
</notes>
<notes notes-type="funding-statement" id="notes-5"><p>This work was
supported by the MOST 106-2628-M-005-001-MY3.</p>
</notes>
<notes notes-type="COI-statement" id="NOTES-d7e1093-autogenerated"><p>The authors declare no
competing financial interest.</p>
</notes>
<ack><title>Acknowledgments</title>
<p>We
thank the National Synchrotron Radiation Research Center
(Taiwan) and the synchrotron radiation facility SPring-8 (Japan) for
X-ray diffraction data collection.</p>
</ack>
<glossary id="dl1"><def-list><title>Abbreviations</title>
<def-item><term>PPIs</term>
<def><p>protein–protein interactions</p>
</def>
</def-item>
<def-item><term>MERS-CoV</term>
<def><p>Middle East
Respiratory Syndrome Coronavirus</p>
</def>
</def-item>
<def-item><term>NTD</term>
<def><p>N-terminal domain</p>
</def>
</def-item>
<def-item><term>CTD</term>
<def><p>C-terminal domain</p>
</def>
</def-item>
<def-item><term>N</term>
<def><p>nucleocapsid</p>
</def>
</def-item>
<def-item><term>β-CoVs</term>
<def><p>betacoronavirus</p>
</def>
</def-item>
<def-item><term>RNP</term>
<def><p>ribonucleoprotein</p>
</def>
</def-item>
<def-item><term>SAXS</term>
<def><p>small-angle X-ray scattering</p>
</def>
</def-item>
<def-item><term>MR</term>
<def><p>molecular replacement</p>
</def>
</def-item>
<def-item><term>BSA</term>
<def><p>buried surface
areas</p>
</def>
</def-item>
<def-item><term>TPSA</term>
<def><p>topological
polar surface areas</p>
</def>
</def-item>
<def-item><term>CC<sub>50</sub>
</term>
<def><p>cytotoxic concentration</p>
</def>
</def-item>
<def-item><term>EC<sub>50</sub>
</term>
<def><p>effective concentration</p>
</def>
</def-item>
<def-item><term><italic>D</italic>
<sub>max</sub>
</term>
<def><p>maximum dimension</p>
</def>
</def-item>
<def-item><term><italic>R</italic>
<sub>g</sub>
</term>
<def><p>radius of gyration</p>
</def>
</def-item>
<def-item><term>MHV</term>
<def><p>mouse hepatitis virus</p>
</def>
</def-item>
</def-list>
</glossary>
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