Aromatic Guanylhydrazones for the Control of Heme-Induced Antibody Polyreactivity
Identifieur interne : 000001 ( Pmc/Corpus ); précédent : 000000; suivant : 000002Aromatic Guanylhydrazones for the Control of Heme-Induced Antibody Polyreactivity
Auteurs : Nina Božinovi ; Vladimir Ajda I ; Jelena Lazic ; Maxime Lecerf ; Victoria Daventure ; Jasmina Nikodinovic-Runic ; Igor M. Opsenica ; Jordan D. DimitrovSource :
- ACS Omega [ 2470-1343 ] ; 2019.
Abstract
In a healthy immune repertoire, there exists a fraction of polyreactive antibodies that can bind to a variety of unrelated self- and foreign antigens. Apart from naturally polyreactive antibodies, in every healthy individual, there is a fraction of antibody that can gain polyreactivity upon exposure to porphyrin cofactor heme. Molecular mechanisms and biological significance of the appearance of cryptic polyreactivity are not well understood. It is believed that heme acts as an interfacial cofactor between the antibody and the newly recognized antigens. To further test this claim and gain insight into the types of interactions involved in heme binding, we herein investigated the influence of a group of aromatic guanylhydrazone molecules on the heme-induced antibody polyreactivity. From the analysis of SAR and the results of UV–vis absorbance spectroscopy, it was concluded that the most probable mechanism by which the studied molecules inhibit heme-mediated polyreactivity of the antibody is the direct binding to heme, thus preventing heme from binding to antibody and/or antigen. The inhibitory capacity of the most potent compounds was substantially higher than that of chloroquine, a well-known heme binder. Some of the guanylhydrazone molecules were able to induce polyreactivity of the studied antibody themselves, possibly by a mechanism similar to heme. Results described here point to the conclusion that heme indeed must bind to an antibody to induce its polyreactivity, and that both π-stacking interactions and iron coordination contribute to the binding affinity, while certain structures, such as guanylhydrazones, can interfere with these processes.
Url:
DOI: 10.1021/acsomega.9b01548
PubMed: 31858028
PubMed Central: 6906781
Links to Exploration step
PMC:6906781Le document en format XML
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<author><name sortKey="Lazic, Jelena" sort="Lazic, Jelena" uniqKey="Lazic J" first="Jelena" last="Lazic">Jelena Lazic</name>
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<front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="ao9b01548_0009" id="ab-tgr1"></graphic>
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<p>In a healthy immune repertoire, there exists a fraction of polyreactive
antibodies that can bind to a variety of unrelated self- and foreign
antigens. Apart from naturally polyreactive antibodies, in every healthy
individual, there is a fraction of antibody that can gain polyreactivity
upon exposure to porphyrin cofactor heme. Molecular mechanisms and
biological significance of the appearance of cryptic polyreactivity
are not well understood. It is believed that heme acts as an interfacial
cofactor between the antibody and the newly recognized antigens. To
further test this claim and gain insight into the types of interactions
involved in heme binding, we herein investigated the influence of
a group of aromatic guanylhydrazone molecules on the heme-induced
antibody polyreactivity. From the analysis of SAR and the results
of UV–vis absorbance spectroscopy, it was concluded that the
most probable mechanism by which the studied molecules inhibit heme-mediated
polyreactivity of the antibody is the direct binding to heme, thus
preventing heme from binding to antibody and/or antigen. The inhibitory
capacity of the most potent compounds was substantially higher than
that of chloroquine, a well-known heme binder. Some of the guanylhydrazone
molecules were able to induce polyreactivity of the studied antibody
themselves, possibly by a mechanism similar to heme. Results described
here point to the conclusion that heme indeed must bind to an antibody
to induce its polyreactivity, and that both π-stacking interactions
and iron coordination contribute to the binding affinity, while certain
structures, such as guanylhydrazones, can interfere with these processes.</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">ACS Omega</journal-id>
<journal-id journal-id-type="iso-abbrev">ACS Omega</journal-id>
<journal-id journal-id-type="publisher-id">ao</journal-id>
<journal-id journal-id-type="coden">acsodf</journal-id>
<journal-title-group><journal-title>ACS Omega</journal-title>
</journal-title-group>
<issn pub-type="epub">2470-1343</issn>
<publisher><publisher-name>American Chemical Society</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">31858028</article-id>
<article-id pub-id-type="pmc">6906781</article-id>
<article-id pub-id-type="doi">10.1021/acsomega.9b01548</article-id>
<article-categories><subj-group><subject>Article</subject>
</subj-group>
</article-categories>
<title-group><article-title>Aromatic Guanylhydrazones for the Control of Heme-Induced
Antibody Polyreactivity</article-title>
</title-group>
<contrib-group><contrib contrib-type="author" corresp="yes" id="ath1"><name><surname>Božinović</surname>
<given-names>Nina</given-names>
</name>
<xref rid="cor1" ref-type="other">*</xref>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath2"><name><surname>Ajdačić</surname>
<given-names>Vladimir</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
</contrib>
<contrib contrib-type="author" id="ath3"><name><surname>Lazic</surname>
<given-names>Jelena</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
</contrib>
<contrib contrib-type="author" id="ath4"><name><surname>Lecerf</surname>
<given-names>Maxime</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath5"><name><surname>Daventure</surname>
<given-names>Victoria</given-names>
</name>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<contrib contrib-type="author" id="ath6"><name><surname>Nikodinovic-Runic</surname>
<given-names>Jasmina</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
<xref rid="aff3" ref-type="aff">§</xref>
</contrib>
<contrib contrib-type="author" id="ath7"><name><surname>Opsenica</surname>
<given-names>Igor M.</given-names>
</name>
<xref rid="aff2" ref-type="aff">‡</xref>
</contrib>
<contrib contrib-type="author" corresp="yes" id="ath8"><name><surname>Dimitrov</surname>
<given-names>Jordan D.</given-names>
</name>
<xref rid="cor2" ref-type="other">*</xref>
<xref rid="aff1" ref-type="aff">†</xref>
</contrib>
<aff id="aff1"><label>†</label>
<institution>Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot</institution>
, F-75006 Paris,<country>France</country>
</aff>
<aff id="aff2"><label>‡</label>
<institution>University of Belgrade—Faculty of Chemistry</institution>
, Studentski trg 16, P.O. Box 51, 11158 Belgrade,<country>Serbia</country>
</aff>
<aff id="aff3"><label>§</label>
Institute of Molecular Genetics and Genetic Engineering,<institution>University of Belgrade</institution>
, Vojvode Stepe 444a, 11000 Belgrade,<country>Serbia</country>
</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>*</label>
E-mail: <email>nina.bozinovic@inserm.fr</email>
. Tel: <phone>+33 1 44 27 82 01</phone>
. Fax: <fax>+33 1 44 27 81
94</fax>
(N.B.).</corresp>
<corresp id="cor2"><label>*</label>
E-mail: <email>jordan.dimitrov@crc.jussieu.fr</email>
, <email>jordan.dimitrov@inserm.fr</email>
. Tel: <phone>+33 1 44
27 82 06</phone>
. Fax: <fax>+33 1 44 27 81 94</fax>
(J.D.D.).</corresp>
</author-notes>
<pub-date pub-type="epub"><day>22</day>
<month>11</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="collection"><day>10</day>
<month>12</month>
<year>2019</year>
</pub-date>
<volume>4</volume>
<issue>24</issue>
<fpage>20450</fpage>
<lpage>20458</lpage>
<history><date date-type="received"><day>27</day>
<month>05</month>
<year>2019</year>
</date>
<date date-type="accepted"><day>06</day>
<month>08</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2019 American Chemical Society</copyright-statement>
<copyright-year>2019</copyright-year>
<copyright-holder>American Chemical Society</copyright-holder>
<license><license-p>This is an open access article published under an ACS AuthorChoice <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_termsofuse.html">License</ext-link>
, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.</license-p>
</license>
</permissions>
<abstract><p content-type="toc-graphic"><graphic xlink:href="ao9b01548_0009" id="ab-tgr1"></graphic>
</p>
<p>In a healthy immune repertoire, there exists a fraction of polyreactive
antibodies that can bind to a variety of unrelated self- and foreign
antigens. Apart from naturally polyreactive antibodies, in every healthy
individual, there is a fraction of antibody that can gain polyreactivity
upon exposure to porphyrin cofactor heme. Molecular mechanisms and
biological significance of the appearance of cryptic polyreactivity
are not well understood. It is believed that heme acts as an interfacial
cofactor between the antibody and the newly recognized antigens. To
further test this claim and gain insight into the types of interactions
involved in heme binding, we herein investigated the influence of
a group of aromatic guanylhydrazone molecules on the heme-induced
antibody polyreactivity. From the analysis of SAR and the results
of UV–vis absorbance spectroscopy, it was concluded that the
most probable mechanism by which the studied molecules inhibit heme-mediated
polyreactivity of the antibody is the direct binding to heme, thus
preventing heme from binding to antibody and/or antigen. The inhibitory
capacity of the most potent compounds was substantially higher than
that of chloroquine, a well-known heme binder. Some of the guanylhydrazone
molecules were able to induce polyreactivity of the studied antibody
themselves, possibly by a mechanism similar to heme. Results described
here point to the conclusion that heme indeed must bind to an antibody
to induce its polyreactivity, and that both π-stacking interactions
and iron coordination contribute to the binding affinity, while certain
structures, such as guanylhydrazones, can interfere with these processes.</p>
</abstract>
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</front>
<body><sec id="sec1"><title>Introduction</title>
<p>Specificity and diversity of adaptive immune responses are critical
features for the proper functioning of the immune system. These properties
of the immune response originate from antigenic receptors on T and
B cells and circulating immunoglobulins. It is estimated that the
human immune system can generate 10<sup>26</sup>
possible B-cell receptor
sequences from V, D, and J region recombination and somatic hypermutations,
providing enormous binding diversity.<sup><xref ref-type="bibr" rid="ref1">1</xref>
,<xref ref-type="bibr" rid="ref2">2</xref>
</sup>
In the early
days of immunology research, it was believed that each antibody is
highly specific for a single antigen. It is now a well-established
fact that the significant fraction of B-cell receptors and antibodies
in a healthy immune repertoire is capable of binding to numerous structurally
unrelated self- or foreign antigens.<sup><xref ref-type="bibr" rid="ref3">3</xref>
−<xref ref-type="bibr" rid="ref7">7</xref>
</sup>
This phenomenon often referred to as antibody polyreactivity, contributes
to the diversification of immune specificities and facilitates the
recognition of pathogens in the early stages of infection.</p>
<p>Apart from polyreactivity occurring naturally, there exist cryptic
polyreactivity that can be induced post-translationally. Exposure
to physiologically relevant redox-active substances, such as iron
ions, reactive oxygen species, and heme causes the appearance of polyreactivity
in a fraction of human immunoglobulins.<sup><xref ref-type="bibr" rid="ref8">8</xref>
−<xref ref-type="bibr" rid="ref12">12</xref>
</sup>
There is evidence suggesting that heme (iron protoporphyrin
IX) induces antibody polyreactivity by direct binding to the variable
region of immunoglobulin molecules. The binding site of heme most
probably overlaps with the antigen-binding site and heme is engaged
in binding to newly recognized antigens.<sup><xref ref-type="bibr" rid="ref13">13</xref>
,<xref ref-type="bibr" rid="ref14">14</xref>
</sup>
Heme molecules contain aromatic pyrrole rings and other hydrophobic
groups, as well as polar, anionic carboxylate groups and an iron ion
capable of coordinative interactions. Such structure provides numerous
possibilities for noncovalent interactions with both immunoglobulins
and possible new antigens.<sup><xref ref-type="bibr" rid="ref15">15</xref>
</sup>
However, the
molecular mechanism of the induced antibody polyreactivity is not
well understood. Additionally, there is no strategy so far for the
control of this phenomenon under physiopathological conditions where
the massive release of intracellular heme occurs such as in hemolytic
diseases.<sup><xref ref-type="bibr" rid="ref9">9</xref>
</sup>
Thus, compounds that have the
capacity to inhibit the effect of heme on antibodies may demonstrate
therapeutic activity for amelioration of the negative proinflammatory
effects of extracellular heme in disorders such as malaria, sickle
cell disease, and autoimmune hemolytic anemia.</p>
<p>To gain an insight into the type of interactions involved, we examined
the effect of a series of heterocyclic guanylhydrazone (iminoguanidine)
molecules on the ability of heme to induce polyreactivity of a prototypic
human monoclonal IgG1. This antibody (Ab21) was previously identified
to gain the capacity to bind with high affinity to structurally different
protein antigens upon heme exposure.<sup><xref ref-type="bibr" rid="ref14">14</xref>
,<xref ref-type="bibr" rid="ref16">16</xref>
,<xref ref-type="bibr" rid="ref17">17</xref>
</sup>
The guanylhydrazone series of molecules investigated
here were chosen based on their expected ability to bind heme by noncovalent
interactions. The studied molecules differed in the number of aromatic
rings and positively charged guanylhydrazone groups, thus providing
different possibilities for coordinative, ionic and π-stacking
interactions with heme and heme-binding site on the immunoglobulin.
We hypothesized that with the gradual increase in the number of structural
elements capable of interacting with heme, the molecules will exhibit
a stronger inhibiting effect on the heme-induced antibody polyreactivity.
Over the past few years, various compounds containing the guanylhydrazone
(iminoguanidine) moiety have attracted much attention for their versatile
biological activities. These compounds were extensively studied for
their antiviral,<sup><xref ref-type="bibr" rid="ref18">18</xref>
,<xref ref-type="bibr" rid="ref19">19</xref>
</sup>
antiparasitic,<sup><xref ref-type="bibr" rid="ref20">20</xref>
</sup>
antibacterial,<sup><xref ref-type="bibr" rid="ref21">21</xref>
−<xref ref-type="bibr" rid="ref23">23</xref>
</sup>
and antifungal activities.<sup><xref ref-type="bibr" rid="ref24">24</xref>
−<xref ref-type="bibr" rid="ref26">26</xref>
</sup>
The ability of the compounds to inhibit or induce antibody polyreactivity
was investigated using standard immunoassays and their interactions
with heme were followed by UV–vis spectroscopic measurements.
We compared the influence of our compounds on the heme-induced polyreactivity
with that of the antimalarial drug chloroquine, which is known to
bind to heme and perturb its interactions. Chloroquine inhibits the
crystallization of toxic ferriprotoporphyrin IX to nontoxic crystalline
hemozoin by binding to monomeric or dimeric forms of free heme, depending
on the chemical environment.<sup><xref ref-type="bibr" rid="ref27">27</xref>
</sup>
</p>
</sec>
<sec id="sec2"><title>Results and Discussion</title>
<p>To study the molecular interactions involved in antibody–heme
binding, we treated Ab21 with a series of guanylhydrazones prior to
heme exposure and followed the changes in the Ab binding potential
to protein antigens. Aromatic guanylhydrazones (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
) were chosen based on their structures that
can provide the possibility for different types of interactions with
heme. They possess positively charged guanylhydrazone groups that
can interact with carboxylates and aromatic rings (benzene, thiophene,
furan, or thiazole) that can form noncovalent interactions with pyrrole
rings. Furthermore, a number of heteroatoms in their structure can
potentially coordinate with an iron ion in the center of the protoporphyrin
IX macrocyclic core. The studied molecules differed in the number
and type of aromatic rings and positively charged guanylhydrazone
groups. The simplest studied molecule is guanabenz, an approved drug
that is used as an antihypertensive agent.<sup><xref ref-type="bibr" rid="ref28">28</xref>
</sup>
It is composed of only one substituted benzene ring and one guanylhydrazone
group (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref>
</xref>
). Other
synthetic iminoguanidines used in this study were previously determined
to exhibit significant antifungal activity.<sup><xref ref-type="bibr" rid="ref24">24</xref>
−<xref ref-type="bibr" rid="ref26">26</xref>
</sup>
They were divided
into three groups based on the structure complexity. The first group
of compounds (compounds <bold>1</bold>
–<bold>8</bold>
) contained
one positively charged iminoguanidine group and two hydrophobic aromatic
rings. Molecules in the second group (<bold>9</bold>
–<bold>11</bold>
) were composed of two positively charged iminoguanidine
groups and two hydrophobic aromatic rings. The third group (<bold>12</bold>
and <bold>13</bold>
) had one additional benzene ring compared
to the second group.</p>
<fig id="fig1" position="float"><label>Figure 1</label>
<caption><p>Structure of aromatic guanylhydrazones.</p>
</caption>
<graphic xlink:href="ao9b01548_0001" id="gr1" position="float"></graphic>
</fig>
<sec id="sec2.1"><title>Acquired Polyreactivity of Ab21</title>
<p>To confirm heme-sensitivity
of Ab21, the binding potential to protein antigens of native and heme-treated
antibody was compared. The selected antibody acquires a strong binding
potential for both human and bacterial antigens after heme treatment
(<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
). After exposure
to two times molar excess of heme, there was substantial binding of
Ab21 to human factor VIII, human C3, and human factor H, as seen in
ELISA experiments (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
A–C). Immunoblot analysis of the interaction of native
and heme-exposed Ab21 with immobilized <italic>Bacillus anthracis</italic>
proteins is in agreement with the conclusion that initially nonreactive
Ab21 acquires the ability to bind to diverse antigens (<xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref>
</xref>
D).</p>
<fig id="fig2" position="float"><label>Figure 2</label>
<caption><p>Heme-induced Ab21 polyreactivity by immunosorbent assay (A–C)
and immunoblot (D).</p>
</caption>
<graphic xlink:href="ao9b01548_0002" id="gr2" position="float"></graphic>
</fig>
</sec>
<sec id="sec2.2"><title>Inhibition of Heme-Induced Antibody Polyreactivity</title>
<p>We evaluated the efficacy of guanylhydrazones to inhibit heme-induced
polyreactivity of Ab21. The antibody was treated with a 30-fold molar
excess of tested compounds, followed by the addition of a 15-fold
molar excess of heme. Binding of native and heme-treated antibody
to three human antigens was examined by ELISA. We hypothesized that
the increase in the complexity will result in an increase of the inhibiting
potential of a given molecule since there is a higher possibility
of it to interact with heme and hence prevent heme from binding to
the IgG. The experiment demonstrated that all tested guanylhydrazones,
except for guanabenz, were able to reduce heme-induced antibody polyreactivity.
Most of the compounds were considerably more efficient inhibitors
than the well-known heme-binding compound, chloroquine. However, contrary
to our prediction, there is a trend of decrease of inhibitory potential
with an increase of the molecular complexity (<xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref>
</xref>
). This may be explained by the lower possibility
for the molecules with higher complexity to adopt a planar conformation
that is necessary for the aromatic moieties to be employed in interactions
with the porphyrin ring. Under experimental conditions applied here,
compounds <bold>1</bold>
–<bold>5</bold>
were able to completely
inhibit acquired polyreactivity of Ab21. To determine more precisely
and compare inhibiting potentials of the two most potent compounds <bold>3</bold>
and <bold>4</bold>
, Ab21 was treated with decreasing concentrations
of <bold>3</bold>
or <bold>4</bold>
in the presence of a constant
concentration of hemin. The binding of treated and non-treated antibody
was followed by ELISA and western blot (<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
A,C). In both experiments, compound <bold>3</bold>
was revealed to be more efficient, given that a lower concentration
was required to completely neutralize the activating potential of
heme (approximately equimolar concentration to heme). The estimated
half-maximal inhibitory concentrations (IC<sub>50</sub>
) of compounds <bold>3</bold>
and <bold>4</bold>
were 3.0 and 5.5 μM, respectively.
An important point can be made for compound <bold>11</bold>
that has
methyl substituents on the carbon of the iminoguanidine group, compared
to compound <bold>10</bold>
that has hydrogen in the same position.
Steric hindrance adjacent to nitrogen atoms decreases the inhibiting
potential of the compound, indicating that these nitrogen atoms play
an important role in the mechanism of the inhibition of the induced
polyreactivity, possibly by coordinating iron ion of heme. This observation
points to the conclusion that guanylhydrazones interact directly with
heme and block its interaction with antibody and/or antigen. Our conclusion
is further supported by the fact that compounds <bold>3</bold>
and <bold>4</bold>
were also able to completely inhibit heme-induced polyreactivity
of pooled immunoglobulin G, which is obtained from plasma of a large
number of healthy donors (IVIg), as can be seen from the ELISA experiment
(<xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref>
</xref>
B). Although
we cannot completely exclude the possibility that the compounds interact
with immunoglobulins, the fact that they inhibit heme-induced polyreactivity
of many different immunoglobulins in the mixture is in accordance
with the conclusion that the inhibitory effect of the compounds is
the consequence of their interactions with heme, rather than with
somatically maturated Ab21.</p>
<fig id="fig3" position="float"><label>Figure 3</label>
<caption><p>Results of the study of inhibition of heme-induced Ab21 polyreactivity
by immunosorbent assay. Binding to particular antigen was expressed
as a percentage of the binding of the heme-exposed Ab21. Binding of
native Ab21 is represented by the blue line and binding of Ab21 exposed
to chloroquine and heme is represented by the red line.</p>
</caption>
<graphic xlink:href="ao9b01548_0003" id="gr3" position="float"></graphic>
</fig>
<fig id="fig4" position="float"><label>Figure 4</label>
<caption><p>Inhibitory potential of compounds <bold>3</bold>
and <bold>4</bold>
determined by immunosorbent assay (left) and immunoblot (right).
Ab21 (A) or IVIg (B) was treated with an excess of hemin and with
increasing concentrations of guanylhydrazones.</p>
</caption>
<graphic xlink:href="ao9b01548_0004" id="gr4" position="float"></graphic>
</fig>
</sec>
<sec id="sec2.3"><title>Spectroscopic Investigation of Interactions of Heme with Compounds <bold>1</bold>
–<bold>13</bold>
</title>
<p>The interaction of heme
with different molecules results in the changes in the Soret region
of the UV–vis absorbance spectrum of oxidized heme.<sup><xref ref-type="bibr" rid="ref29">29</xref>
</sup>
We hypothesized that guanylhydrazones prevent
the binding of heme to antibodies by direct interaction through π-stacking
interactions and coordination of the iron ion. To test this assumption,
we estimated the influence of the tested compounds on the UV–vis
absorbance spectrum of hemin. The spectrum of 20 μM hemin in
PBS displayed the characteristic maximum in the Soret region, at 382
nm. The addition of five times excess of guanylhydrazones resulted
in a red shift of the absorbance maximum and a decrease of the absorbance
intensity. Differential spectra were obtained by subtraction of the
individual spectra of hemin and the compound from the spectra of their
mixture (<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
,
left). Next, the percentage of the inhibition of heme-induced antibody
binding to factor VIII was plotted against the absolute value of Δ<italic>A</italic>
at the absorbance minimum of each differential spectrum
(<xref rid="fig5" ref-type="fig">Figure <xref rid="fig5" ref-type="fig">5</xref>
</xref>
, right).
A general trend can be observed of the increase of inhibiting potential
with the increase of the absorbance intensity change (<italic>R</italic>
<sup>2</sup>
= 0.43, <italic>p</italic>
= 0.016). The obtained data
suggest that the direct interaction of guanylhydrazones with heme
is to a significant extent responsible for the diminished capacity
of heme to induce antigen-binding polyreactivity of the antibody.</p>
<fig id="fig5" position="float"><label>Figure 5</label>
<caption><p>Left: the spectroscopic study of 20 μM hemin with 100 μM
guanylhydrazones. Differential spectra were obtained by subtraction
of the individual spectra of hemin and the compound from the spectrum
of their mixture. Right: the percentage of the inhibition of heme-induced
antibody binding to factor VIII plotted against the absolute value
of Δ<italic>A</italic>
at the absorbance minimum of each differential
spectrum. Each dot represents one compound.</p>
</caption>
<graphic xlink:href="ao9b01548_0005" id="gr5" position="float"></graphic>
</fig>
</sec>
<sec id="sec2.4"><title>Spectroscopic Investigation of the Influence of Compound <bold>3</bold>
on Antibody–Heme Interactions</title>
<p>It was previously
demonstrated that the binding of heme to Ab21 can be followed by absorbance
spectroscopy.<sup><xref ref-type="bibr" rid="ref14">14</xref>
</sup>
Here, we confirmed that
the exposure of 2 μM Ab21 to 16 μM hemin results in an
increase in the absorbance intensity of hemin and a substantial red
shift in the high energy region (black line in <xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref>
</xref>
, left), which is consistent with binding
of heme and coordination of its central iron ion to the antibody molecule.
To test the influence of compound <bold>3</bold>
on antibody–heme
binding, we followed the changes in the UV–vis absorbance spectrum
of the Ab21 and the hemin mixture in the presence of the increasing
concentration of compound <bold>3</bold>
. Differential spectra (<xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref>
</xref>
, left) were obtained
by subtracting the spectra of the heme–compound mixture from
the spectrum of the heme–antibody–compound mixture at
the same concentration of the compound. It is clear that the compound
has a strong effect on antibody–heme binding. However, due
to the complexity of possible interactions in the tri-component mixture,
it is difficult to deduce the nature of the observed effect. This
is partially because the binding of Ab21 has a stronger effect on
the hemin spectrum than the binding of the compound. The value of Δ<italic>A</italic>
at the absorbance maximum of each differential spectrum
was plotted against the concentration of compound <bold>3</bold>
(<xref rid="fig6" ref-type="fig">Figure <xref rid="fig6" ref-type="fig">6</xref>
</xref>
, right). It is possible
that at the lower concentration of the compound, the dominant influence
on the hemin spectrum is the blocking of the antibody–hemin
binding (negative values of Δ<italic>A</italic>
). As the concentration
of <bold>3</bold>
approaches that of hemin, the spectral shift becomes
the consequence of compound–hemin binding.</p>
<fig id="fig6" position="float"><label>Figure 6</label>
<caption><p>Left: the spectroscopic study of 16 μM hemin and 2 μM
Ab21, titrated with the increasing concentration (0, 1, 2, 4, 8, 16,
and 32 μM) of compound <bold>3</bold>
. Differential spectra
were obtained by subtracting the spectra of the heme–compound
mixture from the spectrum of the heme–antibody–compound
mixture at the same concentration of the compound. Right: the value
of Δ<italic>A</italic>
at the absorbance maximum of each differential
spectrum plotted against the concentration of compound <bold>3</bold>
.</p>
</caption>
<graphic xlink:href="ao9b01548_0006" id="gr6" position="float"></graphic>
</fig>
</sec>
<sec id="sec2.5"><title>Induction of Ab21 Polyreactivity by Guanylhydrazones</title>
<p>Unexpectedly, two of the compounds
studied for the inhibition of heme-mediated Ab polyreactivity were
able to trigger antigen-binding polyreactivity of Ab21 themselves.
The antibody was treated with a 30-fold molar excess of tested compounds,
and the binding of the native and treated antibody to three human
antigens was evaluated by ELISA (<xref rid="fig7" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref>
</xref>
A–C). Compounds <bold>12</bold>
and <bold>13</bold>
were inducing Ab21 binding to the studied antigens between
25 and 35% of the binding observed after exposure to heme. It is known
that the IgG exposure to reactive oxygen species can also lead to
the development of polyreactivity.<sup><xref ref-type="bibr" rid="ref10">10</xref>
</sup>
When
irradiated with visible light, protoporphyrin IX is known to produce
these activated forms of oxygen.<sup><xref ref-type="bibr" rid="ref30">30</xref>
</sup>
Indeed,
we demonstrated that the ability of protoporphyrin IX to induce antibody
binding to factor VIII increases when the experiment is repeated under
ambient light. In contrast, the efficacy of compound <bold>13</bold>
to influence Ab–protein binding did not depend on the presence
of light (<xref rid="fig7" ref-type="fig">Figure <xref rid="fig7" ref-type="fig">7</xref>
</xref>
D),
which excludes the formation of ROS as its mechanism of action. Next,
we wanted to investigate whether <bold>13</bold>
gains polyreactivity-inducing
ability because it forms a coordination complex with iron ions present
in traces in the buffer solutions. To this end, Ab21 was exposed to
increasing equimolar concentrations of compound <bold>13</bold>
and
an iron salt (FeSO<sub>4</sub>
or FeCl<sub>3</sub>
), independently
and in the mixture, and the interaction with human factor VIII was
elucidated by ELISA. The curve representing the sum of absorbance
values obtained for compound <bold>13</bold>
and iron alone was compared
with that of the mixture (<xref rid="fig8" ref-type="fig">Figure <xref rid="fig8" ref-type="fig">8</xref>
</xref>
). The addition of Fe(II) or Fe(III) ions did not significantly
influence the polyreactivity-inducing potential of compound <bold>13</bold>
, indicating that the presence of iron ions is not needed
for the effect. The results point to the conclusion that the induction
of polyreactivity occurs by the direct interaction of a compound with
the sensitive antibody, possibly by the same mechanism as that of
heme.</p>
<fig id="fig7" position="float"><label>Figure 7</label>
<caption><p>(A–C) Results of the study of induction of Ab21 polyreactivity
by the immunosorbent assay. The binding to particular antigen was
expressed as a percentage of the binding of the heme-exposed Ab21.
(D) Induction of Ab21 polyreactivity by protoporphyrin IX and compound <bold>13</bold>
with and without irradiation. Binding of native Ab21 is
represented by the blue line.</p>
</caption>
<graphic xlink:href="ao9b01548_0007" id="gr7" position="float"></graphic>
</fig>
<fig id="fig8" position="float"><label>Figure 8</label>
<caption><p>Effect of iron(III) and iron(II) ions on the induction of Ab21
polyreactivity by compound <bold>13</bold>
. The sum of absorbance
values for Ab21 treated with compound <bold>13</bold>
and Ab21 treated
with iron ions is shown in red circles. Black triangles represent
absorbance values for Ab21 treated with increasing concentrations
of an equimolar mixture of compound <bold>13</bold>
and iron ions.</p>
</caption>
<graphic xlink:href="ao9b01548_0008" id="gr8" position="float"></graphic>
</fig>
</sec>
<sec id="sec2.6"><title>Cytotoxicity Assessment of Compounds <bold>3</bold>
and <bold>4</bold>
</title>
<p>Since these molecules are very efficient in preventing
heme from binding to antibodies, they can serve as leads for the development
of the new class of therapeutics to be used in the treatment of complications
related to hemolytic diseases. Previous studies have demonstrated
that this class of compounds is moderately toxic.<sup><xref ref-type="bibr" rid="ref25">25</xref>
</sup>
Here, the antiproliferative activity of two most potent
inhibitors was tested on human lung fibroblasts by the MTT assay.
The concentration of the compound inhibiting cell growth by 50% (IC<sub>50</sub>
) was 6.8 μM for compound <bold>3</bold>
, and 8.4 μM
for compound <bold>4</bold>
. Compared to IC<sub>50</sub>
of the widely
used drug chloroquine (58.0 μM), in the assay used, compounds
were 8.5 and 6.9 times more toxic, suggesting that structural changes
should be introduced to reduce the toxicity while keeping heme binding
capacity, before the compounds can be considered for therapeutic use.</p>
</sec>
</sec>
<sec id="sec3"><title>Conclusions</title>
<p>Previous data suggested that the necessary condition for heme-mediated
antibody polyreactivity is the direct binding of heme to the antibody.
To further test this premise, fourteen aromatic guanylhydrazones have
been investigated for their ability to interact with heme and block
the binding of the latter to the selected antibody (Ab21). Tested
guanylhydrazones differ in the number of positively charged iminoguanidine
groups and aromatic rings. Indeed, most of the studied molecules were
able to inhibit the induction of antigen-binding polyreactivity to
various extents. The inhibiting potential was shown to be decreasing
with the increasing complexity of the molecules. Such result points
to the conclusion that guanylhydrazones of less complex structures
interact with heme by establishing π-stacking interactions;
additional groups make it more difficult for the molecule to adopt
planar conformation necessary for the efficient interaction. Other
mechanisms for the more complex molecules can be considered. Steric
hindrance in the proximity of iminoguanidine nitrogen atoms leads
to the loss of the large part of the inhibiting potential, suggesting
that the nitrogen atom is involved in an important interaction with
heme, possibly the coordination of the central metal ion. Investigation
of the influence of guanylhydrazones on the absorbance spectra of
heme demonstrated that there was a red shift of the absorbance maximum
in the Soret region and significant lowering of the absorbance intensity.
The extent of the change is in accordance with the hypothesis that
strong coordinative interactions with iron are formed. The correlation
between the spectral change and the inhibiting potential of the compounds
implies that they inhibit heme-induced Ab polyreactivity by binding
to heme, thus preventing the interaction with antibodies. It is noteworthy
that the inhibitory capacity of most of the studied compounds was
substantially higher than that of chloroquine. The interaction of
heme with chloroquine is a well-elucidated archetypical case of molecular
recognition of heme by a low molecular weight heterocyclic drug.</p>
<p>The surprising revelation was that two of the iminoguanidines were
able to induce antibody polyreactivity without the presence of heme.
We excluded the possibility that they generate reactive oxygen species
under described experimental conditions or that the polyreactivity
induction is assisted by iron ions present in solution. Thus, we concluded
that they bind directly to the antibody and act as interfacial cofactors
between the antibody and the antigen, similarly as heme does. Significantly
lower inducing potential compared to heme highlights the importance
of the unique heme structure and a possible contribution of coordination
of heme’s iron to the overall binding affinity to the antibody.</p>
<p>Additionally, the ability of guanylhydrazones to inhibit antibody
polyreactivity induced by heme provides a possibility to control the
biological effects of the release of large quantities of heme into
circulation, related to hemolytic diseases. Previously found to be
relatively nontoxic, these molecules can serve as leads for the development
of a new class of therapeutics to be used in the treatment of hemolytic
diseases and autoimmunity. Furthermore, the fact that they are more
efficient in preventing heme from binding to antibodies than the well-known
heme-inhibiting antimalarial agent chloroquine, suggests that their
antimalarial activity should also be investigated.</p>
</sec>
<sec id="sec4"><title>Experimental Section</title>
<p>Hemin, Fe(III)-protoporphyrin IX chloride, was obtained from Frontier
Scientific, Inc. (Logan, UT). Guanabenz, chloroquine diphosphate,
DMSO, and protoporphyrin IX were obtained from Sigma-Aldrich (St.
Louis, MO). All chemicals were with the highest available purity.
The synthesis of compounds <bold>1</bold>
–<bold>13</bold>
was
previously described.<sup><xref ref-type="bibr" rid="ref24">24</xref>
−<xref ref-type="bibr" rid="ref26">26</xref>
</sup>
In brief, twelve aromatic aldehydes and one ketone
were synthesized using the Suzuki–Miyaura reaction of an aryl
halide and an appropriate boronic acid, and the final products were
obtained by a one-step condensation reaction of aminoguanidine hydrochloride
and the corresponding carbonyl compound. Details about the production
of Ab21 are published elsewhere.<sup><xref ref-type="bibr" rid="ref31">31</xref>
,<xref ref-type="bibr" rid="ref32">32</xref>
</sup>
Briefly, this
is a human monoclonal IgG1 antibody whose variable genes encoding
the immunoglobulin heavy and light chains were amplified from a B-cell
by single-cell PCR from synovial tissue of rheumatoid arthritis patients,
cloned in an expression vector containing the genes encoding the constant
Fc-γ1 or κ chain, respectively, and expressed using HEK293.
Ab21 was thoroughly dialyzed against PBS containing 10% sucrose and
stored before use at −20 °C at a concentration of 12 mg/mL.
Human pooled immunoglobulin G (IVIg, Endobulin, Baxter) was dialyzed
against PBS and stored before use at −20 °C at a concentration
of 80 mg/mL. Hemin was dissolved in DMSO to a final concentration
of 2 mM. Guanylhydrazones were dissolved in DMSO to a final concentration
of 10 mM. The treatment of Ab21 was always performed with freshly
prepared heme, at dim light conditions, unless stated otherwise. Measurements
of heme–compound interactions were performed using following
experimental setting: the UV–vis absorbance spectra were recorded
by a Cary-300 spectrophotometer (Agilent Technologies, Santa-Clara,
CA) using 1 mL quartz optical cells (Hellma, Jena, Germany) with a
1 cm optical path. The spectra were recorded in the wavelength range
of 300–700 nm with a spectral resolution of 1 nm and a bandwidth
set at 2 nm. The absorbance background derived by the buffer was only
subtracted from each reading. Measurements were performed at room
temperature.</p>
<sec id="sec4.1"><title>Immunosorbent Assay for Heme-Induced Ab21 Polyreactivity</title>
<p>Ninety-six-well polystyrene plates (Nunc MaxiSorp) were coated with
human factor VIII, human C3, and human factor H and diluted to 2 μg/mL
in PBS. After incubation for 2 h at room temperature, the residual
binding sites on plates were blocked by PBS containing 0.25% Tween
20. For treatment, Ab21 was diluted to 500 μg/mL (3.33 μM)
and exposed to 6.67 μM final concentration of hemin. After 10
min of incubation on ice, native and heme-exposed Ab21 was first diluted
with PBS-T (0.05% Tween 20) to 100 μg/mL. It was further serially
diluted with PBS-T in the range of 0.097–100 μg/mL (dilution
factor of 2) and incubated for 1 h at room temperature with plates
coated with the studied proteins. After incubation with antibodies,
plates were washed extensively with PBS-T and incubated with a peroxidase-conjugated
mouse anti-human IgG (clone JDC-10, Southern Biotech, Birmingham,
AL) for 1 h at room temperature. Immunoreactivity of Ab21 was revealed
by measuring the absorbance at 492 nm after the addition of peroxidase
substrate, <italic>o</italic>
-phenylenediamine dihydrochloride (Sigma-Aldrich)
and stopping the reaction by the addition of 2 M HCl. Measurement
of the absorbance was performed with a microplate reader (Infinite
200 Pro, Tecan).</p>
<p>Following sections describe variations of the
procedure. Initial and final steps of the experiments are identical
to those described above.</p>
<sec id="sec4.1.1"><title>Immunosorbent Assay for the Study of Inhibition of Heme-Induced
Ab21 Polyreactivity</title>
<p>Ab21 was diluted to 100 μg/mL and
exposed to 20 μM final concentration of tested compounds. After
10 min of incubation on ice, compound-treated Ab21 was exposed to
10 μM of hemin. As a positive control, Ab21 at 100 μg/mL
was treated only with 10 μM of hemin. After 15 min of incubation
on ice, native and compound and/or heme-exposed Ab21 was first diluted
with PBS-T (0.05% Tween 20) to a final concentration of 10 μg/mL
and incubated for 1 h at room temperature with plates coated with
the studied proteins.</p>
</sec>
<sec id="sec4.1.2"><title>Comparison of Inhibitory Potential of Compounds <bold>3</bold>
and <bold>4</bold>
by Immunosorbent Assay</title>
<p>Ab21 was diluted
to 100 μg/mL (0.67 μM) and IVIg was diluted to 1 mg/mL
(6.67 μM). Antibody solutions were exposed to decreasing concentrations
(85.76, 42.88, 21.44, 10.72, 5.36, 2.68, 1.34, 0.67, 0.33, and 0 μM)
of compound <bold>3</bold>
or <bold>4</bold>
. After 10 min of incubation
on ice, compound-treated antibodies were exposed to 10 μM of
hemin. After 15 min of incubation on ice, native and compound and/or
heme-exposed antibodies were first diluted with PBS-T (0.05% Tween
20) to the final concentrations of 10 μg/mL for Ab21 and 100
μg/mL for IVIg, and incubated for 1 h at room temperature with
a plate coated with the studied proteins.</p>
</sec>
<sec id="sec4.1.3"><title>Immunosorbent Assay for the Study of Induction of Ab21 Polyreactivity</title>
<p>Ab21 was diluted to 100 μg/mL and exposed to 20 μM
final concentration of tested compounds. As a positive control, Ab21
at 100 μg/mL was treated with 10 μM of hemin. After 10
min of incubation on ice, native and compound exposed Ab21 was first
diluted with PBS-T (0.05% Tween 20) to a final concentration of 10
μg/mL and incubated for 1 h at room temperature with plates
coated with the studied proteins.</p>
</sec>
<sec id="sec4.1.4"><title>Immunosorbent Assay for the Study of the Effect of Iron Ions
on the Induction of Ab21 Polyreactivity by Compound <bold>13</bold>
</title>
<p>Ab21 was diluted to 100 μg/mL (0.67 μM) and
exposed to decreasing concentrations (85.76, 42.88, 21.44, 10.72,
5.36, 2.68, 1.34, 0.67, 0.33, and 0 μM) of compound <bold>13</bold>
, iron salt (FeCl<sub>3</sub>
or FeSO<sub>4</sub>
), or the mixture
of compound and iron. After 15 min of incubation on ice, native and
compound and/or iron-exposed Ab21 was first diluted with PBS-T (0.05%
Tween 20) to a final concentration of 10 μg/mL and incubated
for 1 h at room temperature with the plate coated with the studied
proteins.</p>
</sec>
</sec>
<sec id="sec4.2"><title>Comparison of Inhibitory Potential of Compounds <bold>3</bold>
and <bold>4</bold>
by Western Blot</title>
<p>Bacterial antigens from <italic>B. anthracis</italic>
cell lysate were first separated by
SDS-PAGE (Novex NuPAGE 4–12% Bis-Tris gel, 1.0 mm, Life Technologies)
and then transferred onto nitrocellulose membranes using an iBlot
dry transfer system (Life Technologies). After incubation at 4 °C
overnight with PBS-Tween 0.1%, membranes were mounted in the Miniblot
apparatus (Immunetics, Boston, MA). Ab21 was treated at a 100 μg/mL
concentration in PBS, with hemin solubilized in DMSO (final heme concentration
2.5 μM) and with decreasing concentrations of guanylhydrazones
(100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0 μM).
After 15 min of incubation, native and treated Ab21 was diluted 10-fold
in PBS-Tween 0.1% and loaded on Miniblot channels. After 1 h of incubation
at room temperature, nitrocellulose membranes were removed from the
Miniblot apparatus, washed for 1 h with PBS-Tween 0.1% and incubated
with alkaline phosphatase-conjugated goat anti-human IgG antibody
(Southern Biotech). The enzymatic reaction was performed using the
BCIP/NBT substrate (Sigma-Aldrich).</p>
</sec>
<sec id="sec4.3"><title>Spectroscopic Analyses of Interaction of Heme with Guanylhydrazone
Compounds</title>
<p>Hemin was diluted to 20 μM in PBS. The UV–vis
spectra in the absence and presence of 100 μM final concentration
of compounds were recorded. The UV–vis spectra of 100 μM
final concentration of compounds in PBS were recorded. Differential
spectra were obtained after subtraction of spectra of heme and compound
alone from the spectrum the heme–compound mixture.</p>
</sec>
<sec id="sec4.4"><title>Spectroscopic Analyses of the Influence of Guanylhydrazone Compound <bold>3</bold>
on Antibody–Heme Interactions</title>
<p>A mixture
of Ab21 (2 μM) and hemin (16 μM) in PBS was titrated with
the increasing concentrations of compound <bold>3</bold>
(0, 1, 2,
4, 8, 16, and 32 μM). Hemin alone at 16 μM in PBS was
titrated with the same concentrations of compound <bold>3</bold>
.
Differential spectra were obtained after subtraction of spectra of
heme and compound from the spectra of the heme–antibody–compound
mixture at the same concentration of the compound.</p>
</sec>
<sec id="sec4.5"><title>Antiproliferative Activity of Chloroquine and Compounds <bold>3</bold>
and <bold>4</bold>
</title>
<p>Antiproliferative activity of
the compounds was tested on human lung fibroblasts (MRC5; ATCC collection)
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The cells were incubated in the media containing test compounds
at concentrations ranging from 0.1 to 250 μg/mL and the cell
viability was measured after 48 h. The MRC5 cell line was cultured
in RPMI-1640 medium supplemented with 100 μg/mL streptomycin,
100 U/mL penicillin and 10% (v/v) fetal bovine serum (FBS) (all from
Sigma, Munich, Germany). The cells were maintained as a monolayer
(1 × 10<sup>4</sup>
cells per well) in RPMI-1640 and grown in
a humidified atmosphere of 95% air and 5% CO<sub>2</sub>
at 37 °C.
The extent of MTT reduction was determined by measuring the absorbance
at 540 nm with a microplate reader (Infinite 200 Pro, Tecan) and the
cell survival was expressed as a percentage of the control (untreated
cells). Cytotoxicity is expressed as the concentration of the compound
inhibiting growth by 50% (IC<sub>50</sub>
).</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/acsomega.9b01548?goto=supporting-info">https://pubs.acs.org/doi/10.1021/acsomega.9b01548</ext-link>
.<list id="silist" list-type="simple"><list-item><p>Differential spectra for compounds <bold>1</bold>
–<bold>13</bold>
obtained by subtraction of the individual spectra of hemin
and the compound from the spectrum of their mixture (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acsomega.9b01548/suppl_file/ao9b01548_si_001.pdf">PDF</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="ao9b01548_si_001.pdf"><caption><p>ao9b01548_si_001.pdf</p>
</caption>
</media>
</supplementary-material>
</sec>
<notes notes-type="COI-statement" id="NOTES-d7e830-autogenerated"><p>The authors declare no
competing financial interest.</p>
</notes>
<ack><title>Acknowledgments</title>
<p>This work was supported by the European Research Council (grant
ERC-StG-678905 CoBABATI to J.D.D.), the INSERM, the Ministry of Education,
Science and Technological Development of Serbia (Grant No. 172008),
and the PHC French-Serbian partnership project Pavle Savić
(451-03-01963/2017-09/03).</p>
</ack>
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