Discovery of a New Inhibitor of Myeloid Differentiation 2 from Cinnamamide Derivatives with Anti-Inflammatory Activity in Sepsis and Acute Lung Injury

Article abstract of DOI:10.1021/acs.jmedchem.5b01574

Acute inflammatory diseases, including acute lung injury and sepsis, remain the most common life-threatening illness in intensive care units worldwide. Cinnamamide has been incorporated in several synthetic compounds with therapeutic potentials including anti-inflammatory properties. However, the possible mechanism and direct molecular target of cinnamamides for their anti-inflammatory effects were rarely investigated. In this study, we synthesized a series of cinnamamides and evaluated their anti-inflammatory activities. The most active compound, 2i, was found to block LPS-induced MD2/TLR4 pro-inflammatory signaling activation in vitro and to attenuate LPS-caused sepsis and acute lung injury in vivo. Mechanistically, we demonstrated that 2i exerts its anti-inflammatory effects by directly targeting and binding MD2 in Arg90 and Tyr102 residues and inhibiting MD2/TLR4 complex formation. Taken together, this work presents a novel MD2 inhibitor, 2i, which has the potential to be developed as a candidate for the treatment of sepsis, and provides a new lead structure for the development of anti-inflammatory agents targeting MD2.

Full text of DOI:10.1021/acs.jmedchem.5b01574

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Article
Discovery of a new inhibitor of myeloid differentiation 2 from cinnamamide
derivatives with anti-inflammatory activity in sepsis and acute lung injury
Gaozhi Chen, Yali Zhang, Xing Liu, Qilu Fang, Zhe Wang, Lili Fu,
Zhiguo Liu, Yi Wang, Yunjie Zhao, Xiaokun Li, and Guang Liang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01574 • Publication Date (Web): 03 Mar 2016
Downloaded from http://pubs.acs.org on March 6, 2016
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Discovery of a new inhibitor of myeloid differentiation 2 from
cinnamamide derivatives with anti-inflammatory activity in sepsis
and acute lung injury
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Gaozhi Chen, Yali Zhang, Xing Liu, Qilu Fang, Zhe Wang, Lili Fu, Zhiguo Liu, Yi Wang, Yunjie
Zhao, Xiaokun Li, Guang Liang*
Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China
*
Corresponding authors:
Guang Liang, Ph.D, Professor
Director, Chemical Biology Research Center
School of Pharmaceutical Sciences, Wenzhou Medical University
Tel: (+86)ꢀ577ꢀ86699396; Fax: (+86)ꢀ577ꢀ86699396
Eꢀmail: wzmcliangguang@163.com
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Abstract: Acute inflammatory diseases, including acute lung injury and sepsis, remain the most
common lifeꢀthreatening illness in ICU worldwide. Cinnamamide has been incorporated in
several synthetic compounds with therapeutic potentials including antiꢀinflammatory properties.
However, the possible mechanism and direct molecular target of cinnamamides for their
antiꢀinflammatory effects were rarely investigated. In this study, we synthesized a series of
cinnamamides and evaluated their antiꢀinflammatory activities. The most active compound, 2i,
was found to block LPSꢀinduced MD2/TLR4 proꢀinflammatory signaling activation in vitro and
to attenuate LPSꢀcaused sepsis and acute lung injury in vivo. Mechanistically, we demonstrated
that 2i exerts its antiꢀinflammatory effects by directly targeting and binding MD2 in Arg90 and
Tyr102 residues and inhibiting MD2/TLR4 complex formation. Taken together, this work
presents a novel MD2 inhibitor, 2i, which has the potential to be developed as a candidate for
the treatment of sepsis, and provides a new lead structure for the development of
antiꢀinflammatory agents targeting MD2.
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Keywords: Acute inflammatory diseases, Cinnamanides, MD2, TLR4, Acute lung injury
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Introduction
Acute inflammatory diseases such as acute lung injury (ALI) and sepsis, which occur as a
result of local or systemic inflammation, remain the most common cause of death in intensive
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care units worldwide . Lipopolysaccharide (LPS) is a main stimulator of the acute inflammation
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that leads to sepsis and ALI . Microbial LPS binds to a surface receptor complex consisting of
myeloid differentiation 2 (MD2) and tollꢀlike receptor 4 (TLR4) in innate immune cells through
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ancillary proteins such as LPSꢀbinding protein (LBP) and cluster of differentiation 14 (CD14) .
The LPSꢀTLR4/MD2 complex activates downstream proꢀinflammatory signaling pathways,
including the transcription factor nuclear factor kappaꢀlightꢀchainꢀenhancer of activated B cells
(
NFꢀκB) and mitogenꢀactivated protein kinases (MAPKs), triggering the production of a variety
of inflammatory cytokines. The excessive release of various proꢀinflammatory cytokines result
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in the acute cellular and/or organic injury to form sepsis or ALI . Sepsis remains the leading
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cause of mortality in ICU and leads to millions of deaths globally each year , and the outcomes
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of ALI also remain dismal with a morbidity and mortality rate around 40% .
So far, there is no specific and effective therapeutic strategy available to treat these acute
inflammatory diseases. A novel and effective therapeutic agent is urgently required. A series of
agents that target TLR4 have been investigated for the treatment of sepsis and ALI but have
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failed in preꢀclinical study or in clinical trials . In fact, although TLR4 is the central signaling
receptor for LPS, LPS binds directly to the chaperone protein MD2, rather than directly to
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TLR4 . Recognition of LPS by TLR4 needs MD2. It has been demonstrated that MD2 is an
essential protein for the intracellular distribution of TLR4 and recognition between TLR4 and
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LPS, and that MD2 knockout significantly reversed the LPSꢀinduced endotoxic shock in mice .
Thus, increasing evidence has revealed that MD2 could be an important target for the discovery
of antiꢀinflammatory agents.
The discovery of novel antiꢀinflammatory agents derived from natural active products has
attracted a lot of attention from medicinal chemists. Cinnamic acid is a natural organic acid in
plants with high safety and a variety of pharmacological activities, such as antiꢀoxidant,
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antimicrobial, antiꢀcancer, and antiꢀinflammatory activities
. Due to their common occurence
in plants and their low toxicity, cinnamic acid derivatives have been evaluated as
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pharmacologically active compounds . Among the derivatives, cinnamamide (Scheme 1)
constitutes an interesting scaffold within medicinal chemistry thus it has been incorporated in
several synthetic compounds with therapeutic potentials including neuroprotective,
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antimicrobial, antiꢀnociceptive, and antiꢀinflammatory properties
. We speculate that
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cinnamamide derivatives may represent beneficial pharmacological enhancements in
antiꢀinflammatory action.
While a few derivatives of cinnamamides exhibited antiꢀinflammatory and/or analgesic
activity, the current results of pharmacological evaluation did not lead to certain conclusions
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about the preferred type of substitution
. The structureꢀactivity relationship (SAR) among
cinnamamides for their antiꢀinflammatory activity remains unclear. In addition, although
previous studies performed the antiꢀinflammatory evaluation in LPSꢀstimulated cells or
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carrageenanꢀinduced mouse paw edema , the possible mechanism and direct molecular targets
by which cinnamanides exerted the antiꢀinflammatory effects were rarely investigated.
In the continuation of our study on the discovery of new antiꢀinflammatory agents from
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natural skeletons
, we proposed to find new cinnamanides with antiꢀinflammatory property
for the treatment of acute inflammatory diseases, and to demonstrate the possible mechanism
and molecular target. In this work, 34 cinnamamide derivatives were prepared and biologically
evaluated (Scheme 1). Among these compounds, 2i showed the strongest antiꢀinflammatory
activities in vitro. Furthermore, we identified the antiꢀinflammatory mechanism, molecular
target, and in vivo pharmacology of 2i in mice with sepsis and ALI. Our results demonstrate that
the new cinnamamide, 2i, is a novel MD2 inhibitor that exhibits the potential to treat sepsis and
ALI.
Please insert Scheme 1
Results
Chemistry
Cinnamamide derivatives were prepared by the synthetic routes depicted in Scheme 1, and
their structures are shown in Table 1. The intermediate products used in this method are the
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appropriately substituted cinnamic acids (7wꢀ7z). The cinnamic acids were either commercially
available (7w) or were prepared from the corresponding substituted benzaldehydes (5xꢀ5z) and
malonic acid (6) via the Knoevenagel reaction. Target compounds were obtained through
oneꢀstep amidation using substituted cinnamic acid and corresponding amines under the
catalytic condition of EDCꢀHCl and HOBt. After stirred at 0–8°C for 1 h, the reaction mixture
was maintained at room temperature for 4–8 h to generate a high yield of cinnamamides (Table
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), except for the dimerꢀtype 1iꢀ3i and 1k–3k. For 1iꢀ3i and 1kꢀ3k, two folds of substituted
cinnamic acid were stirred with amines at 0ꢀ8°C for 20h. All of the compounds were identified
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by spectral analysis. By way of nuclear magnetic resonance spectra analysis ( H NMR), the E
configuration of the compounds was clearly demonstrated by the value of the vicinal coupling
constant of the vinylic protons, which indicated a trans relationship between the two hydrogens.
Pure compounds were obtained by either reꢀcrystallization from CHCl /EtOH or by
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3 3
chromatography over silica gel column, and then HPLC with CH CN/CH OH as the solvent
system was used to confirm the purity above 96.0%.
Please insert Table 1
Cinnamamides inhibited TNFꢀα and ILꢀ6 production induced by LPS in macrophages
Thirty four synthesized cinnamamides were tested for their ability to inhibit LPSꢀinduced
release of tumor necrosis factor–α (TNFꢀα) in mouse primary peritoneal macrophages (MPMs).
As shown in Figure 1A, while a majority of these compounds showed inhibition against
LPSꢀinduced TNFꢀα production to various degrees, only 4 compounds exhibited >50%
inhibitory abilities compared to LPS alone group (1h, 2i, 3b and 3j). Based on the bioꢀscreening
results, we may observe some preliminary SAR conclusions. For example, (1) substitutions of
the amide group with longꢀchain aliphatic substituents resulted in compounds with increased
antiꢀinflammatory activity compare to the cyclic substituents; (2) in series 1, an
electronꢀdonating substitute in the amino moiety may decrease its inhibitory activity against
TNFꢀα release, such as 1h, 1g and 1f; (3) in contrast, an electronꢀwithdrawing halogen at the
same site significantly increases the inhibitory effect; (4) in series 2, an electronꢀdonating
substitute in the amino moiety may increase the inhibitory activity against TNFꢀα secretion,
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such as 2h, 2g and 2f; (5) a dimer structure with diamino group as a linker also maintained the
antiꢀinflammatory properties. The quantitative SAR was also performed and described in the
supporting information (Figure S1). We further tested the cytotoxicity of these compounds in
MPMs. As shown in Figure S2, all cinnamamides showed no significant cytotoxicity in MPMs
after 24h treatment. These four compounds were chosen for the next multiꢀdose evaluation in
MPMs. As shown in Figure 1B and 1C, all of four compounds significantly suppressed the
LPSꢀinduced production of interleukinꢀ6 (ILꢀ6) and TNFꢀα. Especially, 3b at four concentrations
inhibited TNFꢀα expression in a comparable degree. Although the response was barely
doseꢀdependent, the multiꢀdose data validated the antiꢀinflammatory ability of these four
compounds. Of these four, 2i exhibited the strongest inhibition against both TNFꢀα and ILꢀ6
expression.
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Please insert Figure 1
The active compound 2i prevented LPSꢀinduced gene expression of proꢀinflammatory
cytokines and activation of MAPKs/NFꢀκB signaling pathways
Having excellent antiꢀinflammatory activity, 2i was further biologically evaluated (Figure
2
A). We determined the inhibitory effect of 2i on the mRNA transcription of important
proꢀinflammatory proteins, including TNFꢀα, ILꢀ1β, ILꢀ6, COXꢀ2, and PGES in LPSꢀstimulated
macrophages. As shown in Figure 2B, realꢀtime qPCR analysis revealed that 2i at 10 ꢁM
potently decreased the LPSꢀinduced upꢀregulation of the mRNAs of these cytokines. In
LPSꢀTLR4/MD2 proꢀinflammatory signaling pathway, NFꢀκB and MAPKs (including p38, JNK,
and ERK) have important roles in regulation of target cytokine gene expression. We investigated
the possible involvement of NFꢀκB/MAPKs signaling in the antiꢀinflammatory action of 2i.
While LPS elevated levels of p38, JNK, and ERK phosphorylation in MPMs, 2i pretreatment
decreased LPSꢀinduced phosphorylation of these three kinases in a doseꢀdependent manner
(Figure 2C–E). NFꢀκB activation requires IκB degradation, causing NFꢀκB p65 submit
translocation from the cytoplasm to the nucleus. We determined the effect of 2i on IκBα
degradation and p65 nuclear translocation. The immunostaining data showed that LPS enhanced
NFꢀκB p65 nuclear translocation (red point in blue nucleus), while in macrophages with 2i
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pretreatment, nuclear p65 was dispersed and transferred outside the nuclei, suggesting that 2i
inhibited p65 translocation (Figure 2FꢀH). In addition, we tested the inhibitory activity of 2i
against NFꢀκB using a Luciferase Reporter system. As show in Figure S3, 2i could
doseꢀdependently inhibit the transcriptional activity of NFꢀκB. Furthermore, LPS reduced IκBα
levels and 2i preꢀtreatment reversed the degradation of IκBα in a doseꢀdependent manner
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(
Figure 2I). Pretreatment of ERK or p38 inhibitor (PD98059 and SB239063, respectively) did
not affect LPSꢀinduced IκBα degradation, indicating that NFꢀκB and MAPKs signaling
pathways are independent in LPSꢀTLR4/MD2 cascade (Figure 2I). These results indicate that
the inhibition of MAPKs/NFꢀκB activation may mediate the antiꢀinflammatory effects of 2i.
Please insert Figure 2
Identification of MD2 as the molecular target of compound 2i
To identify the possible direct molecule target of 2i in LPSꢀTLR4/MD2 pathway (Figure
S4), we investigated whether 2i could directly bind and inhibit the MAPKs and IKKβ, which is
the kinase that regulates IκB activation and degradation. Recombinant kinases, including JNK2,
ERK1, P38α, P38β, and IKKβ, were used to test the direct kinaseꢀinhibitory activity of
compound 2i in a cellꢀfree system by Caliper Mobility Shift Assay. As shown in Table S1, 2i did
not significantly inhibit any of these five kinases, indicating that an upstream target of
MAPK/NFꢀκB might exist for the antiꢀinflammatory action of 2i.
TLR4 and MD2 are important proteins involved in LPSꢀinduced inflammatory signaling.
Upon binding to LPS, TLR4 proceeds to a physical receptor multimer consisting of two copies
of the TLR4/MD2 complex. We next tested whether 2i directly targets TLR4 or MD2. The
interaction between 2i and recombinant TLR4 or MD2 protein was determined by SPR
experiments. As shown in Figure 3A and 3B, 2i did not interact with TLR4, but could
doseꢀdependently bind to recombinant MD2 protein, with a KD value of 34 µM, suggesting that
MD2 may be the direct target of 2i. We also tested the interaction of MD2 with 1f, a
representative cinnamamide derivative without antiꢀinflammatory activity. The result in Figure
S5 showed no interactions between MD2 and 1f. Hence, a series of biochemical experiments
were designed and performed to validate this hypothesis. The fluorescent probe bisꢀANS has
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been used to test the lipidꢀbinding sites of several proteins, including MD2
. Figure 3C
showed that fluorescence levels of bisꢀANS were remarkably increased when binding to rhMD2
protein. However, coꢀincubation with 2i doseꢀdependently inhibited the intensity of bisꢀANS
fluorescence, indicating a displacement of bisꢀANS by 2i from the rhMD2 pocket. Competition
between 2i and LPS in binding to rhMD2 was detected by a biotinꢀstreptavidinꢀbased ELISA
method. Figure 3D shows that biotinꢀLPS in the plates bound to rhMD2, while the coꢀincubation
with 2i doseꢀdependently prevented the interaction between rhMD2 and biotinꢀLPS, suggesting
that the binding sites for 2i on MD2 may overlap the binding sites for LPS. To demonstrate the
effect of 2i on LPS binding to MD2 at the cellular level, macrophages were treated with
FITCꢀlabeled LPS (FITCꢀLPS) in the presence or absence of 2i and then proceeded to flow
cytometric analysis. Preꢀincubation with 2i significantly inhibited FITCꢀLPS binding to the cell
surface (Figure 3E). In addition, an immunoprecipitation assay showed that 2i pretreatment
potently inhibited LPSꢀinduced formation of TLR4/MD2 complex in macrophages, indicating
that 2i inhibited the interaction between TLR4 and secreted MD2 in macrophages (Figure 3F).
MD2 is a chaperone protein of TLR4 and is not associated with the activation of TLR2
proꢀinflammatory signaling. Thus, a specific activator of TLR2, Pam3CK, was used to further
investigate whether 2i specifically targets MD2 and inhibits the TLR4 signaling pathway. As
shown in Figure 3G and 3H, compared to CUꢀCPT22, a specific TLR2 inhibitor, 2i failed to
inhibit the production of ILꢀ6 and TNFꢀα induced by the TLR2 agonist Pam3CK, indicating that
2i does not target TLR2 and the common downstream signaling of TLR2/TLR4. Taken together,
these results are consistent with the observation that 2i binds directly to MD2 and antagonizes
the formation of the LPSꢀTLR4/MD2 complex.
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Please insert Figure 3
Study on 2i and MD2 binding sites
We then investigated the possible binding model and site of 2i binding to MD2. Since this
is the first cinnamamide compound showing to target MD2 protein and no references could be
used to speculate the binding mode, a computerꢀassistant structural simulation of the 2iꢀMD2
complex was firstly carried out as described in Method Section. The docking simulation showed
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the binding model and sites of compound 2i with LPS/MD2/TLR4 complex and MD2 (Figure
4A). In consistent with the experimental data, the docking model reveals that 2i binds to the
same hydrophobic pocket of MD2 to LPS, In the flexible binding simulation, two residues of
MD2, Arg90, and Tyr 102, could form hydrogen bonds with the smallꢀmolecule ligand to
stabilize the binding conformation. These two residues showed no interaction with the structure
of LPS buried in the MD2 pocket. Thus, the next step is to confirm the roles of Tyr102 and
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R90A
Y102A
Arg90 in 2i binding to MD2. Two recombinant mutants, MD2
and MD2
, were
constructed and used in further assays. As shown in Figure S6AꢀC, either R90A or Y102A
mutation did not affect the LPSꢀrhMD2 interaction. However, Figure 4B shows that 2i failed to
R90A
inhibit the binding between BiotinꢀLPS and MD2
. SPR assay revealed that the ability of 2i
R90A
to directly bind MD2
was abolished (Figure 4C). Similar results were observed when
Tyr102 was mutated to Ala (Figure 4D and 4E). These results suggest the possible binding
mechanism and sites for 2i with MD2 protein.
Please insert Figure 4
Effect of 2i administration on the survival rate of LPSꢀinduced septic mice
To determine whether 2i was able to attenuate the development of endotoxic shock by
inhibiting the LPSꢀinduced inflammatory response, LPS at 20mg/kg was intraperitoneally (i.p.)
injected into the mice with or without 2i preꢀtreatment. The mice were then monitored for 7 days.
As shown in Figure 5, all mice in the LPS alone group died within 48 h as a result of sepsis.
Conversely, 100% of the animals that received 2i at 10 mg/kg before LPS injection survived.
Thus, 2i showed excellent antiꢀinflammatory activity in vivo.
Please insert Figure 5
Compound 2i attenuated LPSꢀinduced acute lung injury in mice
ALI was induced in mice by intratracheal LPS instillation to further evaluate the protective
ability of 2i against LPSꢀinduced acute inflammation in vivo. As shown in Figure 6A, a marked
increase in the protein concentration in mice BALF was observed after LPS instillation and the
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increase was significantly inhibited by 2i treatment. The lung wet/dry weight ratio, which is an
index of lung edema, was significantly increased in the LPS alone group when compared to both
the control saline group and the LPS + 2i group, indicating that 2i also suppressed LPSꢀinduced
lung edema (Figure 6B). Recruitment of neutrophils into the pulmonary compartment is an
important feature of ALI. As shown in Figure 6C and 6D, LPS challenge caused a strong
increase in the number of neutrophils and total cells in the bronchial alveolar lavage fluid
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(BALF), while treatment with 2i significantly decreased the numbers of both neutrophils and
total cells in BALF from ALI mice. MPO activity in tissues is an indicator of neutrophil
infiltration. Figure 6E shows that treatment with 2i significantly decreased LPSꢀinduced MPO
activity in mice lung tissues. To evaluate the histological changes, mice lung tissues were
proceeded to hematoxylin and eosin (H&E) staining (Figure 6F). Mice in the control group
exhibited a normal structure of lung tissues under a light microscope, while LPS instillation
resulted in significant pulmonary congestion, infiltration of inflammatory cells, thickening of the
alveolar wall, and interstitial edema. These pathological changes induced by LPS were
remarkably reduced by 2i treatment. Figure 6G further validated the inhibitory effect of 2i
against macrophage infiltration into the lung, as evidenced by the reduced CD68 expression in
LPS + 2i group.
LPS exposure also increased the mRNA expression of inflammatory cytokines such as ILꢀ6,
TNFꢀα, and ILꢀ1β in mice lung tissues, which were all significantly reduced by 2i treatment
(Figure 6Hꢀ6J). We also observed the antiꢀinflammatory effect of 2i in cultured human lung cells.
As shown in Figure S7, preꢀtreatment with 2i reduced the LPSꢀinduced transcription of ILꢀ6,
TNFꢀα, ILꢀ1β, and COXꢀ2 in the human lung epithelial Beasꢀ2B cells. Finally, we investigated
whether the pharmacological activity of 2i was associated with its inhibition of MD2 and
subsequent TLR4/MD2 complex formation. As shown in Figure 6K, compared with the control
group, LPS treatment significantly induced the formation of the TLR4/MD2 complex in mouse
lung tissues, while 2i administration reduced the level of TLR4/MD2 complex. These data
indicate that the MD2 inhibitor 2i exerts an antiꢀinflammatory and antiꢀALI activity in
LPSꢀinduced mice lungs.
Please insert Figure 6
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Discussion and conclusions
Acute inflammatory diseases remain the main cause of death in intensive care units
worldwide and still lack of effective therapy in the clinical treatment. Steroidal and
nonꢀsteroidal antiꢀinflammatory drugs have been found to be therapeutically limited in the
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treatment of sepsis and ALI
. Cinnamamide is a unique template, which has been reported to
11,
possess a variety of biological activities, including antiꢀinflammatory and antiꢀcancer activities
1
5, 24
. The safety and convenience of preparation also contribute to the feasibility of developing
25
bioactive molecules from cinnamamideꢀbased compounds . In this study, we present a series of
novel cinnamamides with potent antiꢀinflammatory activities both in vitro and in vivo, and more
importantly, that we demonstrat the biological mechanism and target for the antiꢀinflammatory
action of dimerꢀtype cinnamamide 2i.
Although there are several examples in the literature of the antiꢀinflammatory activity of
1
5
cinnamamide derivatives , the structureꢀactivity relationship (SAR) remains limited. Here,
antiꢀinflammatory effects of 34 cinnamamideꢀstructural MACs were evaluated in
LPSꢀstimulated macrophages and the preliminary SAR conclusions were obtained. In addition,
we also performed a quantitative SAR analysis as described in the Supporting Information. The
relationship between the antiꢀinflammatory activity and Some structural descriptors of
compounds were found to be able to relate with the antiꢀinflammatory activity. Shown in Figure
S1 is the scatter plot for experimental and computational value of each compound, with a highly
2
squared regression coefficient (R adj = 0.71), which can describe more than 71% of the variance
observed in the experimental activity. The QSAR result indicates that the size and shape of
molecules, as well as their steric properties, play important roles in the SAR of cinnamamides.
Among these synthetic cinnamamides, a compound contain a dimer motif, 2i, was
identified as the most active compound. 2i was able to significantly inhibit the mRNA
expression of inflammatory cytokines, NFꢀκB activation, and MAPKs phosphorylation in
LPSꢀstimulated macrophages. In addition, we tested the pharmacokinetic (PK) profiles of
compound 2i in rats (Figure S10AꢀC). With 20 mg/kg oral administration, the peak
concentration of 2i reached 0.27ꢁg/ml and decreased to 0.04 ug/ml after 12h (Figure S10B);
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with 10mg/kg intravenous administration, the peak concentration of 2i reached 0.55ug/ml and
decreased to 0.04ug/ml after 24h (Figure S10C). These data imply that 2i could be potentially
developed as a novel antiꢀinflammatory agent for the treatment of acute inflammatory diseases.
Thus, it is important to investigate and identify the possible molecular target of 2i. For
smallꢀmolecule drugs, which comprise most of the medicines available today, the identification
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of the molecular targets underlying the therapeutic effect of the drug and/or their adverse side
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effects remains a key challenge
. After finding the inhibition of 2i on cytokine proteins and
mRNAs, we employed a backstepping method to find the molecular target of 2i in
LPSꢀTLR4/MD2 proꢀinflammatory cascade. MAPKs and NFꢀκB are important signaling
components in the LPSꢀTLR4/MD2 proꢀinflammatory pathway, which is activated upon LPS
stimulation in a MyD88ꢀdependent manner, and subsequently trigger the transcription of
proꢀinflammatory genes. Although our data in Figure 2 showed the ability of 2i in inhibiting the
activation of MAPKs and NFꢀκB, Table S1 showed that 2i failed to directly inhibit the activity
of these recombinant kinases, suggesting that 2i may act on the upstream target in the
LPSꢀTLR4/MD2 pathway. We then tested the direct interaction between 2i and TLR4 or MD2,
respectively, and found that 2i could bind to a recombinant MD2 protein with a KD value of 34
ꢁM. Subsequently, some experiments at both molecular and cellular levels were designed to
further confirm this finding. Fluorescence measurements validated the direct binding of 2i to
rhMD2 protein, and ELISA confirmed the effect of 2i on the binding of LPS and rhMD2.
Immunoprecipitation and flow cytometry showed that 2i affected LPSꢀMD2 interactions and the
formation of MD2ꢀTLR4 complex. In addition, our results in Figure 3F and 6K showed that,
without LPS stimulation, TLR4 and MD2 can bind to each other in a low degree, while LPS
stimulation promote the formation of a lot of TLR4/MD2 complex. Thus, we presume that LPS
induces the formation of TLR4 and MD2 and 2i inhibits the formation of TLR4 and MD2
complex in macrophages. However, other group found that TLR4 and MD2 could form complex
1
0
in Golgi apparatus without LPS stimulation in embryonic fibroblasts . We may consider that 2i
inhibits the interaction between TLR4 and secreted MD2 in macrophages and that there may
exist cellꢀtype difference in the TLR4/MD2 signaling regulation. We also found that 2i showed
no inhibitory effect against TLR2ꢀmediated cytokine expression which is independent of MD2
activity. Taken together, these data clearly show that 2i is a novel inhibitor of MD2.
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Recently, a few natural and synthetic molecules have been found to target and inhibit MD2
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8ꢀ30
protein and then to prevent LPSꢀinduced inflammatory responses
. This is the first time to
find cinnamamide could binds MD2 protein, while some small molecules, which contain the
cinnamaldehyde skeleton and α,βꢀunsaturated carbonyl moiety in structure, were found to bind
MDꢀ2. So far, more than seven cinnamaldehyde skeletonꢀcontaining MDꢀ2 inhibitors were
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identified, including JSH , CAPE , curcumin , xanthohumol , 1D10G , and two compounds
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L6H21 and L48H37 found by our group
. Their chemical structures are shown in Figure S8.
They directly interact MDꢀ2 pocket, inhibiting the activation of LPSꢀMD2/TLR4 signaling and
preventing LPSꢀinduced inflammatory response and septic shock. These inhibitors share similar
chemical structure, cinnamaldehyde moiety, with cinnamamides. This note also strengthens our
confidence on the fact that cinnamamide 2i directly binds MD2 and inhibits MD2ꢀLPS
interaction. In addition, we also compared 2i with two previously reported natural MD2
35
inhibitors, curcumin and xanthohumol (Figure S9), two compounds with the cinnamaldehyde
structure. The KD values of 2iꢀMD2, xanthohumolꢀMD2, and curcuminꢀMD2 are 0.000034M,
35
0
.00046M , and 0.000379M, respectively. Our results showed 2i has a stronger interaction than
curcumin and xanthohumol towards rhMD2 protein.
Until now, no crystal structure of the complex of smallꢀmolecule inhibitor and MD2 protein
has been reported. The previous Xꢀray diffractionꢀbased structural characterization showed that
a β cup fold and two antiparallel β sheets, form a hydrophobic pocket in mature MD2 containing
36, 37
143 amino acid residues
. The MD2 pocket is deep and narrow, with a volume of about 1700
3
Å . The hydrophobic residues are coated on the internal surface of the pocket, and some residues
3
6, 37
with positive charges are located near the opening rim of the cavity
. In the flexible binding
simulation, 20 conformations of 2i formed two hydrogen bonds with Arg90 and Tyr102 residues
of MD2, indicating their importance in the molecular interaction. Then, two mutated forms of
R90A
Y102A
MD2, MD2
and MD2
, were employed to validate this finding. Previously, some
residues Lys122, Tyr102, Gly123, Ser120, Lys130, and Phe126 in the MD2 protein pocket have
been predicted as key binding sites in the interactions between MD2 and nonꢀlipid inhibitors
29
38
30
such as JSH , taxanes , and xanthohumol . In addition, Cys133 was considered to form
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covalent bonds via a Michaelꢀtype reaction when CAPE binds MD2 . However, it seems to be
28
of no role in the interaction between MD2 and curcumin , despite that both CAPE and
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curcumin have the α,βꢀunsaturated carbonyl moiety. Our previous studies revealed the
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importance of Arg90 and Tyr102 residues in the binding of L6H21 and L48H37 with MD2
.
This study demonstrates that Arg90 and Tyr102 are also important residues involved in the
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iꢀMD2 interaction.
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Both pharmacological and genetic MD2 inhibition has been reported to inhibit
LPSꢀinduced inflammatory responses and the development of sepsis. Acute inflammatory
diseases such as sepsis induced by endotoxin have received increased awareness due to their
prevalence and poor therapy options. Therapies through neutralizing both inflammatory
39
cytokine TNFꢀα and signaling proteins CD14 and TLR4 already failed in clinical trials .
Therefore, to validate the clinical efficacy of agents targeting MD2 is very important. A
lipidꢀlike TLR4/MD2 antagonist eritoran was evaluated in clinical study for the treatment of
1
sepsis. However, the Phase III clinical study showed that eritoran failed to cure severe sepsis .
Also, none of smallꢀmolecule inhibitors of MD2 have been pursued in clinical trials. Our study
found that the MD2 inhibitor 2i could prevent LPSꢀinduced septic shock at an unbelievable 100%
level, indicating that 2i may be a potential candidate for sepsis therapy.
More importantly, we demonstrate for the first time, that MD2 inhibition could
significantly attenuate ALI in animal models. ALI is a critical illness that is characterized by
noncardiogenic edema, pulmonary inflammation, severe systemic hypoxemia, decreased
40
neutrophil apoptosis in the lung, and multiple organ failure . Numerous pharmacological
therapies for established ALI, including corticosteroids and steroids, have failed to show benefit
4
1
in multicenter clinical trials . Thus, new agents are still urgently needed for ALI therapy. Since
LPS is a common cause of ALI, the TLR4/MD2 receptor complex could be considered as a
therapeutic target of ALI. Hadina et al. showed that MD2ꢀnull mice do not respond to nasal
42
aspiration of LPS from Neisseria meningitides . 2i significantly attenuated LPSꢀinduced lung
injury as well pulmonary inflammation, accompanied with reduced MD2/TLR4 complex
formation in lung tissue. These results show that 2i inhibited the development of LPSꢀinduced
ALI by targeting MD2 and inhibiting the MD2/TLR4ꢀdependent inflammatory responses in
mouse lung (Figure 7). Therefore, it is also suggested that MD2 inhibition is a potential
therapeutic strategy for ALI in the future.
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Please insert Figure 7
In conclusion, 34 cinnamamide derivatives were synthesized, their bioactivities were
evaluated against LPSꢀinduced inflammation. The most active compound 2i, which contain a
dimer motif, was further investigated for the molecular target and antiꢀinflammation activity
both in vitro and in vivo. Our data shows that 2i significantly blocked LPSꢀinduced
proꢀinflammatory gene expression and MAPKs/NFꢀκB activation in vitro and prevented
LPSꢀcaused septic shock and ALI in vivo. Mechanistically, these antiꢀinflammatory actions of 2i
seem to be mediated by directly targeting and inhibiting MD2. Arg90 and Tyr102 in the MD2
protein are key residues in the interaction between 2i and MD2 protein. Taken together, this
study presents a new MD2 inhibitor, 2i, which deserves further development as a potential
candidate for the therapy of sepsis and ALI, and more importantly, this work finds the first
cinnamamideꢀstructural MD2 inhibitor and suggests a new lead structure for the development of
antiꢀinflammatory agents targeting MD2.
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Experimental Section
Reagents
Chemical reagents, ERK inhibitor PD98059, p38 inhibitor SB239063, and LPS were
purchased from Sigma (Louis, MO). Recombinant human TLR4 (rhTLR4) protein and TLR2
inhibitor CUꢀCPT22 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).
Recombinant human MD2 (rhMD2) and mutated rhMD2 was obtained using the methods
34
described in our previous publication .
Chemical synthesis
Melting points were determined on a FisherꢀJohns melting apparatus and were uncorrected.
1
H NMR spectra were recorded on a Bruker AVANCE III 600 spectrometer. The chemical shifts
were presented in terms of parts per million with TMS as the internal reference. Electronꢀspray
ionization mass spectra in positive mode (LCꢀESIꢀMS) data were recorded on a Bruker Esquire
HCT spectrometer. Column chromatography purifications were carried out on Silica Gel 60 (E.
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Merck, 70–230 mesh). The purity of all target compounds was confirmed to be greater than 96%
by HPLC analysis conducted on an Agilent 1260 liquid chromatograph system. The
chromatography was carried out at room temperature using a ZORBAX SBꢀC18 column (150 ×
4
.6 mm, 5 ꢁm) and a gradient of 10−90% acetonitrile in methanol in 20 min. The detection
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wavelength was set at 254 nm.
The general procedure for synthesis of cinnamamides was two steps including knoevenagel
reaction and amidation. Briefly, an amount of 20 mmol different substituted arylaldehyde and 24
mmol malonic acid were mixed with 0.5mmol amidobenzene to give corresponding cinnamic
o
acid. The mixture was stirred at 95 C and monitored with TLC (CH
3
COOEt/CHCl
3
). When the
reaction was finished, 3mol/L HCl was dropped into the residue until no new solid was
precipitated. The precipitate was filtered and washed with water and cold ethanol, and dried in
vacuum. The intermediate was purified after recrystallization. The target compounds were
prepared by condensation of the corresponding cinnamic acids with various amines utilizing
1
1
ꢀ(3ꢀdimethylaminoꢀpropyl)ꢀ3ꢀethylcarbodiimide
hydrochloride
(EDC)
and
ꢀhydroxybenzotriazole (HOBT) as catalyst in methylene chloride. An amount of 1.65mmol
o
EDC·HCl and 1.95mmol HOBt was stirred with the 1.50 mmol cinnamic acid at 0ꢀ8 C. After
o
1
0 min, the 1.50 mmol amine was added,and the mixture was stirred at 0ꢀ8 C for 1h before
room temperature reaction (for dimerꢀtype series 1iꢀ3i and 1kꢀ3k, 0.75 mmol amine was added
and stirred at 0ꢀ8°C for 20h). The procedure was monitored with TLC until the reaction was
3 2 2
finished. The residue was washed with saturated NaHCO solution and extracted with CH Cl .
After solvent evaporation, the solid was purified by chromatography over silica gel to yield
compounds. Their structures were characterized by spectral data from MS and 1H NMR. The
spectral data of novel or unreported compounds are listed as the following.
(E)-3-(2,3-Dichlorophenyl)-1-morpholinoprop-2-en-1-one(1a): Yellow powder, 85.3%
1
yield, mp 125.3ꢀ127.1°C. HꢀNMR (CDCl
3
): δ (ppm) 3.74 (8H, br.s,ꢀCH
2
ꢀ×4), 6.79 (1H, d, J =
6
5
15.0 Hz, =CHꢀC=O), 7.19ꢀ7.25 (1H, m, ArꢀH ), 7.43ꢀ7.48 (1H, m, ArꢀH ), 7.43ꢀ7.48 (1H, m,
4
+
+
ArꢀH ), 8.01 (1H, d, J = 15.0 Hz, ArꢀCH=C). ESIꢀMS m/z: 286.2(M) , 308.0 (M+Naꢀ1) , calcd
NO : 286.15.
for C13
H
13Cl
2
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(E)-N-butyl-3-(2,3-Dichlorophenyl)acrylamide(1b): Yellow powder, 83.2% yield, mp
1
107.0ꢀ108.8°C. HꢀNMR (CDCl ): δ (ppm) 0.94 (3H, t, J = 7.2 Hz, CꢀCH ), 1.35ꢀ1.42 (2H, m,
3
3
ꢀCH
2
ꢀC), 1.53ꢀ1.58 (2H, m, ꢀCH
2
ꢀCꢀC), 3.37ꢀ3.41 (2H, m, NꢀCH
2
ꢀ), 6.37 (1H, d, J = 15.6 Hz,
6
5
4
=
CHꢀC=O), 7.16ꢀ7.19 (1H, m, ArꢀH ), 7.42ꢀ7.44 (1H, m, ArꢀH ) ,7.42ꢀ7.44 (1H, m, ArꢀH ), 7.95
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+
+
(
1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 272.1 (M) , 294.0 (M+Naꢀ1) , calcd for
13 2
C H15Cl
NO: 272.17.
(
E)-N-Benzyl-3-(2,3-dichlorophenyl)acrylamide(1c): Yellow powder, 79.6% yield, mp
1
1
75.7ꢀ177.1°C. HꢀNMR (CDCl
3
2
): δ (ppm) 4.58 (2H, d, J = 5.4 Hz, ArꢀCH ), 6.40 (1H, d, J =
6
15.6 Hz, =CHꢀC=O), 7.17ꢀ7.20 (1H, m, ArꢀH ), 7.26ꢀ7.36 (5H, m, ArꢀH×5), 7.44ꢀ7.45 (1H, m,
5
4
ArꢀH ), 7.44ꢀ7.45 (1H, m, ArꢀH ) , 7.99(1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 306.6
+
+
(M) , 308.4(M+2) , calcd for C H Cl NO: 306.19.
16 13 2
(
E)-3-(2,3-Dichlorophenyl)-N-phenethylacrylamide(1d): Yellow powder, 82.1% yield,
1
mp 151.3ꢀ153.0°C. HꢀNMR (CDCl ): δ (ppm) 2.90 (2H, t, J = 6.6 Hz, ArꢀCH ), 3.65ꢀ3.69 (2H,
3
2
6
m, ArꢀCꢀCH
2
ꢀ.), 6.30 (1H, d, J = 15.6 Hz, =CHꢀC=O), 7.16ꢀ7.19 (1H, m, ArꢀH ), 7.25ꢀ7.43 (5H,
5
4
m, ArꢀH×5), 7.43ꢀ7.44 (1H, m, ArꢀH ), 7.43ꢀ7.44 (1H, m, ArꢀH ) , 7.94 (1H, d, J = 15.6 Hz,
+
+
2
ArꢀCH=C). ESIꢀMS m/z: 320.2 (M) , 322.1 (M+2) , calcd for C17H15Cl NO: 320.21.
(E)-3-(2,3-Dichlorophenyl)-N-(3-phenylpropyl)acrylamide(1e): Yellow powder, 79.6%
1
yield, mp 89.6ꢀ91.1°C. HꢀNMR (CDCl
3
): δ (ppm) 1.88ꢀ1.92 (2H, m, ArꢀCꢀCH
2
ꢀ), 2.68 (2H, t, J
=
7.2 Hz, ArꢀCH ꢀ), 3.40ꢀ3.43 (2H, m, ArꢀCꢀCꢀCH ꢀ), 6.38 (1H, d, J = 15.6 Hz, =CHꢀC=O),
2 2
6
5
7
.13ꢀ7.15 (1H, m, ArꢀH ), 7.15ꢀ7.28 (5H, m, ArꢀH×5), 7.40ꢀ7.42 (1H, m, ArꢀH ), 7.40ꢀ7.42 (1H,
4
+
+
m, ArꢀH ) , 7.93 (1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 334.2 (M) , 356.1 (M+Naꢀ1) ,
calcd for C H Cl NO: 334.24.
18
17
2
(
E)-3-(2,3-Dichlorophenyl)-N-(3,4-dimethoxybenzyl)acrylamide(1f): Yellow powder,
1
4
3 3
83.5% yield, mp 169.4ꢀ170.5°C. HꢀNMR (CDCl ): δ (ppm) 3.89 (3H, s, ArꢀOCH ), 3.90 (3H,
3
3
s, ArꢀOCH
), 4.54 (2H, d, J = 5.4 Hz, ArꢀCH
2
), 6.41 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.85 (1H,
2
5
2
6
2
2
d, J = 8.4 Hz, Ar ꢀH ), 6.90 (1H, d, J = 8.4 Hz, Ar ꢀH ), 6.91 (1H, s, Ar ꢀH ), 7.20ꢀ7.22 (1H, m,
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ArꢀH ), 7.46ꢀ7.48 (1H, m, ArꢀH ), 7.46ꢀ7.48 (1H, m, ArꢀH ), 8.03 (1H, d, J = 15.6 Hz,
+
+
ArꢀCH=C).ESIꢀMS m/z: 366.1 (M) , 388.0 (M+Naꢀ1) , calcd for C H Cl NO : 366.24.
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(E)-3-(2,3-Dichlorophenyl)-N-(4-methoxyphenethyl)acrylamide(1g): Yellow powder,
0
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1
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8
9
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
3
6
7
9.6% yield, mp 159.0ꢀ160.5°C. HꢀNMR (CDCl ): δ (ppm) 2.83 (2H, t, J = 6.6 Hz, ArꢀCH ),
3
2
.61ꢀ3.64 (2H, m, ArꢀCꢀCH
2
ꢀ.), 3.79 (3H, s, ArꢀOCH
3
), 6.30 (1H, d, J = 15.6 Hz, =CHꢀC=O),
2
3,5
2
2,6
6
.86 (2H, d, J = 9.0 Hz, Ar ꢀH ), 7.14 (2H, d, J = 9.0 Hz, Ar ꢀH ), 7.17ꢀ7.19 (1H, m, ArꢀH ),
5
4
.42ꢀ7.44 (1H, m, ArꢀH ), 7.42ꢀ7.44 (1H, m, ArꢀH ),7.95(1H, d, J = 15.6 Hz, ArꢀCH=C).
+
+
2 2
ESIꢀMS m/z: 350.2 (M) , 372.1 (M+Naꢀ1) , calcd for C18H17Cl NO : 350.24.
(E)-3-(2,3-Dichlorophenyl)-N-(4-fluorobenzyl)acrylamide(1h): Yellow powder, 73.3%
1
yield, mp 169.5ꢀ171.3°C. HꢀNMR (CDCl ): δ (ppm) 4.57 (2H, d, J = 6.0 Hz, ArꢀCH ꢀ), 6.41
3
2
2
3,5
6
(
1H, d, J = 15.6 Hz, =CHꢀC=O), 7.04ꢀ7.07 (2H, m, Ar ꢀH ), 7.20ꢀ7.22 (1H, m, ArꢀH ),
2
2,6
5
4
7
.32ꢀ7.34 (2H, m, Ar ꢀH ), 7.45ꢀ7.48 (1H, m, ArꢀH ), 7.45ꢀ7.48 (1H, m, ArꢀH ), 8.04(1H, d, J
+
+
=
15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 324.2 (M) , 346.1 (M+Naꢀ1) , calcd for C H Cl FNO:
1
6
12
2
3
24.18.
(2E,2'E)-N,N'-(Ethane-1,2-diyl)bis(3-(2,3-dichlorophenyl)acrylamide)(1i):
Yellow
1
2
powder, 54.3% yield, mp 239.8ꢀ241.4°C. HꢀNMR (DMSO): δ (ppm) 3.16 (4H, s, ꢀCH ꢀ×2),
6
6.68 (2H, d, J = 15.6 Hz, =CHꢀC=O×2), 7.41ꢀ7.43 (2H, m, ArꢀH ×2), 7.66ꢀ7.67(2H, m,
5
4
ArꢀH ×2), 7.66ꢀ7.67(2H, m, ArꢀH ×2) , 7.74 (2H, d, J = 15.6 Hz, ArꢀCH=C×2), 8.46(2H, s,
+
ꢀ
NHꢀ×2). ESIꢀMS m/z: 458.9 (M+1) , calcd for C H Cl N O : 458.17.
2
0
16
4
2
2
(E)-N-(4-Chlorobenzyl)-3-(2,3-dichlorophenyl)acrylamide(1j): Yellow powder, 76.2%
1
yield, mp 205.7ꢀ207.5°C. HꢀNMR (DMSO): δ (ppm) 4.38 (2H, d, J = 5.4 Hz, ArꢀCH ), 6.73
2
2
2,6
(
1H, d, J = 15.6 Hz, =CHꢀC=O), 7.31 (2H, d, J = 8.4 Hz, Ar ꢀH ), 7.39 (2H, d, J = 8.4 Hz,
2
3,5
6
5
4
Ar ꢀH ),7.40ꢀ7.42 (1H, m, ArꢀH ), 7.65ꢀ7.67 (1H, m, ArꢀH ), 7.65ꢀ7.67 (1H, m, ArꢀH ),
+
7.75(1H, d, J = 15.6 Hz, ArꢀCH=C), 8.80 (1H, s, ꢀNHꢀ). ESIꢀMS m/z: 340.0 (M) ,calcd for
16 3
C H12Cl NO: 340.63.
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(2E,2'E)-N,N'-(Hexane-1,6-diyl)bis(3-(2,3-dichlorophenyl)acrylamide)(1k):
Yellow
1
powder, 44.5% yield, mp 199.3ꢀ201.1°C. HꢀNMR (DMSO): δ (ppm) 1.30 (4H, m,
ꢀNꢀCꢀCꢀCH
2
×2), 1.45(4H, m, ꢀNꢀCꢀCH
2
×2), 3.17(4H, m, ꢀNꢀCH
2
×2), 6.67 (2H, d, J = 15.6 Hz,
6
5
=
CHꢀC=O×2), 7.38ꢀ7.41 (2H, m, ArꢀH ×2), 7.63ꢀ7.64 (2H, m, ArꢀH ×2), 7.63ꢀ7.64(2H, m,
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59
60
4
ArꢀH ×2) , 7.68 (2H, d, J = 15.6 Hz, ArꢀCH=C×2), 8.24(2H, t, J = 5.4 Hz, ꢀNHꢀ×2). ESIꢀMS
+
m/z: 514.9 (M+1) , calcd for C24
H
24Cl
4
N
2
O
2
: 514.27.
(E)-3-(2,4-Dimethoxyphenyl)-1-morpholinoprop-2-en-1-one(2a): Yellow powder, 84.3%
1
yield, mp 60.6ꢀ62.3°C. HꢀNMR (CDCl
3
): δ (ppm) 3.71 (8H, br.s, ꢀCH
2
ꢀ×4), 3.82 (3H, s,
4
2
3
5
ArꢀOCH
3
), 3.85 (3H, s, ArꢀOCH
3
), 6.44 (1H, s, ArꢀH ), 6.48 (1H, d, J = 8.4 Hz, ArꢀH ), 6.86
6
(1H, d, J = 15.6 Hz, =CHꢀC=O), 7.40 (1H, d, J = 8.4 Hz, ArꢀH ), 7.85 (1H, d, J = 15.6 Hz,
+
+
ArꢀCH=C). ESIꢀMS m/z: 278.1 (M+1) , 300.1 (M+Na) , calcd for C H NO : 277.32.
15 19
4
(E)-N-Butyl-3-(2,4-dimethoxyphenyl)acrylamide(2b): Yellow powder, 76.4% yield, mp
1
9
9.0ꢀ100.8°C. HꢀNMR (CDCl ): δ (ppm) 0.94 (3H, t, J = 7.2 Hz, CꢀCH ), 1.35ꢀ1.39 (2H, m,
3
3
4
ꢀCH
2
ꢀC), 1.51ꢀ1.54 (2H, m, ꢀCH
2
ꢀCꢀC), 3.35ꢀ3.38 (2H, m, NꢀCH
2
3
ꢀ), 3.82 (3H, s, ArꢀOCH ),
2
3
5
3
1
.84 (3H, s, ArꢀOCH
3
), 6.41 (1H, s, ArꢀH ), 6.45 (1H, d, J = 8.4 Hz, ArꢀH ), 6.47 (1H, d, J =
6
5.6 Hz, =CHꢀC=O), 7.37 (1H, d, J = 8.4 Hz, ArꢀH ), 7.76 (1H, d, J = 15.6 Hz, ArꢀCH=C).
+
+
3
ESIꢀMS m/z: 264.0 (M+1) , 286.0 (M+Na) , calcd for C15H21NO : 263.33.
(E)-3-(2,4-Dimethoxyphenyl)-N-phenethylacrylamide(2d): Yellow powder, 81.1% yield,
1
mp 102.3ꢀ103.8°C. HꢀNMR (CDCl ): δ (ppm) 2.87 (2H, t, J = 7.2 Hz, ArꢀCH ꢀ), 3.64 (2H, m,
3
2
4
3
2
3
ArꢀCꢀ CH
2
ꢀ), 3.81 (3H, s, ArꢀOCH
), 3.82 (3H, s, ArꢀOCH
), 6.38 (1H, d, J = 15.6 Hz,
3
5
=
CHꢀC=O), 6.43(1H, s, ArꢀH ), 6.45 (1H, d, J = 8.4 Hz, ArꢀH ), 7.21ꢀ7.32 (5H, m, ArꢀH×5),
6
7
3
.37 (1H, d, J = 8.4 Hz, ArꢀH ) , 7.78 (1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z:
+
+
3
12.1(M+1) , 334.0 (M+Na) , calcd for C19H21NO : 311.37.
(E)-3-(2,4-Dimethoxyphenyl)-N-(3-phenylpropyl)acrylamide(2e): Yellow powder, 75.1%
1
yield, mp 69.5ꢀ71.2°C. HꢀNMR (CDCl
3
): δ (ppm) 1.86ꢀ1.91 (2H, m, ArꢀCꢀCH
2
ꢀ), 2.68 (2H, t, J
4
=
7.2 Hz, ArꢀCH
2
ꢀ), 3.39ꢀ3.42 (2H, m, ArꢀCꢀCꢀCH
2
ꢀ), 3.81 (3H, s, ArꢀOCH
3
), 3.83 (3H, s,
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ArꢀOCH
3
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ArꢀH ), 7.16ꢀ7.28 (5H, m, ArꢀH×5), 7.37 (1H, d, J = 8.4 Hz, ArꢀH ) , 7.76 (1H, d, J = 15.6 Hz,
+
+
3
ArꢀCH=C). ESIꢀMS m/z: 326.1 (M+1) , 348.0 (M+Na) , calcd for C20H23NO : 325.40.
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(
E)-N-(3,4-Dimethoxybenzyl)-3-(2,4-dimethoxyphenyl)acrylamide(2f): Yellow powder,
1
4
7
3 3
0.1% yield, mp 159.0ꢀ160.8°C. HꢀNMR (CDCl ): δ (ppm) 3.82 (3H, s, ArꢀOCH ), 3.84 (3H, s,
2
2
4
2
3
3
ArꢀOCH
3
), 3.86 (3H, s, Ar ꢀOCH
3
), 3.88 (3H, s, Ar ꢀOCH
2
), 4.49(1H, d, J = 5.4 Hz, ArꢀCH ),
3
5
6
.44 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.45(1H, s, ArꢀH ), 6.47 (1H, d, J = 8.4 Hz, ArꢀH ), 6.81
2
5
2
6
2
2
(
1H, d, J = 8.4 Hz, Ar ꢀH ), 6.86 (1H, d, J = 8.4 Hz, Ar ꢀH ), 6.88 (1H, s, Ar ꢀH ), 7.38 (1H, d, J
6
+
= 8.4 Hz, ArꢀH ) , 7.80 (1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 358.1 (M+1) , 380.0
+
(M+Na) , calcd for C20
H23NO
5
: 357.40.
(
E)-3-(2,4-Dimethoxyphenyl)-N-(4-methoxyphenethyl)acrylamide(2g): Yellow powder,
1
6
9.5% yield, mp 121.5ꢀ123.0°C. HꢀNMR (CDCl
3
2
): δ (ppm) 2.81 (2H, t, J = 6.6 Hz, ArꢀCH ),
2
4
3
.58ꢀ3.61 (2H, m, ArꢀCꢀCH ꢀ), 3.79 (3H, s, Ar ꢀOCH ), 3.81 (3H, s, ArꢀOCH ), 3.83 (3H, s,
2
3
3
2
3
3
ArꢀOCH ), 6.35 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.43 (1H, s, ArꢀH ), 6.46 (1H, d, J = 8.4 Hz,
5
2
3,5
2
2,6
ArꢀH ), 6.85 (2H, d, J = 8.4 Hz, Ar ꢀH ), 7.13 (2H, d, J = 8.4 Hz, Ar ꢀH ), 7.37 (1H, d, J = 8.4
6
+
+
Hz, ArꢀH ), 7.76 (1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 342.1 (M+1) , 364.0 (M+Na) ,
calcd for C20 : 341.40.
H23NO
4
(
E)-3-(2,4-Dimethoxyphenyl)-N-(4-fluorobenzyl)acrylamide(2h): Yellow powder, 80.1%
1
4
yield, mp 152.2ꢀ154.1°C. HꢀNMR (CDCl ): δ (ppm) 3.82 (3H, s, ArꢀOCH ), 3.84 (3H, s,
3
3
2
ArꢀOCH
3
2
), 4.52 (2H, d, J = 6.0 Hz, ArꢀCH ), 6.44 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.47 (1H, s,
3
5
2
3,5
2
2,6
ArꢀH ), 6.48 (1H, d, J = 8.4 Hz, ArꢀH ), 6.99ꢀ7.02 (2H, m, Ar ꢀH ), 7.28ꢀ7.30 (2H, m, Ar ꢀH ),
6
7
.38 (1H, d, J = 8.4 Hz, ArꢀH ), 7.80 (1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 316.1
+
+
(
3
M+1) , 338.0 (M+Na) , calcd for C18H18FNO : 315.34.
(2E,2'E)-N,N'-(Ethane-1,2-diyl)bis(3-(2,4-dimethoxyphenyl)acrylamide)(2i): Yellow
1
powder, 35.6% yield, mp 194.2ꢀ194.8°C. HꢀNMR (DMSO): δ (ppm) 3.74 (4H, s, ꢀCH
2
×2),
4
2
3
.78 (6H, s, ArꢀOCH
3
×2), 3.84 (6H, s, ArꢀOCH
3
×2), 6.50 (2H, d, J = 15.6 Hz, =CHꢀC=O×2),
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6.57 (2H, d, J = 8.4 Hz, ArꢀH ×2), 6.60 (2H, s, ArꢀH ×2), 7.44 (2H, d, J = 8.4 Hz, ArꢀH ×2) ,
+
7.58 (2H, d, J = 15.6 Hz, ArꢀCH=C×2), 8.10(2H, s, ꢀNHꢀ×2). ESIꢀMS m/z: 441.1 (M+1) , calcd
28 2 6
for C24H N O : 440.29.
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60
(
E)-N-(4-Chlorobenzyl)-3-(2,4-dimethoxyphenyl)acrylamide(2j): Yellow powder, 78.4%
1
4
yield, mp 171.4ꢀ173.1°C. HꢀNMR (CDCl
3
): δ (ppm) 3.82 (3H, s, ArꢀOCH
3
), 3.84 (3H, s,
2
ArꢀOCH
3
), 4.52(2H, d, J = 5.4 Hz, ArꢀCH ), 6.44 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.47 (1H, s,
2
3
5
2
2,6
ArꢀH ), 6.49 (1H, d, J = 8.4 Hz, ArꢀH ), 7.25 (2H, d, J = 7.8 Hz, Ar ꢀH ), 7.29 (2H, d, J = 7.8
2
3,5
6
Hz, Ar ꢀH ), 7.38 (1H, d, J = 8.4 Hz, ArꢀH ), 7.82 (1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z:
+
332.1 (M+1) , calcd for C18
H18ClNO
3
: 331.79.
(2E,2'E)-N,N'-(Hexane-1,6-diyl)bis(3-(2,4-dimethoxyphenyl)acrylamide)(2k): Yellow
1
powder, 43.4% yield, mp 203.7ꢀ205.3°C. HꢀNMR (DMSO): δ (ppm) 1.29 (4H, m,
4
3
ꢀ
NꢀCꢀCꢀCH
2
×2), 1.43(4H, m, ꢀNꢀCꢀCH
2
×2), 3.14(4H, m, ꢀNꢀCH
2
×2), 3.80 (6H, s, ArꢀOCH
×2),
2
3
3
.83 (6H, s, ArꢀOCH ×2), 6.51 (2H, d, J = 15.6 Hz, =CHꢀC=O×2), 6.56 (2H, d, J = 8.4 Hz,
5
3
6
ArꢀH ×2), 6.59 (2H, s, ArꢀH ×2), 7.43 (2H, d, J = 8.4 Hz, ArꢀH ×2) , 7.55 (2H, d, J = 15.6 Hz,
+
ArꢀCH=C×2), 8.10(2H, t, J = 5.4 Hz,ꢀNHꢀ×2). ESIꢀMS m/z: 497.2 (M+1) , calcd for
28 36 2 6
C H N O : 496.60.
(E)-N-Benzyl-3-(4-hydroxy-3-methoxyphenyl)acrylamide(3c)：: Yellow powder, 70.1%
1
yield, mp 88.9ꢀ90.2°C. HꢀNMR (CDCl
3
): δ (ppm) 3.89 (3H, s, ArꢀOCH
3
), 4.57 (2H, d, J = 5.4
Hz, ArꢀCH ), 5.93 (1H, br.s, ArꢀOH), 6.27 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.89 (1H, d, J = 8.4
2
5
2
6
Hz, ArꢀH ), 6.97 (1H, d, J = 1.8Hz, ArꢀH ), 7.05 (1H, dd, J
1
= 8.4 Hz, J
2
= 1.8 Hz, ArꢀH ),
+
7
.28ꢀ7.35 (5H, m, ArꢀH×5), 7.58 (1H, d, J = 15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 284.1 (M+1) ,
+
3
06.1 (M+Na) , calcd for C H NO : 283.32.
17
17
3
(E)-N-(3,4-Dimethoxybenzyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide(3f): Yellow
1
2
4
3
powder, 68.4% yield, mp 63.7ꢀ65.9°C. HꢀNMR (CDCl
3
): δ (ppm) 3.80 (3H, s, Ar ꢀOCH
),
2
3
3
3.84 (3H, s, Ar ꢀOCH
), 3.88 (3H, s, ArꢀOCH
3
), 4.49 (2H, d, J = 6.0 Hz, ArꢀCH
2
), 6.13 (1H,
2
5
br.s, ArꢀOH), 6.29 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.79 (1H, d, J = 9.0 Hz, Ar ꢀH ), 6.84 (1H, d,
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J = 9.0 Hz, Ar ꢀH ), 6.85 (1H, s, Ar ꢀH ), 6.88 (1H, d, J = 8.4 Hz, ArꢀH ), 6.96 (1H, d, J = 1.2
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Hz, ArꢀH ), 7.02 (1H, dd, J = 8.4 Hz, J = 1.2 Hz, ArꢀH ), 7.57 (1H, d, J = 15.6 Hz, ArꢀCH=C).
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ESIꢀMS m/z: 344.3 (M+1) , 366.2 (M+Na) , calcd for C19H21NO : 343.37.
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(
E)-N-(4-Fluorobenzyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide(3h):
Yellow
1
powder, 73.2% yield, mp 65.0ꢀ66.7°C. HꢀNMR (CDCl ): δ (ppm) 3.86 (3H, s, ArꢀOCH ), 4.51
3
3
(
2H, d, J = 6.0 Hz, ArꢀCH
2
), 6.12 (1H, br.s, ArꢀOH), 6.29 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.88
5
2
(
1H, d, J = 8.4 Hz, ArꢀH ), 6.95 (1H, d, J = 1.2 Hz, ArꢀH ), 6.98 (1H, dd, J
1
= 8.4 Hz, J
2
= 1.2
6
2
3,5
2
2,6
Hz, ArꢀH ), 7.01ꢀ7.03 (2H, m, Ar ꢀH ), 7.26ꢀ7.28 (2H, m, Ar ꢀH ),7.57 (1H, d, J = 15.6 Hz,
+
+
3
ArꢀCH=C). ESIꢀMS m/z: 302.2 (M+1) , 324.1 (M+Na) , calcd for C17H16FNO : 301.31.
(
E)-N-(4-Chlorobenzyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide(3j):
Yellow
1
powder, 79.0% yield, mp 58.7ꢀ60.3°C. HꢀNMR (CDCl ): δ (ppm) 3.86 (3H, s, ArꢀOCH ), 4.51
3
3
(
2H, d, J = 6.0 Hz, ArꢀCH
2
), 6.12 (1H, br.s, ArꢀOH), 6.29 (1H, d, J = 15.6 Hz, =CHꢀC=O), 6.88
5
2
(
1H, d, J = 8.4 Hz, ArꢀH ), 6.95 (1H, d, J = 1.8 Hz, ArꢀH ), 7.03 (1H, dd, J = 8.4 Hz, J = 1.8
1
2
6
2
2,6
2
3,5
Hz, ArꢀH ), 7.23 (2H, d, J = 8.4 Hz, Ar ꢀH ), 7.27 (2H, d, J = 8.4 Hz, Ar ꢀH ), 7.57 (1H, d, J
+
=
15.6 Hz, ArꢀCH=C). ESIꢀMS m/z: 318.1 (M+1) , calcd for C17
H
16ClNO
3
: 317.77.
(2E,2'E)-N,N'-(Hexane-1,6-diyl)bis(3-(4-hydroxy-3-methoxyphenyl)acrylamide)(3k):
1
Yellow powder, 24.7% yield, mp 214.9ꢀ215.9°C. HꢀNMR (DMSO): δ (ppm) 1.30 (4H, m,
ꢀNꢀCꢀCꢀCH
2
×2), 1.44 (4H, m, ꢀNꢀCꢀCH
2
×2), 3.15 (4H, m, ꢀNꢀCH
2
×2), 5.95 (1H, br.s, ArꢀOH),
5
6
.50 (2H, d, J = 15.6 Hz, =CHꢀC=O×2), 6.88 (2H, d, J = 8.4 Hz, ArꢀH ×2), 6.96 (2H, d, J = 1.8
2
6
Hz, ArꢀH ×2), 7.03 (2H, dd, J
1
2
= 8.4 Hz, J = 1.8 Hz,, ArꢀH ×2), 7.54 (2H, d, J = 15.6 Hz,
+
+
ArꢀCH=C×2), 8.03(2H, t, J = 5.4 Hz,ꢀNHꢀ×2). ESIꢀMS m/z: 469.1 (M+1) , 491.1 (M+Na) ,
calcd for C H N O : 468.54.
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Animals
C57BL/6 (B6) mice weighing 18ꢀ22 g were obtained from the Animal Center of Wenzhou
Medical University (Wenzhou, China). Animals were housed at a constant room temperature
with a 12:12 hour lightꢀdark cycle, and fed with a standard rodent diet and water. The animals
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were acclimatized to the laboratory for at least 7 days before use in experiments. Protocols
involving the use of animals were approved by the Wenzhou Medical University Animal Policy
and Welfare Committee (Approval documents: 2013/APWC/0361).
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Cells and treatment
Mouse RAW 264.7 macrophages were obtained from the American Type Culture
Collection (ATCC, USA). Mouse primary peritoneal macrophages (MPMs) were prepared and
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cultured from C57BL/6 mice using the method described in our previous paper . Both RAW
64.7 macrophages and MPMs were incubated in DMEM media (Gibco) supplemented with 10%
FBS,100 U/ml penicillin, and 100 mg/ml streptomycin at 37 ºC with 5% CO . Compounds were
added into cell cultural medium in DMSO solution with the final concentration of DMSO is
.1%.
2
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Determination of TNFꢀα and ILꢀ6
After treatment of cells with compounds and LPS or Pam3CK, the TNFꢀα and ILꢀ6 levels
in media were determined with an enzymeꢀlinked immunosorbent assay (ELISA) kit
(BioScience, San Diego, CA) according to the manufacturer’s instructions. The total amount of
the inflammatory factor in the media was normalized to the total protein quantity of the viable
cell pellets.
Realꢀtime quantitative PCR
Cells were homogenized in TRIZOL kit (Invitrogen, Carlsbad, CA) for extraction of RNA
according to each manufacturer’s protocol. Both reverse transcription and quantitative PCR
were carried out using a twoꢀstep MꢀMLV Platinum SYBR Green qPCR SuperMixꢀUDG kit
(Invitrogen, Carlsbad, CA). Eppendorf Mastercycler ep realplex detection system (Eppendorf,
Hamburg, Germany) were used for qꢀPCR analysis. The primers of genes including TNFꢀα, ILꢀ6,
PGES, ILꢀ1β, COXꢀ2, and βꢀactin were synthesized by Invitrogen. The primer sequences used
are shown in Table S2. The amount of each gene was determined and normalized by the amount
of βꢀactin.
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Western Immunoblot analysis
The treated cells were collected and lysated, then 30 ꢁg of the whole cell lysates were
separated by 10% sodium dodecyl sulfateꢀpolyacrylamide gel electrophoresis and
electrotransferred to a nitrocellulose membrane. Each membrane was preincubated for 1 h at
room temperature in Trisꢀbuffered saline, pH 7.6, containing 0.05% tweenꢀ20 and 5% nonꢀfat
milk. Each nitrocellulose membrane was incubated with specific antibodies against pꢀp38, p38,
pꢀJNK, JNK, pꢀERK, ERK, IκB, or βꢀActin (Santa Cruz, CA). Immunoreactive bands were then
detected by incubating with secondary antibody conjugated with horseradish peroxidase and
visualized using enhanced chemiluminescence reagents (BioꢀRad, Hercules, CA).
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Assay of cellular NFꢀκB pꢀ65 translocation
The cells were immunofluorescenceꢀlabeled according to the manufacturer’s instructions
using a Cellular NFꢀκB pꢀ65 Translocation Kit (Beyotime Biotech, Nantong, China). P65
protein and nuclei fluoresce red and blue, respectively, and can be simultaneously viewed by
fluorescence microscope (Nikon, Tokyo, Japan) at an excitation wavelength of 350 nm for DAPI,
and 540 nm for Cy3. To create a twoꢀcolor image, the red and blue images were overlaid,
producing purple fluorescence in areas of coꢀlocalization.
Docking of 2i to the MDꢀ2 structural model
In order to get further insight into the interactions mode between compound 2i and MDꢀ2,
flexible ligand rigid protein docking was performed using AutoDock version 4.2. The crystal
structure of human MDꢀ2ꢀlipid IVa complex (PDB code 2E59) was derived from Protein Data
Bank as the receptor in current docking model. The AutoDock Tools version 1.5.6 package was
employed to generate the docking input files and to analyze the docking results. A grid box size
of 60×60×60 points with a spacing of 0.375 Å between the grid points was implemented and
covered almost the entire proteinꢀbinding site. The affinity maps of MDꢀ2 were calculated using
AutoGrid. One hundred Lamarckian Genetic Algorithm runs with default parameter settings
were performed. The interactions of complex proteinꢀligand conformations hydrogen bonds and
bond lengths were analyzed
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Fluorescence measurements of bisꢀANS dispalcment
Fluorescence measurements were performed with a spectraMax M5 (Molecular Devices,
Sunnyvale, CA). All measurements were done at 25 °C in a 1 cm pathꢀlength quartz cuvette.
Briefly, 1,1’ꢀBis(anilino)ꢀ4,4’ꢀbis(naphthalene)ꢀ8,8’ꢀdisulfonate (bisꢀANS, 5 ꢁM) and rhMDꢀ2
protein (5 nM) were mixed in PBS (pH 7.4) and incubated to reach stable relative fluorescence
units (RFUs) emitted at 430–590 nm under excitation at 385 nm. Nonꢀfluorescent 2i (at 2.5, 5,
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0, 20, 40 ꢁM) was then treated for 5 min, and followed by measuring relative fluorescence
units (RFUs) emitted at 430ꢀ590 nm.
Surface Plasmon Resonance (SPR) Analysis
The binding affinity of 2i was determined using a ProteOn XPR36 Protein Interaction
Array system (BioꢀRad Laboratories, Hercules, CA) with a HTE sensor chip(ProteOn™,
#
176ꢀ5033). Briefly, MDꢀ2 and TLR4 protein (in acetate acid buffer pH 5.5) was loaded to the
sensors which activated with 10 mM NiSO . And the 2i samples (at 100, 50, 25, 12.5 and 6.25
M) were prepared with runing buffer (PBS, 0.1% SDS, 5% DMSO, and 0.05% Tweenꢀ20).
4
ꢁ
Sensor and sample plate were placed on the instrument. The 2i samples were then captured in
flow cells 1, leaving the second flow cell as a blank. Five concentrations were injected
simultaneously at a flow rate of 30 ꢁM /min for 120 s of association phase, followed with 120 s
of dissociation phase at 25 °C. The final graphs were obtained by subtracting blank sensorgrams
from the duplex or quadruplex sensorgrams. Data were analyzed with ProteOn manager
software. KD was calculated by global fitting of the kinetic data from various concentrations of
2
i using 1:1 Langmuir binding model.
In vitro assay for LPS binding to MD2 (ELISA)
R90A
The MDꢀ2 antibody was coated to 96ꢀwell at 4 °C overnight. RhMDꢀ2, MDꢀ2
and
Y102A
MDꢀ2
(4 µg/mL) in 10 mM TrisꢀHCl buffer was added to the preꢀcoated plate for 1.5 h at
room temperature. After washing with PBST, biotinꢀlabeled LPS was added to the plate with or
without the presence of 2i (1 ꢁM). BiotinꢀLPS binding to MDꢀ2 was determined by ELISA,
representing as absorbance values at 450 nm (A450). Data are mean values (±SD) of 3 separate
experiments, each performed in duplication. *P<0.05, **P<0.01 vs. buffer aloneꢀadded group.
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Flow Cytometric Analysis
Cellular binding of fluorescein isothiocyanateꢀlabeled LPS (LPSꢀFITC, from E.coli 055:B5,
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Sigma, St. Louis, MO) was measured as described previously . Briefly, HUVEC304 cells
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1×10 ) were incubated with LPSꢀFITC (50 ꢁg/mL) for 30 min with or without the presence of
2
i. After washing, the cells with bound LPSꢀFITC were analyzed by flow cytometry.
Immunoprecipitation
Cells or mouse lung tissues were lysed by an extraction buffer (containing mammalian
protein extraction reagent supplemented with protease and phosphatase inhibitor cocktails) and
centrifuged at 12,000 rpm for 10 min at 4 °C. Sufficient amount of MDꢀ2 antibody was added
into 400 µg proteins and gently rotated at 4 °C overnight. The immunocomplex were collected
with protein A+G agarose (Beyotime Biotech, Nantong, China) and the precipitates were
washed four times with iceꢀcold PBS. Then, proteins were released by boiling in sample buffer,
followed by Western blot analysis as described above.
LPSꢀinduced inflammatory mortality in B6 mice
Compound 2i was firstly dissolved with macrogol 15 hydroxystearate (a nonionic
solubilizer for injection, from BASF [Ludwigshafen, Germany]) with or without mediumꢀchain
triglycerides (MCT, from BASF) in a water bath at 37°C. The concentration of 2i was 2 mg/mL.
The concentration of solubilizer was 5%–10%, and MCT 0.5%–2.0% in final solution. For the
vehicle, the mixture of solubilizer and MCT was prepared at 10% and 2%, respectively. Male
C57BL/6 mice weighing 18–22 g were treated with 2i solution (200 ꢁL, 10 mg/kg) by
intravenous injection 15 minutes before the intraperitoneal injection of LPS (20 mg/kg). Control
animals received a similar volume (200 ꢁL) of vehicle. Bodyweight change and mortality were
recorded for 7 days.
LPSꢀinduced ALI
Male B6 mice were randomly divided into three groups, designated control (n = 6), LPS (n
=
6) and 2i+LPS (n = 6). Prior to intratracheal injection of LPS (10 mg/kg), the mice were
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treated orally with 2i at a dosage of 20 mg/kg for one week. Mice were then euthanized with
ketamine 6 hours after LPS induction. The chest cavity of each animal was carefully opened and
the collection of bronchoalveolar lavage fluid (BALF) was performed.
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BALF analysis
The collected BALF was centrifuged at 1000 rpm for 10 min at 4 °C, the supernatant was
used for protein concentration detection and subsequent cytokine determinations. The
precipitation was resuspended using 50 ꢁL physiological saline. The total number of cells on
BALF was detected by cell counting instrument. The number of neutrophils on BALF was
examined using WrightꢀGimesa stain.
Lung Wet/Dry ratio
To observe the pulmonary edema, the lung wet/dry weight ratio was calculated. After the
middle lobe of right lung was collected, the wet weight was recorded. Lung were then heated in
a thermostatic oven at 65 °C for 72 h and weighed to determine the baseline lung dry mass
levels.
Histopathological study
The superior lobe of right lung was collected and fixed in 4% paraformaldehyde, then
embedded in paraffin and cut into 5 ꢁm sections. The sections were stained with hematoxylin
and eosin using standard protocol for light microscopy examination.
Immunohistochemistry
Tissue sections (5 ꢁm thickness) were prepared, deparaffinized in xylene, and hydrated
using an ethanol gradient. Pressureꢀcooker was used for antigen retrieval. After treatment with
3
0% of hydrogen peroxide, all the sections were blocked in 5% bovine serum albumin (BSA)
and incubated with primary AntiꢀCD68 antibody for overnight at 4 °C. The slides were then
incubated with HRPꢀlabeled secondary antibody for 10 min. After the sections were stained with
3,3ꢀdiaminobenzidine tetrahydrochloride (DAB), the slides were evaluated under microscope.
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Statistical analysis
The results are presented as means ± SD. The statistical significance of differences between
groups was obtained by the student’s t test or ANOVA multiple comparisons in GraphPad Pro5.0
(GraphPad, San Diego, CA). P values less than 0.05 (p<0.05) were considered indicative of
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significance. All experiments were repeated at least three times.
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Acknowledgement
Financial support was provided by the National Natural Science Foundation of China (81472307,
1272179, and 81503123), and Natural Science Funding of Zhejiang Province (LY14C050004,
2
LQ14H310003, and LQ15H120005).
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Author Information Notes
Corresponding Author
wzmcliangguang@163.com; Tel: (+86)ꢀ577ꢀ86699396
Notes
The authors declare no competing financial interest.
Associated Content
Supporting Information. Additional experiments, Table S1ꢀS2 and Figure S1ꢀS10.
List of nonstandard abbreviations and acronyms
MD2: Myeloid Differentiation 2; LPS: Lipopolysaccharides;TLR4: Tollꢀlike Receptor 4; ALI:
Acute Lung Injury; CD14: Cluster of Differentiation 14; MAPKs: MitogenꢀActivated Protein
Kinases; NFꢀκB: Nuclear Factor Kappaꢀlightꢀchainꢀenhancer of activated B cells; EDCꢀHCl:
1ꢀ(3ꢀDimethylaminopropyl)ꢀ3ꢀethylcarbodiimide hydrochloride; TNF–α: Tumor Necrosis
Factor–α; HOBT: Nꢀhydroxybenzotriazole; MPMs: Mouse Primary Peritoneal Macrophages;
ILꢀ6: Interleukinꢀ6; SAR: StructureꢀActivity Relationship; ILꢀ1β: Interleukinꢀ1β; PGE:
Prostaglandin E; COXꢀ2: CycloꢀOxygenꢀase 2; JNK: cꢀJun Nꢀterminal Kinase; ERK:
Extracellular Regulated protein Kinases; IκB: Inhibitor of NFꢀΚb; FITC: Fluorescein
isothiocyanate isomer I; BALF: Bronchoalveolar lavage fluid; MPO: Myeloperoxidase
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