Treatment of Sepsis Pathogenesis with High Mobility Group Box Protein 1-Regulating Anti-inflammatory Agents

Article abstract of DOI:10.1021/acs.jmedchem.6b00954

Sepsis is one of the major causes of death worldwide when associated with multiple organ failure. However, there is a critical lack of adequate sepsis therapies because of its diverse patterns of pathogenesis. The pro-inflammatory cytokine cascade mediate

Full text of DOI:10.1021/acs.jmedchem.6b00954

Article
pubs.acs.org/jmc
Treatment of Sepsis Pathogenesis with High Mobility Group Box
Protein 1‑Regulating Anti-inflammatory Agents
Wansang Cho,†,# Ja Young Koo,†,# Yeonju Park,‡ Keunhee Oh,§ Sanghee Lee,† Jin-Sook Song,∥
Myung Ae Bae,∥ Donghyun Lim,⊥ Dong-Sup Lee,‡ and Seung Bum Park*,†,⊥
†CRI Center for Chemical Proteomics, Department of Chemistry, Seoul National University, Seoul 08826, Korea
‡Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Korea
§Transplantation Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea
∥Korea Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon, 34114, Korea
⊥Department of Biophysics and Chemical Biology, Seoul National University, Seoul 08826, Korea
S
* Supporting Information
ABSTRACT: Sepsis is one of the major causes of death worldwide when associated with multiple organ failure. However, there
is a critical lack of adequate sepsis therapies because of its diverse patterns of pathogenesis. The pro-inflammatory cytokine
cascade mediates sepsis pathogenesis, and high mobility group box proteins (HMGBs) play an important role as late-stage
cytokines. We previously reported the small-molecule modulator, inflachromene (1d), which inhibits the release of HMGBs and,
thereby, reduces the production of pro-inflammatory cytokines. In this context, we intraperitoneally administered 1d to a cecal
ligation and puncture (CLP)-induced mouse model of sepsis and confirmed that it successfully ameliorated sepsis pathogenesis.
On the basis of a structure−activity relationship study, we discovered new candidate compounds, 2j and 2l, with improved
therapeutic efficacy in vivo. Therefore, our study clearly demonstrates that the regulation of HMGB1 release using small
molecules is a promising strategy for the treatment of sepsis.
in the U.S.7−9 Although numerous therapeutic approaches to
INTRODUCTION
■
sepsis have been advanced, the mortality rate of severe sepsis is
Sepsis, a systemic inflammatory response caused by infectious
nearly 20%.10 Furthermore, the annual hospital healthcare cost
processes, is one of the major causes of death worldwide.1−3
for patients with severe sepsis in the U.S. is the highest among
External pathogenic molecules known as pathogen-associated
all diseases and was around $20 billion.11 The biggest obstacle
molecular patterns (PAMPs) are recognized by pattern
to the discovery of therapeutics for sepsis is its diverse etiology
recognition receptors (PRRs) in innate immune cells. The
specific recognition of PAMP by the PRRs activates immune
signaling, resulting in the production of various pro-
inflammatory cytokines that mediate the inflammatory
among patients.12 The heterogeneous patterns of sepsis
pathogenesis depend on the pathogenic organisms and sites
of infection.7,12 The diverse characteristics of sepsis patho-
responses.4,5 Sepsis occurs when immune responses are
genesis have prompted researchers to study the molecular
mechanism of sepsis progression based on systemic inflamma-
overactivated, including intractable inflammatory responses
associated with imbalanced cytokine production. In addition,
the subsequently unmanaged pro-inflammatory cytokine
tory responses.
The emerging role of various pro-inflammatory cytokines in
cascade results in whole body shock.1,6,7 When the pathophysi-
the progression of sepsis has been examined by a series of
ology of sepsis is associated with hypoperfusion, hypotension,
and organ dysfunction, it is termed as severe sepsis,1 which
Received: July 5, 2016
occurs in approximately 10% of all intensive care unit patients
© XXXX American Chemical Society
A
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scientific studies.13−15 Furthermore, tumor necrosis factor
(TNF)-α and interleukin-(IL)-1β are two major pro-inflam-
matory cytokines involved in sepsis and their secretion is
regulated by positive feedback during systemic inflammatory
responses.15,16 When the secreted cytokines circulate in the
whole body, considerable amounts of cytokines are produced,
resulting in septic shock and secondary multiple organ
dysfunction.16 Therefore, therapeutic approaches to antagonize
the production of these cytokines have been used for the
treatment of sepsis.17,18 For example, an anti-TNF-α antibody
underwent large-scale clinical trials but was discontinued
because it lacked efficacy.7,19,20 Literature reports suggest
these failures might have been caused by two reasons. First,
because the production of TNF-α and IL-1β peaks at the early
stage of systemic inflammation, it is difficult to reverse the
enhanced cytokine production without inhibiting the early
stage of pathogenesis. Second, the continuous inflammatory
stimuli unexpectedly restored the TNF-α levels after treatment
with the antagonizing anti-TNF-α antibody.19−21
On the other hand, high mobility group box protein 1
(HMGB1) has been identified as a late-stage mediator of
inflammatory responses.22 HMGB1 is a DNA-binding protein
that is associated with the nuclear genomic DNA. When
damaged cells undergo necrotic death, HMGB1 is released into
the extracellular milieu, alerting adjacent cells to the damage.
Interestingly, in immune cells such as macrophages, HMGB1 is
actively secreted as a pro-inflammatory cytokine during
systemic inflammatory responses.23−26 Previous reports have
indicated that secreted HMGB1 plays an important role in
regulating the production of other pro-inflammatory cyto-
kines.27 In this context, it has been reported that the inhibition
of HMGB1 secretion successfully ameliorated the pathogenesis
of sepsis in an in vivo cecal ligation and puncture (CLP) mouse
model, the gold standard animal model in the field of sepsis
study.28,29 Briefly, the CLP mouse model is established by
performing a surgical procedure to the cecum, which is the
organ where the resident enteric microbiome exists. Ligating
the cecum and puncturing the end with a needle causes
endotoxemia by inducing polymicrobial contamination in the
abdominal cavity toward the circulatory system. This action
subsequently initiates a systemic inflammatory response,
namely sepsis.
Regarding the role of HMGB1 in sepsis, Tracey and co-
workers reported that the anti-HMGB1 antibody abrogated the
lethality of the CLP-induced mouse model by inhibiting the
release of HMGB1.30 Endogenous small molecules such as
cholinergic agonists have also inhibited HMGB1 secretion,
thereby exhibiting the therapeutic effects in sepsis.31−33
Furthermore, a known autophagy modulator, (−)-epigalloca-
techin-3-gallate (EGCG), has been studied for the prevention
of lipopolysaccharide (LPS)-induced HMGB1 release by
triggering the degradation of cytosolic HMGB1, thereby
boosting the autophagic process.34−36
Figure 1. Structure and in vivo therapeutic effect of inflachromene
(ICM, 1d) in cecal ligation and puncture (CLP) model of sepsis. (A)
During sepsis, the extracellular secretion of high-mobility group box
protein 1 (HMGB1) occurs in activated macrophages. The small-
molecule ICM (1d) inhibits the release of HMGB1, thereby
suppressing immune responses. (B) Chemical structure of ICM. (C)
Survival rate of vehicle- and ICM-treated CLP mouse model. ICM was
administered intraperitoneally at a daily dose of 10 mg/kg for 9 days.
translational modifications.40 On the basis of the fact that
microglia in the central nervous system is a cell type sharing the
same ontology with peripheral macrophages,41,42 we hypothe-
sized that ICM can inhibit the secretion of HMGB1 and
HMGB2 in macrophages, reduce the production of additional
pro-inflammatory cytokines, and thereby exhibit its therapeutic
effect on the systemic inflammatory response. To test our
hypothesis, we examined whether in vivo administration of
ICM to CLP-induced mice inhibits sepsis pathogenesis by
measuring the survival rate. On the basis of the initial proof-of-
concept in vivo study, we investigated the enhancement of the
anti-inflammatory efficacy of ICM in sepsis treatment by
conducting a systematic structure−activity relationship study.
After synthesizing approximately 30 ICM analogues, we
examined their anti-inflammatory activities in Raw264.7
macrophage-like cells. Then, candidate compounds were
selected based on a series of biological evaluations including
the Griess assay, enzyme-linked immunosorbent assay (ELISA),
microsomal stability testing, and pharmacokinetics (PK) as well
as in vivo studies using a CLP mouse model. Finally, we
discovered new candidate compounds with enhanced ther-
apeutic efficacy against sepsis.
Previously, we reported the discovery of a novel small
molecule, inflachromene (ICM, 1d, Figure 1B), that inhibits the
activation of BV-2 microglia-like cells, via screening a pDOS-
based drug-like compound library.37,38 On the basis of our
efforts on target identification using the fluorescence difference
in two-dimensional gel electrophoresis (FITGE) method,39 we
revealed HMGB1 and HMGB2 as the target proteins of ICM,
and the subsequent biochemical and biophysical studies
confirmed that ICM inhibits the secretion of HMGB1 and
HMGB2 in microglia via the modulation of their post-
RESULT AND DISCUSSION
■
In Vivo Therapeutic Effect of ICM in CLP-Induced
Mouse Model of Sepsis. When pathogens invade a host
organism, an acute inflammatory response is initiated in the
body. Macrophages, one of the key immune cells that mediate
B
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the inflammatory responses, are activated following their
recognition of pathogens, which subsequently induces the
secretion of various pro-inflammatory cytokines including
HMGBs, TNF-α, and IL-6 (Figure 1A).4−6 At the basal state,
HMGBs are associated with genomic DNA within the nucleus.
After activation of macrophage upon recognition of LPS,
HMGBs are translocated from nucleus to cytosol and
subsequently released to the extracellular milieu.43 These pro-
inflammatory cytokines activate other adjacent macrophages
and initiate a positive feedback mechanism of immune
response.44 We previously reported that ICM (1d, Figure
1B) inhibits the translocation of HMGBs by blocking their
post-translational modifications and subsequently reduces the
secretion of HMGBs in microglia, which led to the suppression
of neuroinflammatory response.40
Table 1. Relationship between Structural Modifications in
Benzopyranyl Core and Percent (%) Inhibition of Released
Nitric Oxide (NO) Level
Therefore, we envisaged that 1d would be potentially
efficacious for the treatment of sepsis. On the basis of this
assumption, we adopted a murine CLP model, which is
considered as the gold standard model of in vivo sepsis.28 The
intraperitoneal (ip) injection in the CLP-induced mice of 10
mg/kg of 1d for 9 days significantly increased the survival rate
compared to that of the vehicle-treated group (Figure 1C). The
measurement of the serum cytokines after euthanizing the mice
revealed that IL-6 levels were highly correlated with their
survival (Supporting Information, Figure S1). Based on these
data, we concluded that 1d exhibited a potential therapeutic
effect in sepsis.
Synthesis and Biological Evaluation of ICM (1d)
Analogues to Discover New Compounds with Enhanced
Anti-inflammatory Effects. To improve the therapeutic
efficacy of 1d, we first comprehensively explored the
structure−activity relationship of its anti-inflammatory effects.
Starting from the benzopyranyl core structure of 1d, we
designed and synthesized a series of analogues to clarify the
importance of each structural motif. To measure the inhibitory
effects on immune response, Raw264.7 cells were treated with
the synthesized analogues in the presence of LPS, which is
widely used to activate macrophages45 because it is one of the
major components of external membrane of Gram-negative
bacteria.45−47 LPS-activated macrophages produce various pro-
inflammatory cytokines48,49 and nitric oxide (NO), which was
selected as the molecular marker to monitor the activation level
of macrophages. We quantitatively measured the secreted NO
level in the culture medium by using the Griess assay as an
efficient high-throughput screening system.40,50
Our original hit compound (1d) inhibited the NO release by
87% in Raw264.7 cells compared to the vehicle. As shown in
Table 1, the systematic modification of the R1, R2, R3, and R4
groups of the benzopyranyl core structure revealed that most of
R1, R2, and R3 substituents (1a−1h) reduced the inhibition
potency on NO release respect to 1d: in particular substitution
of the R2 hydroxyl group with a methoxy one was very
unfavorable. Even when both methyl groups of 1d at the R4
position were modified as in 1i−1j analogues, the inhibition of
cellular NO release was significantly reduced. These results
indicate that the R1−R4 groups’ combination of 1d is essential
for the anti-inflammatory effects. Similarly, the shift or removal
of the double bond of the tetrahydropyridazine ring failed to
induce inhibitory effects on NO release (Supporting
Information, Figure S2).
a
Percentage inhibition of NO by 10 μM ICM and its analogues was
b
measured in Raw264.7 cells by using Griess assay. ICM, Cpd.,
compound; NO inh, nitric oxide inhibition.
reaction of the intermediate 3 with N-substituted 1,2,4-
triazoline-3,5-diones. First, the replacement of the phenyl
group of 1d with a methyl one (2a) caused the lowest
inhibitory effect (45%) on NO release, being reduced also with
benzyl (2b) and cyclohexyl (2c) substitutions. Then we
incorporated various substituents at the ortho, meta, and para
positions of the original phenyl ring. The ortho-substituted
compounds (2d, 2e) exhibited little inhibitory effects on NO
release. In contrast, a methoxy group at the meta (2f) and para
(2g) positions enhanced the inhibition of NO release. To
examine the relationship between the position of the methoxy
group on the phenyl ring and the inhibition of NO release, we
synthesized ICM analogues containing 3,4-(methylenedioxy)-
phenyl (2k) and 3,5-dimethoxyphenyl (2l) as the R groups. A
series of para-substituted (2g−j, 2m−o) analogues were also
synthesized, and their activity was evaluated by using the Griess
assay. The screening results indicated that compounds 2g−2l
effectively inhibited the NO release compared to 1d while 2m,
2n, and 2o showed cytotoxicity at the higher concentration,
which led us to discontinue their further biological evaluation.
To confirm the anti-inflammatory activity of our synthesized
compounds, we measured their inhibitory effect on the
production of another cytokine, TNF-α. As shown in Table
2, compounds 1d and 2f−2l inhibited the TNF-α release in
Raw264.7 cells. The amount of TNF-α released was
quantitatively measured by using ELISA. On the basis of
these results, we selected compounds 2i, 2j, 2k, and 2l as our
improved candidate compounds for further biological evalua-
tion.
Inhibition of HMGB1 and IL-6 Release by Initial Hit
Compounds. For the secondary confirmation of biological
activities, we first examined whether our initial hit compounds
could inhibit the translocation of HMGB1. As mentioned
Next, we prepared a series of 1d analogues by introducing
various N-substituents of the tetracyclic benzopyranyl core,
indicated as the R group in Table 2, via hetero-Diels−Alder
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covalent UV cross-linking with adjacent proteins and a
bioorthogonal alkyne moiety for fluorophore conjugation via
click reaction.39 We previously used ICM-BP for the
identification of HMGBs as the target protein of 1d and
demonstrated its competitive binding toward HMGBs with 1d
in the cellular system.40 With this ICM-BP in hand, we
evaluated the relative binding affinity of candidate compounds
1d, 2j, or 2l with HMGB1 by comparing the competitive
labeling of fluorophores toward HMGB1 (Figure 2D). As
shown in Figure 2E, we clearly observed the reduced labeling of
Cy5 in the presence of either 1d, 2j, or 2l in a dose-dependent
manner. But the negative compound 2a did not show any dose-
dependent competition, which confirms the specific compet-
itive binding event of ICM-BP with candidate compounds. On
the basis of this experiment, we could confirm that 2j and 2l
bind more tightly than 1d, which suggests that 2j and 2l might
have enhanced activities in the regulation of HMGB1.
Moreover, on the basis of our preliminary docking simulation
with HMGB1, 2j and 2l showed lower binding energies to
HMGB1 than the original ligand 1d did (Supporting
Information, Figure S3). Therefore, we selected 2j and 2l as
candidate compounds with enhanced anti-inflammatory effect
for further in vivo evaluation.
Inhibition of Inflammatory Signaling Pathway by
Candidate Compounds (1d, 2j, and 2l). Intracellular
inflammatory signaling pathways play important roles in
propagating immune responses in the activated macrophage.
Mitogen-activated protein kinase (MAPK) and nuclear factor
kappa-light-chain enhancer in B cells (NF-κB) complex are two
of the most important inflammatory signaling systems that
regulate the release of HMGBs and other cytokines in
macrophages.4,51,52 There are three distinct types of MAPKs:
p38 MAPK (p38), c-jun N-terminal kinases (JNKs), and
extracellular signal-regulated kinase (ERK). Following the
activation of macrophage, the MAPKs are phosphorylated
and subsequently trigger the transduction of inflammatory
signals within the cells.52,53 The Western blot data showed that
our compounds 1d, 2j, and 2l clearly inhibited the
phosphorylation of MAPK including p38, JNK, and ERK
(Figure 3A,B).
Table 2. Effects of Structural Modifications Relative to the
N-Substituents of the Triazolinedione Ring Structure of 1d
on the Percentage (%) Inhibition of Nitric Oxide (NO) and
Tumor Necrosis Factor (TNF)-α Release, As Well As on the
Cell Viability (%)
% NO
a
%
viab.
% TNF-α
b
c
Cpd.
R
inh.
inh.
d
1d
phenyl
87
45
70
63
48
66
96
89
87
94
93
92
94
85
99
99
105
114
106
117
109
115
118
127
109
130
111
121
121
43
e
2a
2b
2c
2d
2e
2f
methyl
benzyl
e
cyclohexyl
2-iodophenyl
e
e
2-methoxyphenyl
3-methoxyphenyl
4-methoxyphenyl
4-fluorophenyl
e
38
44
44
45
68
46
60
2g
2h
2i
4-methylphenyl
2j
4-acetylphenyl
2k
21
2m
2n
2o
3,4-(methylenedioxy)phenyl
3,5-dimethoxyphenyl
4-(trifluoromethyl)phenyl
4-tert-butylphenyl
4-n-butylphenyl
f
f
f
106
105
50
a
Inhibition of NO release (%) by each compound (10 μM) was
measured by using Griess assay in Raw264.7 cells. Cpd., compound;
WST, water-soluble tetrazolium; ELISA, enzyme-linked immunosorb-
b
ent assay. Relative cell viability (%) of each compound (10 μM) in
the presence of LPS was measured by using WST assay, and compared
c
to vehicle. Inhibition of TNF-α release (%) by each compound (10
d
e
μM) was measured by using ELISA. ICM. Excluded by low NO
f
inhibition (%). Excluded due to the cellular toxicity observed at the
higher concentration.
earlier, HMGBs are translocated from the nucleus to cytosol in
activated macrophage before they are released into the
extracellular milieu. Therefore, we measured the cellular
localization of HMGB1 upon LPS treatment in the absence
and presence of our hit compounds using immunohistochem-
istry (TRITC-labeled secondary antibody). As shown in Figure
2A, our initial hit compounds (2i, 2j, 2k, 2l, and 1d) inhibited
the translocation of HMGB1 from the nucleus to the cytosol
induced by LPS-treatment. Because we observed a drastic
decrease of IL-6 levels in the surviving CLP-induced mice
treated with 1d (Supporting Information, Figure S1), we
measured the secreted IL-6 levels in Raw264.7 cells. The
Western blot analysis confirmed that the secreted IL-6 levels
were significantly decreased upon treatment with 1d, 2i, 2j, 2k,
and 2l in LPS-activated macrophages (Figure 2B). Interestingly,
we also observed the reduction of intracellular IL-6 levels upon
treatment with hit compounds, which indicates that the positive
feedback of cytokine production was inhibited by our
molecules. Furthermore, among the initial hit compounds, 2j
and 2l showed superior inhibitory efficacy on IL-6 secretion
(Figure 2B) and TNF-α release (Table 2).
The NF-κB pathway is another key signaling pathway that
regulates inflammatory responses. NF-κB is a complex of two
different protein domains, p50 and p65. In the basal state, the
activity of the NF-κB complex is inhibited by nuclear factor of
kappa-light polypeptide gene enhancer in B-cells inhibitor
(IκB). When the activation signal reaches the macrophages, IκB
kinase (IKK) phosphorylates IκB, which is subsequently
degraded leading to the activation of the NF-κB complex.
The activated NF-κB then translocates to the nucleus and acts
as a transcription factor to produce pro-inflammatory cytokines.
In this signaling pathway, the cellular translocation of the NF-
κB complex is the key event in the activation of the
macrophages.51,54 As expected, the nuclear translocation of
the NF-κB was inhibited by treatment with our candidate
compounds 1d, 2j, and 2l, as evidenced by immunofluor-
escence assays on p65 subunit (Figure 3C and Supporting
Information, Figure S5). On the basis of these observations, we
concluded that our candidate compounds 1d, 2j, and 2l
regulated the inflammatory signaling pathways in activated
macrophages.
To confirm the improved activity of 2j and 2l, we performed
in vitro binding competition assay using ICM-BP (Figure 2C).
ICM-BP has a photoactivatable benzophenone moiety for
Pharmacokinetics of ICM and Selected Compounds.
Before investigating the selected compounds, 1d, 2j, and 2l in
the in vivo CLP-induced mouse model, we measured their
D
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Figure 2. Inhibition of high mobility group box protein B1 (HMGB1) translocation and interleukin (IL)-6 release upon treatment with 1d, 2i, 2j, 2k,
and 2l. (A) Cellular immunofluorescence images of HMGB1 after pretreatment with 20 μM of 1d, 2i, 2j, 2k, and 2l for 1 h, followed by 500 ng/mL
lipopolysaccharide (LPS) treatment. Dimethyl sulfoxide (DMSO) was the control. (B) Western blot analysis of released IL-6 after pretreatment with
10 μM of 1d, 2i, 2j, 2k, and 2l for 1 h, followed by 100 ng/mL LPS treatment of Raw264.7 cells. Images shown represent at least three independent
experiments. (C) Structure of ICM-BP. Purple, benzophenone moiety; orange, alkyne moiety. (D) Schematic figure of in vitro binding competition
assay. After UV cross-linking of ICM-BP, Cy5-azide was incorporated via bioorthogonal click reaction. (E) Fluorescence gel image of purified
HMGB1 protein labeled with Cy5-azide. The same amount of HMGB1 was loaded into each lane. Competitor 1d, 2j, and 2l were treated to the
purified HMGB1 protein in a dose-dependent manner prior to the treatment of ICM-BP (5 μM). 2a was treated as a negative control, of which data
exhibit no dose-dependency. Cpd., compound; Lys, cell lysate; CM, culture medium; Conc., concentration.
aqueous solubility,55 liver microsomal stabilities, and PK
profiles (Table 3). Compared to 1d, compounds 2j and 2l
showed improved solubility in aqueous media and improved
liver microsomal stability in both mice and humans. Probably,
the PK data revealed that although the area under the curve
(AUC) values of 2j and 2l were lower than that of 1d, their
comparable (2j) or longer (2l) half-life was expected to
contribute to a better efficacy in the in vivo sepsis model. The
1d, 2j, and 2l were all easily administered by ip injection and
retained their activities for several hours. Along with their
biological activities found in previous biological experiments,
these results indicate that the compounds 1d, 2j, and 2l all
performed adequately in the in vivo mouse model following ip
administration.
CONCLUSION
■
In this study, we explored the therapeutic efficacy in the
treatment of sepsis of a previously reported small molecule,
ICM (1d), which regulated the secretion of HMGB1 and
HMGB2 in activated microglia.40 Pro-inflammatory cytokines,
such as TNF-α and IL-1β, have shown limited clinical
usefulness because of their characteristic early stage mediator
effects,7,19−21 while the clinical relevance of antagonizing
HMGBs in sepsis has been proposed and reported in several
studies.30−36 Indeed, as late-stage mediators of systemic
inflammatory responses, HMGBs regulate the production of
other pro-inflammatory cytokines, thereby modulating the
systemic inflammatory response. Therefore, the inhibition of
HMGB1 release could downregulate the production of pro-
inflammatory cytokines.30,31 With this in mind, we evaluated
Enhanced in Vivo Therapeutic Effect of 2j and 2l in
CLP-Induced Mouse Model of Sepsis. Finally, we compared
our novel HMGB1-regulating compound, ICM (1d), in an in
the in vivo therapeutic effect of 1d, 2j, and 2l in a CLP-induced
vivo CLP-induced mouse model, finding that it efficiently
mouse model. The surgical procedure used to perform the CLP
is illustrated in Figure 4A. Briefly, after excising the abdomen,
the exposed cecum was ligated just below the ileocecal valve
and then punctured using a surgical needle. Then, the excised
abdomen was closed. The CLP-induced mice were treated with
our candidate compounds for 21 days by daily ip injection,
observing that the survival rate of the 1d-, 2j-, and 2l-treated
mice was significantly improved compared to that of the
vehicle-treated mice (Figure 4B). Compared to 1d with a 45%
survival rate, 74% and 56% were found for 2j- and 2l-treated
mice, respectively, while only 10% of the vehicle-treated mice
survived. Furthermore, serum IL-6 levels were significantly
decreased in the survived mice (Supporting Information, Figure
S6), which is consistent with our previous observation in Figure
2B and Supporting Information, Figure S1. Therefore, we
successfully enhanced the therapeutic effect of 1d by structural
optimization obtaining 2j and 2l as potential drug candidates
for the treatment of sepsis.
ameliorated the sepsis pathogenesis.
We further sought to improve the efficacy of 1d by
synthesizing 30 analogues, and their anti-inflammatory activities
were evaluated by monitoring the inhibition of NO release in
the Griess assay. On the basis of our structure−activity
relationship study and secondary biological evaluations, we
selected 2j and 2l, with different substituents on the 1d phenyl
ring, as our final candidates. Both 2j and 2l inhibited the
translocation of HMGB1 from the nucleus to the cytosol,
improved the inhibition of IL-6 release, and regulated the
downstream inflammatory signaling pathways. Furthermore, 2j
and 2l also showed better liver microsomal stabilities and
reasonable PK profiles compared to that of 1d. Finally, the daily
ip administration of 2j and 2l in the in vivo CLP-induced
mouse model showed enhanced therapeutic effects and 2j
exhibited the best therapeutic efficacy against sepsis. Therefore,
this research study has provided considerable evidence
supporting the use of selective regulation of HMGB1 release
E
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Griess Assay and WST Assay. Griess assay reagent was generated
prior to the assay with the following contents listed below: N-(1-
naphthyl)ethylenediamine dihydrocholoride, 0.1% (m/v); sulfanila-
mide, 1% (m/v); phosphoric acid (85%); 5% (v/v) in deionized water.
Raw264.7 cell was seeded in transparent 96-well plate and maintained
for 1 day. Media was aspirated, and each well was washed with serum-
free DMEM for twice. Each well was refilled with serum-free DMEM.
Then 100 ng/mL LPS and compounds were treated afterward and
cultured for 24 h. After the incubation, the supernatant culture media
was transferred to new 96-well plate and mixed with 50 μL of Griess
assay reagent. Blank values were measure by mixing 50 μL of Griess
assay reagent with 10% FBS and 1% antibiotic-antimycotic containing
DMEM. Four wells each were used for the control experiment, LPS
and DMSO (without any compounds) treated well, and DMSO-only
treated well. Without LPS, Raw264.7 cell does not release the nitric
oxide in measurable concentration. Cell-seeded plate was conserved
for further WST assay. Media−reagent mixture was incubated at room
temperature for several minutes. Absorbance was read at 548 nm.
Immediately after transferring media, cell-seeded wells were refilled
with 100 μL of 10% FBS and 1% antibiotic-antimycotic containing
DMEM. Then 10 μL of Ez-cytox WST assay reagent was used to treat
each well, following the manufacturer’s protocol. After incubating 20
min in a 5% CO2 incubator at 37 °C with humidified atmosphere,
absorbance was read at 450 nm. Blank values were obtained from the
well only treated with 10% FBS and 1% antibiotic-antimycotic
containing DMEM. All experiments were independently performed at
least three times.
ELISA Assay. For the quantification of secreted TNF-α, Raw264.7
cell was seeded in transparent 96-well plate and maintained for 1 day.
Media was aspirated after 1 day and replaced to serum-free DMEM
with 1% (v/v) antibiotic−antimycotic solution. LPS and compounds
were treated in a final concentration of 100 ng/mL and 10 μM. After 4
h, media were transferred to new transparent 96-well plate and diluted
with 1% BSA in PBS solution. ELISA assay was performed following
the manufacturer’s protocol. Standard curve was plotted by the dose-
dependent treatment of the control TNF-α protein provided with the
ELISA kit. Blank value was measured by adding solutions without
TNF-α substrate. All experiments were independently performed at
least three times.
For the quantification of serum IL-6 level in CLP-induced C57BL/6
mice, blood was collected from mice at 24 h after surgery. ELISA
plates (Costar, Cambridge, MA) were coated overnight with anti-IL-6
monoclonal antibody (eBioscience, San Diego, CA) at 4 °C. Wells
were blocked with PBS containing 1% bovine serum albumin (Sigma-
Aldrich, St. Louis, MO) for 1 h at room temperature (RT) and
incubated with serum overnight at 4 °C. After washing, plates were
incubated with biotinylated anti-IL-6 antibody (eBioscience) for 1 h at
RT, followed by streptavidin-conjugated horseradish peroxidase
(eBioscience) for 1 h at RT. Color was developed using TMB
solution (eBioscience), and absorbance was read at 450 nm with a
Multiskan Ascent (Labsystems, Kennett Square, PA). All assays were
performed in triplicate and were independently repeated three times.
Figure 3. Inhibition of inflammatory signaling pathways in Raw264.7
cells. (A) Western blot analysis relative to the phosphorylation (p) of
p38, JNK, and ERK after pretreatment with 20 μM 1d, 2j, or 2l in
Raw264.7 cells, followed by 100 ng/mL LPS treatment. DMSO was
the control. (B) Quantification of phosphorylation level of p38, JNK,
and ERK after compound treatment by Western blot analysis. (C)
Cellular NF-κB p65 immunofluorescence images after pretreatment
with 20 μM 1d, 2j, or 2l in Raw264.7 cells, followed by 500 ng/mL
LPS treatment. Data represent at least three independent experiments.
Cpd., compound; GAPDH, glyceraldehyde 3-phosphate dehydrogen-
ase; LPS, lipopolysaccharide; DMSO, dimethyl sulfoxide; pERK,
phosphorylated extracellular signal-regulated kinase; pJNK, phos-
phorylated c-Jun N-terminal kinase; pp38, phosphorylated p38; NF-
κB, nuclear factor kappa-light chain enhancer in B cells.
as a promising therapeutic strategy for the treatment of sepsis
by using small molecules.
EXPERIMENTAL SECTION
■
Cell Culture. Raw264.7 macrophage cell was cultured in Dulbecco
Modified Eagle Medium (DMEM) with 10% (v/v) fetal bovine serum
(FBS) and 1% (v/v) antibiotic-antimycotic solution. Cell was
maintained in a 100 mm cell culture dish, in a 5% CO2 incubator at
37 °C with humidified atmosphere.
a
Table 3. Aqueous Solubility, Liver Microsomal Stability, and Pharmacokinetic (PK) Data of 1d, 2j, and 2l
Cpd.
solubility
1d
2j
2l
aqueous (μg/mL)
mouse (%)
23.5 1.02
0.41 0.10
0.82 0.29
0.14 0.10
387 122
327 68.7
3.87 0.84
162.5 2.12
2.46 1.83
30.2 2.81
0.25 0.00
100 3.72
202 28.1
3.45 1.00
65.1 2.74
20.2 1.78
41.2 4.33
0.14 0.10
282 87.0
218 51.8
5.00 0.98
microsomal stability
human (%)
Tmax (h)
PK data
Cmax (μg/mL)
AUC (μg/mL·h)
T1/2 (h)
a
Equilibrium aqueous solubility of 1d, 2j, and 2l wes measured by μsol method. PK data were obtained after 10 mg/kg of each compound was
administrated by intraperitoneal (ip) injection. Among PK parameters, Cmax, Tmax, AUC, and T1/2 were measured. Liver microsomal stability was
measured by quantifying percentage remaining compound after 30 min. Data are means
standard deviation (SD, n = 3). Cmax, maximum
concentration; Tmax, time to achieve Cmax; AUC, area under the curve; T1/2, half-life.
F
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Journal of Medicinal Chemistry
Article
Figure 4. Therapeutic effect of selected compounds in in vivo CLP-induced mouse model. (A) Cecal Ligation and puncture procedure. Severity of
sepsis can be controlled by the position of ligation. (B) Percentage survival rate of vehicle- and compound 1d-, 2j-, and 2l-treated CLP-induced mice.
1d, 2j, and 2l were administered intraperitoneally in a daily dose of 10 mg/kg for 21 days, starting from 1 h prior to CLP induction. Data shown is
the accumulation of three independent experiments, each trial with at least N = 5. (C) Chemical structure of 2j and 2l.
Western Blot. Protein sampling was done in 4 °C. Cells were
harvested in RIPA lysis buffer containing protease inhibitor cocktail
and phosphatase inhibitor. After centrifugation at 15000 rpm for 20
min, supernatant was transferred to new 1.5 mL tube. Protein
concentration was normalized with a Micro BCA protein assay kit.
Prepared protein samples were analyzed with SDS-PAGE and
following Western blot procedure. Protein was transferred into
PVDF membrane after SDS-PAGE. Membrane was blocked after
transfer step, with 2% BSA in TBST for 1 h in room temperature.
Primary antibodies were treated overnight at 4 °C or 1 h in room
temperature, followed by washing with TBST. HRP-conjugated
secondary antibodies were treated for 1 h in room temperature.
Antibodies were diluted in 1% BSA in TBST solution, with the
concentration indicated in antibody manufacturer’s protocol. After
washing with TBST, membrane was developed by Amersham ECL
prime solution. Chemiluminescent signal was measured by ChemiDoc
MP imaging system, and relative quantification were followed using
ImageLab 4.0 software. Quantification result was processed with
GraphPad Prism 5.0 software.
Immunofluorescence. Raw264.7 cell was seeded in Nunc Lab-
Tek II chambered coverglass and maintained for 1 day. Then 20 μM of
compounds were treated 1 h prior to 500 ng/mL LPS treatment for 2
h. After aspirating the media and washing with PBS, 200 μL of 4%
paraformaldehyde solution was treated and incubated at room
temperature for 20 min. Then 4% paraformaldehyde solution was
aspirated and the sample was washed with PBS three times. For the
permeabilization to enable antibody binding, 200 μL of methanol was
treated and incubated at −20 °C for 20 min. Methanol was then
removed and the sample was washed with PBS three times. Sample
was blocked with 2% BSA in PBS solution at room temperature for 1
h. Then 2% BSA in PBS solution was removed and primary antibody
containing 1% BSA in PBS was treated at 4 °C overnight. Antibody
concentration was optimized according to the manufacturer’s protocol.
Primary antibody solution was removed and sample was washed with
PBS three times. Secondary antibody containing 1% BSA in PBS
solution was treated at room temperature for 1 h. The secondary
antibody used was goat antirabbit TRITC labeled antibody from
Abcam. After 1 h, antibody solution was aspirated and the sample was
washed with PBS three times. For nucleus staining, Hoechst 33342
was diluted in PBS, and each well was treated and incubated at room
temperature for 1 h. Hoechst 33342 was then removed and the
chamber was filled with PBS, continued for imaging using DeltaVision
microscope.
DeltaVision Imaging. DeltaVision Elite imaging system was used
for the immunfluorescence imaging of HMGB1 and p65 translocation
in Raw264.7 cell. Chamber was maintained with constant temperature
of 25 °C. Image was obtained with 60× scale, using DAPI/DAPI and
TRITC/TRITC filter set: DAPI (excitation, 390/18 nm; emission,
435/48 nm), and TRITC (excitation, 542/27 nm; emission, 597/45
nm). Images were analyzed and merged with SoftWorks deconvolu-
tion software.
In Vitro Fluorescence Binding Competition Assay. Purified
HMGB1 protein from R&D Biosystems were reconstituted in
deionized water. Then 1 μg of HMGB1 solution was transferred to
a 20 μL PCR tube (Axygen). Compounds were diluted in deionized
water from 20 mM DMSO stock solution. The final concentration of
DMSO was adjusted to 0.8%. Diluted competitors were pretreated for
60 min, and then ICM-BP was treated for additional 20 min. After
compound treatment, 365 nm UV light from BLAK-Ray (B-100AP)
UV lamp (UVP, USA) was irradiated to each sample for 5 min on ice.
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The samples were then subject to click reaction with Cy5-azide
(Lumiprobe, USA) (40 μM) in the solution of tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine (TBTA) (Sigma, USA) (100 μM), CuSO4
(Sigma, USA) (1 mM), tris(2-carboxyethyl)phosphine (TCEP) (TCI,
Japan) (1 mM), and tBuOH (Sigma, USA) (5%) for 90 min. Then 5×
Lammeli buffer was added to the sample and incubated for 5 min at
room temperature. Samples were loaded in 12.5% polyacrylamide gel
for 1D gel electrophoresis. Finally, fluorescence was scanned by
Typhoon Trio (Amersham Bioscience, USA).
Computational Calculation. Prior to the docking simulation
study, the conformers of each ligand were generated by Vconf2.0 using
Tork conformational analysis method.56 The binding modes and
binding energies of ICM-derived ligands in HMGB1 were predicted by
docking simulation using the Discovery Studio 1.7 program. Prediction
of binding modes were calculated from cavities in X-ray crystal
structure of HMGB1 (PDB 1HMF). Graphical analyses were
performed with UCSF Chimera package version 1.8. Chimera
developed by the Resource for Biocomputing, Visualization, and
Informatics at the University of California, San Francisco.
Liver Microsomal Stability and Pharmacokinetics. Liver
metabolic stability of compounds was examined in both mouse and
human liver microsomes. Compounds were mixed with mouse or
human liver microsomes (Gentest, BD Biosciences) in 100 mM
potassium phosphate buffer (pH 7.4) and incubated at 37 °C for 5
min. Initiation of reaction was done by NADPH regeneration solution
(BD Biosciences), and termination was performed by adding ice-cold
acetonitrile (three times volume), with imipramine (80 ng/mL) as an
internal standard at single time-point 30 min. After pretreating the
biological samples with vortex and centrifuge, samples were analyzed
by LC/MS/MS system. Pharmacokinetic data was obtained by
following procedure. The candidate compound 1d, 2j, and 2l were
administered by ip injection (10 mg/kg) in ICR male mice (n = 5). 1d,
2j, and 2l were prepared as a solution (DMSO, PEG400, and distilled
water at 5:40:55, v:v:v %). Blood samples were taken at 10 and 30 min
and at 1, 4, 8, and 24 h after injection. After the centrifugation of
plasma for purification, the concentrations of candidate compound 1d,
2j, and 2l were analyzed using Agilent 6460 LC/MS/MS system
(Agilent) using electron spray ionization and a reverse-phase column
(Hypersil GOLD C18, 50 mm × 2.1 mm, Thermo Scientific).
Pharmacokinetic parameters were obtained after the analysis of plasma
concentration−time plot with WinNonlin software (Pharsight).
Mice and Cecal Ligation and Puncture (CLP) Experiments.
C57BL/6 (B6) mice were obtained from The Jackson Laboratory (Bar
Harbor, ME). All mice were bred and maintained in specific pathogen-
free conditions at the animal facility of Seoul National University
College of Medicine. All animal experiments were performed with the
approval of the Institutional Animal Care and Use Committee
(IACUC) at Seoul National University (authorization no.
SNU05050203). For CLP induction, after anesthesia, a 15 mm
midline incision was performed in the abdomen to expose the cecum.
The exposed cecum was ligated with 6−0 prolene just below the
ileocecal valve (5.0 mm from the cecal tip). The cecal stump was
punctured once with a 20-gauge needle, and small amount of stool (1
mm) was extruded. The cecum was placed back into its normal intra-
abdominal position, and the abdomen was closed with a running
suture of 6−0 prolene. Mice were treated with candidate compounds
for a daily dose of 10 mg/kg/day for 21 days by intraperitoneal
injection, starting from 1 h prior to CLP induction.
Preparation of Compounds 1a−j, 2a−o. Preparation and
characterization of all new compounds (2e, 2j, 2k, 2l, 2m, and 2o)
were described. Preparation and characterization of 1a−j, 2a−d, 2f−i,
2n, and diene-intermediate 3 were previously reported.57
Detailed Synthetic Procedure for the Preparation of 2e, 2j,
2k, 2l, 2m, and 2o. To a solution of 3 (0.9 mmol) in anhydrous
toluene (5 mL), a red-colored solution of appropriate 4-substituted-
1,2,4-triazoline-3,5-dione (1.5 equiv) in THF (5 mL) was added.
(Preparation of appropriate 4-substituted-1,2,4-triazoline-3,5-dione
was previously reported.54) The reaction mixture was stirred at
room temperature for 1 to 6 h until the complete consumption of
starting material 3. After the reaction completion monitored by TLC,
the resulting mixture was concentrated in vacuo and purified with
silica-gel flash column chromatography to provide the desired product,
TIPS-protected ICM derivative. Yields: 59%, TIPS-2e; 44%, TIPS-2j;
41%, TIPS-2k; 49%, TIPS-2l; 35%, TIPS-2m; 73%, TIPS-2o.
Finally, the resulting TIPS-protected ICM derivative (0.5 mmol)
was resolved in in 3 mL of HF/pyridine/THF (1:1:18, v/v) and
stirred for 6 h at room temperature in a plastic falcon tube. After the
reaction completion, the remaining fluoride source was quenched with
excess ethoxytrimethylsilane (6 mL). The mixture was concentrated in
vacuo, and the resultant product was purified with silica-gel flash
column chromatography with ethyl acetate/hexane or dichloro-
methane/methanol conditions to provide the desired ICM derivative
product. Yields: 79%, 2e; 80%, 2j; 65%, 2k; 93%, 2l; 92%, 2m; 93%,
2o.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00954.
Procedures for synthetic and biological experiments, and
spectroscopic data of all new compounds (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: +82-2-880-9090. Fax: +82-2-884-4025. E-mail:
sbpark@snu.ac.kr.
■
ORCID
Wansang Cho: 0000-0002-5262-9543
Seung Bum Park: 0000-0003-1753-1433
Author Contributions
#W.S. and J.Y.K. equally contributed to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Creative Research Initiative
Grant (2014R1A3A2030423) and the Bio & Medical
Technology Development Program (2012M3A9C4048780)
through the National Research Foundation of Korea (NRF)
funded by the Korean Government (Ministry of Science, ICT
& Future Planning). W.C. is grateful for the NRF-2016-
Fostering Core Leaders of the Future Basic Science Program/
Global Ph.D. Fellowship Program (2016H1A2A1908141).
J.Y.K. and S.L. are grateful for their fellowship awarded by
the BK21 Plus Program.
Purity Determination from HPLC Analysis. Reverse-phase
HPLC analysis was performed with VP-ODS C-18 column (150
mm × 4.6 mm) at a flow rate of 1.0 mL/min. This HPLC system was
equipped with a Shimadzu LC-6AD pump and a SPD-10A detector
(Japan). HPLC solvents consist of water containing 0.1% trifluoro-
acetic acid (TFA) (solvent A) and acetonitrile containing 0.1% TFA
(solvent B). Solvent A:B ratio was gradually increased, from 5:95 to
100:0 for 30 min, then 100:0 for at least 15 min. HPLC spectra were
obtained based on the absorption wavelengths of 210 and 254 nm for
the confirmation of ≥95% purity based on the area percentage. HPLC
spectra and purity values of 2e, 2j, 2k, 2l, 2m, and 2o are in the
Supporting Information.
ABBREVIATIONS USED
■
HMGB, high mobility group box protein; TNF-α, tumor
necrosis factor-alpha; IL-1β, interleukin-1-beta; IL-6, interleu-
kin-6; ERK, extracellular signal-regulated kinase; JNK, c-jun N-
H
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terminal kinase; p38, p38 MAPK; NF-κB, nuclear factor kappa-
light chain enhancer in B cells; PAMP, pathogen associated
molecular pattern; PRR, pattern recognition receptor; ICM,
inflachromene; EGCG, (−)-epigallocatechin-3-gallate; ELISA,
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