Peperomins as anti-inflammatory agents that inhibit the NF-κB signaling pathway

Article abstract of DOI:10.1016/j.bmcl.2009.06.029

The transcription factor nuclear factor κB (NF-κB) induces the expression of various inflammatory genes. In the common NF-κB signaling pathway, peperomin E and 2,6-didehydropeperomin B inhibited IκB degradation upon stimulation with TNF-α or interleukin-1

Full text of DOI:10.1016/j.bmcl.2009.06.029

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4084–4087
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peperomins as anti-inflammatory agents that inhibit the NF-
signaling pathway
jB
Chieko Tsutsui a, Yuriko Yamada a, Masayoshi Ando b,§, Daisuke Toyama c, Jian-lin Wu c,
,
Liyan Wang c,à, Shigeru Taketani a, Takao Kataoka a,
*
a Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
b Department of Chemistry and Chemical Engineering, Faculty of Engineering, Niigata University, Ikarashi 2-8050, Niigata 950-2181, Japan
c Graduate School of Science and Technology, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2009
Revised 2 June 2009
Accepted 3 June 2009
Available online 13 June 2009
The transcription factor nuclear factor
j
B (NF-
j
B) induces the expression of various inflammatory genes.
In the common NF-
radation upon stimulation with TNF-
2,6-didehydropeperomin B blocked the TNF-
effect on the I B kinase activity. Our present results clearly demonstrate that peperomins inhibit the NF-
B signaling pathway by blocking I B kinase activation.
j
B signaling pathway, peperomin E and 2,6-didehydropeperomin B inhibited I
or interleukin-1. Consistent with these results, peperomin E and
-induced activation of I B kinase, while they had no direct
jB deg-
a
a
j
j
j
j
Keywords:
Peperomin
Secolignan
Ó 2009 Elsevier Ltd. All rights reserved.
NF-jB
IjB kinase
Inflammatory cytokines, such as tumor necrosis factor-
and interleukin-1 (IL-1), play an essential role in inflammation
a
(TNF)-
I
j
B that interacts with the NF-
phosphorylated I B is ubiquitinylated and immediately hydro-
lyzed by proteasome.5–7 The NF-
B heterodimer becomes free
jB heterodimer in the cytosol, and
a
j
and induce a variety of genes responsible for inflammatory re-
sponses, such as intercellular adhesion molecule-1 (ICAM-1;
CD54).1 During inflammation, the inducible expression of ICAM-1
on the vascular endothelium is regulated mainly by inflammatory
cytokines, thereby facilitating the adhesion and subsequent trans-
migration of leukocytes.2,3 The ICAM-1 expression is predomi-
j
and translocates to the nucleus where it activates a variety of genes
responsible for inflammation as well as cancer development and
progression.8
We have isolated 13 secolignans from the extract of Peperomia
dindygulensis and investigated their SAR with focus on anti-
inflammatory and anticancer activities.9 Among these secolignans,
peperomin E (Fig. 1A)9 and its structural derivatives were found to
inhibit cell-surface ICAM-1 expression induced by inflammatory
cytokines.9 Except for its anti-inflammatory activity,9 multidrug-
resistant reversal activity,9 and anticancer activity,9,10 the biolog-
ical activities of peperomins are little understood. In this study,
we further investigated the anti-inflammatory activities of
peperomin E and its structural derivatives at the cellular and
molecular levels.
nantly upregulated by the transcription factor nuclear factor
(NF-
B).1
The NF-
including inflammatory cytokines. Upon TNF-
jB
j
j
B signaling pathway is activated by various stimuli
stimulation, TNF
a
receptor 1 recruits several adaptor proteins to form a membrane-
bound complex.4 This complex is prerequisite to activating the
Ij
B (inhibitor of
j
B) kinase (IKK) complex containing two catalytic
subunits, IKK and IKKb, and a regulatory subunit IKK
a
c
/NEMO.5,6
On the other hand, IL-1 receptor recruits different sets of adaptor
proteins to its cytoplasmic domain, leading to the activation of
the IKK complex.7 Once activated, the IKK complex phosphorylates
Peperomin A (1),9–12 peperomin B (2),9–12 and peperomin E
(3)9–11 were isolated from the dried material of P. dindygulensis
as previously described.9 As 2,6-didehydropeperomin B (4) was
not obtained from natural sources, 4 was synthesized from 2 as
shown in Scheme 1 (experimental details in Supplementary data).
The introduction of a double bond at C-2(6) position of 2 with a
known method was unsuccessful, because the C-2 position of 2 is
sterically hindered by the bulky substituent at C-3 of 2. Enolsilyla-
tion of 2 with TMSOTf and Et3N in CH2Cl2 and subsequent treat-
ment of the resulting silyl enol ether with phenylselenyl chloride
* Corresponding author. Tel./fax: +81 75 724 7752.
E-mail address: takao.kataoka@kit.ac.jp (T. Kataoka).
Present address: Department of Chemistry, Hong Kong Baptist University,
SCT1207 Hong Kong.
Present address: Department of Pharmacy Engineering, College of Chemistry and
Chemistry Engineering, Qiqihar University, 42 Wenhuadajie, Qiqihar, Heilongjiang
Sheng 161006, China.
à
§
Present address: 22-20 Keiwa-machi, Taihaku-ku, Sendai 982-0823, Japan.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.029

C. Tsutsui et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4084–4087
4085
Figure 1. Peperomin E and 2,6-didehydropeperomin B inhibit cell-surface ICAM-1 expression induced by TNF-
were pretreated with various concentrations of peperomin A (open circles), peperomin B (open squares), peperomin E (filled circles), and 2,6-didehydropeperomin B (filled
squares) for 1 h, and then incubated with TNF- (2.5 ng/mL) (B) or IL-1 (0.25 ng/mL) (C) for 6 h in the presence or absence of peperomins. Cell-surface ICAM-1 expression was
a or IL-1. (A) Structures of peperomins. (B and C) A549 cells
a
measured by the Cell ELISA assay. Data points represent means SD of triplicate cultures. (D) A549 cells were incubated with various concentrations of peperomin A (open
circles), peperomin B (open squares), peperomin E (filled circles), and 2,6-didehydropeperomin B (filled squares) for 6 h. Cell viability (%) was measured by the MTT assay.
Data points represent means SD of triplicate cultures.
Scheme 1. Synthesis of 2,6-didehydropeperomin B.
afforded a mixture of stereoisomers of phenylseleno lactones, the
HPLC separation of which gave -isomer 5 and b-isomer 6 in 17%
eromin E and 2,6-didehydropeperomin B also inhibited the cell-
surface expression induced by TNF- or IL-1 as strongly as in
a
a
and 47% yields, respectively. The structures of 5 and 6 were con-
firmed by the analyses of spectral data, including 1H- and 13C-
NMR spectra and HREIMS. The oxidative syn elimination of 5 with
A549 cells, while peperomin A and peperomin B exerted approxi-
mately 10-fold weaker inhibitory effects (Supplementary Fig. 1A
and B). Therefore, it seems that the a-methylene c-lactone moiety
26% H2O2 gave undesired endo-unsaturated
yield. On the contrary, the oxidative syn elimination of 6 with
26% H2O2 gave desired 4 in 97%.
Peperomin E and 2,6-didehydropeperomin B are structural ana-
logues of peperomin A and peperomin B in which
c
-lactone 7 in 90%
in peperomin E and 2,6-didehydropeperomin B is important for
their inhibitory activities. Indeed, the inhibitory concentration of
peperomin E and 2,6-didehydropeperomin B on the IL-1-induced
ICAM-1 expression was comparable with that of santonin-related
a-methylene
c-
compound 2 harboring the a-methylene-c
-lactone moiety.14 As
lactone groups are replaced by -methyl -lactone groups, respec-
a
c
judged by the MTT assay,13 while peperomin E and 2,6-didehydro-
peperomin B did not reduce cell viability of MCF7 cells up to
tively (Fig. 1A). Human lung carcinoma A549 cells were pretreated
with various concentrations of peperomins for 1 h and then incu-
100 lM (Supplementary Fig. 1C), they partially decreased cell via-
bated with TNF-
a
or IL-1 for 6 h in the presence of peperomins.
bility of A549 cells (Fig. 1D). Therefore, peperomin E and 2,6-dide-
Cell-surface expression of ICAM-1 was measured by the Cell ELISA
hydropeperomin B may exert some nonspecific cytotoxic effects at
concentrations higher than 100 lM.
assay.13 Peperomin A and peperomin B did not reduce cell-surface
ICAM-1 expression up to a concentration of 100
By contrast, peperomin E and 2,6-didehydropeperomin B inhibited
the cell-surface ICAM-1 expression induced by TNF- or IL-1 in a
l
M (Fig. 1B and C).
It has been shown that ICAM-1 expression is highly NF-jB-
dependent in A549 cells.15 The amounts of cell-surface and
intracellular proteins expressed in A549 cells were measured by
Western blotting.16 The expression of ICAM-1 was barely detect-
a
dose-dependent manner and at almost equivalent concentrations
(Fig. 1B and C). In human breast adenocarcinoma MCF7 cells, pep-
able in unstimulated A549 cells, while stimulation with TNF-
a or

4086
C. Tsutsui et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4084–4087
IL-1 induced a marked increase in ICAM-1 expression. Peperomin E
and 2,6-didehydropeperomin B substantially decreased ICAM-1
(Fig. 3 and Supplementary Fig. 3). Time-course experiments re-
vealed that I is phosphorylated within 5 min following stimu-
lation with TNF- or IL-1, and then degraded to barely detectable
levels within 10 min. Peperomin E prevented I phosphoryla-
tion as well as its subsequent degradation upon stimulation with
TNF- or IL-1 (Fig. 3 and Supplementary Fig. 4). The expression
of two IKK catalytic subunits, IKK and IKKb, was not obviously
jBa
expression at concentrations higher than 10
pletely decreased the expression at 100 M, whereas peperomin
A and peperomin B were inactive up to 100 M (Fig. 2 and Supple-
mentary Fig. 2). It is known that cyclooxygenase-2 (Cox-2) expres-
sion is induced by inflammatory cytokines in an NF- B-dependent
manner.8 The expression of Cox-2 was markedly increased when
A549 cells were stimulated with TNF- or IL-1 (Fig. 2). Likewise,
lM and almost com-
a
l
jBa
l
a
j
a
influenced by peperomin E during TNF-
a
and IL-1 stimulation
a
(Fig. 3 and Supplementary Fig. 4).
peperomin E and 2,6-didehydropeperomin B, but neither pepero-
min A nor peperomin B, reduced the expression of Cox-2 to back-
The IKK complex directly phosphorylates IjBa at N-terminal
two serine residues (Ser-32 and Ser-36).6 The IKK complex was
pulled down from cell lysates by immunoprecipitation using
ground levels at 100
results indicate that peperomin E and 2,6-didehydropeperomin B
generally inhibit NF- B-dependent gene expression induced by
TNF- or IL-1. It should be noted that the inhibitory effects of pep-
lM (Fig. 2 and Supplementary Fig. 2). These
anti-IKKa antibody and its kinase activity toward IjBa (1–54)
j
fused to glutathione S-transferase (GST) was measured as de-
scribed in supplementary data. A549 cells were pretreated with
a
eromin E and 2,6-didehydropeperomin B on ICAM-1 expression are
approximately 10-fold stronger than those on Cox-2 expression.
Since ICAM-1 is a cell-surface glycoprotein, it might be possible
that these compounds influence the ICAM-1 processing and/or its
transport from the endoplasmic reticulum to the cell-surface.
peperomins or not pretreated for 1 h, and stimulated with TNF-
for 15 min in the presence or absence of peperomins. The IKK com-
plex exhibited strong activity toward GST-I (1–54) in an ATP-
dependent manner, as well as only when A549 cells were stimu-
lated with TNF- (Fig. 4A and Supplementary Fig. 5A). The IKK
activity was completely blocked when A549 cells were pretreated
with peperomin E or 2,6-didehydropeperomin B prior to TNF-
a
jBa
a
TNF-
phorylated I
tion.5–7 A549 cells constitutively expressed I
with TNF- or IL-1 induced rapid I degradation. Peperomin E
and 2,6-didehydropeperomin B, but neither peperomin A nor pep-
eromin B, inhibited the I degradation induced by TNF- or IL-1
a
and IL-1 induce rapid phosphorylation of I
immediately undergoes proteasomal degrada-
, and stimulation
jBa, and phos-
jBa
a
jBa
stimulation (Fig. 4A and Supplementary Fig. 5A). It seems that pep-
eromin E and 2,6-didehydropeperomin B do not primarily disrupt
a
jBa
the interaction of IKKb with IKKa, as IKKb was co-immunoprecip-
jBa
a
itated with IKK even in the presence of peperomin E and 2,6-dide-
a
Figure 2. Peperomin E inhibits expression of ICAM-1 and Cox-2 upon stimulation with TNF-
or peperomin E for 1 h, and then incubated with (+) or without (À) TNF- (2.5 ng/mL) (A) or IL-1 (0.25 ng/mL) (B) for 6 h in the presence (+) or absence (À) of peperomin A or
peperomin E. Expression of ICAM-1, Cox-2, and b-actin was analyzed by Western blotting.
a or IL-1. A549 cells were pretreated with various concentrations of peperomin A
a
Figure 3. Peperomin E inhibits I
concentrations of peperomin A or peperomin E for 1 h, and then incubated with (+) or without (À) TNF-
(+) or absence (À) of peperomin A or peperomin E. I and b-actin expression was analyzed by Western blotting. (C and D) A549 cells were pretreated with peperomin E
(100 (2.5 ng/mL) (C) or IL-1 (0.25 ng/mL) (D) for the indicated times in the presence (+) or
M) or not pretreated for 1 h, and then incubated with (+) or without (À) TNF-
absence (À) of peperomin E. The expression of I , phospho-I , IKK , IKKb, and b-actin was analyzed by Western blotting. The amounts of protein bands (C and D) were
quantified by the ImageJ image processing program, and relative intensity compared with b-actin was shown in Supplementary Figure 4A and B, respectively.
jB
a
phosphorylation and its subsequent degradation upon stimulation with TNF-a or IL-1. (A and B) A549 cells were pretreated with various
a
(2.5 ng/mL) (A) or IL-1 (0.25 ng/mL) (B) for 15 min in the presence
jBa
l
a
jB
a
jB
a
a

C. Tsutsui et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4084–4087
4087
as cysteine residues, of proteins via the Michael addition reaction.
IKK and IKKb have a cysteine residue (Cys-179) within their acti-
vation loop.17,19 15-Deoxy- 12,14-PGJ2 as well as 2-cyano-3,12-
a
D
dioxooleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me)
or 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole
(CDDO-Im) contains an
a,b-unsaturated carbonyl moiety that can
form reversible adducts with reactive thiol groups. It has been
shown that 15-deoxy-D
12,14-PGJ2, CDDO-Me, and CDDO-Im inhibit
IKKb activity through direct modification of Cys-179.17,18,20,21 Sim-
ilar to these compounds, the sesquiterpene lactone parthenolide
contains an
a-methylene c-lactone group and inhibits IKKb by
covalently binding to Cys-179.22 The Cys-179 residue was also
shown to be essential for the inactivation of IKKb in the case of
arsenite and a gold compound, auranofin.19,23 However, in contrast
to these previous studies, under the experimental conditions in
which 15-deoxy-D
12,14-PGJ2 directly inhibited IKKb activity, pep-
eromin E and 2,6-didehydropeperomin B were unable to inhibit
the IKKb activity. Therefore, it seems likely that peperomin E and
2,6-didehydropeperomin B do not primarily block IKKb activity
via the modification of Cys-179, but may target other protein(s) re-
quired for IKK activation.
Acknowledgments
We are grateful to Dr. J. Tschopp for the gift of the IKKb expres-
sion vector. This work was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT), Japan.
Supplementary data
Figure 4. Peperomin
E and 2,6-didehydropeperomin B inhibit TNF-a-induced
activation of the IKK complex. (A) A549 cells were pretreated with various
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.029.
concentrations of peperomins for 1 h, and then incubated with (+) or without (À)
TNF-a (2.5 ng/mL) for 15 min in the presence of peperomins. The IKK complex was
immunoprecipitated with anti-IKK
tates were used for the kinase reaction using GST-I
presence (+) or absence (À) of ATP (10 M). Phospho-GST-I
by Western blotting using anti-phospho-I B antibody. The amounts of IKK
IKKb in the immunoprecipitates were determined by Western blotting. The
amounts of protein bands were quantified, and relative intensity of phospho-I
(1–54) and IKKb compared with IKK was shown in Supplementary Figure 5A. (B)
a
antibody and the resultant immunoprecipi-
(1–54) as substrate in the
(1–54) was detected
and
jBa
References and notes
l
jBa
j
a
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deoxy-
Phospho-GST-I
B antibody. The amounts of FLAG-IKKb in the reaction mixtures were determined
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12,14-PGJ2 (50
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Ij
intensity of phospho-GST-I
Supplementary Figure 5B.
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11. Govindachari, T. R.; Kumari, G. N. K.; Partho, P. D. Phytochemistry 1998, 49,
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hydropeperomin B (Fig. 4A and Supplementary Fig. 5A). IKKb is the
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B signaling pathway.5,6 To obtain active IKKb, FLAG-IKKb was
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body. It has been shown that 15-deoxy-
(15-deoxy-
12,14-PGJ2) directly inhibits IKKb activity.17,18 Consis-
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12,14-PGJ2 strongly inhib-
D
12,14-prostaglandin J2
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D
ited IKKb activity (Fig. 4B and Supplementary Fig. 5B). However,
under the same experimental conditions, peperomin E and 2,6-
didehydropeperomin B had no effect on IKKb activity (Fig. 4B and
Supplementary Fig. 5B). These data indicate that peperomin E
and 2,6-didehydropeperomin B inhibit IKK complex activation in
a manner distinct from 15-deoxy-
Studies of SAR revealed that the
is important for peperomin E and 2,6-didehydropeperomin B to ex-
hibit the inhibitory activity. It is known that the -methylene
lactone moiety can bind covalently to reactive thiol residues, such
D
a
12,14-PGJ2.17,18
-methylene c-lactone moiety
a
c-