1. DOI: 10.1002/cbic.201100635
The Myxobacterial Compounds Spirangien A and
Spirangien M522 are Potent Inhibitors of IL-8 Expression
Marc RenØ Reboll,[a]
Birgit Ritter,[b]
Florenz Sasse,[a]
Jutta Niggemann,[c]
Ronald Frank,[a]
and
Mahtab Nourbakhsh*[d]
Introduction
Initially characterized for its chemotactic activity, interleukin-8
(IL-8) is now known to possess tumorigenic and proangiogenic
properties as well.[1]
This chemokine is secreted by a variety of
cell types, including monocytes/macrophages, Tcells, neutro-
phils, fibroblasts, endothelial cells, and various tumor cell lines,
in response to inflammatory stimuli such as interleukin-1 (IL-
1).[2]
Numerous studies indicate the crucial roles of IL-8 in vari-
ous pathological conditions, such as chronic inflammation,
cancer, and metastasis, and suggest IL-8 as a novel therapeutic
target.[1c,3]
IL-1 increases the level of IL-8 mRNA in cells, and IL-8 protein
synthesis and secretion are both directly related to the cellular
levels of IL-8 mRNA.[4]
IL-8 mRNA levels are maintained at a
steady state by the transcriptional rate of the IL-8 gene and
the IL-8 mRNA half-life.[5]
IL-8 gene transcription is regulated
by its 5’-flanking region, which contains binding sites for tran-
scriptional modulators such as activator protein 1 (AP-1), nucle-
ar factor kB (NF-kB), and CCAAT enhancer binding protein/nu-
clear factor for interleukin-6 (C-EBP/NF-IL-6).[5b,6]
NF-kB binding
is required for transcriptional activity at the IL-8 promoter in all
cell types, whereas AP-1 and C-EBP/NF-IL-6 binding contribute
to transcriptional activity in a cell-dependent manner.[5a]
One of the critical regulators of IL-1-induced NF-kB activity
is the phosphorylation status of IkB, its natural cytosolic inhibi-
tor. Phosphorylation of two adjacent serines on IkBa by IkB
kinase-a (IKK-a) and IKK-b leads to proteasomal degradation of
IkBa and to release of the p50/p65 heterodimeric NF-kB into
the nucleus to initiate transcription.[7]
IKK-a and -b are activat-
ed as a result of their phosphorylation by MAP kinase kinase 1
(MEKK1), which strongly enhances transcriptional activity at
the IL-8 promoter.[8]
IL-8 mRNA stability is also influenced by
MEKK1, MAP kinase kinase 6 (MKK6), and p38 MAP kinase.[9]
Thus, activation of NF-kB, stress-activated protein kinase c-Jun
N-terminal kinase (JNK), and p38 MAP kinases all contribute to
increases in IL-8 mRNA levels, either through increased tran-
scription rates or through increased transcript stability. MEKK1
activates these three signaling mediators and likely induces
the maximal IL-8 levels.
Humanized monoclonal antibodies against IL-8, as well as
small chemical antagonists to IL-8 receptors, have previously
been developed and are now in clinical trials conducted by
several companies.[10]
To reduce side effects in future therapeu-
tic treatments, there is a need for products that could be com-
bined in multitherapy treatments and optimized for individual
patients, depending on their particular signaling pathways and
metabolism. By using a highly specific cell-based assay for
screening compound libraries, our study identified spirangiens
Elevated expression of interleukin-8 (IL-8) has been implicated
in inflammatory diseases, in tumor growth, and in angiogene-
sis. The aim of this study was to identify natural or synthetic
compounds that suppress IL-8 production in response to inter-
leukin-1 (IL-1), the natural inflammatory stimulus of the IL-8
gene. We therefore developed an IL-1-inducible cell-based
screening assay by stable integration of an IL-8 reporter gene
into HeLa S3 cells. The screening of heterogeneous compound
libraries revealed several compounds that displayed an inhibi-
tory effect on the reporter gene expression. Following hit vali-
dation, we focused on the most efficient compound, spiran-
gien A, and its chemical derivate spirangien M522. Detailed
analysis shows that both compounds are potent inhibitors of
the endogenous IL-8 gene transcription. Furthermore, both
compounds decelerate the phosphorylation and degradation
of IkBa, the key regulator of the IL-1-stimulated NF-kB signal-
ing pathway. Our study has identified the two spirangiens A
and M522 as potent inhibitors of IL-1/NF-kB-mediated IL-8
gene expression.
[a] Dr. M. R. Reboll, Dr. F. Sasse, Dr. R. Frank
Department of Chemical Biology
Helmholtz Centre for Infection Research
38124 Braunschweig (Germany)
[b] B. Ritter
Institute of Virology, Hannover Medical School
30625 Hannover (Germany)
[c] Dr. J. Niggemann
Department of Medicinal Chemistry
Helmholtz Centre for Infection Research
38124 Braunschweig (Germany)
[d] Dr. M. Nourbakhsh
Collaborative Research Centre CRC 566
Hannover Medical School
Carl-Neuberg-Strasse 1/OE 6790, 30625 Hannover (Germany)
E-mail: mnourbakhsh@hotmail.com
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100635.
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2. A and M522 as inhibitors of IL-8 promoter activity. Subsequent
experiments characterized the effects of spirangiens A and
M522 on cellular signaling pathways and IL-8 gene transcrip-
tion.
Results
IL-8 reporter cell line
We first established a stable reporter cell line to monitor the
IL-8 gene activity throughout compound screening experi-
ments. Previous studies had demonstrated that the steady-
state level of IL-8 mRNA in cells is primarily defined by the
transcriptional rate at the IL-8 promoter. Therefore, we used a
reporter gene construct (pIL-8 Lucdel) that contains the firefly
luciferase reporter gene under the control of the IL-8 promoter
(À302 to À1) encompassing binding sites for AP-1, NF-kB, and
C-EBP/NF-IL-6 proteins (Figure 1A).[5a,11]
The reporter gene con-
struct was transfected into HeLa S3 cells together with a puro-
mycin-selection construct.[12]
Following selection, single, resist-
ant clones were tested for reporter gene activity, response to
IL-1 (Figure 1), and the number of integrated copies of the re-
porter gene construct (Figure S1A in the Supporting Informa-
tion). A single clone was selected; it showed the highest re-
porter gene response but only contained a single copy of the
reporter construct that included the full-length reporter region
(HeLa S3 IL-8 Luc). We next performed Northern blot analysis
to compare the time course of the reporter mRNA and the en-
dogenous IL-8 mRNA expression. As shown in Figure 1B, firefly
luciferase and IL-8 mRNAs were induced upon IL-1 stimulation
in the reporter cell line. The highest levels of both mRNAs
were achieved within 2 h. However, the firefly luciferase mRNA
was detectable for a longer time (up to 3.5 h) than the endog-
enous IL-8 mRNA. For compound screening, the IL-8 reporter
assay was implemented in a 96-well cell culture plate format.
IL-1-stimulated cells showed a significant increase in firefly luci-
ferase relative to unstimulated cells (Figure S1B).
Compound screening and hit validation
For screening, HeLa S3 IL-8 Luc cells were stimulated with IL-1
and simultaneously treated with 1900 compounds from the
Helmholtz Center for Infection Research (HZI) compound libra-
ries, which include a collection of myxobacterial metabolites[13]
and a highly diverse collection of natural and synthetic com-
pounds. Cells were harvested 16 h after treatment, and the
levels of luminescence in cellular extracts were determined im-
mediately. Of the 1900 compounds, only those that led to at
least 70% decrease in luciferase expression were considered as
hits (Figure S2). Of the six identified hits, spirangien A was the
most potent inhibitor of firefly luciferase reporter expression.
Spirangien A is a spiroacetal secondary metabolite derived
from the myxobacterium Sorangium cellulosum (strain
So ce90).[14]
We note that no other compounds containing sim-
ilar scaffolds are included in the libraries tested here. In a previ-
ous study, a chemical derivate including the spiroacetal core of
spirangien A was prepared and designated by its molecular
mass as spirangien M522.[15]
The chemical structures of spiran-
gien A and its derivate are shown in Scheme 1. The effects of
both compounds were also tested in unstimulated and IL-1-
stimulated cells; these data show that both compounds effi-
ciently reduced the luciferase expression in IL-1-stimulated
cells to the level found in unstimulated cells (Figure 2). More
importantly, the compounds showed no inhibitory effect on
the low levels of firefly luciferase in unstimulated cells; thus,
both compounds specifically inhibited IL-1-induced reporter
gene expression.
We performed additional reporter gene experiments with a
wide range of different concentrations of spirangiens A and
M522 to characterize their effects (Figure 3A). HeLa S3 IL-8 Luc
cells were stimulated with IL-1 and simultaneously treated
with spirangiens A and M522 at concentrations ranging from
0.1 nm to 10 mm. The calculated IC50 values indicate that spiran-
gien A is a highly potent inhibitor (IC50 =7.2Æ0.9 nm). Spiran-
gien M522 is less potent, but still has an IC50 value of about
90.7 nm (IC50 =90.7Æ19.8 nm).
Next, cell viability assays were performed to monitor possi-
ble cytotoxic effects of spirangiens A or M522 that might cause
decreases in reporter gene expression (Figure 3B). HeLa S3 IL-
8 Luc cells were treated with different concentrations of the
compounds for 16 h, based on the incubation times in the re-
porter gene assays. These data show that the viabilities of the
cells were not affected by spirangiens A or M522 or by their
solvent (methanol) over the entire range of concentrations, so
Figure 1. Construction of a stable IL-8 reporter cell line. A) Schematic dia-
gram of the stable transfected reporter gene construct. The IL-8 reporter
gene construction is described in detail in the Experimental Section. The
construct contains a truncated human IL-8 promoter from positions À302 to
À1. Several transcription factor binding sites are indicated within this pro-
moter section (AP-1, C/EBP, p65). After a short linker sequence of 24 nucleo-
tides, the firefly luciferase coding sequence was inserted. An EcoRI site was
used to detect single integration clones in Southern blots (Figure S1) and is
indicated in the luciferase sequence. B) Induction of IL-8 and firefly luciferase
mRNAs due to IL-1 stimulation. Stably transfected HeLa S3 IL-8 Luc cells
were treated with IL-1 for the indicated times, and subsequent Northern
blot analysis was performed with isolated total RNA (20 mg). Firefly lucifer-
ase, IL-8, and TFR (control) mRNAs were detected by using radioactively la-
beled firefly luciferase, IL-8 and TFR probes, respectively, followed by auto-
radiography. Firefly luciferase, IL-8, and TFR mRNAs are indicated by arrows
on the left. The data are representative for three independent experiments.
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M. Nourbakhsh et al.

3. the crucial inhibitory effects of spirangiens A and M522 on IL-8
reporter gene expression were not based on general effects on
cell viability.
Dose-dependent reduction of IL-8 expression by spirangiens
A and M522
The inhibitory effects of spirangiens A and M522 on IL-8 re-
porter gene expression in Figures 2 and 3A imply equal inhibi-
tion of endogenous IL-8 expression in HeLa S3 IL-8 Luc cells. To
confirm this suggestion, HeLa S3 IL-8 Luc cells were stimulated
with IL-1 and treated with a range of different concentrations
of spirangiens A and M522 for 16 h. Subsequently, levels of IL-
8 protein in the supernatants and IL-8 mRNA from cellular ex-
tracts were quantitated (Figure 4). Similarly to the IL-8 reporter
gene experiments, spirangiens A and M522 decreased the IL-8
protein levels in the supernatants of HeLa S3 IL-8 Luc cells in a
dose-dependent manner (Figure 4A). The calculated IC50 values
(3.6Æ0.7 and 41.7Æ5.1 nm, respectively) were almost half
those obtained in the IL-8 reporter gene experiments in Fig-
ure 3A. Moreover, we performed real-time PCR experiments to
determine whether the significant reductions in IL-8 protein
were based on comparable declines in IL-8 mRNA levels. The
relative IL-8 mRNA levels were measured at compound con-
centrations near the estimated IC50 values. IL-1 stimulation led
to a strong increase in the relative IL-8 mRNA levels in HeLa S3
IL-8 Luc cells. Spirangiens A and M522 efficiently reduced the
high levels of IL-8 mRNA in a dose-dependent manner (Fig-
ure 4B). The inhibitory effect of spirangien A on IL-8 mRNA
Scheme 1. The spirangien compounds. The chemical structures of the
spiroketal compound spirangien A and of the truncated derivate spiran-
gien M522, containing the chemical core structure of spirangien A, are
shown.
Figure 2. Effects of spirangiens on reporter gene expression. HeLa S3 IL-
8 Luc cells were stimulated with IL-1 and treated with screening concentra-
tions (1.6 mm) of spirangiens A or M522 for 16 h. Cells were lysed and firefly
luciferase activities were measured. Firefly luciferase activities are shown
here as the meanÆSEM of three independent experiments. The firefly luci-
ferase activities for IL-1-stimulated cells were set as 100% in each experi-
ment.
Figure 3. Validation of the hit compounds spirangien A and spirangien
M522. A) Spirangiens A (*) and M522 (*) efficiently reduce luciferase expres-
sion in a dose-dependent manner. HeLa S3 IL-8 Luc cells were simultaneous-
ly treated with IL-1 and the compounds for 16 h. Following cell lysis, firefly
luciferase activities were measured in the lysates. Firefly luciferase activities
are shown here as the meanÆSEM of at least three independent experi-
ments and were set as 100% for IL-1-stimulated cells. B) The effects of spi-
rangiens A (*) and M522 (*) on cell viability were monitored by an MTT
assay. HeLa S3 IL-8 Luc cells were treated with both compounds and metha-
nol (solvent control, !) for 16 h. The absorbances at 595 nm were deter-
mined and are presented as the meanÆSEM of three independent experi-
ments. The absorbance of untreated cells was set as 100%.
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Inhibitors of IL-8 Expression

4. and protein levels occurred at similar concentrations of the
compound, but spirangien M522 inhibited IL-8 mRNA and pro-
tein levels at significantly different concentrations, of approxi-
mately 810 and 41.7 nm, respectively. This might be indicative
of disparate mechanisms of IL-8 inhibition by spirangien A and
by spirangien M522.
Spirangiens A and M522 lead to delayed phosphorylation
and degradation of IkBa
Activation of IL-8 gene expression involves several intracellular
signaling pathways. The substantial inhibition of IL-8 expres-
sion by spirangiens A and M522 led us to investigate the ef-
fects of these compounds on the activation of signal proteins
that result from IL-8 gene induction. HeLa S3 IL-8 Luc cells
were therefore treated with spirangiens A or M522 for 16 h,
stimulated with IL-1, and subjected to Western blot analysis to
examine an important inhibitor of NFkB proteins: IkBa, which
is one of the downstream targets of the IKK complex. Follow-
ing phosphorylation by IKK complex, IkBa has been shown to
be rapidly degraded in proteasomes.[7]
The phosphorylation
and degradation of IkBa were shown by Western blot analysis
to occur within 30 min of IL-1 stimulation (Figure 5). These ex-
periments were performed at a constant concentration of the
compounds known to result in strong inhibition of IL-8 expres-
sion (Figure 4). The antibody against IkBa phosphorylated at
Ser32 indicates that IkBa was phosphorylated within 5 min of
IL-1 stimulation. The degradation of IkBa was clearly detecta-
ble after 15 min of IL-1 stimulation by an antibody against
IkBa. Notably, spirangien A slowed the time course of phos-
phorylation and degradation (Figure 5A, 15 min). These results
were substantiated by densitometric analyses of several inde-
pendent experiments (Figure S3A). These same experimental
methods were used to measure the effects of spirangien M522
on the phosphorylation and degradation of IkBa. Albeit at a
significantly higher final concentration than spirangien A, spi-
rangien M522 exerts almost identical inhibitory effects on IkBa
phosphorylation and degradation (Figure 5B and Figure S3B).
To verify the specific effects of spirangiens A and M522, we
also analyzed the phosphorylation of a number of related sig-
naling proteins and transcription factors. However, neither
compound showed detectable effects on the phosphorylation
of TAK1, IKKa/b, JNK, p38, p65, or c-Jun, which is a vital part of
Figure 4. Effect of spirangien compounds on endogenous IL-8 expression.
A) HeLa S3 IL-8 Luc cells were treated with IL-1 as well as with spirangiens A
(*) or M522 (*) for 16 h. The concentration-dependent reductions in IL-8
protein levels in the cell supernatants were monitored by use of an IL-8-spe-
cific ELISA. IL-8 protein levels are shown as the meanÆSEM of three inde-
pendent experiments. IL-8 protein levels for IL-1-stimulated cells were set
as 100% in each experiment. B) Spirangiens A () and M522 () efficiently
reduce IL-8 mRNA levels. HeLa S3 IL-8 Luc cells were treated with the indi-
cated concentrations of spirangien A or M522 for 16 h and stimulated with
IL-1 for 2.5 h. Total RNA was isolated and subjected to an RT reaction and
real-time PCR. Relative IL-8 mRNA levels were calculated as percentages of
the control mRNA (GAPDH). The relative IL-8 mRNA levels are presented as
the meanÆSEM of at least three independent experiments. The relative IL-8
mRNA level for IL-1-stimulated cells was set as 100% in each experiment.
Figure 5. Spirangiens A and M522 cause delayed phosphorylation and deg-
radation of IkBa. A) HeLa S3 IL-8 Luc cells were treated with spirangien A
(195 nm) for 16 h and stimulated with IL-1 for the times indicated. Subse-
quently, cellular protein samples were subjected to Western blot analysis
with antibodies against IkBa Ser32 or IkBa and a rabbit polyclonal antibody
against GAPDH as a control. IkBa Ser32, IkBa, and GAPDH bands are indicat-
ed on the left. The data are representative for three independent experi-
ments. B) HeLa S3 IL-8 Luc cells were treated with spirangien M522 (1.5 mm)
for 16 h and stimulated with IL-1 for the times indicated. The experimental
procedure is the same as described in (A). The data are representative for
three independent experiments.
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M. Nourbakhsh et al.

5. the AP-1 complex (Figure S4A and B). These results were con-
firmed at a range of different concentrations upon 30 min of
IL-1 stimulation (Figure S3A and B).
In summary, our study has identified the natural compound
spirangien A and its truncated variant spirangien M522 as
highly efficient inhibitors of IL-8 expression. Both compounds
decrease the activation of IL-8 promoter by slowing the phos-
phorylation and degradation of IkBa and thereby interfering
with the NFkB signaling pathway. Thus spirangiens A and
M522 could serve as anti-inflammatory drug candidates.
Discussion
There is strong evidence that elevated IL-8 levels contribute to
a number of inflammatory diseases and tumor growth.[1d,16]
It
is important to consider new therapeutic approaches to antag-
onizing IL-8 activity in the clinical setting. This study has identi-
fied myxobacterial spirangien A and its derivate M522 as
potent inhibitors of IL-8 expression. The spirangiens, originally
described in 1993,[14]
possess more than ten stereocentres with
a spiroketal core structure. In addition to antifungal activity in
agar fusion assays, spirangien A has been found to inhibit the
proliferation of mouse fibroblasts following prolonged treat-
ment over five days.[15]
However, the effects of spirangiens A
and M522 on cellular signaling pathways and gene expression
have not yet been studied. Our current data demonstrate that
spirangiens A and M522 interfered with IL-1-mediated signal-
ing pathways and inhibited the transcriptional activity of IL-8
gene. Other than their specific impact on gene regulation, the
compounds did not show any acute cytotoxic effects on HeLa
cells.
The use of HeLa S3 IL-8 Luc cells enabled us to identify spi-
rangiens A and M522 from a diverse compound library (Fig-
ure S2). This stable cell line was carefully designed to reduce
deviations in the screening experiments. Furthermore, we
excluded cells harboring multiple copies of the reporter that
might lower the response to the test compounds and cause
false negative signals. In general, the ability to identify hits in
screening experiments is largely dependent on the quality of
the reporter assay setup. A coefficient, called the Z’ factor, had
previously been defined to evaluate the quality of an assay.[17]
The Z’ factor is a dimensionless value that represents the varia-
bility and the dynamic range between two data sets, which
here were IL-1-stimulated and untreated control cells. For our
cell-based screening, we calculated a Z’ factor of 0.51Æ0.04,
which indicates a high-quality assay with a wide separation be-
tween signal and background and low data variability. More
importantly, the reporter gene and the endogenous IL-8 gene
were equally inducible by IL-1 and were correspondingly inhib-
ited by two closely related compounds: spirangiens A and
M522.
Despite their structural and functional similarities, we ob-
tained significantly higher IC50 values for spirangien M522, thus
indicating a higher potency of spirangien A. Unlike M522, spi-
rangien A contains a pentane side chain with a terminal car-
boxylic acid group, which can have a rather destabilizing effect
on its chemical structure. The higher potency of spirangien A
in IL-8 inhibition is therefore unlikely to be based on a higher
stability than M522, and so we assume that the higher potency
of spirangien A in IL-8 inhibition might be due to a more effi-
cient uptake by the cells and/or a higher binding affinity to
the target molecule than in the case of spirangien M522. Inter-
estingly, unlike spirangien A, spirangien M522 showed differen-
tial potencies in inhibition of IL-8 protein and IL-8 mRNA ex-
pression. An concentration of spirangien M522 approximately
20 times higher was required to exert an inhibition ratio com-
parable with that achieved with spirangien A on IL-8 mRNA
levels. This difference might hint at a unique mechanism of
action for spirangien M522, which would need to be confirmed
experimentally.
Here, we report several interesting observations based on
the evaluation of IL-1-activated signaling pathways in the pres-
ence of spirangiens A and M522 (Figure 6). Firstly, there was a
significant delay in IkBa phosphorylation and degradation in
the presence of the compounds. Among numerous inhibitors
of the NF-kB pathway (http://www.bu.edu/nf-kb/physiological-
mediators/inhibitors), a similar effect was previously observed
for the IKKb inhibitor SC-514;[18]
however, in contrast to the spi-
rangiens, SC-514 was also shown to inhibit the phosphoryla-
tion and translocation of p65 into the nucleus.[18]
SC-514 and
the spirangiens therefore likely target disparate regulatory
mechanisms. Secondly, the compounds did not inhibit the
phosphorylation and activation of TAK1 or the IKK complex,
suggesting that spirangiens A and M522 inhibit the activity of
the IKK complex without affecting the phosphorylation of IKKs.
Thirdly, neither c-Jun nor p65 phosphorylation were affected
Figure 6. A simplified overview of IL-1-dependent IL-8 gene expression. The
signal transduction cascades triggered by stimulation of the IL-1 receptor
are shown.
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Inhibitors of IL-8 Expression

6. by spirangiens A or M522, so spirangiens A and M522 can ef-
fectively reduce the level of IL-8 gene expression without influ-
encing p65 Ser536 or c-Jun phosphorylation.
Finally, spirangiens A and M522 showed a greater impact on
the level of phosphorylated IkBa than on the total amount of
IkBa protein (Figure S3A and B). It is conceivable that phos-
phorylation of IkBa contributes to IL-8 gene expression by reg-
ulatory mechanisms other than the regulation of IkBa protein
levels by degradation. Indeed, a number of previous studies
led to similar conclusions and suggested disparate regulatory
functions of the phosphorylated IkBa protein in gene activa-
tion.[19]
The two spirangiens A and M522 act as potent inhibitors of
IL-1-stimulated IL-8 gene expression at relatively low concen-
trations without obvious cytotoxic effects. Although both com-
pounds selectively slow the IkBa phosphorylation at Ser32,
many additional effects might also contribute to the inhibition
of the IL-8 gene. Besides a possible effect of spirangiens A and
M522 on still unknown regulatory mechanisms, slower degra-
dation of IkBa can disturb the orchestrated recruitment of
transcription factors to the IL-8 promoter, which is crucial for
the effective transcription initiation. In general, IKK signaling
has been recognized as a promising therapeutic target for
combating chronic inflammatory diseases.[20]
Besides consider-
ation as potential therapeutics, spirangiens A and M522 might
also be useful tools to elucidate the regulatory function of
IkBa phosphorylation in future studies.
Experimental Section
Plasmid constructions: The reporter gene vector pIL8-Lucdel was
derived from the IL8-LUC reporter plasmid[21]
and contains the
À302/+40 bp region (corresponding to nucleotides 1184 to 1526
of GenBank accession number M28130) of the human IL-8 gene
driving the firefly luciferase coding region. The puromycin selection
plasmid pSV2PAC was described previously.[12,22]
Cell lines and transfection: HeLa S3 (CCL-2.2, LGC) cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM) with
fetal calf serum (FCS; 10%). The cells were cotransfected with the
pIL8-Lucdel reporter vector and the puromycin selection plasmid
pSV2PAC with use of calcium phosphate coprecipitation[23]
to
create the stable cell line HeLa S3 IL-8 Luc. Following introduction
of the DNA plasmids, cells were selected with puromycin, and sev-
eral independent clones were cultured. HeLa S3 IL-8 Luc cells were
maintained in DMEM with FCS (10%) and puromycin (0.4 mgmLÀ1
).
For IL-1 stimulation, cells were treated with IL-1b (10 ngmLÀ1
) for
the times indicated.
Screening procedure: A cell-based assay to screen for IL-8-inhibit-
ing compounds was established with the HeLa S3 IL-8 Luc cells.
Cells were seeded in 96-well plates (Nunc) in DMEM (100 mL per
well) containing FCS (10%). After 24 h of incubation, the cells were
stimulated with IL-1 and incubated with compounds (160 nL) from
the HZI compound libraries for 16 h. Following cell lysis (1” pas-
sive lysis buffer, Promega), cell lysates were transferred to white
96-well plates (Nunc) and subjected to luminescence detection. All
compounds used for screening were pure compounds and were
dissolved in DMSO at concentrations of 1 mm for the collection of
myxobacterial metabolites[13]
and 5 mm for the highly diverse HZI
natural and synthetic compound collection. For the entire cell-
based screening of all compounds, a Z’ factor[17]
of 0.51Æ0.04 was
calculated.
Luciferase assay: Firefly luciferase activities were determined with
the aid of the Dual-Luciferase reporter system (Promega Corpora-
tion) as described in the manufacturer’s protocol. The lumines-
cence measurements were carried out in 96-well format with a
DTX 880 multimode detector (Beckman Coulter).
Cell viability assay: The effects of the compounds on cell viability
were determined with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; Sigma). The assay was performed as de-
scribed previously.[24]
Enzyme-linked immunosorbent assay: IL-8 protein levels in cell
supernatants were determined with a specific enzyme-linked im-
munosorbent assay (ELISA) against human IL-8 (RD Systems) ac-
cording to the manufacturer’s instructions.
Real-time PCR: Total RNA was extracted from cells by use of
RNeasy spin columns (Qiagen) and RNA quality was verified with
an RNA Nano 6000 Kit (Agilent) by standard protocols. Subsequent-
ly, the RNA samples were treated with DNase (Promega) and tran-
scribed into cDNA by use of oligo dT primers and a first strand
cDNA synthesis kit (NEB) according to the manufacturer’s protocol.
For real-time PCR, a LightCycler 480 SYBR green kit (Roche) was
used according to the manufacturer’s instructions. The PCR reac-
tions were performed with a LightCycler 480 (Roche) instrument in
96-well plates (Roche) by using the SYBR green template program
(Roche). The primer sequences used for IL-8 were 5’-AGA CAG CAG
AGC ACA CAA GC-3’ and 5’-ATG GTT CTT TCC GGT GGT-3’. The
primer sequences used for GAPDH were 5’-AGC CAC ATC GCT CAG
ACA C-3’ and 5’-GCC CAA TAC GAC CAA ATC C-3’.
Northern blot analysis: Total RNA from cells was isolated with the
aid of TRIZOL reagent (Gibco/BRL, Life Technologies) according to
the manufacturer’s instructions. Northern blot analysis was per-
formed as described previously.[25]
The specific probes for detection
of IL-8, firefly luciferase, and transferrin receptor (TFR) RNAs have
been described previously.[26]
Southern blot analysis: DNA was isolated from different HeLa S3
IL-8 Luc cell clones and digested with EcoRI. Southern blot analysis
was performed by standard protocols.[27]
The specific probe for
detection of the firefly luciferase coding region was obtained as
described previously.[26b]
Western blot analysis: Western blots were performed as described
previously[28]
with use of antibodies against transforming growth
factor b activated kinase 1 (TAK1), TAK1 Thr184/187, IKK-b, IKK-a/b
Ser176/180, p65 Ser536, JNK, JNK Thr183/Tyr187, c-Jun Ser63, p38,
p38 Thr180/Tyr182, IkBa, IkBa Ser32 (all from Cell Signaling Tech-
nology), and p65 and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH; both from Santa Cruz Biotechnology). Secondary polyclo-
nal goat anti-rabbit and rabbit anti-mouse antibodies were ob-
tained from Dako.
Statistics: Results are presented as meansÆSEM values. The data
set was analyzed for statistical significance by analysis of variance.
Reported P values were derived from the combined data from at
least three repetitions of each experiment. P values 0.05 were
considered to indicate significant differences.
Abbreviations: AP-1: activator protein 1. C-EBP: CCAAT enhancer
binding protein. GADPH: glyceraldehyde 3-phosphate dehydrogen-
ase. IkB: inhibitor of kB. IKK: IkB kinase. IL-1: interleukin-1. IL-6: in-
terleukin-6. IL-8: interleukin-8. JNK: c-Jun N-terminal kinase. MAPK:
mitogen-activated protein kinase. MAP3K: mitogen-activated pro-
6 www.chembiochem.org 2012 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim ChemBioChem 0000, 00, 1 – 8
ÝÝ These are not the final page numbers!
M. Nourbakhsh et al.

7. tein kinase kinase kinase. MEKK1: MAP kinase kinase 1. MKK6: MAP
kinase kinase 6. NF-IL-6: nuclear factor for IL-6. NF-kB: nuclear fac-
tor kB. NRF: NF-kB repressing factor. TAK1: transforming growth
factor beta activated kinase.
Acknowledgements
We thank Katrin Finger and Jürgen Ruland for initial discussions.
This work was supported by the Deutsche Forschungsgemein-
schaft (CRC 566) and the European Research Council (ERC) (PIOF-
GA-2008-221359).
Keywords: cell-based screening · inflammation · signal
transduction · spirangien A · spirangien M522
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Received: October 11, 2011
Published online on , 0000
ChemBioChem 0000, 00, 1 – 8 2012 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim www.chembiochem.org 7
These are not the final page numbers! ÞÞ
Inhibitors of IL-8 Expression

8. FULL PAPERS
M. R. Reboll, B. Ritter, F. Sasse,
J. Niggemann, R. Frank, M. Nourbakhsh*
–
The Myxobacterial Compounds
Spirangien A and Spirangien M522 are
Potent Inhibitors of IL-8 Expression
The screening of heterogeneous com-
pound libraries identified the myxobac-
terial compound spirangien A and its
derivate spirangien M522 as potent in-
hibitors of IL-8 expression. Both com-
pounds decelerate the phosphorylation
and degradation of IkBa, the key regu-
lator of the IL-1-stimulated NF-kB signal-
ing pathway.
8 www.chembiochem.org 2012 Wiley-VCH Verlag GmbH Co. KGaA, Weinheim ChemBioChem 0000, 00, 1 – 8
ÝÝ These are not the final page numbers!

9. Supporting Information
Copyright Wiley-VCH Verlag GmbH Co. KGaA, 69451 Weinheim, 2012
The Myxobacterial Compounds Spirangien A and
Spirangien M522 are Potent Inhibitors of IL-8 Expression
Marc RenØ Reboll,[a]
Birgit Ritter,[b]
Florenz Sasse,[a]
Jutta Niggemann,[c]
Ronald Frank,[a]
and
Mahtab Nourbakhsh*[d]
cbic_201100635_sm_miscellaneous_information.pdf

10. 1
SUPPLEMENTAL MATERIAL
SUPPLEMENTAL FIGURE LEGENDS
SUPPLEMENTAL FIGURE 1. A) Different HeLa S3 cell clones were checked for
single integration of the reporter gene construct using Southern blot analysis. Therefore, DNA
was isolated from stable transfected HeLa S3 cell clones, digested with EcoRI and subjected
to Southern blot analysis. The DNA blot was hybridized with firefly luciferase specific probe
and detected by autoradiography. The result of three different clones is presented. The clones
2 and 3 show the expected two fragments detected by firefly luciferase probe. A DNA marker
is indicated at the left. B) The induction of Firefly luciferase following IL-1 stimulation in 96-
Well format. Stable transfected HeLaS3 IL-8Luc cells were treated with IL-1 for 16 hours
where indicated. The induction of firefly luciferase was determined using luciferase assay.
Therefore, cells were lysated and luciferase activities were measured. Firefly luciferase
activities are shown here as the mean ± SEM of six independent experiments. Firefly
luciferase activities for untreated cells were set as 1.
SUPPLEMENTAL FIGURE 2. Summary of screening data. HeLa S3 reporter cells
were seeded in 96 well plates and incubated for 24 hours. Simultaneously, cells were IL-1
stimulated and compounds were added. Following incubation for 16 hours, cells were lysated.
Firefly luciferase activities were measured in the lysates. On each plate, firefly luciferase
activity for IL-1 stimulated cells treated with DMSO was set as 100%. Relative firefly
luciferase activity of all tested compounds is presented. Compounds which reduce relative
firefly luciferase activity at least to 30% are considered as hits. Hit compounds are depicted in
the hatched area of the diagram. The best hit, Spirangien A, is indicated with an asterisk.

11. 2
SUPPLEMENTAL FIGURE 3. Densitometric analysis of IκBα phosphorylation
and degradation. A) The effects of Spirangien A on IκBα phosphorylation and degradation
were determined. The experiments were carried out as described in the legend of figure 5. The
densitometric analysis of detected bands is presented as the mean ± SEM from three
independent experiments. IκBα and IκBα Ser 32 bands were quantified and normalized to
corresponding GAPDH signals, respectively. Relative phosphorylation of IκBα at Ser 32 in
untreated cells was set as 1 and the relative IκBα protein level in untreated cells was set as
100% in each experiment. The p-values 0.05 and 0.01 are indicated by (*) and (**),
respectively. B) The densitometric analysis was performed as described in section A. Instead
of Spirangien A, cells were treated with Spirangien M522. A p-value 0.05 is indicated (*).
SUPPLEMENTAL FIGURE 4. The effects of Spirangien A and Spirangien M522
on IL-1 dependent cellular signaling. A) HeLa S3 cells were treated with indicated
concentrations of Spirangien A for 16 hours. Following IL-1 stimulation for 30 min, cellular
lysates were prepared and equal amounts of protein were subjected to Western blot analysis.
The protein level and phosphorylation status of several signal transducers were detected and
are indicated at the left. The GAPDH protein level was detected as loading control. B) The
experiments were performed as described in section A. Instead of Spirangien A, indicated
concentrations of Spirangien M522 were used here.

12. 3
SUPPLEMENTAL FIGURES
EcoRI
specific
fragments
clones
1 2 3
150
200
250
300
400
500
A
B
Supplemental Figure 1
-foldfireflyluciferaseinduction
0
1
2
3
4
5
Control IL-1

13. 4
Supplemental Figure 2
Compounds
0 200 400 600 800 1000 1200 1400 1600 1800 2000
Relativefireflyluciferaseexpression[%]
10
100
1000
Hits ≤ 30%

14. 5
Supplemental Figure 3A
A
IL-1 stimulation [min]
control 5 15 30
relativeIkBalphaphosphorylation
0
10
20
30
40
50
unstimulated
IL-1 + Spirangien A
IL-1
*
IL-1 stimulation [min]
control 5 15 30
relativeIkBalphaproteinlevel[%]
0
20
40
60
80
100
120
140
unstimulated
IL-1 + SpirangienA
IL-1
**
A

15. 6
Supplemental Figure 3B
B
IL-1 stimulation [min]
control 5 15 30
relativeIkBalphaphosphorylation
0
2
4
6
8
10
12
14
16
unstimulated
IL-1 + Spirangien M522
IL-1
*
IL-1 stimulation [min]
control 5 15 30
relativeIkBalphaproteinlevel[%]
0
20
40
60
80
100
120
unstimulated
IL-1 + Spirangien M522
IL-1
*
B

16. 7
Supplemental Figure 4A
Spirangien A [nM]
IL-1 - + + + + + +
- - 2.5 7 21 65 195
α- TAK1 Thr 184/187
α- p65 Ser536
α-JNK Thr 183, Tyr 187
α- c-Jun Ser 63
α- GAPDH
α- TAK1
α- p65
α- IKK α/β Ser 176/180
α- IKKβ
α-JNK
α-p38 Thr 180, Tyr 182
α-p38
A

17. 8
Supplemental Figure 4B
Spirangien M522 [nM]
IL-1 - + + + + + + +
- - 10 30 90 270 810 2430
α- IKK α/β Ser 176/180
α- IKK β
α- JNK Thr 183, Tyr 187
α- GAPDH
α- TAK1 Thr 184/187
α- TAK1
α- cJun Ser 63
α-p38 Thr180/Tyr182
α-p65 Ser 536
α-p65
α- JNK
α-p38
B