Synthetic Indolactam V Analogues as Inhibitors of PAR2-Induced Calcium Mobilization in Triple-Negative Breast Cancer Cells

Article abstract of DOI:10.1002/cmdc.201700640

Human proteinase-activated receptor 2 (PAR2), a transmembrane G-protein-coupled receptor (GPCR), is an attractive target for a novel anticancer therapy, as it plays a critical role in cell migration and invasion. Selective PAR2 inhibitors therefore have potential as anti-metastatic drugs. Knowing that the natural product teleocidin A2 is able to inhibit PAR2 in tumor cells, the goal of the present study was to elaborate structure–activity relationships and to identify potent PAR2 inhibitors with lower activity against the adverse target, protein kinase C (PKC). For this purpose, an efficient gram-scale total synthesis of indolactam V (i.e., the parent structure of all teleocidins) was developed, and a library of derivatives was prepared. Some compounds were indeed found to exhibit high potency as PAR2 inhibitors at low nanomolar concentrations with improved selectivity (relative to teleocidin A2). The pseudopeptidic fragment bridging the C3 and C4 positions of the indole core proved to be essential for target binding, whereas activity and target selectivity depends on the substituents at N1 or C7. This study revealed novel derivatives that show high efficacy in PAR2 antagonism combined with increased selectivity.

Full text of DOI:10.1002/cmdc.201700640

Accepted Article
Title: Synthetic Indolactam V Analogs as Inhibitors of PAR2 Induced
Calcium Mobilization in Triple Negative Breast Cancer Cells
Authors: Jan Stein, Sonja Stahn, Jörg-Martin Neudörfl, Julia Sperlich,
Hans-Günther Schmalz, and Nicole Teusch
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To be cited as: ChemMedChem 10.1002/cmdc.201700640
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700640
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ChemMedChem
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FULL PAPER
Synthetic Indolactam V Analogs as Inhibitors of PAR2 Induced
Calcium Mobilization in Triple Negative Breast Cancer Cells
[
a]
[b]
[a]
[b]
[a]
Jan Stein , Sonja Stahn , Jörg-M. Neudörfl , Julia Sperlich , Hans-Günther Schmalz *, Nicole
[
b]
Teusch *
[
[
a]
b]
Dr. J. Stein, Dr. J.-M. Neudörfl, Prof. Dr. H.-G. Schmalz
University of Cologne
Department of Chemistry
Greinstrasse 4, D-50939 Koeln, Germany
E-mail: schmalz@uni-koeln.de
Dr. S. Stahn, J. Sperlich, Prof. Dr. N. Teusch
Technische Hochschule Koeln
Bio-Pharmaceutical Chemistry & Molecular Pharmacology
Faculty of Applied Natural Sciences
Kaiser-Wilhelm-Allee, G. E39, D-51373 Leverkusen, Germany
E-mail: nicole.teusch@th-koeln.de
*
H.-G. S. and N. T. share last authorship
Supporting information for this article is given via a link at the end of the document.
Abstract: Human proteinase-activated receptor
2
(PAR2),
a
perspective, elevated PAR2 expression in biopsy and metastatic
tissue could be linked to an increased malignancy grade and
transmembrane G-Protein-coupled receptor (GPCR), represents an
attractive target for a novel anti-cancer therapy as it plays a critical
role in cell migration and invasion. Thus, selective PAR2 inhibitors
possess a potential as anti-metastatic drugs. Knowing that the
natural product teleocidin A2 is able to inhibit PAR2 in tumor cells,
the goal of the present study was to elaborate structure-activity
relationships and to identify potent PAR2 inhibitors with lower activity
against the adverse target protein kinase C (PKC). For this purpose,
an efficient gram scale total synthesis of indolactam V (i.e. the
parent structure of all teleocidins) was developed and a library of
derivatives was prepared. Some compounds indeed exhibited high
potency as PAR2 inhibitors at low nanomolar concentrations with
improved selectivity (as compared to teleocidin A2). The pseudo-
peptidic fragment bridging the C-3 and the C-4 positions of the
indole core proved to be essential for target binding, while activity
and target selectivity depends on the substituents at N-1 or C-7. The
study revealed novel derivatives depicting high efficacy in PAR2
antagonism combined with increased selectivity.
[
6]
subsequently to a reduced overall survival rate . Whereas initial
PAR2 inhibiting compounds were mostly limited in potency or
displayed agonistic properties at higher concentrations, recent
medicinal chemistry approaches have led to more efficacious
[
7,8]
small molecule PAR2 antagonists
. Overall, the distinct role of
PAR2 in tumor biology illustrates the need for low molecular
weight PAR2 inhibitors providing the basis for the development
[
9,10]
of a novel therapeutic strategy in cancer treatment
.
In the course of our previous work, we recently demon-
strated that some marine natural products of the indolactam/
[
2]
teleocidin family exhibit significant effects as PAR2 inhibitors .
Prior to this, the natural products (Figure 1), which were all
isolated from marine organisms, had been reported to exhibit
strong biological activities including tumor-promoting effects,
[
11–14]
mainly due to the activation of protein kinase C (PKC)
.
However, as a solely PKC-mediated effect cannot explain the
remarkable efficacy of teleocidin on PAR2-mediated signaling, it
remains elusive whether the ability to activate PKC is
mechanistically linked to the inhibition of PAR2-induced
[
2]
signaling .
Introduction
Cancer is one of the diseases with high unmet medical need
raising most cases of death after cardiovascular diseases in the
industrialized countries. Notably, the dissemination of cancer
from the primary tumor is the main reason of cancer-related
mortality and represents an enormous clinical challenge. Among
the different types of malignant tumors, breast cancer is the
OH
OH
OH
H
N
H
N
H
N
N
N
N
O
O
O
N
H
N
H
N
H
[
1]
leading cause of cancer death in females worldwide .
Proteinase activated receptor 2 (PAR2), a member of the
Indolactam V (1)
Teleocidin A2 (TC-A2)
Teleocidin B1 (TC-B1)
seven transmembrane
G protein-coupled receptor (GPCR)
Figure 1. Structure of indolactam V (1) and teleocidins A2 and B1
superfamily, has been shown to play a major role in cancer
[
2,3]
progression. Numerous in vitro studies including breast
,
Biosynthetically, the teleocidins are derived from (L)-trypto-
[
4]
[5]
[15–17]
pancreatic and lung adenocarcinoma cells revealed a critical
role of PAR2 signaling in cancer cell migration and invasion
correlating with increased metastatic potential. From a clinical
phane via indolactam V
. All compounds of this class display
a 3,4-disubstituted indole nucleus with a characteristic (L)-N-
methyl-valine-containing bridge forming a 9-membered ring. Due
1
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to their powerful biological activity and their limited accessibility
from natural sources, several total syntheses of indolactam V
by reduction of the nitro group of
hydrogenation. The resulting aniline 8 was then converted into
the valine derivative 9 by S 2-alkylation in virtually quantitative
7 through catalytic
[
18–31]
[32–38]
(
1)
and teleocidins
have been developed, and a
N
[
23]
number of studies employing total synthetic analogs have
contributed to the profiling of indolactam V-related or -derived
yield . The acid-catalyzed hydrolysis of the cyclic N,O-acetal
afforded the desired product 11 besides significant amounts of
the double deprotected by-product 10 which, however, was
[
13,39]
compounds with respect to PKC activation
.
In contrast to these previous studies, the major goal of the
present work was to synthesize and investigate novel indolac-
easily re-protected with Boc
2
O/NEt
3
to afford 11 in 91%
combined yield from 9. After protecting the primary alcohol
functionality as a TBDPS ether, both the N-Boc group and the
benzyl ester protecting groups, were cleaved off under standard
conditions. Treatment of the resulting intermediate 14 with
HATU as a coupling agent in the presence of DIPEA smoothly
afforded the cyclized product 15. In comparison with other
indolactam V syntheses, the Eschweiler-Clark methylation of the
secondary amino group in 15 was achieved in very high yield
[
2]
tam V analogs with respect to their PAR2-inhibiting properties .
Our approach was aimed at deriving distinct structure-activity
relationships (SAR) and to design derivatives depicting potent
PAR2 antagonism combined with selectivity against PKC, the
originally described adverse target of teleocidin A2.
Results and Discussion
under appropriate conditions (CH
2
O, NaCNBH
3
, AcOH, MeCN,
[
42]
0
°C) . Treatment of the resulting compound 16 with TBAF
finally afforded indolactam V (1) in high overall yield.
Synthesis
By means of a single crystal X-ray analysis of the hydro-
tetrafluoroborate salt of 1 the relative and absolute configuration
of the synthetic compound was unambiguously confirmed. The
high-resolution structure (Figure 2) shows the 9-membered ring
preferring a twisted conformation with a virtually planar s-cis-
configurated peptide unit positioned almost perpendicular
Our first task was to prepare substantial amounts (gram
scale) of indolactam V (1) as a precondition for the planed
structural variation. Careful analysis of the published syntheses
[
21-34]
[23]
of 1
prompted us to consider the strategy of Kogan and
[
28]
Xu
as most promising (Scheme 1). Key elements of this
(
orthogonal) to the plane defined by the indole ring system. This
approach are the assembly of the nitro-indole derivative 4 from
[
31]
characteristic 3D structure of the indole moiety with the polar
unit bridging C-3 and C-4 appears to be the essential structural
motif ensuring an effective interaction (binding) with the
respective protein partners.
building blocks 5 and 6
group into a valinyl moiety and closure of the 9-membered ring
followed by conversion of the nitro
[
23]
through intramolecular peptide coupling
.
H
N
HN
OPG
HN
CO2H
OPG
NH2
O
1
N
N
PG
PG
2
3
NO2
O
NO2
I
O
O
+
BocN
BocN
NH2
N
H
6
5
4
Scheme 1. Retrosynthesis of indolactam V (1) according to Kogan and Xu.
The preparation of the 4-nitro-tryptophanol derivative 4 (by
Pd-catalyzed condensation of 2-iodo-3-nitroanililin 6 with the L-
glutamine-derived aldehyde 5 in the presence of DABCO as a
Figure 2. Structure of the hydro-tetrafluoroborate salt of indolactam V (1-
4
HBF ) in the crystalline state.
[
28]
base)
proceeded smoothly on a multi-gram scale with 85%
Having successfully developed an efficient synthetic scheme,
yield. However, our initial attempts to perform the subsequent
planned steps according to the reported protocols were
hampered by the instability of intermediates and low yields. We
thus realized that a proper protection of the indole nitrogen
appeared to be absolutely essential. After first evaluating both,
tosyl and Boc, we identified the SES group (2-trimethylsilyl-
allowing us to prepare indolactam V (1) in gram amounts, we
next turned our attention to the synthesis of various derivatives
[30,43–45]
(
Scheme 3)
. For this purpose, we preferentially used the
TBS-protected compound 17a as a safe (biologically inactive)
key intermediate. In a first set of experiments we prepared a
small set of N1 derivatives by simply reacting the indole 17a with
different acylating and benzylating reagents and subsequent
fluoride-induced cleavage of the silyl protecting group (Scheme
[
40,41]
ethansulfonyl)
as a superior protecting group for our
purposes. In this case, all intermediates proved to be stable and
readily isolated and, even more importantly, all steps could be
reproducibly performed on a multi gram scale with high yield.
As shown in Scheme 2, the resulting greatly improved
synthesis of 1 commenced with the SES protection of 4 followed
3
). The compounds (20-25) thus prepared are listed in Chart 1
together with some other indolactam V derivatives obtained in
the course of the synthesis development.
2
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OH
HN
CO Bn
2
e
NH2
c
21%
N
SES
NO2
NH2
HN
CO Bn
2
O
O
O
10
b
TfO
CO Bn
2
d
99%
BocN
BocN
BocN
OH
9
9%
94%
N
R
N
SES
N
SES
HN
CO Bn
2
8
9
4
7
(R = H)
(R = SES)
a
96%
NHBoc
71%
N
SES
11
f
98%
OTBDPS
OTBDPS
H
N
H
N
OTBDPS
OTBDPS
NHBoc
N
HN
HN
CO R
HN
CO Bn
2
2
O
N
O
N
k
j
i
g
1
NH2
9
9%
96%
93%
98%
N
SES
N
SES
SES
SES
4
9%
1
6
15
12
overall
13 (R = Bn)
h
90%
1
4 (R = H)
2
Scheme 2. Improved synthesis of indolactam V (1) performed on a gram scale. Reagents and conditions: a) NaH, SES-Cl, THF, -20 °C, 30 min; b) Pd/C, H ,
MeOH, rt, 12 h; c) 2,6-lutidine, C
2
H
4
Cl
2
, 80 °C, 12 h; d) pTsOH.H
2
O, CH
2
Cl
2
, rt, 4 h; e) Boc N, MeCN, rt; f) TBDPS-Cl, imidazole, DMF, rt, 3 h; g) TFA,
2
O, Et
3
CH
2
Cl
2
, 0 °C -> rt, 6 h; h) Pd/C, H
2
, MeOH, rt, 3 h; i) HATU, DIPEA, THF, rt, 48 h; j) CH
2
O, NaCNBH , AcOH, MeCN, 0 °C -> rt, 1 h; k) TBAF, THF, 0 °C to rt, 2 h.
3
2
4 h.
As also shown in Scheme 3, a variety of C-7-substituted
derivatives were synthesized starting from TBS-protected
OR
H
OH
OH
H
H
N
N
N
[
30,43–45]
N
N
HN
indolactam V (17a). Using the iodinated compound 26
a key intermediate Pd-catalyzed cross-coupling and carbonyla-
as
O
O
O
[
46]
tion methods
were applied to introduce different substituents
N
H
N
R
N
R
at C-7. As before, the silyl protecting group was cleaved off only
in the final step under standard conditions (TBAF). The allylation
product 27 was synthesized employing allyl-Bpin under Suzuki
1
1
7a (R = TBS)
7b (R = TBDPS)
18a (R = Ac)
18b (R = Ts)
19a (R = H)
19b (R = Ts)
[
47,48]
type conditions
. Compounds 31a-31e in turn were smoothly
OH
H
N
OH
OH
[
49]
H
N
H
N
obtained under Sonogashira conditions . Two of the product
alkynes (31a and 31b) were hydrogenated in the presence of
Pd/C to the corresponding alkanes 32a and 32b, respectively.
Carboylative Suzuki coupling of 26 with phenyl boronic acid
afforded the ketone 28. In a related fashion, the alkoxy-
carbonylated products 30a and 30b and the amides 30c and
N
N
N
O
N
O
N
O
N
O
O
MeO
O
N
3
0d, respectively, were prepared by Pd-catalyzed reaction of 26
2
0
21
22
[
50,51]
with different alcohols and amines under CO atmosphere
.
R
OH
OH
OH
H
N
H
N
Compound 29 was obtained by carbonylative cross-coupling
[52]
N
N
N
using phenylacetylene
and subsequent TBAF-induced 6-
O
N
O
O
N
endo-dig cyclisation. The structure of 29 was again
unambiguously proven by X-ray crystal structure analysis
N
R
(
Figure 4).
R
2
2
2
2
4a (R = 2-F-Ph)
4b (R = 3-F-Ph)
4c (R = 4-F-Ph)
25 (R = 4-F-Ph)
2
3
4d (R = 3-MeO-Ph)
Chart 1. Various derivatives of indolactam V prepared according to Scheme 3.
3
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H
N
H
N
H
N
N
OH
N
OH
N
OH
O
O
O
N
R
N
H
N
H
Ph
O
2
0-24 (see Chart 1)
27
28
f,c
b,c
e,c 27%
8
6%
H
N
H
N
H
N
N
OTBS
N
OTBS
N
OH
O
O
N
O
N
a
d
g,c
53%
1
8
7%
9
2%
N
H
H
I
O
Ph
1
7a
26
2
9
i,c
h,c
H
N
H
N
H
N
N
OH
N
OH
N
OH
O
O
O
j,c
N
H
N
H
N
H
O
R
R
3
3
3
3
3
1a (R = Ph)
1b (R = n-Bu)
1c (R = tert-Bu)
53%
86%
40%
30a (R = OMe) 28%
3
3
2a (R = Ph)
2b (R = n-Bu) 63%
64%
R
30b (R = OEt)
7
1%
0c (R = NHMe) 34%
0d (R = NHEt) 51%
3
3
1d (R = cyclopropyl) 50%
1e (R = 4-F-Ph)
62%
Scheme 3. Preparation of various derivatives of indolactam V (1). Reagents and conditions: a) TBSCl, imidazole, TBAI, DMF, rt, 12 h; b) NaH, R-X, DMF, -20 °C
to rt, 40 min – 12 h; c) TBAF, THF, 0 °C to rt, 2 h; d) I , pyridine, 1,4-dioxane, 0 °C to rt, 2.5 h; e) allyl-Bpin, cat. Pd(PPh , CsF, THF, reflux, 24 h; f) PhB(OH)
CO, cat. PdCl (PPh , 1,4-dioxane, Et N, 60 °C, 24 h; g) phenylacetylene, CO, cat. PdCl (PPh , CuI, 1,4-dioxane, Et N, rt, 12 h; h) ROH or RNH , CO, cat.
PdCl (PPh , 1,4-dioxane, Et N, 60 °C, 12 h; i) alkyne, cat. PdCl (PPh , CuI, 1,4-dioxane, Et N, rt, 12 h; j) Pd/C, H , rt, 5 h.
2
3
)
4
2
,
2
3
)
2
3
2
3
)
2
3
2
2
3
)
2
3
2
3
)
2
3
2
Biological investigations
The various compounds synthesized as described above
were biologically evaluated with respect to (1) inhibition of
2
+
PAR2-induced Ca mobilization and (2) intracellular PKC
activation.
2
+
First, physiological Ca release induced by the PAR2
specific tethered ligand SLIGKV-NH was determined in the
2
human breast adenocarcinoma cell line MDA-MB 231 which
is categorized triple negative due to the lack of expression of
hormone receptors or HER2. Indolactam V derivatives were
tested in the presence of the EC50 determined for the
agonistic ligand SLIGKV-NH
2
(7.46 µM) to achieve IC50
values for PAR2 inhibition. The data shown in Table 1
indicate that indolactam V (1) itself displays a 4-fold potency
2
+
loss in inhibiting PAR2 induced Ca release (IC50 = 72.1 ±
4
.4 nM) compared to teleocidin (IC50 = 18.1 ± 1.7 nM).
Changes at the pseudopeptidic bridge (N-demethylation
Figure 3. Structure of the cyclized indolactam V derivative 29 in the
crystalline state.
or O-protection with a bulky silyl group) led to inactive
compounds. Derivatization at the indole nitrogen caused
significant effects: Small acyl substituents such as acetyl
(
18a) (IC50 = 26.1 ± 8.0 nM) or methoxylcarbonyl (22) (IC50
5.3 ± 6.3 nM) enhanced activity, as did fluorinated benzylic
residues (e.g. 24c) (IC50 = 24.0 ± 5.9 nM). Interestingly,
=
2
4
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2
substituents at C-7 of the indole nucleus (R ) also caused
significant changes.
Table 1. IC50 values of indolactam V analogues for inhibition of PAR2
induced intracellular Ca mobilization in MDA-MB 231 breast cancer cells.
2
+
While some groups (benzoyl, alkynyl or aminocarbonyl)
did not improve activity, the allylated compound 27 proved to
be quite active (IC50 = 2.25 ± 0.39 nM). Excellent results,
however, were observed for the alkoxycarbonylated
indolactam V derivatives 30a and 30b, the latter inhibiting the
H
N
R3
N
OR4
O
N
2
+
PAR2 induced intracellular Ca mobilization at single-digit
nanomolar concentrations (IC50 = 1.57 ± 0.90 nM). Thus,
compared to the starting compound indolactam V (1) the
R2 R1
derivative 30b displayed
a
46-fold efficacy increase in
1
2
3
4
[a]
R
R
R
R
PAR2 IC50 [nM]
inhibiting PAR2-induced intracellular signalling. Comparable
concentration-dependent inhibition curves of PAR2-induced
(mean± SEM)
TC-A2
1
H
H
H
tert.-alkyl
Me
Me
Me
Me
Me
H
H
H
18.1 ± 1.7
72.1 ± 4.4
>10000
26.1 ± 8.0
791 ± 48
> 1000
2
+
Ca mobilization by indolactam V (1) and indolactam V
analogs 18a, 22 and 30b in MDA-MB 231 breast cancer cells
are displayed in Figure 4. Furthermore, cytotoxicity
assessment of selected derivatives revealed no notable effect
on cell viability (Supplemental Figure 1).
H
H
H
H
H
H
H
17b
TBDPS
1
1
1
1
2
8a
8b
9a
9b
0
Ac
H
H
H
H
H
Ts
H
Ts
i-butyryl
H
Me
> 1000
206 ± 56
2
2
1
2
benzoyl
Moc
H
H
Me
Me
H
H
116 ± 11
25.3 ± 6.3
2
2
3
Bn
H
H
Me
Me
H
H
78.8 ± 18.3
205 ± 54
4a
2-F-Bn
2
2
2
4b
4c
4d
3-F-Bn
4-F-Bn
H
H
H
Me
Me
Me
H
H
H
26.3 ± 6.1
24.0 ± 5.9
45.1 ± 8.9
3-MeO-
Bn
2
2
2
3
3
3
3
5
4-F-Bn
H
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
2450 ± 430
2.25 ± 0.39
> 2000
7
H
H
H
H
H
H
H
allyl
9
cyclyzed
0a
0b
0c
0d
CO
CO
2
2
Me
Et
6.96 ± 1.73
1.57 ± 0.90
1994 ± 562
235 ± 29
2
+
Figure 4. Concentration-dependent inhibition of PAR2 induced Ca
mobilization by indolactam V (black) and indolactam V derivative 18a (blue),
2 (red) and 30b (green) in MDA-MB 231 breast cancer cells. Data points
CONHMe
CONHEt
alkynyl
2
represent mean values ± SEM from a minimum of three independent
experiments performed in triplicates and data of each experiment was
31e
18.3 ± 2.1
2
+
2+
2+
normalized to PAR2 activating peptide SLIGKV-NH
2
induced Ca release.
[a] Intracellular Ca mobilization assay. PAR2 induced intracellular Ca
mobilization was induced with the determined EC50 concentration of the
2
PAR2 activating peptide SLIGKV-NH . Data represent mean +SEM of a
minimum three independent determinations performed in triplicates.
Statistical analysis of specificity of single analogues was performed using
an unpaired Student’s t-Test.
Intracellular PKC activation: To date, teleocidin
derivatives are mainly categorized as tumor-promoting agents.
Thus, within our medicinal chemistry approach aiming at
targeting PAR2 with indolactame-derived compounds,
putative PKC activation represents an undesirable off-target
effect. To characterize the potential of indolactam
a
V
derivatives to activate intracellular PKC, phosphorylation
induction of the specific endogenous PKC substrate
MARCKS was determined in the presence of the respective
compounds (Figure 5). As expected, the positive control PMA
(
10 nM) induced a strong phosphorylation up to 3.5-fold
compared to DMSO control representing a significant PKC
activation. Furthermore, MARCKS was phosphorylated up to
4
-fold compared to the untreated control in the presence of 25
nM teleocidin A2. In contrast to teleocidin, the level of
MARCKS phosphorylation induced by indolactam V (1) was
comparable to the level of the DMSO control (Figure 5).
Importantly, the novel indolactam V derivatives 18a and
2
4c did not activate PKC, whereas the analogs 22 and 24b
Figure 5. Phosphorylation of PKC substrate MARCKS in MDA-MB 231
cells by derivatives of indolactam V. Cells were treated for 10 min with
displayed moderate activation of intracellular PKC. While the
2
5nM of each compound. The solvent DMSO represents the control.
respective PKC activation potential of analogs 24a, 24d and
Gö6983 is a commercially availbale PKC inhibitor. Data represent mean
values ±SEM from three independent experiments performed in triplicates,
‘ns’ not statistically significant, **P < 0.01, ***P < 0.001.
2
7 is comparable to teleocidin, compounds 30a and 30b were
able to phosphorylate MARCKS by
compared to negative control.
a 3-fold increase
5
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ChemMedChem
10.1002/cmdc.201700640
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A)
Moreover, PAR2 inhibition data combined with selectivity
data concerning PKC activation (represented by MARCKS
phosphorylation) were visualized in
a
two-dimensional
efficacy plot (Figure 6). IC50 values reflecting inhibition of
PAR2, our target of interest, are shown on the x-axis,
whereas the fold increase in MARCKS phosphorylation,
reflecting the PKC activating potential, is depicted on the y-
axis. Potent derivatives (IC50 PAR2 < 30 nM) with no or
moderate (up to 2-fold compared to DMSO control) off-target
activity towards PKC are depicted in green, whereas highly
potent PAR2 inhibitors with more pronounced PKC activity
(
up to 3-fold induction of MARCKS phosphorylation) are
displayed in yellow. Indolactam V (1) is illustrated in yellow
based on its IC50 for PAR2 which is above 70 nM. Moreover,
derivatives with unfavourable selectivity profiles comparable
to teleocidin (> 3-fold induction of MARCKS phosphorylation)
are visualized in red.
B)
2
+
Figure 6. Scatterplot summarizing the inhibition of PAR2 induced Ca
release and the activation of PKC by selected analogues of indolactam V.
IC50 values versus PAR2 activating peptide SLIGKV-NH are shown on the
2
x-axis. y-axis depicts fold increase in phosphorylation of endogenous
MARCKS protein, representing PKC activation. See Table 1 for exact IC50
values for PAR2 inhibition.
With respect to further selectivity assessment, our previous
2
+
work revealed that teleocidin markedly reduced Ca
mobilization of the Gq-coupled GPCR PAR1, however, with a
[
2]
2
0fold potency loss compared to PAR2 inhibition .
Noteworthy, in this study novel indolactam derivatives were
identified confrming a clear preference for PAR2 inhibition
Figure 7. Indolactam V derivative 30b inhibits migration of triple negative
breast cancer cells. A) Image of the scratch wound assay in MDA-MB-231
cells. First row depicts control cells treated with DMSO and second row
shows treatment with 30b at t=0 and t=24h, respectively. The initial scratch
wound mask is marked with blue lines. Scratch wound after 24h is marked
in yellow. Indolactam V derivative 30b was applied at a concentration of
(
Supplemental Figure 2.
Finally, the effect on cancer cell migration, a notorious
cause for the poor prognosis of triple negative breast cancer,
was evaluated. After 24 hours of incubation of the most
potent indolactam derivative 30b (IC50 for PAR2 1.57 ± 0.90
nM) migration of triple negative MDA-MB-231 cells was
inhibited by 25% (Figure 7).
1
00 nM. B) The quantification of migration is shown in a bar graph. x-axis
shows time after inserting the initial scratch wound and y-axis depicts
relative wound density. P-value of 0.0002 was calculated using Sidak’s
multiple comparisons test. Data represent mean +SEM of quadruplicates of
three independent experiments.
Conclusions
To investigate the potential of novel derivatives of the
natural product teleocidin as inhibitors of the human
proteinase-activated receptor
2 (PAR2), we have first
6
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ChemMedChem
10.1002/cmdc.201700640
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developed (1) a powerful total synthetic access to indolactam
V and (2) novel derivatization options such as carbonylative
transformations of C-7-iodinated intermediates. From a small
library of synthetic compounds we could then derive distinct
structure-activity relationships with respect to the inhibition of
PAR2. Furthermore, we found a particularly active compound
The PathScan® Phospho-MARCKS (Ser152/156) Sandwich ELISA
(
Cell Signaling Technology, catalog no. 7251) was used to measure
the level of cellular PKC activation in MDA-MB 231 cells according to
the manufacturer’s information. The ELISA detects the specific
endogenous phosphorylation of MARCKS at serine residues 152 and
[53]
6
1
56 by PKC as described . Cells were seeded overnight at 1.8 x 10
cells in FCS-free Leibovitz’s L15 culture medium to 10 cm dishes
(Greiner Bio-One) and treated with the compounds at their determined
(
30b) with the potential to significantly inhibit tumor cell
2
+
migration. Moreover, derivatives with IC50 values for PAR2
blockade in the two-digit nanomolar range also exhibiting a
good selectivity towards the tumor-promoting target PKC
have been identified. Importantly, the present study demon-
strates that activation of PKC is not mandatory for inhibiting
PAR2 signaling. Thus, our medicinal chemistry approach has
IC50 values for inhibition of PAR2 induced Ca release for exactly
10min. Cell lysates of controls and compound-treated cells were
generated
using
lysis
buffer
supplemented
with
1mM
phenylmethanesulfonyl
fluoride
(PMSF). Lysate
protein
concentrations were determined with Bradford protein assay and
-
1
adjusted to 0.3 mg mL with sample diluent. Phosphorylated
MARCKS protein was detected colorimetrically by biotinylated
phospho-MARCKS antibody, Horseradish peroxidase (HRP) coupled
Streptavidin and HRP substrate tetramethylbenzidine (TMB) using
Tecan Infinite M200 Pro microplate reader.
revealed novel indolactam
V derivatives depicting high
efficacy and promising selectivity in PAR2 antagonism
providing the basis for the development of a novel therapeutic
anti-cancer strategy.
Scratch Wound Assay
4
MDA-MB-231 cells were seeded at a density of 6 x 10 cells per well
Experimental Section
in an image lock 96 well plate (IncuCyte®, Essen Bioscience, Ann
Arbor, USA) and incubated over night at 37° C. Scratch wound was
inserted with IncuCyte® Woundmaker from Essen Bioscience and
cells were washed twice with PBS. Automatic image acquisition and
analysis were done with IncuCyte® ZOOM over twenty-four hours.
Chemical Synthesis
Detailed synthetic procedures and characteristic compound data are
given in the Supporting Information.
Statistics
Biological evaluation
Cell culture
Presented data were obtained from a minimum of three independent
experiments and expressed as mean ± SEM. Statistical analysis of
data was held with equal sample values using one-way ANOVA
followed by Dunnett’s post hoc test, when groups were compared with
a control and/ or comparison of mean values of only two groups, an
unpaired t-Test was applied. Two-way ANOVA was chosen when
comparing two different groups in a grouped analysis followed by
Sidak's multiple comparisons test. P < 0.05 was chosen to announce
statistically significant difference. Data analysis was done with
GraphPad Prism v. 6.07 (GraphPad Software).
Human breast adenocarcinoma MDA-MB 231 (European Collection of
Cell cultures, catalog no. 92020424) was used to examine the effect
of indolactam V derivatives on endogenous PAR2 and PAR1. Cells
were grown in Leibovitz’s L15 (Biochrom) containing 15% fetal calf
-
1
-1
serum (FCS), 2mM glutamin, 100units ml Penicillin and 100µg ml
Streptomycin and cultivated in humidified atmosphere at 37°C.
Trypsin-free Cell Dissociation Buffer (Life Technologies) was applied
for cell dissociation during passaging and seeding.
Intracellular calcium mobilization
Acknowledgements
Kinetic measurements of mobilization of intracellular calcium in MDA-
The PhD training of S.S. and J.S. was financed by the
graduate program in Pharmacology and Experimental
Therapeutics at the University of Cologne which is financially
and scientifically supported by Bayer. We would like to thank
Ina-Maria Albrecht for excellent technical assistance.
MB 231 cells were performed in black 96-well clear bottom plates
4
(
Greiner Bio-One). Cells were seeded overnight at 2 x 10 cells per
well and on the day of the experiment loaded with the indicator dye
Quest Fluo-8TM AM (4µM) (AAT Bioquest) in HBSS and 1% FCS. To
2
+
examine inhibitory effects on PAR2 induced Ca mobilization, dilution
series (1:5) of derivatives in HHBS were prepared starting at
concentrations of 2µM. Wells contained HHBS, 2mM Probenecid
Keywords: Indole alkaloids, Teleocidins, Triple negative
(
AAT Bioquest) and compounds in
Stimulation of the named receptors was performed with the PAR2-
activating peptide SLIGKV-NH (Sigma-Aldrich) at its determined EC50
a total volume of 100µL.
breast cancer, Proteinase-activated receptor 2, Intracellular
+
2
2
Ca mobilization, Total synthesis
using the injector unit from Tecan Infinite M1000 Pro microplate
reader after 16s of baseline measurement. Fluorescence was
measured using excitation at λ = 490nm and emission at λ = 525nm.
Calcium ionophore A23187 (Sigma-Aldrich) was used to generate the
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Entry for the Table of Contents
Synthetic Indolactam V Analogs as Inhibitors of PAR2 Induced Calcium Mobilization in Triple
Negative Breast Cancer Cells
Dr. J. Stein, Dr. S. Stahn, Dr. Jörg-M. Neudörfl, J. Sperlich, Prof. H.-G. Schmalz and Prof. N. Teusch
H
N
Me
total%
synthesis
N
OH
biological
testing
O
N
R2
R1
Derivatives of the natural product indolactam V were prepared by total synthesis and their potential as inhibitors of the human
proteinase-activated receptor 2 (PAR2) was investigated. Distinct structure-activity relationships were derived, also with respect to
the activation of protein kinase C (PKC) as an undesirable side effect. Importantly, the study revealed that activation of PKC is not
mandatory for inhibiting PAR2 signaling. Some highly active and selective compounds were identified providing a potential basis for
the development of a novel therapeutic anti-cancer strategy.
9
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