Sulfonamide Inhibitors of β-Catenin Signaling as Anticancer Agents with Different Output on c-MYC

Article abstract of DOI:10.1002/cmdc.202000594

The Wnt/β-catenin pathway is often found deregulated in cancer. The aberrant accumulation of β-catenin in the cell nucleus results in the development of various malignancies. Specific drugs against this signaling pathway for clinical treatments have not b

Full text of DOI:10.1002/cmdc.202000594

Accepted Article
Title: Sulfonamide Inhibitors of ß-Catenin Signaling with
Different Output on c-MYC as Anticancer Agents
Authors: Romano Silvestri, Laura Di Magno, Fiorella Di Pastena,
Michela Puxeddu, Giuseppe La Regina, Antonio Coluccia,
Alessia Ciogli, Simone Manetto, Marella Maroder, Gianluca
Canettieri, and Marianna Nalli
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000594
Link to VoR: https://doi.org/10.1002/cmdc.202000594
01/2020

10.1002/cmdc.202000594
ChemMedChem
COMMUNICATION
The Wnt/-catenin pathway is often found deregulated in
cancer. The aberrant accumulation of -catenin in the cell nucleus
boosts the transcription of many oncogenes, such as Axin-2, c-
Myc and Cyclin-D1, resulting in the development of various types
of carcinoma, including colon cancer, hepatocellular carcinoma,
pancreatic cancer, lung cancer and ovarian cancer.[4,5] Current
inhibitors of the Wnt/-catenin pathway fall into five classes (small
molecules, peptides, antibodies, RNA interference and natural
compounds) that target Wnt ligand, its receptor, -catenin
subcellular localization or -catenin transcriptional complex.[4]
Despite efforts to discover Wnt/-catenin targeting agents,
specific drugs against this signaling pathway for the clinical
treatments have not been approved so far.
Sulfonamide Inhibitors of -
Catenin Signaling with Different
Output on c-MYC as Anticancer
Agents
Laura Di Magno,#[a] Fiorella Di Pastena,#[a]
Michela Puxeddu,#[b] Giuseppe La Regina,[b]
Antonio Coluccia,[b] Alessia Ciogli,[b] Simone
Manetto,[b] Marella Maroder,[a] Gianluca
Canettieri,*[a] Romano Silvestri,*[b] Marianna
Nalli*[b]
Recently, we discovered novel -catenin/TCF-dependent
mechanisms of colorectal cancer (CRC) carcinogenesis that
provided preclinical proof of concept for combining -catenin and
NHERF1 pharmacological inhibitors as a novel strategy to
enhance the apoptotic cell death of CRC resistant to current
Wnt/-catenin-targeted drugs. In this paper, we used sulfonamide
1 (FH535) as an inhibitor of TCF/-catenin signaling pathway
since it was reported to suppress β-catenin/TCF-mediated
transcription without affecting β-catenin levels,[6] and to induce
CRC cell cycle arrest without significant apoptosis[7]. We then
sought to identify novel NHERF1 PDZ1 domain inhibitors to be
used in combination with antagonists of -catenin. For this work,
we synthesized 4-((4-bromophenyl)sulfonamido)benzoate (3), as
a new -catenin antagonist that combined with NHERF1 inhibitors
was expected to enhance the CRC cell apoptotic response.
Indeed, compound combinations of the most potent NHERF1
inhibitors with 3 inhibited the CRC cells at submicromolar
concentrations, and showed higher effectiveness than
combinations of the same NHERF1 inhibitors with 1.[8]
[a]
Dr. L. Di Magno, Dr. F. Di Pastena, Prof. M. Maroder, Prof. G.
Canettieri
Laboratory affiliated to Istituto Pasteur Italia – Fondazione Cenci
Bolognetti, Department of Molecular Medicine
Sapienza University of Rome
Viale Regina Elena 291
I-00161 Rome, Italy
E-mail: gianluca.canettieri@uniroma1.it
Dr. M. Puxeddu, Prof. G. La Regina, Dr. A. Coluccia, Prof. R. Silvestri,
Prof. M. Nalli
Laboratory affiliated to Istituto Pasteur Italia – Fondazione Cenci
Bolognetti, Department of Drug Chemistry and Technologies
Sapienza University of Rome
Piazzale Aldo Moro 5
I-00185 Rome, Italy
E-mail: romano.silvestri@uniroma1.it, marianna.nalli@uniroma1.it,
#These authors contributed equally to this work.
Supporting Information for this article is given via a link at the end of the
document.
[b]
Abstract: The Wnt/-catenin pathway is often found deregulated in
cancer. The aberrant accumulation of -catenin in the cell nucleus
results in the development of various malignancies. Specific drugs
against this signaling pathway for clinical treatments have not been
approved yet. Here we report inhibitors of -catenin signaling of
potential therapeutic value as anticancer agents. Compound 14
inhibits the effect on Wnt reporter with IC50 of 7.0 μM, significantly
reduces c-MYC levels, inhibits HCT116 colon cancer cell growth with
IC50 of 20.2 M, does not violate the Lipinski and Veber rules and
shows predicted Caco-2 and MDCK cell permeability Papp >500 nm/s.
Compound 14 seems to have potential for the development of new
anticancer therapies.
Cl
ring B
O
O
O
O
4
S
S
H3C
4’
HN
NO2
HN
Cl
O
H3C
ring A
CH3
O
2 (MSAB)
1 (FH535)
O
O
S
O
O
O
O
Br
R1
S
HN
HN
4-
R2
CH3
17
3
Introduction
Figure 1. Structures of compounds 1−17 (see Table 1).
Wnt/-catenin signaling pathway plays a key role in regulating
tissue homeostasis and is found deregulated in a variety of human
diseases. In the absence of Wnt ligand, -catenin is
phosphorylated in the cytoplasm through the destruction complex.
After dissociation from the complex, -catenin is ubiquitinated by
-transducing repeats-containing proteins (-TrCP) and then
degraded by the proteasome. Alternatively, the destruction
complex phosphorylates -catenin followed by -TrCP
ubiquitination. In the presence of a Wnt ligand, the interaction with
frizzled receptor triggers downstream events which lead -catenin
to translocate into the nucleus, where it prompts the expression
of TCF/LEF target genes through the recruitment of
Handeli and Simon discovered 1 through the screening of a
chemical library using a cell-based -catenin/TCF-responsive
reporter.[7] Compound 1 showed to suppress both Wnt/-catenin
and peroxisome proliferator-activated receptors PPAR and
PPAR and was the best -catenin/TCF inhibitor among four
analogs characterized by the presence of a nitro group located at
the position 4’ or 3’. Later, Kril and co-workers replaced the 2,5-
dichlorophenyl group of 1 with dihalogen patterns.[9] Among them,
only derivatives bearing the 2,6-dihalogenation pattern were more
active than 1; other analogues showed comparable or slightly
improved effect on the TCF/-catenin signaling (TOPFlash
reporter assay) as well as on [3H]-thymidine incorporation assay.
transcriptional
coactivators
and
changes
to
histone
modifications.[1-3]
1
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10.1002/cmdc.202000594
ChemMedChem
COMMUNICATION
Hwang et al. conducted high-throughput screening to
identify modulators of -catenin-mediated Wnt signaling in
HCT116 cancer cells. These studies led to identify 2 (MSAB) as
an inhibitor of Wnt/-catenin signaling with selective antitumor
effect on Wnt-dependent cancer cells in vitro and in mouse cancer
models.[10] In comparing the structure-activity relationships
(SARs) by Kril and Hwang, we found some inconsistency. In
particular, Kril reported that the dihalogenation pattern at the ring
A correlated with an effective inhibition of Wnt/β-catenin signaling
pathway. On the contrary, Hwang reported that the introduction of
a chlorine or fluorine atom at position 4 of this ring resulted in loss
of inhibitory activity towards the Wnt/β-catenin signaling pathway.
Moreover, analogues of MSAB bearing substitutions at position 4’
of the ring B were not reported by Hwang (Figure 1).
Results and Discussion
Inhibition of -catenin signaling. Compounds 4-14 inhibited the -
catenin with IC50 values ranging from 7.0 (14) to 18.3 M (8). In
the luciferase-based TOPFlash assay four analogs (4, 11, 12 and
14) were more effective than compound 3 (Table 1). Derivatives
with the methoxy (11), chlorine (12) or trifluoromethyl (14) at
position 4 and the ethyl ester at position 4’ showed the highest
inhibition of -catenin pathway with IC50 values of 9.7, 10.7 and
7.0 M, respectively. Notably, shifting of the ethyl ester from
position 4’ to 2’ resulted in an improvement of the -catenin
inhibitory activity (compare 3, IC50 = 14.1 M with 4, IC50 = 11.1
M). As an inhibitor of -catenin, the methyl ester 10 (IC50 = 14.8
M), bearing the same ester function of MSAB, showed slightly
decreased activity than 3 (IC50 = 14.1 M). On the contrary, the
methyl ester 5 (IC50 = 15.1 M) was more potent than the corre-
sponding ethyl ester 6 (IC50 = 16.9 M). We analyzed the data
from Table 1 comparing the -catenin inhibitory concentrations
(μM) with the predicted logP values (Table 2S, Supporting
Information). Compounds 6-8 bearing the ethyl ester group at
position 3’ showed the logP values in the 2.49-3.36 range. The
two most potent inhibitors of -catenin within the series, 14 (IC50
= 7.0 M) and 11 (IC50 = 9.7 M), showed logP values of 3.35 and
2.41, respectively. The solubility in phosphate buffered saline
(PBS) at pH 7.4 of 4 (5.6 M), 11 (44.6 M) and 14 (63.6 M) was
notably lower than that of the corresponding carboxylic acids 15-
17 (PBS solubility of 179.1, 187.7 and 119.1 M, respectively; see
Table 3S, Supporting Information) which were weak inhibitors of
-catenin. With the exception of 11, the presence of a methoxy or
a nitro group had a detrimental effect on the inhibition of -catenin.
These data suggest that both the R1 substituent and the nature
and position of the group at the ring B play key roles in the
inhibition of -catenin.
To further explore this class of Wnt/β-catenin inhibitors, we
carried out the synthesis of a small test set of compounds,
numbered 4-17, by changing the R1 substituents at the ring A and
the position of the R2 groups at the ring B. Sulfonamides 4-14
were easily obtained by reaction of
a methyl or ethyl
aminobenzoate with an appropriate benzenesulfonyl chloride in
anhydrous pyridine at 80 °C for 1 h, Acids 15-17 were prepared
by lithium hydroxide hydrolysis of esters 4, 11 and 14 in aqueous
tetrahydrofuran at 40 °C for 16 h (See Supporting Information).
Table 1. Inhibition of -catenin by Compounds 2-17.
Compd
R1
R2
-Catenin Inhib.[a]
IC50 (M)[b]
3[c]
Br
4’-COOC2H5
2’-COOC2H5
3’-COOCH3
3’-COOC2H5
3’-COOC2H5
3’-COOC2H5
4’-COOCH3
4’-COOCH3
4’-COOC2H5
4’-COOC2H5
4’-COOC2H5
4’-COOC2H5
2’-COOH
14.1
11.1
15.1
16.9
14.0
18.3
15.9
14.8
9.7
4
Br
5
CH3O
CH3O
Br
6
7
8
NO2
CH3O
Br
9
10
11
CH3O
Cl
12
10.7
14.7
7.0
13
NO2
CF3
Br
14
15
50.0
737.9
55.8
14.2
16
CH3O
CF3
CH3
4’-COOH
17
4’-COOH
2 (MSAB)
3’-COOCH3
[a] HCT116 cells were transfected with luciferase-based vectors and treated with
LiCl (50 mM) together with increasing concentrations of the indicated
compounds. Cells were harvested 24 h post treatment and assayed for
luciferase activity. Inhibitions were calculated as the Luciferase/Renilla ratio of
the treated samples vs the Luciferase/Renilla ratio of the untreated (control)
samples and plotted against the log of the compound concentration using
GraphPad Prism software (version 6.0) to generate the IC50 values. [b] Standard
deviations (SDs) went from ± 5% to ± 10% of the indicated IC50 values. [c] Lit.[8]
Figure 2. Effects on cell viability of 4 and 14 in HCT116 cells. The cells were
treated with LiCl (50 mM) and 4 or 14 at the indicated concentrations for 24 h.
Results are expressed as % of proliferation activity compared to control sample
and IC50 values are shown. Data represent the mean ± SD of three independent
experiments, each performed in triplicate.
2
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10.1002/cmdc.202000594
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COMMUNICATION
Inhibition of HCT116 colon cancer cells. We performed cell
growth curve analysis of HCT116 colon cancer cells with
derivatives 4 and 14. We selected 14 since it is the most potent
inhibitor of -catenin within this series; compound 4 is also an
effective inhibitor of -catenin featuring the ethoxycarbonyl group
at position 2’. Both compounds inhibited cell proliferation with IC50
values of 20.2M ± 1.1 (14) and 28.2 ± 1.6 M (4) (Figure 2).
Effect on the Wnt/-catenin signaling. To test the ability of
compounds 4 and 14 to affect Wnt/-catenin signaling, we
transfected human embryonic kidney (HEK) 293T cells with a
reporter vector (M50 Super 8x TOPFlash) containing 8 repeats of
TCF/LEF binding sites, or its negative control containing the
mutated TCF/LEF binding sites (M51 Super 8x FOPFlash).[7] After
transfection, cells were treated with the GSK3 inhibitor LiCl to
activate the Wnt pathway[11] and incubated with increasing
concentrations of 4 or 14. Both compounds displayed a significant
inhibitory effect on Wnt reporter activity in the 293T cells with IC50
values of 7.0 ± 0.5 M (14) and 11.1 ± 0.3 M (4). Conversely,
the two drugs did not affect the activity of FOP reporter, indicating
the specificity of the effect (Figure 3).
Figure 4. Compounds 4 and 14 specifically inhibit Wnt target genes in HCT116
colon cancer cells. HCT116 cells were treated with LiCl (50 mM) and 4 or 14 at
the indicated concentrations for 24 h. -catenin and c-MYC protein levels were
analyzed by western blot. Vinculin was used as a loading control. c-Myc mRNA
levels were measured by qPCR and normalized to the expression of -actin
mRNA and expressed as fold change relative to control sample. Data represent
the mean ± SD of three independent experiments, each performed in triplicate.
**p <0.01, ***p <0.001 as determined by ANOVA
Figure 3. 293T cells were transfected with luciferase-based reporter plasmids
and treated with LiCl (50 mM) and the indicated compounds for 24 h. Luciferase
activity was measured and expressed as % of the control sample. IC50 values
of each compound were determined. Data are represented as means ± SD of
three independent experiments, each performed in triplicate.
Effect on Wnt target genes. Both 4 and 14 inhibited
endogenous -catenin protein levels in HCT116 cells, likely
reflecting the ability of the two compounds to enhance -catenin
proteasomal degradation. In keeping with the decrease of -
catenin protein, mRNA levels of c-Myc, a transcriptional target of
-catenin-TCF/LEF axis, were similarly inhibited in a dose-
dependent fashion (Figure 4).
Figure 5. Compounds 4 and 14 specifically inhibit Wnt target genes in HCT116
colon cancer cells. HCT116 cells were treated with LiCl (50 mM) and 10 M 4
or 10 M 14 for 24 h. c-Myc, Fgf20 and Sall4 (Wnt target genes) mRNA levels
were measured by qPCR and normalized to the expression of β-actin mRNA.
Results are expressed as fold change relative to control sample and represent
the mean ± SD of three independent experiments, each performed in triplicate.
**p <0.01, ***p <0.001 as determined by ANOVA.
3
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10.1002/cmdc.202000594
ChemMedChem
COMMUNICATION
We report new sulfonamides, to further explore this class of
Wnt/β-catenin inhibitors. In particular, compounds number 4 and
14 have also been tested for the capability to inhibit colon cancer
cell growth by affecting β-catenin and c-MYC expression levels.
Both revealed great ability, but it was more pronounced for
compound 14. These findings prompt the synthesis of new
analogs and the set-up of combined experiments with NHERF1
inhibitors. Since sulfonamides have been reported to inhibit
tubulin polymerization,[19] it is possible that this additional off-
target activity may affect the results reported here. The results
deserve to be further addressed in future studies.
Surprisingly, however, the protein levels of c-MYC appeared
to be differently regulated by the two compounds. Indeed, while
14 almost completely abrogated c-MYC protein expression at 100
M concentration, the effect of 4 was barely detectable at the
same dose and required 1 mM concentration to induce c-MYC
downregulation. Hence, this latter evidence suggested that,
besides its effect on -catenin protein stability, 14 could operate
through an additional post-transcriptional mechanism. To address
this possibility, we incubated the cells with the protein synthesis
inhibitor cycloheximide in the absence or presence of the two
compounds for different time points. As shown in Figure 6, c-MYC
degradation rate was not modified by any of the two compounds,
thus suggesting that the lower levels of c-MYC observed after
incubation with compound 14 are related to the ability of this drug
to affect c-MYC biosynthesis rather than its stability.
Experimental Section
The synthesis of compound 4-17 and the biochemical assays
have been described in the Supporting Information.
Acknowledgements
This work was supported by Italian PRIN 2015 (2015FCHJ8E to
R.S.), Sapienza University grants RP11715C7D1CF0D1 to G.L.R.
and RG11816428A9B4D5 to R.S, and by AIRC-IG 17575, AFM-
Telethon
grant
#21025,
Sapienza
University
grant
RM1181642798C54A to G.C. We also gratefully thank Istituto
Pasteur Italia – Fondazione Cenci Bolognetti. Co-authors from
Department of Drug Chemistry and Technologies acknowledge
the Italian Ministry of Education, Universities and Research -
Dipartimenti di Eccellenza - L. 232/2016.
Figure 6. c-MYC is not targeted for degradation by compounds 4 and 14 in CRC
cells. HCT116 cells were treated with 4 (50 M) or 14 (50 M) for 2 h.
Cycloheximide (100 μg/mL) was added and the cells were harvested at the
indicated time points. c-MYC and vinculin (as a loading control) protein levels
were analyzed by immunoblot. Graph represent percentage of remaining c-
MYC after cycloheximide addition. Data represent the mean ± SD of three
independent experiments, each performed in triplicate. DMSO as a control.
Conflict of Interest
The authors declare no conflict of interest.
The drug-like properties of compounds 4 and 14 were
predicted through most representative descriptors. According to
the Lipinski[12] and Veber[13] rules, both compounds were
predicted to have a good absorption after oral administration.
Furthermore, the predicted Caco-2 and MDCK cell permeability
was in the range of recommended values (Papp >500 nm/s)[14]
(Table 3S, Supporting Information). Therefore, these compounds
are expected to be well absorbed.
Keywords: -catenin • c-MYC • colorectal cancer • sulfonamide
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Table of Contents
A small test set of sulfonamides was synthesized to further explore this class of Wnt/β-catenin inhibitors. Compound 14 inhibits
HCT116 colon cancer cell growth with IC50 of 20.2 μM and the effect on Wnt reporter with IC50 of 7.0 μM in HEK293T cells, and
significantly reduces c-MYC levels. Compound 14 shows to have potential for the development of new anticancer agents
6
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