Small molecule modulators of Wnt/β-catenin signaling

Article abstract of DOI:10.1016/j.bmcl.2013.01.101

The Wnt signal transduction pathway is dysregulated in many highly prevalent diseases, including cancer. Unfortunately, drug discovery efforts have been hampered by the paucity of targets and drug-like lead molecules amenable to drug discovery. Recently, we reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling by a unique mechanism, though the target responsible remains unknown. We interrogated the mechanism and structure-activity relationships to understand drivers of potency and to assist target identification efforts. We found inhibition of Wnt signaling by Niclosamide appears unique among the structurally-related anthelmintic agents tested and found the potency and functional response was dependent on small changes in the chemical structure of Niclosamide. Overall, these findings support efforts to identify the target of Niclosamide inhibition of Wnt/β-catenin signaling and the discovery of potent and selective modulators to treat human disease.

Full text of DOI:10.1016/j.bmcl.2013.01.101

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2187–2191
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Small molecule modulators of Wnt/b-catenin signaling
Robert A. Mook Jr. a, , , Minyong Chen a, , Jiuyi Lu a, Larry S. Barak b, H. Kim Lyerly c, Wei Chen a,
⇑
⇑
a Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States
b Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, United States
c Department of Surgery, Duke University Medical Center, Durham, NC 27710, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The Wnt signal transduction pathway is dysregulated in many highly prevalent diseases, including can-
cer. Unfortunately, drug discovery efforts have been hampered by the paucity of targets and drug-like
lead molecules amenable to drug discovery. Recently, we reported the FDA-approved anthelmintic drug
Niclosamide inhibits Wnt/b-catenin signaling by a unique mechanism, though the target responsible
remains unknown. We interrogated the mechanism and structure–activity relationships to understand
drivers of potency and to assist target identification efforts. We found inhibition of Wnt signaling by
Niclosamide appears unique among the structurally-related anthelmintic agents tested and found the
potency and functional response was dependent on small changes in the chemical structure of Niclosa-
mide. Overall, these findings support efforts to identify the target of Niclosamide inhibition of Wnt/
b-catenin signaling and the discovery of potent and selective modulators to treat human disease.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 14 November 2012
Revised 15 January 2013
Accepted 22 January 2013
Available online 30 January 2013
Keywords:
Wnt signaling inhibitor
Wnt signaling activator
Small molecule
Mechanism of action
Target identification
Niclosamide
The Wnt signaling pathway is a key signal transduction path-
way involved in tissue development and homeostasis, stem cell
maintenance and renewal, and is dysregulated in many can-
cers.1–3 The pathway is of immense importance in biology and
holds promise in the treatment of a number of highly prevalent
diseases.4,5 For example, in colorectal cancer (CRC) more than
80% of all sporadic and hereditary cancers show hyperactivation
of the pathway due to mutations in the adenomatous polyposis coli
(APC) or the b-catenin gene.1,2 As a result, inhibitors of the
pathway are highly sought-after as the basis of a new generation
of targeted therapeutic agents.
A mechanistic understanding of the canonical Wnt/b-catenin
signaling pathway has evolved over the past decade.6 Briefly, Wnt
proteins are secreted glycoproteins that bind and activate the seven
transmembrane receptor Frizzled. Wnt binding to Frizzled results in
activation of cytosolic proteins called dishevelled, leading to inter-
nalization of the Frizzled receptor.7,8 Downstream signaling events
resulting from Wnt binding include the stabilization and transloca-
tion of cytosolic b-catenin proteins into the nucleus, activation of the
transcription factor LEF/TCF and transcription of Wnt/b-catenin
target genes. Overall, the Wnt signal transduction pathway consists
of many protein–protein interactions that traditionally have been
difficult to target with small drug-like molecules.1,9 The barrier to
progress in the field stems from the lack of targets in the pathway
amenable to the design of small drug-like molecules, good chemical
startingpoints, and a gap in our ability to design drugs that penetrate
cells and target intracellular protein–protein interactions. Recent
publications, including our own, have begun to provide insights into
mechanisms and chemical starting points1,10–30 though no approved
drugs specifically targeting the pathway have been identified.1,2,30
Reasoning plasma membrane proteins play critical roles in signal
transduction and offer good targets for therapeutic intervention to
treat human disease, we developed a novel cell-based high-through-
put screening assay capable of detecting inhibitors or activators of
Wnt signaling using a chimeric Frizzled1-GFP receptor (Fig. 1).7,10
In this assay, called the Frizzled internalization assay, a U2OS cell
line stably expressing Frizzled1-GFP (Fzd1GFP-U2OS) localizes Friz-
zled1-GFP predominantly to the plasma membrane with almost no
internalized vesicles present until stimulated with Wnt ligands.
Upon stimulation and visualization with confocal microscopy, a
punctate pattern is observed in cells indicative of receptor transloca-
tion. Upon screening in 384-well format, we discovered the FDA-ap-
proved anthelmintic drug Niclosamide inhibits Wnt/b-catenin
signaling,10 a finding since confirmed by others.31,32 Niclosamide
produces robust internalization of the receptor (Fig. 1B), and despite
expectations that internalization of Frizzled receptor by Niclosa-
mide would activate the pathway, Niclosamide inhibits the pathway
Abbreviations: APC, adenomatous polyposis coli; CRC, colorectal cancer; Dvl,
dishevelled; LEF/TCF, lymphoid enhancer factor/T cell factor; Fzd1, Frizzled1; GFP,
green fluorescent protein; GSK3, glycogen synthase kinase 3; SAR, structure–
activity relationships.
⇑
Corresponding authors. Tel.: +1 919 684 8717 (R.A.M.); tel.: +1 919 684 4433
(W.C.).
E-mail addresses: robert.mook@duke.edu (R.A. Mook), w.chen@dm.duke.edu
(W. Chen).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.101

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R. A. Mook Jr. et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2187–2191
A
B
DMSO
Niclosamide
Wnt
membrane
Fzd1
GFP
DVL
Frizzled1-GFP
GSK3β
C
90
80
70
60
50
40
30
20
10
0
LEF/TCF
-8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0
Log10 [Niclosamide]
Figure 1. Niclosamide promotes Frizzled1-GFP internalization and inhibits Wnt3A-induced Wnt/b-catenin signaling. (A) Illustration of Frizzled-GFP internalization assay
with canonical Wnt signaling. (B) Confocal image of U2OS cells harboring Frizzled1-GFP treated with DMSO or Niclosamide (12.5 M) for 6 h. Punctuate structure is the
internalized vesicles of Frizzled1-GFP. (C) Niclosamide inhibits the activity of Wnt3A-induced TOPflash reporter assay. RLU: relative luciferase unit.
l
A
B
Frizzled
Internalization
(12.5μM)
Compound
Name
Structure
Niclosamide
Oxyclozanide
Rafoxanide
Closantel
+++
-
-
-
-
n.s.
C
150
140
130
120
110
100
90
*
80
70
60
50
Tribromosalicy-
anilide
40
30
20
10
0
Wnt3A
Figure 2. Structures of anthelmintic derivatives tested and their activity in the Frizzled1-GFP internalization and the Wnt3A-stimulated TOPflash b-catenin reporter assay.
(A) Chemical structures of salicylanilide anthelmintics tested and amount of punctate observed after 6 h with different anti-helminthic compounds at 12.5 M in the
l
Frizzled1-GFP internalization assay. Punctate observed in confocal images of Frizzled1-GFP USOS cells were scored visually and with Metamorph software (Molecular
Devices, LLC). 10 to 20 cells per sample were analyzed to reach statistical significance. (B) Confocal images of Frizzled1-GFP U2OS stable cells treated with different anti-
helminthic compounds at 12.5 lM for 6 h; and (C) Wnt-3A stimulated TOPflash reporter activity of salicylanilide anthelmintics. HEK293 cells harboring TOPflash reporter
were treated with DMSO (control) or with Wnt3A-conditional medium and different anthelmintic compounds at 12.5 lM for 8 h. RLU: relative luciferase unit. n.s., not
significant (P >0.05); ⁄P <0.05 (t tests).

R. A. Mook Jr. et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2187–2191
2189
Inhibition
β-catenin
transcription
Frizzled
Internalization TOPFlash
@12.5 μM
(IC50, μM)
Compound
R1
R2
R3
R4
Niclosamide OH
Cl
Cl
Cl
Cl
Br
Br
Cl
Cl
H
Cl
Cl
H
H
Cl
H
4-NO2
4-NH2
2-NO2
3-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-N3
+++
-
-
0.4
>12
>12
2.5
0.26
1.2
1.2
0.44
2.4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
(a)
(a)
+
+++
+++
+++
+++
+++
++
-
H
OCH3
Br
Cl
Cl
Cl
Cl
Cl
H
H
Cl
1.8
>12
>12
0.89
>12
3.4
(a)
(a)
OCH3 Cl
-
OH
OH
OH
OH
OH
Cl
Cl
Cl
Cl
Ph
+++
ND
ND
ND
ND
4-CO2H
4-CN
4-NHAc
(b)
(a)
Cl
Cl
>12
1.3
4-NO2
OH
Cl O-PEG-N3c
4-NO2
++
2.1
a No inhibition of luciferase signal was observed; b low solubility in assay;
c PEG is -(CH2CH2-O)2-CH2CH2- ; ND = not determined
Figure 3. Structure–activity relationships in the Frizzled1-GFP internalization and the TOPflash b-catenin reporter assays. Punctate produced by derivatives in Frizzled1-GFP
U2OS stable cell line were evaluated by confocal imaging and visually scored by the amount of punctate observed: comparable to control (À); trace (+); moderate (++); strong
(+++); or ND (not determined).
by decreasing levels of cytosolic dishevelled2 and b-catenin, and
decreasing Wnt/b-catenin genetranscription(see Fig. 1C).10 Though
the specific molecular target of Niclosamide remains unknown, a
number of studies now indicate the novel mechanism of Niclosa-
mide-mediated inhibition of Wnt/b-catenin signaling can be
exploited to treat CRC.31–33 For example, preclinical studies using
well-known human colon cancer cell lines and patient derived colo-
rectal cancer cells isolated from surgically resected metastatic dis-
ease, as well as studies using patient derived tumor explants in
immunodeficient mice all indicate Niclosamide decreases dishev-
elled and b-catenin levels and inhibits Wnt/b-catenin signaling even
in the presence of APC mutations; findings which strongly support
its use to clinically evaluate its efficacy to treat CRC.33
enabling target identification studies and assessing the potential
to optimize the chemical series for CRC clinical studies. To better
understand the mechanism of Wnt inhibition, we acquired com-
mercially available anthelmintic compounds structurally-related
to Niclosamide—all in the same salicylanilide drug class and all with
reported mechanisms of action involving uncoupling of oxidative
phosphorylation.36,38 We then tested these anthelmintic agents
side-by-side in the Frizzled internalization assay and in the Wnt/
b-catenin gene transcription assay (TOPflash). With the exception
of Niclosamide, none of the compounds tested produced a robust
punctate pattern indicative of Frizzled internalization nor did they
inhibit Wnt3A-stimulated gene transcription in the Wnt/b-catenin
TOPflash gene transcription assay (see Fig. 2). Given these
compounds are all anthelmintic agents and share a common mech-
anism of action, these findings suggest the mechanism responsible
for Niclosamide inhibition of Wnt signaling appears different from
its mechanism of anthelmintic activity.
Intrigued that a mechanism with a novel target may exist, we
purchased and synthesized focused libraries of compounds to con-
duct pilot SAR studies that could enable target identification and
lead optimization efforts (Fig. 3)52 (see also Supplementary data).
In all cases studied, we found a good correlation between Friz-
zled1-GFP receptor internalization and inhibition of Wnt3A-stimu-
lated inhibition in the TOPflash assay (see Supplementary Fig. 1 for
confocal images). Given this correlation, we focused on the TOP-
flash assay to develop the SAR. In the A-ring, methylation of the hy-
droxyl group (R1) (compound 11) or substitution of the hydroxyl
group with an H-atom (compound 10) yielded a loss of activity
Niclosamide is a member of the salicylanilide class of anthel-
mintic agents and is currently listed on the World Health Organiza-
tion’s list of essential medicines.34 It has been used as an
anthelmintic agent in livestock since the early 1960s and was ap-
proved by the FDA for use in humans to treat tapeworm infections
in 1982.35,36 Niclosamide exerts its anthelmintic effect via a mech-
anism involving uncoupling of oxidative phosphorylation, though
no specific target has been identified and the mechanism has not
been well-delineated.37,38 In the years since its discovery, addi-
tional biological activities have been reported.31,32,39–51 However,
in nearly every case, the interaction of Niclosamide with a specific
molecular target was not identified.
Given its intriguing mechanism and promise in treating CRC,10,33
we initiated pilot studies to interrogate the mechanism and
structure-activity relationships of the chemotype with the goal of

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R. A. Mook Jr. et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2187–2191
Figure 4. The Niclosamide derivative DK3-1 synergistically activates TOPflash reporter activity in presence of Wnt-3A ligand. (A) Chemical structure of DK3-1. (B) DK3-1
synergizes with Wnt3A and activates TOPflash reporter activity. HEK293 cells harboring TOPflash reporter were treated without or with Wnt3A conditional medium and
DK3-1 at the concentration indicated for 8 h. ⁄p <0.05, ⁄⁄p <0.001, compared to 5% Wnt3A only sample by t test. (C) Confocal images of Frizzled1-GFP U2OS stable cells treated
with DMSO, Niclosamide (12.5 lM), DK3-1 (12.5 lM), 5% Wnt3A conditioned medium, and combination of 5%Wnt3A and DK3-1 (12.5 lM).
in Wnt/b-catenin signaling. Substituting the 4-chloro substituent
(R2) with a hydrogen atom (compound 8 and 9) generally resulted
in a slight decrease in activity, while substitution by bromine pro-
duced a compound with slightly greater potency (compound 4). In
the B-ring, substitution of the 2-chloro group (R3) with a hydrogen
atom at the B-2 position produced an active compound with a
slight loss in activity (compound 6), while replacement by a meth-
oxy group produced a compound of similar potency (compound 7).
The overall SAR at this position indicated inhibitory potency was
fairly insensitive to substitution and suggested to us that substitu-
tion at this position with a peg-ether tethering group may provide
access to affinity reagents to help identify the target. In the event,
we were pleased to find that a peg-substituted derivative useful to
prepare affinity reagents for target identification studies resulted
in an active inhibitor (compound 17).
The SAR at the para position in the B-ring was particular interest-
ing (Fig. 3). Here, a substituent change within the chemotype not
only effected the potency of inhibition but could also change the
inhibitory functional response to Wnt3A stimulation to an activa-
tion functional response. A dramatic loss of inhibitory potency re-
sulted when the nitro group at B-4 (R4) in the anilide ring was
substituted by an amine (compound 1) or an acetamide (compound
15). Substitution with cyano (compound 14) resulted in comparable
activity to the similarly substituted nitro derivative (compound 6).
To our delight, replacement of the nitro group with an azide group
that could provide access to photo-affinity reagents and assist target
and binding site identification produced an active compound (com-
pound 12). Moving the nitro group to the ortho or meta position re-
sulted in reduced activity (compound 2 and 3, respectively).
Remarkably, substitution of the nitro group with an aryl sulfon-
amide (DK3-1) produced the opposite functional response in the
TOPflash assay and increased b-catenin transcriptional activity
(Fig. 4). This change in functional response was interesting. Upon
further evaluation, we found that DK3-1 synergistically increased
b-catenin gene transcription in a dose dependent manner in the
presence of Wnt3A-ligand (Fig. 4B).53 Compound DK3-1 alone does
not appear to increase the punctate pattern of Frizzled1-GFP in the
Frizzled internalization assay. However, it does produce a small
dose-dependent increase in activity in the TOPflash assay
(Fig. 4C). Co-treatment of DK3-1 and 5% Wnt3A-conditioned media
produces a visible punctate phenotype and a very significant in-
crease in functional response. Overall, the SAR findings indicate that
small variation in structure can change not only the Wnt/b-catenin
inhibitory potency, but also change the functional response. This
dynamic SAR supports a hypothesis that the Wnt-modulating activ-
ity in the Niclosamide chemotype may allow optimization.
Our findings to date suggest the mechanism of action by which
Niclosamide inhibits Wnt signaling is distinct from the mechanism
of anthelmintic activity within the salicylanilide class of anthel-
mintic agents. Our pilot SAR studies indicate that a good correla-
tion exists between the Frizzled internalization assay and the
TOPflash b-catenin transcription assay and that a dynamic SAR ex-
ists within the chemotype. Additional SAR, target ID and mecha-
nism studies are underway. These studies offer the opportunity
to identify small molecule modulators of Wnt/b-catenin signaling
that enhance our understanding of Wnt/b-catenin pathway regula-
tion while at the same time providing therapeutic agents to treat
diseases such as colon cancer that are mediated via dysregulation
of Wnt signaling.
Conflicts of interest
None declared.
Acknowledgments
This work was funded in part by 5R01 CA113656-03 (W.C.), the
Pediatric Brain Tumor Foundation (W.C.) and a Clinical Oncology
Research Center Development Grant 5K12-CA100639-08 (R.A.M.,
M.C.). Wei Chen is a V foundation Scholar and an American Cancer
Society Research Scholar. NMR instrumentation in the Duke NMR
Spectroscopy Center was funded by the NIH, NSF, NC Biotechnology

R. A. Mook Jr. et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2187–2191
2191
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