Drug Design and Synthesis of First in Class PDZ1 Targeting NHERF1 Inhibitors as Anticancer Agents

Article abstract of DOI:10.1021/acsmedchemlett.8b00532

Targeted approaches aiming at modulating NHERF1 activity, rather than its overall expression, would be preferred to preserve the normal functions of this versatile protein. We focused our attention on the NHERF1/PDZ1 domain that governs its membrane recruitment/displacement through a transient phosphorylation switch. We herein report the design and synthesis of novel NHERF1 PDZ1 domain inhibitors. These compounds have potential therapeutic value when used in combination with antagonists of β-catenin to augment apoptotic death of colorectal cancer cells refractory to currently available Wnt/β-catenin-targeted agents.

Full text of DOI:10.1021/acsmedchemlett.8b00532

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Letter
Drug Design and Synthesis of First in Class PDZ1
targeting NHERF1 Inhibitors as Anticancer Agents
Antonio Coluccia, Giuseppe La Regina, Valentina Naccarato, Marianna Nalli, viviana orlando, Stefano
Biagioni, Maria Laura De Angelis, Marta Baiocchi, Candice Gautier, Stefano Gianni, Fiorella Di
Pastena, Laura Di Magno, Gianluca Canettieri, Addolorata Maria Luce Coluccia, and Romano Silvestri
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00532 • Publication Date (Web): 14 Jan 2019
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Drug Design and Synthesis of First in Class PDZ1 target-
ing NHERF1 Inhibitors as Anticancer Agents
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†
†
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†
fi
Antonio Coluccia, Giuseppe La Regina, Valentina Naccarato, Marianna Nalli, Viviana Orlando, Stefano
fi
ß
ß
§
§
Biagioni, Maria Laura De Angelis, Marta Baiocchi, Candice Gautier, Stefano Gianni, Fiorella Di
Pastena, Laura Di Magno Gianluca Canettieri, Addolorata Maria Luce Coluccia, Romano Silvestri,
∞
,¢
∞
‡
†,*
†
Department of Drug Chemistry and Technologies, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia –
Fondazione Cenci Bolognetti, Piazzale Aldo Moro 5, I-00185 Roma, Italy
∞
Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena, 291, I-00161 Roma, Italy, Laboratory affiliat-
ed to Istituto Pasteur Italia – Fondazione Cenci Bolognetti, Piazzale Aldo Moro 5, I-00185 Roma, Italy
‡
Department of Biological and Environmental Sciences and Technologies, University of Salento, I-73100 Lecce, Italy
§
Department of Biochemistry, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia – Fondazione Cenci Bolo-
gnetti, Piazzale Aldo Moro 5, I-00185 Roma, Italy
ß
Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
fi
Department of Biology and Biotechnologies “Charles Darwin”, Piazzale Aldo Moro 5, I-00185 Roma, Italy
¢
Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, 00161 Rome, Italy
KEYWORDS: Cancer, NHERF1, PDZ domain, β-Catenin, drug design, synthesis
ABSTRACT: Targeted approaches aiming at modulating NHERF1 activity, rather than its overall expression, would be preferred to preserve
the normal functions of this versatile protein. We focused our attention on the NHERF1/PDZ1 domain that governs its membrane recruit-
ment/displacement through a transient phosphorylation switch. We herein report the design and synthesis of novel NHERF1 PDZ1 domain
inhibitors. These compounds deserve potential therapeutic value when used in combination with antagonists of β-Catenin to augment apop-
totic death of colorectal cancer cells refractory to currently available Wnt/β-Catenin-targeted agents.
NHERF1 (Na+/H+ exchanger 3 regulating factor 1) is an
integral membrane adaptor protein of 358 amino acids
carrying two NH2-terminal PDZ (post-synaptic density
95/discs large/zona occludens 1) tandem domains.1 PDZ1
(11-97 amino acids) and PDZ2 (150-237 amino acids)
show 74% identity to each other and bind to specific
carboxyl-terminal motifs on target proteins, such as β-
Catenin and PTEN, that may have a pivotal role in
tumorigenesis. Oncogenic activity of NHERF1 is strictly
dictated by changes on its subcellular localization.1,2 Whilst
a loss of its physiologic membranous expression usually
occurs in early dysplastic human adenomas, especially
those of colon and breast, NHERF1 is progressively re-
expressed throughout the cytoplasm and nuclei in highly
invasive/metastatic carcinomas. Recent evidence also
earmarked NHERF1 as a major driver of a cytoprotective
autophagic responses elicited by therapeutic inhibition of
the Wnt/β-Catenin signaling cascade which triggers
colorectal cancers (CRC).3 Notably, these studies have
available Wnt-targeted agents, in particular at early stages
of the disease
Previous studies focused on small molecule ligands of the
PDZ1-domain of NHERF1 to prevent its ectopic
cytoplasmic/nuclear mislocalization and oncogenic
function.4-6 These studies predicted the canonical
inactivating binding motif for the NHERF1 PDZ1-domain
on four D/E-(S/T)-X-(L/V/I/M) residues that are commonly
contained at the C-terminal of its specific ligands, such as
DSLL for the β2-adrenergic receptor (β2-AR) or ETWM
for the parathyroid hormone receptor (PTHR).7
The crystal structure of the PDZ1-β2-AR C-terminal
peptide is available at the protein data bank (PDB code
1GQ4).8 The complex was fixed cutting the chimeric
covalent bond between PDZ1 and the substrate (-NDSLL)
and submitted to molecular dynamics (MD) simulations to
obtain the reasonable binding conformations. MD of the
complex PDZ1-PTH1R C-terminal peptide (WETVM) was
also performed (Figures 1S and 2S, Supporting
Information). Trajectory analyses showed that the systems
were stable during simulation time of 50 ns, with an
started to explore
inhibitory strategy as a fruitful approach to augment
apoptotic death of CRC cells refractory to currently
a combined β-Catenin/NHERF1-
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Table 1. Structure and Activity of Compounds 1-14 in
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average PDZ1 Ca RMSD of 1.66 and 1.73 Å for PDZ1-
NDSLL-COOH and PDZ1-WETVM-COOH respectively.
The main interactions between the PDZ1 and the -β2-AR
substrate observed at the crystal structures were retained
during the MD simulations (Supporting Information). The
trajectories were clustered by the RMSDs of the binding
site residues giving 5 clusters for the two studied
complexes, each containing representative structures. The
1
2
3
4
5
6
7
8
Ls174TshβCat Cells Expressing Low (-DOX) or High (+DOX)
Levels of the Protein Target NHERF1.a
Cl
Cl
R
R
N
H
N
H
1-7
8-14
obtained ten structures guided the design of
a
Ls174Tsh
pharmacophore model (Figure 3S, Supporing Information)
that was used to filter out an in-house training set of about
6000 compounds. One out of six selected compounds
showed significant NHERF1 inhibition.3 We herein report
the design and synthesis of new NHERF1 inhibitors 1-14.
In preliminary docking studies, these compounds matched
the requirements of the pharmacophore model, in particular
the interactions at the t-COOH binding pocket.9
9
IC50 (µM)
10
11
12
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Compd
1
R
-Dox
+Dox
Δ
H
N
62 ± 4
24 ± 4
38
O
OH
OH
H
N
2
3
4
66 ± 2
65 ± 2
62 ± 2
25 ± 2
24 ± 5
22 ± 3
41
41
40
O
H
N
OH
OH
O
H
N
O
H
N
5
74 ± 6
12 ± 4
62
O
OH
O
O
H
N
OH
6
7
66 ± 4
64 ± 8
25 ± 2
22 ± 6
41
42
O
O
OH
H
N
O
O
OH
8
9
86 ± 24
82 ± 4
55 ± 21
8 ± 4
31
74
N
H
O
OH
N
H
O
N
H
10
11
84 ± 4
65 ± 6
5 ± 2
79
43
OH
O
O
OH
22 ± 2
N
H
O
OH
12
13
14
66 ± 2
85 ± 2
68 ± 4
25 ± 2
10 ± 2
25 ± 2
41
75
43
N
H
O
O
N
H
OH
OH
O
OH
O
N
H
aLs174T cells stably transfected with Dox-inducible shRNA for β-
Catenin (Ls174Tshβ-Cat) were cultured without or with 2 μg/mL of
Dox (-Dox/+Dox) for 1, 3 or 5 days.
Compounds 1-14 were synthesized starting from 5-chloro-
1H-indole-2-carboxylic acid (18) or its 3-isomer 19
(Scheme 1, Supporting Information). The acids were
transformed into the corresponding acyl chlorides with
thionyl chloride and coupled with an appropriate
aminobenzoate in dichloromethane in the presence of
pyridine to afford carboxamides 20-24. Hydrolysis of the
ester functionality of 20-24 was performed with lithium
hydroxide monohydrate in tetrahydrofuran (5 and 6) or
with 3N sodium hydroxide in ethanol at 25 °C (7, 11 and
12). Alcohol derivatives 1, 3, 4 and 8-10 were obtained by
tetrabutylammonium fluoride cleavage in tetrahydrofuran
at 25 °C of the corresponding tert-butyldimethylsilyl
derivatives 25-30. The latter compound were prepared by
Figure 1. Proposed binding modes. Top panel: derivatives 5 (cyan),
6 (orange) and 7 (magenta); bottom panel: derivatives 9 (cyan) and
12 (orange). Residues involved in interactions are reported as stick.
PDZ1 is reported as cartoon (green). The H-bonds are depicted as
yellow dot lines.
reaction
of
18
or
19
with
tert-butyl
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dimethylsilyloxymethylanilines 31-33. Carboxamides 2,
were involved in H-bond/polar contact with the terminal
COOH binding pocket (Figure 1, bottom panel).
1
2
3
4
5
6
7
8
13, 25-30 and 34 were synthesized from the corresponding
anilines in the presence of tripyrrolidinophosphonium
hexafluorophosphate (PyBOP) and trietylamine in DMF at
25 °C. Compound 14 was obtained after lithium hydroxide
of the intermediate methyl tyrosinate 34.
The binding of 9, 10 and 13 to NHERF1 PDZ1 was
assessed by means of a dansylated peptide corresponding to
the C-terminal sequence of β2-AR (DNDSLL) using a a
fluorescent pseudo-wild type produced by replacing Tyr38
of PDZ1 with a Trp.9 The presence of a constant
concentration of 9, 10 or 13 abolished the binding C-
terminal sequence of β2-AR with an affinity of about 10
µM, thus validating these compounds as specific inhibitors
of NHERF1/PDZ1 domain (Figure 2). To further test the
ability of 9, 10 and 13 to bind NHERF1 PDZ1, we
performed urea-induced denaturation in the presence of the
inhibitors. In fact, the ability to bind the native, rather than
the denatured, state of the protein induces a stabilization of
the domain. Such a stabilization is correlated to the affinity
of the compounds to PDZ1 NHERF1 (Figure 4S,
Supporting Information). The results provide compelling
evidence of the inhibition of NHERF1 PDZ1 by derivatives
9, 10 and 13.
The ability of 1-14 to inhibit NHERF1 was assessed in
Ls174T CRC cells harboring a Doxycycline hyclate (Dox)-
inducible small hairpin RNA (shRNA) for β-Catenin
(Ls174Tshβ-Cat) (Table 1). Our published data indicated
that such CRC cells exhibited low/undetectable NHERF1
levels at baseline (i.e. in the absence of Dox), whilst they
markedly increase the expression of the protein as well as a
NHERF1-mediated survival response under Dox
treatment.3 The relative activity of 1-14 can be esplicited
by Δ values obtained by subtracting the IC50 values of the
Ls174Tshβ-Cat cells cultured in the presence or in the
absence of Dox. Indeed, NHERF1/PDZ-1 ligand inhibitors
are expected to exert their growth-inhibitory effects only on
β-Catenin-depleted CRC cells which highly re-expressed
the protein target NHERF1, thus showing the highest Δ
values with regard to Dox-untreated cells. Introduction of a
methylene spacer between the 2-carboxamide function and
the phenyl group of 1 (-Dox IC50 = 62 µM; +Dox IC50 = 24
µM) to afford 4 gave a slight improvement of activity.
Moving the hydroxymethyl group of 1 from para to meta
(3) or orto (4) only minimally affected the difference (Δ) of
activity between the two cell lines. Replacement of the
hydroxymethyl of 1 with a carboxylic acid function (5) (-
Dox IC50 = 74 µM; +Dox IC50 = 12 µM) improved the Δ
value from 38 to 62, while the corresponding meta (6) or
orto (7) carboxylic acids gave similar results to 2-4. We
synthesized the corresponding analogs 8-14 at position 3 of
9
10
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12
13
14
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the indole. Derivatives 9 (-Dox IC50 = 82 µM; +Dox IC50
=
8 µM) and 10 (-Dox IC50 = 84 µM; +Dox IC50 = 5 µM)
bearing the hydroxymethyl at position meta and orto (Δ of
74 and 79 respectively) were superior to the para analogue
7 as well as to the carboxylic acids 11 and 12. The 3-
Figure 2. Binding of PDZ1 NHERF1 Y38W to the ligand Dansyl-
NDSLL in the presence (black circles) and in the absence of 5 µM of 9
(green), 10 (red) or 13 (blue). Fluorescence data were recorded in the
presence of 50 mM Na phosphate pH 7.2, 300 mM NaCl, 5 mM DTT,
20% DMSO, at 25 °C. Lines are the best fir to a hyperbolic binding
transition. The binding between PDZ1 NHERF1 Y38W to the ligand
Dansyl-NDSLL was abolished in the presence of 9, 10 or 13.
indolecarboxamide 13 (-Dox IC50 = 85 µM; +Dox IC50
=
10 µM) yielded a pleasant Δ value of 75, that was 1.8 fold
superior to the isomeric derivative 1. In sum, compounds 5,
9, 10 and 13 exhibited a remarkable cytotoxicity and the
highest Δ values on Ls174Tshβ-Cat which highly re-
express the specific protein target NHERF1.
The indole NH and the carboxamide oxygen formed H-
bonds with the Leu27 backbone. Hydrophobic contacts
were observed between the phenyl ring and Tyr24, Phe26
and Ile78, as well as between the indole ring and Val75 and
Val76. Noteworthy, the carboxylic group of derivatives 5-7
formed polar contacts with the Arg80 (Figure 1, top panel).
Derivatives 8-14 showed binding modes with different
patterns in H-bonding. In comparing the alcohol 9 and its
corresponding carboxylic acid 12, the indole NH formed an
H-bond with His72 backbone, the amide oxygen atom
formed an H-bond with His27 side chain, the indole ring
established hydrophobic contacts with Leu28 and Val75,
the phenyl ring arranged hydrophobic contacts with Tyr24
and Phe26 and a pi-cation interaction with the guanidine
moiety of Arg80. The alcohol and the carboxyl moieties
Table 2. Cell Growth Inhibition of DLD-1, SW480 and SW620
Cell Lines by Compounds 5, 9, 10 and 13.a
IC50 (µM)
Compd
DLD-1
68 ± 1
82 ± 2
85 ± 2
84 ± 2
SW480
17 ± 1
13 ± 1
15 ± 1
16 ± 1
SW620
22 ± 1
27 ± 1
30 ± 1
31 ± 1
5
9
10
13
aCytotoxic concentrations for the indicated cell lines. Experiments
were performed in triplicate and repeated at least twice.
Derivatives 5, 9, 10 and 13 with the highest Δ values as
inhibitors of Ls174Tshβ-Cat cells were also tested in three
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CRC cell lines, such as DLD-1, SW480 and SW620. It is
concentration, the most potent combination was 15+17
(IC50 = 0.1 µM). Combinations 10+17 and 15+17 showed
the higher effectiveness, 15+17 was the most potent with
IC50 values of 0.1 and 0.5 µM (Table 3).
1
2
3
4
5
6
7
8
known that NHERF1 is expressed at detectable baseline
levels in SW480 and SW620 but not in DLD-1 cells.3 This
relatively explains why DLD-1 cells showed the highest
IC50 values compared to SW480 and SW620 cells (Table
2), at micromolar concentrations quite similar to those
previously obtained in Dox-untreated Ls174Tshβ-Cat cells
(Table 1) (Additional cancer cell lines are reported in Table
1S, Supporting Information).
Cancer stem cells (CSCs) are receiving growing interest
during the last years, because they have proved to be
selectively resistant to chemotherapics, thus giving an
explanation to the relapse that in most cases follows fast
tumor shrinkage induced by standard therapies. In this
frame, the identification of targeted drugs active on CSCs
may give hope of a full tumor eradication. Preliminary
screening of two APC-mutated CSCs indicated that their
sensitivity to compounds 10 and 17 was broadly
comparable to that of standard cell lines (Table 4). Analysis
on a wider panel of CSCs is ongoing, in order to verify a
possible role of different mutational profiles of individual
CSCs on drug sensitivity.
9
We previously reported that 3-benzyl-5-chloro-N-(4-
(hydroxymethyl)-phenyl)-1Hindole-2-carboxamide (15), a
NHERF1 PDZ1 inhibitor with -Dox IC50 = 52 µM; +Dox
IC50 = 8 µM, synergized N-(2-methyl-4-nitrophenyl)-2,5-
dichlorobenzen-sulfonamide (FH535, 16) and pyrvinium
pamoate inhibitor of β-Catenin-dependent transcription in
triggering massive apoptosis of Ls174T and DLD1 CRC
cells, thereby preventing persistent ERK1/2 phospho-
activation mediated by single β-Catenin targeting.3,10,11 We
therefore speculated the potential benefit of a synergic
activity of NHERF1 PDZ1 and β-Catenin inhibitors in
CRC therapy.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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Table 4. Cell Growth Inhibition of CSC13 and CSC8 Cancer
Stem Cell Lines by Compounds 10 and 17.a
IC50 (µM)
Table 3. Synergistic Cell Growth Inhibition of SW480 and
SW620 Cell Lines by Compounds 10, 15, 16 and 17.a,b
Compd
10
17
Inhibitor
NHERF1
β-Cat
CSC13
12 ± 1
22 ± 2
CSC8
8 ± 1
13 ± 2
aCytotoxic concentrations for the indicated CSCs. Experiments
were performed in triplicate repeated at least twice.
IC50 (µM)
Compd
10
15
16
17
10 + 16
10 + 17
15 + 16
15 + 17
Inhibitor
NHERF1
NHERF1
β-Cat
β-Cat
DLD-1
85 ± 2
65 ±11
28 ± 5
21 ± 2
0.8 ± 2
0.1 ± 2
0.5 ± 3
0.1 ± 2
SW480
15 ± 1
8 ± 1
10 ± 2
8 ± 2
3.0 ± 2
0.3 ± 2
0.9 ± 2
0.1 ± 2
SW620
30 ± 1
13 ± 2
19 ± 1
17 ± 1
22 ± 2
12 ± 2
18 ± 1
0.5 ± 2
Since physiological or pathological roles of NHERF1 are
related to its subcellular localization, targeted approaches
aiming at modulating NHERF1 activity, rather than its
overall expression, would be preferred to preserve the
normal functions of this versatile protein. By far, particular
attention has been paid to the NHERF1/PDZ1-domain that
governs its membrane recruitment/displacement through a
transient phosphorylation switch. We herein characterized
novel NHERF1 PDZ1 domain inhibitors with therapeutic
value when used in combination with antagonists of β-
Catenin. This double targeted strategy deserves potential to
augment apoptotic death of CRC cells refractory to
currently available Wnt-targeted agents, particularly at
early stages of the disease.
NHERF1 + β-Cat
NHERF1 + β-Cat
NHERF1 + β-Cat
NHERF1 + β-Cat
aCytotoxic concentrations for the indicated cell lines. Experiments
b
were performed in triplicate repeated at least twice. Compound com-
binations were in 1:1 ratio.
We compared 10, the compound with the highest Δ value
within this series, and 15,3 with two β-Catenin inhibitors,
FH535 (16),12 and 17 (Chart 1S, Supporting Information).
We tested the effect of increasing concentrations of 17 on
the Wnt signaling pathway, by performing Wnt reporter
assays in 293T cells. We transfected TOPFlash (TOP)
reporter plasmid, containing 8 repeats of TCF/LEF binding
site or FOPFlash (FOP) plasmid, where the TFC/LEF
binding sites were mutated as negative control.13 Wnt
pathway was activated using the GSK3beta inhibitor LiCl.
17 inhibited the activity of TOP reporter, with an IC50 of
14.1 µM (Figure 5S, Supporting Information). Conversely,
this compound failed to prevent the activity of FOP
reporter, indicating the specificity of the inhibition of the
Wnt pathway.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Additional information for MD simulations and binding
modes, experimental procedures of synthesis, computational stud-
ies, NHERF1 PDZ1 binding studies, Wnt reporter assay, cell cul-
tures and biological assays, additional cancer cell growth inhibi-
tion”.
Indeed, compound 1:1 combination of a NHERF1 inhibitor
with a β-Catenin inhibitor significantly improved the
growth inhibition of all CRC cells. All combinations were
superior to each single tested compound in DLD-1 and
SW480 cells. Compound combinations 10+17, 15+16 and
15+17 inhibited the SW480 cells at submicromolar
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 06 4991 3800. Fax: +39 06 4991 3133. E-mail:
romano.silvestri@uniroma1.it.
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knockdown promotes NHERF1-mediated survival of colorectal cancer
cells: implications for a double-targeted therapy. Oncogene 2018, 37,
3301-3316.
Author Contributions
1
2
3
4
5
6
7
8
Chemistry: A.C., computational studies; G.L.R., V.N. and M.N., che-
mical synthesis. Biology: V.O., cancer cells; M.B., cancer stem cells;
C.G. and S.G., PDZ domain; F.D.P., L.D.M. and G.C., β-Catenin;
A.M.L.C., NHERF1.
(4) Mayasundari, A.; Ferreira, A. M.; He, L.; Mahindroo, M.; Bashford, D.;
Fujii, N. Rational design of the first small-molecule antagonists of
NHERF1/EBP50 PDZ domains. Bioorg. Med. Chem. Lett. 2008, 18,
942-945.
(5) Du, G.; Gu, Y.; Hao, C.; Yuang, Z.; He, J.; Jiang, W. G.; et al. The cellu-
lar distribution of Na+/H+ exchanger regulatory factor 1 is determined
by the PDZ-I domain and regulates the malignant progression of
breast cancer. Oncotarget 2016, 7, 29440-29453.
(6) Fitzpatrick, J. M.; Pellegrini, M.; Cushing, P. R.; Mierke, D. F. Small
molecule inhibition of Na(+)/H(+) exchange regulatory factor 1 and
parathyroid hormone 1 receptor interaction. Biochemistry 2014, 53,
5916-5922.
(7) Mamonova, T.; Kunikova, M.; Friedman, P. A. Structural basis for
NHERF1 PDZ domain binding. Biochemistry 2012, 51, 3110-3120.
(8) Karthikeyan, S.; Leung, T.; Ladias, J. A. A. Structural determinants of
the Na+/H+ exchanger regulatory factor interaction with the beta 2
adrenergic and platelet-derived growth factor receptors. J. Biol. Chem.
2002, 277, 18973-18987.
Funding Sources
We gratefully acknowledge funding from Italian PRIN 2015
(2015FCHJ8E
to
R.S.),
Sapienza
University
2017
(RP11715C7D1CF0D1 to G.L.R.), AIRC-IG (14236 to A.M.L.C. and
17575 to GC), AFM-Telethon 21025 and Institute Pasteur Italy (to
G.C.), and from European Union’s Horizon 2020 research and innova-
tion program under the Marie Sklodowska-Curie grant agreement No
675341 (to S.G).
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
(9) Gianni, S.; Engström, A.; Larsson, M.; Calosci, N.; Malatesta, F.;
Eklund, L.; Ngang, C. C.; Travaglini-Allocatelli, C.; Jemth, P. The ki-
netics of PDZ domain-ligand interactions and implications for the bin-
ding mechanism. J. Biol. Chem. 2005, 280, 34805-34812.
(10) Handeli, S.; Simon, J. A. A small-molecule inhibitor of Tcf/beta-
M50 Super 8x TOPFlash and FOPFlah plasmids were a gift from Ran-
dall Moon (Addgene plasmid # 12456).
ABBREVIATIONS
NHERF1, Na+/H+ exchanger 3 regulating factor 1; PDZ, post-synaptic
density 95/discs large/zona occludens 1; CRC, colorectal cancer; Dox,
doxycycline.
catenin signaling down-regualtes PPARγ and PPARδ activities. Mol.
Cancer Ther. 2008, 7, 521-529.
(11) Thorne, C. A.; Hanson, A. J.; Schneider, J.; Tahinci, E.; Orton, D.; Cse-
lenyi, C. S.; Jernigan, K. K.; Meyers, K. C.; Hang, B. I.; Waterson, A.
G.; Kim, K.; Melancon, B.; Ghidu, V. P.; Sulikowski, G. A.; La Fleur,
B.; Salic, A.; Lee, L, A.; Miller, D. M. 3rd; Lee, E. Small-molecule inhibi-
tion of Wnt signaling through activation of casein kinase 1α. Nat.
Chem. Biol. 2010, 6, 829-836.
(12) Chen, Y.; Rao, X.; Huang, K.; Jiang, X.; Wang, H.; Teng, L. FH535
inhibits proliferation and motility of colon cancer cells by targeting
Wnt/β-catenin signaling pathway. J. Cancer 2017, 8, 3142-3153.
(13) Veeman, M. T.; Slusarski, D. C.; Kaykas, A.; Louie, S. H.; Moon, R. T.
Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, re-
gulates gastrulation movements. Curr. Biol. 2003, 13, 680-685.
REFERENCES
(1) Vaquero, J.; Nguyen Ho-Bouldoires T. H.; Claperon, A.; Fouassier, L.
Role of the PDZ scaffold protein NHERF1/EBP50 in cancer biology:
from signaling regulation to clinical relevance. Oncogene 2017, 36,
3067-3079.
(2) Georgescu, M. M.; Morales, F. C.; Molina, J. R.; Hayashi, Y. Roles of
NHERF1/EBP50 in cancer. Curr. Mol. Med. 2008, 8, 459-468.
(3) Saponaro, C.; Sergio, S.; Coluccia, A.; De Luca, M.; La Regina, G.; Mo-
logni, L.; Famiglini, V.; Naccarato, V.; Bonetti, D.; Gautier, C.; Gianni,
S.; Vergara, D.; Salzet, M.; Fournier, I.; Bucci, C.; Silvestri, R.; Gamba-
corti Passerini, C.; Maffia, M.; Coluccia, A. M. L. AML. β-Catenin
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Drug design
b
-Catenin
NHERF1 PDZ1 domain
IC50 = 14.1 µM
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5
9
17
10
11
12
13
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15
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Synergic effect
IC50 (µM)
Ls174Tsh
IC50 (µM)
Compd
DLD-1
SW480
SW620
Compd -Dox
+Dox
10 (NHERF1)
15 (NHERF1)
17 (β-Catenin)
79 10 + 17
75 15 + 17
85 ± 2
65 ±11
21 ± 2
0.1 ± 2
0.1 ± 2
15 ± 1
8 ± 1
8 ± 2
0.3 ± 2
0.1 ± 2
30 ± 1
13 ± 2
17 ± 1
12 ± 2
0.5 ± 2
Δ
62
74
5
9
10
74 ± 6
82 ± 4
84 ± 4
85 ± 2
12 ± 4
8 ± 4
5 ± 2
15
NHERF1 inhibitors
Pharmacophore
13
10 ± 2
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