The development of highly potent inhibitors for porcupine

Article abstract of DOI:10.1021/jm400159c

Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It catalyzes the palmitoylation of Wnt proteins, a process required for their secretion and activity. We recently disclosed a class of small molecules (IWPs) as the first reported Porcn inhibitors. We now describe the structure-activity relationship studies and the identification of subnanomolar inhibitors. We also report herein the effects of IWPs on Wnt-dependent developmental processes, including zebrafish posterior axis formation and kidney tubule formation.

Full text of DOI:10.1021/jm400159c

Brief Article
pubs.acs.org/jmc
The Development of Highly Potent Inhibitors for Porcupine
Xiaolei Wang,† Jesung Moon,‡ Michael E. Dodge,§ Xinchao Pan,∥ Lishu Zhang,§ Jordan M. Hanson,†
Rubina Tuladhar,§ Zhiqiang Ma,† Heping Shi,† Noelle S. Williams,† James F. Amatruda,‡,∥
Thomas J. Carroll,∥ Lawrence Lum,§ and Chuo Chen*,†
†Department of Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas
75390, United States
‡Department of Pediatrics, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390,
United States
§Department of Cell Biology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas
75390, United States
∥Department of Internal Medicine and Molecular Biology, The University of Texas Southwestern Medical Center, 5323 Harry Hines
Boulevard, Dallas, Texas 75390, United States
S
* Supporting Information
ABSTRACT: Porcupine is a member of the membrane-bound O-acyltransferase family of proteins. It catalyzes the
palmitoylation of Wnt proteins, a process required for their secretion and activity. We recently disclosed a class of small
molecules (IWPs) as the first reported Porcn inhibitors. We now describe the structure−activity relationship studies and the
identification of subnanomolar inhibitors. We also report herein the effects of IWPs on Wnt-dependent developmental processes,
including zebrafish posterior axis formation and kidney tubule formation.
INTRODUCTION
■
The Wnt genes encode a large family of cell-to-cell signaling
molecules that are essential to embryonic development and the
regeneration of tissues. Aberrant Wnt signaling plays an
important role in the formation and metastasis of tumors.1
Currently, there is no drug targeting this cellular process in
clinical use.2 We recently identified a class of small molecules
that inhibit the production of Wnt proteins and named them
IWPs for inhibitors of Wnt production.3 We further identified
their molecular target to be Porcupine (Porcn), a membrane-
bound O-acyltransferase (MBOAT) family protein.4 This
acyltransferase catalyzes the palmitoylation of Wnt to enable
its exit from the secretory pathway and subsequent activation of
cellular responses. Compromised Porcn activity commonly
results in developmental disorders including focal dermal
hypoplasia (Goltz syndrome), whereas hyperactivity of Porcn is
associated with cancerous cell growth.5 We envision that
Figure 1. The structures and activities of IWPs identified from a high-
throughput screen in cells exhibiting cell-autonomous Wnt signaling.
inhibition of Porcn will be an effective strategy for broadly
suppressing Wnt signaling and thus hold potential in
regenerative medicine and anticancer applications. Although
genetically based targeting of Wnt signaling components
suggests that chemical inhibitors of Wnt signaling may give
rise to toxic effects, Porcn inhibitors have proven to be
remarkably nontoxic in rodents.6 Indeed, we surmise that these
favorable results in preclinical tests were a prerequisite to the
phase I trials underway for LGK974, a novel Porcn inhibitor.2
The four IWP molecules (1−4) identified in the initial screen
of 200000 compounds7 bear similar molecular skeletons
(Figure 1). They all suppress cell-autonomous Wnt signaling
in mouse fibroblasts at nanomolar concentrations.3 We
consider the phthalazinone moiety of IWP-1 (1) and
pyrimidinone moiety of IWP-2−4 (2−4) exchangeable
scaffolding motifs. The benzothiazole moiety appears to be a
conserved motif, and the phenyl group tolerates both electronic
and steric perturbations. On the basis of this information, we
prepared an IWP−biotin conjugate and an IWP−Cy3
conjugate (5) and used them to demonstrate that IWP-2 (2)
directly binds to Porcn.3 We report herein the subsequent
structure−activity relationship (SAR) studies yielding new
Received: January 30, 2013
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
Porcn inhibitors that suppress Wnt signaling at subnanomolar
concentrations.
Table 1. Effects of the Substituent Groups of the Phenyl and
Benzothiazole Groups of IWP-1 (1) and IWP-2 (2)
a
RESULTS AND DISCUSSION
■
We recently identified 13 additional Porcn inhibitors from the
same screen that netted IWP-1−4 (1−4).8 Five of them (6−
10) possess similar molecular skeletons as IWP-1−4 (1−4) and
provided further SAR information. The discovery of 6−10 as
active Porcn inhibitors confirmed that the phthalazinone and
pyrimidinone moieties are scaffolding motifs. Most importantly,
the phenyl and benzothiazole groups of IWP-1−4 (1−4) can
be replaced by an alkyl group and a simple aromatic group,
respectively. We therefore hypothesized that IWPs bind to
Porcn by fitting the phthalazinone/pyrimidinone and the
benzothiazole regions into the binding pocket (Figure 2).
R2
entry
R1
scaffold
H
p-F
o-OMe
p-OMe
1
OMe
A
B
A
B
A
A
A
B
A
B
A
A
210
40
90
250
90
50
60
120
25
2
3
Me
35
100
4
30
40
30
5
H
F
2600
470
30
>25000
10000
25000
6
7
Cl
25
175
90
15
35
8
40
50
30
9
CF3
110
40
10
11
12
100
100
NO2
240
>25000
COOEt
a
EC50 values in nM.
aromatic groups and examined the activity of molecules with
general structure C (Figure 3). We first found that the
benzothiazole group can be replaced by a quinoline group (13)
despite compromised activity. We next found that the
phenylthiazole derivatives (14 and 15) are also active Porcn
inhibitors, and the 5-phenyl derivative 15 has a similar potency
as IWP-2 (2). We subsequently found that weak or nearly no
activity was observed with simple phenyl derivatives (16−18)
that contain a small substituent group at the 4-position.
However, installing a piperidine group resulted in an equal
potent inhibitor (19) as IWP-2 (2). We therefore decided to
explore the potential of biaryl systems as new Porcn inhibitors.
We first found that the 4-biphenyl derivative 20 was 40 times
more potent than IWP-2 (2) while a significant loss of activity
was observed with the 3- and 2-biphenyl derivatives (21 and
22). We next introduced a nitrogen atom to either the outer or
inner phenyl ring (23−27). We observed slight improvement
of activity and with 24, 26, and 27, which are also more soluble
than IWP-2 (2) and 20. Introduction of an additional nitrogen
atom (28−30) did not further improve the activity. We also
attempted to replace the phenyl group with a furan or
thiophene group (32−37) and found the 2-thiophenyl
derivative 35 a highly potent Wnt inhibitor.
Figure 2. The phthalazinone/pyrimidinone and the benzothiazole
moieties of IWPs are important for their binding to Porcn.
Consistent with this model, we prepared 11 and 12 and found
that they both failed to suppress the Wnt signaling at up to 25
μM in L-Wnt-STF cells, potentially due to reduced hydro-
phobic interactions. In addition, effective biotin and Cy3
conjugates (5) were obtained from derivatizing the phenyl
group of IWP-2 (2), a region that is believed to be exposed to
the solvent.
We started our investigation by examining effects of
substituent groups on the benzothiazole and phenyl moieties
(Table 1). As expected, there is no significant difference in
potency for either the IWP-1 (A) or the IWP-2 (B) scaffolds
harboring adducts to these moieties. Exchanging the substituent
patterns observed in IWP-1−4 (1−4) (R1 = OMe or Me; R2 =
H, p-F, o-OMe, or p-OMe) also did not significantly affect the
activity, (entries 1−4). However, there is a steric preference at
the 6-position of the benzothiazole. Removing the substituent
group at the 6-position (R1 = H) of the benzothiazole led to
drastic loss of activity (entry 5), presumably due to reduced
interactions. Introducing a fluorine atom restored the activity
(entry 6). Further increasing the size of the substituent group
improved the activity (entries 7−11), but there is a limit of the
size that can be tolerated (entry 12). Overall, these results
support the hypothesis that the benzothiazole group fits into a
binding pocket of Porcn. We therefore decided to focus on this
region of the molecule for further SAR studies.
Among the five newly identified subnanomolar IWPs, we
selected 27 for further biological evaluations and named 27
IWP-L6. We confirmed that IWP-L6 (27) effectively sup-
pressed the phosphorylation of dishevelled 2 (Dvl2) in
HEK293 cells, a biochemical event associated with many
Wnt-dependent cellular responses (Figure 4).8,9 We further
profiled the in vivo stability of IWP-L6 (27) (Figure 5).
Whereas IWP-L6 (27) was stable in human plasma over 24 h, it
was rapidly metabolized in rat plasma (t1/2 = 190 min), murine
plasma (t1/2 = 2 min), and the murine liver S9 fractions (t1/2
=
Motivated by the Porcn-inhibiting activities associated with
6, 9, and 10, we replaced the benzothiazole group with other
26 min). The major metabolites are the amide cleavage
products (see Supporting Information). Similar species-
B
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Journal of Medicinal Chemistry
Brief Article
Figure 5. Stability of IWP-L6 (27) in plasma.
Figure 6. Inhibition of the regeneration of tailfin of juvenile zebrafish.
posterior axis formation, a Wnt/β-catenin dependent devel-
opmental process, at low micromolar concentrations (Figure
7). IWP-L6 (27) and 35 are therefore at least 10 times more
potent than IWP-12 (7) and 2.5 times more potent than IWR-1
in this in vivo assay.16 While there is only 69% sequence
identity between mouse Porcn and zebrafish Porcn, the in vitro
EC50 values (Figure 3) measured in mouse fibroblasts (L cells)
correlate with the in vivo activity observed in fish but not
linearly.
Figure 3. Effects of the arylamide groups on the Porcn-inhibiting
activity.
We have previously shown that IWP2 (2) specifically and
reversibly blocks Wnt signaling and Wnt mediated branching
morphogenesis in cultured mouse embryonic kidneys.8,17 We
now show that IWP-L6 (27) is 100 times more potent than
IWP-2 (2) in these experiments. We cultured embryonic day
(E) 11.5 kidneys in media containing various doses (1 nM to 1
μM) of IWP-L6 (27). Doses of 10 nM and above significantly
reduced branching morphogenesis relative to DMSO treated
controls (Figure 8). Doses of 50 nM and above completely
blocked branching morphogenesis, indicating a complete
inhibition of Wnt signaling. In comparison, a dose of 5 μM
of IWP-2 (2) was required to obtain similar results.8,17
Figure 4. IWP-L6 (27) blocks the phosphorylation of the cytoplasmic
Wnt pathway effector Dvl2.
dependent metabolitic profiles due to the involvement of
carboxylesterase (CES) have been reported with other drug
candidates.10,11 These observations are consistent with the
elevated activity of CES in mouse and rat but not human.12−14
Despite its modest metabolic stability in mouse-derived
plasma, IWP-L6 (27) was highly active in zebrafish. We had
previously shown that both the tankyrase (Tnks) inhibitor
IWR-1 and the Porcn inhibitor IWP-12 (7) effectively block the
regeneration of the tailfin, a Wnt-dependent process, in adult
and juvenile fish.3,8,16 We herein show that IWP-L6 (27)
exhibited more potent activity (Figure 6). We further show that
IWP-L6 (27) and 35, but not 30 and 32, effectively inhibited
CONCLUSION
■
The SAR data presented herein suggest that two structural
motifs of IWPs interact with Porcn and are important for the
activity. We further showed that optimization of one of these
motifs led to a significant improvement in activity. The newly
discovered IWP-L6 (27) is a subnanomolar Porcn inhibitor. It
C
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Journal of Medicinal Chemistry
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Figure 8. IWP-L6 (27) inhibits Wnt mediated branching morpho-
genesis in cultured embryonic kidneys. Hoxb7Cre;RosaTomato
kidneys were dissected from E11.5 embryos and cultured at the air/
media interface (top).8,17 Media was replaced every 24 h. Images were
captured every 24 h using a Zeiss Lumar V12 fluorescent stereoscope.
Magnitude: 80× at 0 and 24 h; 60× at 48 h. The fold increase in the
number of ureteric bud tips between time = 0 and 48 h for each
treatment was quantified (bottom). Statistics were performed using
Student t test.
Figure 7. IWP-L6 (27) inhibits posterior axis formation. Zebrafish
embryos harboring an mCherry-based reporter of Wnt/β-catenin
signaling15 were treated with increasing concentrations of IWP-L6
(27) in the aquarium water (top). Fluorescence intensity of animals
was quantified as before.8 Activity of the IWP compounds IWP-L6
(27), 30, 32, and 35 was measured using the posterior axis formation
assay (bottom).
dimension 4.6 mm × 150 mm using an Alltech 3300 evaporative light
scattering detector. The purity of all compounds for biologically assays
was determined to be >95% by HPLC.
has good stability in human plasma but compromised stability
in rat and mouse plasma. The in vivo activities of IWP-L6 (27)
were also validated by their ability to disrupt well-established
Wnt-dependent developmental processes of embryonic and
juvenile zebrafish and the branching morphogenesis in cultured
mouse embryonic kidneys. Further tuning of its activity and
stability is underway.
Synthesis of IWP-L6 (27). To
a solution of 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU, 26 mL, 0.17 mol) in methanol
(90 mL) were added methyl thioglycolate (14.4 mL, 0.158 mol) and
acrylonitrile (12 mL, 0.17 mol) at 0 °C. The solution was stirred at 0
°C for 5 h and then at 80 °C overnight. After cooling to room
temperature, the solvent was evaporated, quenched with a saturated
solution of ammonium chloride, and extracted with ethyl acetate. The
organic layer was dried over sodium sulfate, concentrated, and purified
by silica gel column chromatography (40% ethyl acetate/hexanes) to
give 3-amino-2-(methoxycarbonyl)4,5-dihydrothiophene (9.72 g,
EXPERIMENTAL SECTION
■
General. All chemical reactions were performed in glassware under
a positive pressure of argon. The normal-phase flash column
chromatography was performed with EMD silica gel 60 (230−400
mesh ASTM). TLC analyses were performed on EMD 250 μm Silica
Gel 60 F254 plates and visualized by quenching of UV fluorescence
1
39%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 2.86 (t, J =
8.0 Hz, 2H), 3.03 (t, J = 8.0 Hz, 2H), 3.68 (s, 3H).18
A solution of 3-amino-2-(methoxycarbonyl)4,5-dihydrothiophene
(1.00 g, 5.78 mmol) and phenyl isothiocyanate (937 mg, 6.94 mmol)
in pyridine (18 mL) was stirred at 100 °C overnight. The solvent was
then evaporated, and the residue was purified by silica gel column
chromatography (30% ethyl acetate/hexanes then acetone) and then
washed three times with ethyl acetate to give pure 3-phenyl-6,7-
dihydrothieno[3,2-d]pyrimidine-2-thione-4-one (635 mg, 42%). 1H
NMR (400 MHz, DMSO-d6) δ 13.17 (s, 1H) 7.30−7.50 (m, 3H),
7.08−7.20 (m, 2H), 3.20−3.40 (m, 4H).
1
(λmax = 254 nm) or by staining ceric ammonium molybdate. H and
13C NMR spectra were recorded on Varian Inova-400. Chemical shifts
for H and 13C NMR spectra are reported in ppm (δ) relative to the
1
1H and 13C signals in the solvent (CDCl3: δ 7.26, 77.16 ppm; DMSO-
d6: δ 2.50, 39.52 ppm) and the multiplicities are presented as follows:
s, singlet; d, doublet; t, triplet; m, multiplet. Mass spectra were
acquired on Agilent 6120 Single Quadrupole LC/MS. Analytic HPLC
was performed using an Eclipse XDB-C18 5 μm column with
D
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Journal of Medicinal Chemistry
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To a solution of 5-phenylpyridin-2-amine (170 mg, 1.00 mmol) in
benzene/THF (9/1, 10.0 mL) was added a solution of chloroacetyl-
chloride (0.111 mL, 1.40 mmol) in benzene (1.0 mL). The reaction
mixture was then stirred at 50 °C overnight. After cooling to room
temperature, the solution was washed with a saturated solution of
sodium bicarbonate and water, dried over sodium sulfate, and
concentrated to give 2-chloro-N-(5-phenylpyridin-2-yl)acetamide,
which was used directly for next step without purification.
A solution of 3-phenyl-6,7-dihydrothieno[3,2-d]pyrimidine-2-thi-
one-4-one (81 mg, 0.310 mmol), 2-chloro-N-(5-phenylpyridin-2-
yl)acetamide (80 mg, 0.325 mmol), and triethylamine (0.13 mL,
0.93 mmol) in N,N-dimethylformamide (DMF, 3.0 mL) was stirred at
80 °C for 2 h. The reaction was quenched with water, extracted with
ethyl acetate, washed three times each with water and brine, dried over
sodium sulfate, concentrated, and purified by silica gel column
chromatography (30% ethyl acetate/hexanes) to give IWP-L6 (27)
(136 mg, 93%) as white solid. 1H NMR (400 MHz, CDCl3) 10.06 (s,
1H), 8.57 (d, J = 2.1 Hz, 1H), 8.25 (d, J = 8.6 Hz, 1H), 7.95 (dd, J =
8.6, 2.4 Hz, 1H), 7.56−7.63 (m, 5H), 7.48−7.56 (m, 2H), 7.40−7.47
(m, 1H), 7.29−7.35 (m, 2H), 3.85 (s, 2H), 3.54−3.62 (m, 2H), 3.44−
3.52 (m, 2H).; 13C NMR (100 MHz, CDCl3) δ 166.6, 160.2, 159.2,
157.4, 150.5, 146.3, 137.4, 136.7, 135.0, 133.0, 130.6, 130.0, 129.1,
128.6, 127.9, 126.8, 122.3, 113.7, 37.6, 37.0, 29.3. MS(ES)+ calcd for
C25H21N4O2S2 (M + H)+ 473.1, found 473.1.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Procedures for metabolic studies, metabolic profiles of IWP-L6
1
(27), and H and 13C NMR spectra of IWP-L6 (27). This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 214-648-5048. E-mail: chuo.chen@utsouthwestern.
edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(12) Berry, L. M.; Wollenberg, L.; Zhao, Z. Esterase Activities in the
Blood, Liver and Intestine of Several Preclinical Species and Humans.
Drug Metab. Lett. 2009, 3, 70−77.
■
Financial support was provided by the Cancer Prevention and
Research Institute of Texas (RP100119 to L.L. and C.C.),
National Institutes of Health (5R21HD061303 to J.F.A., C.C.,
and L.L., R01CA135731 to J.F.A., and R01DK080004 &
P30DK079328 to T.C.), the Welch Foundation (I-1596 to
C.C., and I-1665 to L.L.), and UT Southwestern. L.L. is a
Virginia Murchison Linthicum Scholar in Medical Research,
and C.C. is a Southwestern Medical Foundation Scholar in
Biomedical Research. X.P. is supported by a postdoctoral
fellowship from the National Kidney Foundation (FLB1686).
(13) Rudakova, E. V.; Boltneva, N. P.; Makhaeva, G. F. Comparative
Analysis of Esterase Activities of Human, Mouse, and Rat Blood. Bull.
Exp. Biol. Med. 2011, 152, 73−75.
(14) Bahar, F. G.; Ohura, K.; Ogihara, T.; Imai, T. Species Difference
of Esterase Expression and Hydrolase Activity in Plasma. J. Pharm. Sci.
2012, 101, 3979−3988.
(15) Moro, E.; Ozhan-Kizil, G.; Mongera, A.; Beis, D.; Wierzbicki, C.;
Young, R. M.; Bournele, D.; Domenichini, A.; Valdivia, L. E.; Lum, L.;
Chen, C.; Amatruda, J. F.; Tiso, N.; Weidinger, G.; Argenton, F. In
Vivo Wnt Signaling Tracing through a Transgenic Biosensor Fish
Reveals Novel Activity Domains. Dev. Biol. 2012, 366, 327−340.
(16) Lu, J.; Ma, Z.; Hsieh, J.-C.; Fan, C.-W.; Chen, B.; Longgood, J.
C.; Williams, N. S.; Amatruda, J. F.; Lum, L.; Chen, C. Structure/
Activity Relationship Studies of Small-Molecule Inhibitors of Wnt
Response. Bioorg. Med. Chem. Lett. 2009, 19, 3825−3827.
(17) Karner, C. M.; Michael Dodge, C. E.; Ma, Z.; Lu, J.; Chen, C.;
Lum, L.; Carroll, T. J. Tankyrase Is Necessary for Canonical Wnt
Signaling during Kidney Development. Dev. Dyn. 2010, 239, 2014−
2023.
ABBREVIATIONS USED
■
CES, carboxylesterase; Dvl2, dishevelled 2; MBOAT, mem-
brane-bound O-acyltransferase; Porcn, porcupine; SAR, struc-
ture−activity relationship; Tnks, tankyrase
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