Identification of a novel Smoothened antagonist that potently suppresses Hedgehog signaling

Article abstract of DOI:10.1016/j.bmc.2012.09.030

The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeostasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform using confocal imaging, based on the role of β-arrestin in Hedgehog signal transduction, that can identify agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a compound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel screening approach in the further development of A8 and related congeners to treat hedgehog related diseases, including the treatment of basal cell carcinoma and medulloblastoma.

Full text of DOI:10.1016/j.bmc.2012.09.030

Bioorganic & Medicinal Chemistry 20 (2012) 6751–6757
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of a novel Smoothened antagonist that potently suppresses
Hedgehog signaling
Jiangbo Wang a, , Robert A. Mook Jr. a, , Jiuyi Lu a, David M. Gooden b, Anthony Ribeiro c, Anchen Guo f,
⇑
,
Larry S. Barak d, H. Kim Lyerly e, Wei Chen a,
⇑
a Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
b Duke Small Molecule Synthesis Facility, Department of Chemistry, Duke University, Durham, NC 27710, USA
c Department of Radiology and Duke NMR Spectroscopy Center, Duke University, Durham, NC 27710, USA
d Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
e Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
f Laboratory of Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, No. 6 Tiantanxili, Dongcheng District, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2012
Revised 31 August 2012
Accepted 10 September 2012
Available online 23 September 2012
The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeo-
stasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this
signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform
using confocal imaging, based on the role of b-arrestin in Hedgehog signal transduction, that can identify
agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the
use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a com-
pound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point
mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel
screening approach in the further development of A8 and related congeners to treat hedgehog related
diseases, including the treatment of basal cell carcinoma and medulloblastoma.
Keywords:
Hedgehog signaling
Smoothened
High-throughput screening
Smo antagonist
Smo mutation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
are observed in cancers of the pancreas,11,12 proximal gastro-
intestinal tract,11 and prostate.13 In mice, about 14–30% of Ptc
The evolutionarily conserved Hedgehog (Hh) signaling pathway
is essential for embryonic development, tissue homeostasis, and
maintenance of self-renewal potential in adult stem cells.1–3 An
increasing body of evidence has shown that key components of
the pathway: Hh protein, its receptor Patched (Ptc) and an effector
receptor Smoothened (Smo), also play pivotal roles in the develop-
ment of numerous cancers.4,5 For example, dysregulation of Hh sig-
naling, resulting from mutations in components of the pathway
has been directly implicated in the development of basal cell carci-
noma and medulloblastoma.6–10 High levels of pathway activity
heterozygous knockout mice develop medulloblastoma14 and the
homozygous deletion of Ptc in GFAP-positive progenitor cells re-
sulted in the development of medulloblastoma in 100% of geneti-
cally engineered mice.15
Several small molecule inhibitors of the pathway that bind the
Smo receptor, such as cyclopamine, IPI-926, and GDC-0449, have
been identified with a number of inhibitors under investigation
in clinical trials.16–21,49 Among these inhibitors, GDC-0449 (Vis-
modegib) was recently approved by the FDA to treat patients with
advanced basal cell carcinoma.22–24 Unfortunately, acquired resis-
tance to GDC-0449 was recently described in which an Asp to
His point mutation (D473H) was found in the Smo gene. The
Smo-D473H mutant receptor is refractory to inhibition by GDC-
0449 due to loss of interaction between the drug and receptor.17,25
Thus, new Smo inhibitors with pharmacological properties capable
of inhibiting wild-type and clinically relevant mutant receptors are
needed to overcome acquired drug resistance and extend the dura-
tion of response.
Abbreviations: Smo, smoothened; Hh, hedgehog; Ptc, patched; Shh, sonic
hedgehog; b arr2, b-arrestin2; b arr2-GFP, b-arrestin2-green fluorescent protein
chimera; Gli, glioma-associated oncogene; GPCR, G-protein-coupled receptor; V2R,
vasopressin2 receptor; HTS, high-throughput screening; WT, wild-type; PBS,
phosphate-buffered saline; GCP, granular cell precursor; HBBS, Hanks balanced
salt solution; DCC, N,N0-dicyclohexylcarbodiimide; TFA, trifluoroacetic acid; HOBt,
N-hydroxybenzotriazole; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene.
⇑
Corresponding authors. Tel.: +1 919 684 8717 (R.A.M.); tel.: +1 919 684 4433
(W.C.).
A mechanistic understanding of the Hh signaling pathway has
evolved over the past decade.26 The Hedgehog family of growth
factor proteins is comprised of three members: Sonic, Desert, and
E-mail addresses: robert.mook@duke.edu (R.A. Mook), w.chen@dm.duke.edu
(W. Chen).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.030

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J. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6751–6757
Indian Hedgehog, each known to bind the transmembrane receptor
Ptc. In the resting, non-ligand bound state, the unoccupied trans-
membrane receptor Ptc inhibits the activity of the transmembrane
protein Smo. Upon binding of Hh ligand to its receptor Ptc, Smo be-
comes activated and transduces signaling by activating Gli tran-
scription factors that results in the modulation of Hh responsive
genes such as Myc and Ptc.
cyclopamine, GDC-0449, LDE-225 or A8 (from 0 to 10
Cells were then washed with binding buffer and the bound
[3H]-cyclopamine was extracted in 200
l of 0.1 N NaOH and neu-
tralized with 200
l of 0.1 N HCl. The amount of [3H]-cyclopamine
in the extracts was measured using a scintillation counter.
Gli-luciferase reporter assay: The Gli-luciferase assay was con-
ducted in Shh-LIGHT2 cells, a clonal NIH3T3 cell line stably incor-
porating Gli-dependent firefly luciferase and constitutive Renilla
luciferase reporters.35 Cells were treated with purified Sonic
Hedgehog protein from StemRD (50 ng/mL) together with the cor-
responding compounds for 2 days. The reporter activity was
determined by using the Dual-Luciferase Reporter Assay System
(Promega).
Cell proliferation: Primary neuronal granular cell precursor
(GCP) cells were obtained from the cerebellum of 7-day postnatal
C57BL/6 mice and labeled with [3H]-thymidine. Proliferation as-
says were performed as previously described.28
Animal studies: Eight-week-old C57BL/6 female mice were
shaved on the dorsal surface and depilated with NairÒ (Carter-
Wallace, New York, New York). Briefly, the bottom half of the
shaved area was treated with Nair for 2 min, and the depilated area
rinsed with water to remove residual Nair. Compound A8 was
dissolved in a vehicle of 95% acetone/5% DMSO at a concentration
lM).
l
l
Activated Smo shares important similarities with canonical G
protein-coupled receptors (GPCRs), including an ability to undergo
GPCR kinase-mediated phosphorylation and to recruit b-arrestin2
(barr2) proteins for endocytosis and signaling. In our previous
work,27 we found that barr2 binds Smo at the plasma membrane
in an activation-dependent manner, and that the Smo antagonist
cyclopamine inhibits the activity of Smo by preventing its phos-
phorylation and interaction with barr2. These findings enabled
the development of a versatile cell-based high-throughput imag-
ing-based screening platform capable of identifying either agonists
or antagonists of the pathway by the presence or absence of cyclop-
amine, respectively, in the assay. These assay formats led to the dis-
covery of Smo agonist activity in a select subset of commonly used
glucocorticoid medications28 and Smo antagonist activity in piper-
onyl butoxide,29 a pesticide synergist present in over 1500 prod-
ucts30 recently associated with delayed learning in children31 and
one of the top 10 chemicals detected in indoor dust.32 Here, we
report the use of this platform to search systematically for Smo
inhibitors in small molecule chemical libraries. This effort resulted
in the discovery of a number of active hits, including a low nanomo-
lar Smo antagonist (compound A8) that binds to Smo receptors,
inhibits the transcriptional activity of Gli, inhibits cell proliferation
of neural precursor cells and prevents Hh signaling dependent hair
growth in mice. In contrast to GDC-0449, compound A8 binds the
Smo mutant D473H recently associated with medulloblastoma dis-
ease progression and resistance to GDC-0449,17,25,33 thereby
providing the basis of a strategy to treat resistant disease.
of 0.5 mM, and 30 ll of A8 solution or the vehicle were applied
topically to the depilated area of mice daily for 2 weeks. Mice were
anesthetized briefly using 3% isoflurane anesthetic inhalant during
all procedures. Five mice were included in each treatment group.
All animals were treated in accordance with protocols approved
by Institutional Animal Care and Use Committee at Duke
University.
NMR spectroscopy: Full NMR structural identification of Tripos
3910 and compound A8 was achieved from 2D NMR data sets
(COSY, TOCSY, HMQC and HMBC) obtained on Agilent 500 and
800 NMR instruments in the Duke NMR Spectroscopy Center.
3. Results
2. Materials and methods
3.1. Identification of compound A8 from screening
Reagents: A library of 5740 compounds (Tripos Gold) were used
for high-throughput screening. b-Arrestin2 green fluorescent
protein (barr2-GFP), wild-type Smo, Smo-633 mutant, and
Gli-luciferase reporter have been previously described.27,28 The
Smo-D473H mutant construct was generated using the Quik-
Change site-directed mutagenesis kit (Stratagene). Purified Sonic
Hedgehog was obtained from StemRD. Cyclopamine was purchased
from Toronto Research Chemicals. [3H]-Cyclopamine (specific
activity = 20 Ci/mmol) was purchased from American Radiolabeled
Chemicals. GDC-0449 (Vismodegib), LDE-225 (NVP-LDE225, Eris-
modegib) and select hits identified from screening were synthe-
sized by the Small Molecule Synthesis Facility at Duke University.
Primary high-throughput screening assay: U2OS cells stably
expressing a chimera Smo-633 receptor and barr2-GFP were used
in HTS screening. Smo-633 was used in this assay because it pro-
duces a stronger signal than WT Smo in the barr2-GFP translocation
assay, but is otherwise pharmacologically similar.27,34 The antago-
nist mode screening protocol used here to identify antagonists of
Smo is similar to the protocol to identify Smo agonists described
previously with the exception that cyclopamine pretreatment was
not used prior to the addition of test compounds.28
To identify novel Smo inhibitors, we screened chemical libraries
using our confocal imaging, cell-based platform assay as the pri-
mary high-throughput screening assay. This assay derived from
our discovery that co-expression of Smo and barr2-GFP in cells re-
sults in an activation-dependent translocation of barr2-GFP into
endocytic vesicles. barr2-GFP distributes homogenously through-
out the cytoplasm when expressed alone in cells (Fig. 1A).28 In
marked contrast, cells co-expressing Smo-633 and barr2-GFP local-
ize barr2-GFP into intracellular vesicles as aggregates (Fig. 1B).
Addition of a Smo antagonist, such as cyclopamine, inhibits the
aggregation of barr2-GFP, as demonstrated by the disappearance
of intra-vesicular aggregates (Fig. 1C). Thus, small molecule inhib-
itors of Smo are identified by visually inspecting the cells for the
loss of the punctate pattern. Upon screening of a library of 5740
compounds from Tripos, Inc. at a concentration of 5 lM, we
identified 32 hit compounds that inhibited the formation of intra-
cellular barr2-GFP aggregates similar to that observed with cyclop-
amine treatment,36 one of which was a screening sample Tripos
3910 discussed later (see supplementary Fig. 1). Hit compounds
in this assay were confirmed by further evaluation in Gli-reporter
and [3H]-cyclopamine competition assays, and by testing new solid
Smo receptor binding: For competitive binding assays, U2OS cells
overexpressing wild-type Smo or Smo-D473H mutant receptors
were grown in 24-well plates and fixed with 4% (v/v) formaldehyde/
PBS for 20 min at room temperature (RT). Cells were subsequently
incubated for 2 h at RT in binding buffer (Hanks balanced salt
solution (HBSS) without Ca2+ and Mg2+) containing 25 nM of
samples of the hit compounds. At 1 lM concentration, the positive
control cyclopamine and hit compounds showed strong inhibition
of the Gli-reporter activity.36
Of the hits obtained from screening, one hit compound (Tripos
3910) (Fig. 2A) synthesized at Duke based on the structure
[3H]-cyclopamine and
a range of different concentrations of

J. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6751–6757
6753
DMSO
Cyc
A8
A
A
B
C
D
Figure 1. Identification of novel Smo inhibitors in U2OS cells. Inhibitors are detected by the homogenous distribution of the green punctate pattern that results when the
intracellular association of barr2-GFP with Smo is inhibited. Confocal images of U2OS cells stably expressing (A) barr2-GFP alone, or (B–D) barr2-GFP co-expressed with Smo-
633. Cells were treated for 6 h with DMSO (B); 5 lM cyclopamine (Cyc) (C); or 5 lM compound A8 (D). Scale bar: 10 lM.
assigned to the material by Tripos, had substantially reduced
Smo antagonist activity compared to the previous test samples.
Reduced activity associated with this structure was confirmed
upon subsequent purification of the Tripos sample in which the
major component in the library sample agreed for structure
and was less active. Instead, the active substance was found to
be a small impurity isolated from the library sample (ca. 1.5–
2.6 area percent by UV at k = 210, 254, 280 nm). Storage of the
active impurity at room temperature in a DMSO or methanolic
solution for 1 week retained activity. Subsequent characterization
of this impurity by high-resolution mass spectrometry (HRMS)
and by extensive NMR analysis allowed assignment of structure
to the impurity as shown for Compound A8 (Fig. 2A) (see Supple-
mentary data). Confirmation of the structural assignment was
achieved by synthesis of authentic material using the route de-
scribed in Fig. 2B (see Supplementary data). Synthesized material
matched the isolated material from the library sample by exten-
sive NMR analysis, HRMS, TLC and HPLC. The activity of the syn-
thesized material was confirmed upon testing the synthesized
material in the primary Smo/barr2-GFP assay (Fig. 1D).
Figure 2. Chemical structures of screening hits and synthesis of A8. (A) Structures of Tripos 3910 and compound A8; (B) synthesis of compound A8.

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J. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6751–6757
3.2. Compound A8 is a competitive antagonist of Smo
GCP cells requires Hh signaling.39 Thus, a mouse GCP proliferation
assay was performed to assess the hedgehog growth-inhibiting ef-
fects of compound A8. We found that compound A8 and GDC-0449
were potent inhibitors of GCP proliferation with IC50s of 16.6 2.3
and 16.4 2.5 nM, respectively (Fig. 4B). Consistent with the
finding that higher concentration of cyclopamine was needed to
inhibit Gli activity compared to A8 and GDC-0449 (Fig. 4A),
cyclopamine was also a less potent inhibitor of GCP proliferation
(IC50 = 414 73nM). Collectively, these results indicate that A8 is
a potent inhibitor of Smo activity and is capable of inhibiting Hh-
dependent Gli transcription and cell proliferation in vitro.
To further characterize the binding of compound A8 to Smo, we
tested the ability of A8 to competitively displace [3H]-cyclopamine
from Smo in U2OS cells overexpressing wild-type Smo. We previ-
ously determined the affinity (Kd) of [3H]-cyclopamine for wild-
type Smo as 12.4 4.2 nM.29 In the current study, we performed
competition binding assays and found cyclopamine, GDC-0449,
LDE-22537 and A8 completely displaced 25 nM of [3H]-cyclop-
amine from Smo with similar affinities, Ki = 12.7 1.7, 16.2 2.1,
6.0 1.4 and 37.9 3.7 nM, respectively (Fig. 3A). Given the impor-
tance of mutations in resistance to anti-cancer therapies, we tested
whether A8 is capable of binding to a mutant Smo receptor (Smo-
D473H) recently associated with clinical resistance and disease
progression to GDC-0449 therapy.17,25,33 Using U2OS cells over-
expressing Smo-D473H receptors, we conducted saturation bind-
ing experiments with [3H]-cyclopamine against the mutant
SmoD473H receptor and determined its Kd as 116 21nM (see
Supplementary Fig. 2). Consistent with previous reports, competi-
tion binding studies with GDC-0449 confirmed it was largely inef-
fective at competing for binding the mutant receptor and only
partially displaced [3H]-cyclopamine at high concentration
3.4. Compound A8 inhibits hair regrowth in mouse
Hedgehog signaling plays a key role in regulating hair follicle
growth.40 To determine the efficacy of the novel Smo inhibitor
A8 in suppressing Hh signaling in vivo, we used a model of hedge-
hog inhibition that examines inhibition of hair-growth.41–43 Eight-
week old female C57BL mice in telogen phase of the hair cycle
were used in these experiments.44 Chemical depilation with NairÒ
induces anagen phase and regrowth of hair by activating the Hh
signaling pathway. In our experiments, most of the hair on the
back of vehicle treated mice grew back 2 weeks after removal with
Nair (Fig. 5). In contrast, Hh-induced hair growth was largely inhib-
ited in the A8 treated group, suggesting that A8 also functions as
an inhibitor of Hh signaling in vivo (Fig. 5).
(10 lM) (Fig. 3B). Another leading Smo antagonist in clinical trials,
LDE-225 (Erismodegib), was also largely ineffective. However,
both A8 and cyclopamine were able to completely displace [3H]-
cyclopamine from Smo-D473H receptors (Kis of 478 123 nM
and 232 53 nM, respectively Fig. 3B). Taken together, these
results suggest that A8 competes with cyclopamine for the same
binding site on Smo and binds both wild-type Smo and the
Smo-D473H mutant receptor.
4. Discussion
Following the discovery of oncogenic Ptc mutations, increasing
numbers of studies have demonstrated hyperactivation of Hh sig-
naling plays a critical role in promoting the development and pro-
gression of various cancers.21 As a result, a number of small
molecule inhibitors of Hh signaling targeting Smo have progressed
into clinical trials, one of which (GDC-0449) was recently ap-
proved. Unfortunately, drug resistance has already been described
in which mutation of the target decreases affinity of the drug to the
target, a common resistance mechanism seen with other recent
anti-cancer drugs. Thus there is a need for potent inhibitors of
wild-type Smo with activity against a spectrum of mutations in
Smo. This need has prompted recent reports of second generation
inhibitors that offer a degree of activity against relevant Smo muta-
tions.45–48
3.3. Compound A8 inhibits Gli activity and proliferation of
mouse cerebellar granular cell precursor (GCP) cells
We next examined the inhibitory effect of compound A8 on Hh
signaling. Since activation of Smo is known to increase the tran-
scriptional activity of Gli, a Gli-luciferase reporter assay was used
to measure inhibition of Smo activation.38 As expected of an inhib-
itor of hedgehog signaling targeting Smo, compound A8 effectively
inhibited Shh-induced Gli-reporter activity (IC50 = 2.6 0.4 nM) in
Shh-LIGHT2 cells (Fig. 4A). Inhibition by A8 was comparable to that
of GDC-0449 (IC50 = 1.5 0.2 nM) and considerably more potent
than Cyclopamine (IC50 = 484 122 nM). Proliferation of cerebellar
Figure 3. Compound A8 competitively displaces [3H]-cyclopamine binding to wild-type Smo and mutant Smo-D473H. Competitive binding of [3H]-cyclopamine with Smo
antagonists was performed in fixed U2OS cells overexpressing wild-type Smo (A) and Smo-D473H (B). Results were normalized to the maximal binding of [3H]-cyclopamine
over baseline and were analyzed by fitting to a one-site competition curve using Graphpad Prism. Data were acquired in duplicate from three independent experiments and
are presented as the mean SEM.

J. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6751–6757
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Figure 4. Compound A8 inhibits Gli-reporter activity and GCP proliferation. (A) Gli-luciferase response in Shh-LIGHT2 cells treated for 30 h with Shh in the absence or
presence of increasing concentrations of cyclopamine (Cyc), GDC-0449, or A8; (B) GCP cells were treated for 48 h with Shh in the absence or presence of increasing
concentrations of Cyc, GDC-0449, or A8. Cells were then exposed to [3H]-thymidine for 16 h and [3H]-thymidine incorporation was measured. Data were fit using Graphpad
Prism (mean SEM, n = 3).
A8 0.5mM
vehicle
Day 14
Day 0
Figure 5. Compound A8 inhibits Hh-dependent hair growth post depilation. Eight-week old female C57BL mice in the telogen phase of the hair cycle were used. Chemical
depilation with Nair activates Hh signaling pathway and induces anagen phase and hair regrowth. This Hh-dependent hair growth is inhibited by daily topical treatment of
30 ll of 0.5 mM Smo antagonist A8 for 2 weeks. The vehicle control is 95% acetone/5% DMSO.
In the work described herein, we utilized a robust and versatile
not inhibited by the Smo antagonist cyclopamine29 or by A8
(Supplementary Fig. 3). This control assay helps ensure the mech-
anism of inhibition is Smo receptor specific and allows molecules
with non-specific mechanisms of inhibition to be ruled-out. Only
compounds that inhibited aggregation of Smo and did not inhibit
aggregation of V2R were evaluated in confirmatory assays. Using
this process, we identified a lead compound (A8) with nanomolar
inhibitory activity against wild-type Smo. This compound also
bound to a mutated from of Smo associated with clinical resistance
(SmoD473H), albeit with a right shift of approximately 13-fold in
affinity. The binding affinity of LDE-225 and GDC-0449 to the mu-
tant receptor was too weak (up to 10 lM) to enable determination
of a Ki value. The right shift in affinity of A8 was similar to a right
shift in affinity of 19 - fold observed for cyclopamine.
The Smo/barr2-GFP assay is a versatile assay platform that pro-
vides the ability to screen for antagonists or agonists by a small
change in the screening protocol. Screening in the antagonist mode
is as described above. Screening in the agonist mode is accom-
cell-based assay platform based on Smo receptor biochemistry
developed in our lab to identify a potent antagonist of Smoothened
that is capable of binding a mutated form of the receptor. The Smo/
barr2-GFP high-throughput assay platform exploits the discovery
that activated wild-type Smo or Smo-633 binds barr2-GFP and
changes its cellular distribution.27,28 Addition of a Smo antagonist,
such as cyclopamine inhibits the aggregation of Smo-633 with
barr2-GFP. Upon screening small molecule chemical libraries at a
concentration of 5 lM, hits were identified by the disappearance
of barr2-GFP intra-vesicular aggregates in cells, similar to the dis-
appearance of aggregates observed with cyclopamine. To control
for receptor specificity and to rule-out non-specific mechanisms,
hits were cross-screened in the same assay format using the
vasopressin2 receptor (V2R), a different seven-transmembrane
receptor. In this control assay, cells transfected with V2R and
barr2-GFP are stimulated with the agonist arginine vasopressin.
Stimulation causes barr2-GFP to aggregate and produces
a
punctate pattern in cells. Aggregation of V2R and barr2-GFP is
plished by the addition of 0.1 lM of cyclopamine to the cells prior

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J. Wang et al. / Bioorg. Med. Chem. 20 (2012) 6751–6757
to screening test libraries.28 In the agonist mode, active compounds
are identified by the appearance of a green punctate pattern in the
cells. The ability to screen cells in an agonist or antagonist mode
provides significant advantages to chemical genetic screening ap-
proaches while also providing a cellular context to identify mole-
cules with unique mechanisms of action. The follow-up assays
used here clearly demonstrate the ability of this innovative assay
format to identify authentic inhibitors of Smo that inhibit hedge-
hog signaling. Structure–activity relationships studies and assays
that delineate the anti-cancer effects of the compound A8 and
congeners are underway.
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5. Conclusions
In summary, a novel high-throughput, cell-based assay plat-
form based on a fundamental finding that activated Smo causes
the translocation of barr2 was capable of identifying Smo antago-
nists in chemical screening libraries. Here, the assay identified an
impurity in a chemical library that is a potent inhibitor of hedge-
hog signaling and is capable of binding wild-type Smo and a
mutated form of Smo associated with clinically resistance in
medulloblastoma. The cell-based nature of this assay provides
the basis of discovering second generation Hedgehog signaling
inhibitors with different binding modes and mechanisms of action
that can address drug resistance issues in cancers with activated
Hedgehog signaling.
Conflicts of interest
None declared.
Acknowledgments
26. Jiang, J.; Hui, C. Dev. Cell 2008, 15, 801.
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This work was funded in part by 5RO1 CA113656-03 (WC), the
Pediatric Brain Tumor Foundation (WC) and a Clinical Oncology
Research Center Development Grant 5K12-CA100639-08 (RAM).
Wei Chen is a V foundation Scholar and an American Cancer Soci-
ety Research Scholar. NMR instrumentation in the Duke NMR Spec-
troscopy Center was funded by the NIH, NSF, NC Biotechnology
Center and Duke University. The authors gratefully acknowledge
this support and the support of Professor Eric Toone and the Duke
Small Molecule Synthesis Facility.
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