Discovery of small-molecule modulators of the sonic hedgehog pathway

Article abstract of DOI:10.1021/ja400034k

The Hedgehog signaling pathway is involved in the development of multicellular organisms and, when deregulated, can contribute to certain cancers, among other diseases. The molecular characterization of the pathway, which has been enabled by small-molecule probes targeting its components, remains incomplete. Here, we report the discovery of two potent, small-molecule inhibitors of the Sonic Hedgehog (Shh) pathway, BRD50837 and BRD9526. Both compounds exhibit stereochemistry-based structure-activity relationships, a feature suggestive of a specific and selective interaction of the compounds with as-yet-unknown cellular target(s) and made possible by the strategy used to synthesize them as members of a stereochemically and skeletally diverse screening collection. The mechanism-of-action of these compounds in some ways shares similarities to that of cyclopamine, a commonly used pathway inhibitor. Yet, in other ways their mechanism-of-action is strikingly distinct. We hope that these novel compounds will be useful probes of this complex signaling pathway.

Full text of DOI:10.1021/ja400034k

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Discovery of Small-Molecule Modulators of the Sonic Hedgehog
Pathway
Giannina I. Schaefer,†,‡ Jose R. Perez,‡ Jeremy R. Duvall,‡ Benjamin Z. Stanton,†,‡,§ Alykhan F. Shamji,‡
́
and Stuart L. Schreiber*,†,‡
†Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United
States
‡Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: The Hedgehog signaling pathway is involved in
the development of multicellular organisms and, when deregu-
lated, can contribute to certain cancers, among other diseases.
The molecular characterization of the pathway, which has been
enabled by small-molecule probes targeting its components,
remains incomplete. Here, we report the discovery of two potent,
small-molecule inhibitors of the Sonic Hedgehog (Shh) pathway,
BRD50837 and BRD9526. Both compounds exhibit stereochemistry-based structure−activity relationships, a feature suggestive
of a specific and selective interaction of the compounds with as-yet-unknown cellular target(s) and made possible by the strategy
used to synthesize them as members of a stereochemically and skeletally diverse screening collection. The mechanism-of-action
of these compounds in some ways shares similarities to that of cyclopamine, a commonly used pathway inhibitor. Yet, in other
ways their mechanism-of-action is strikingly distinct. We hope that these novel compounds will be useful probes of this complex
signaling pathway.
motor protein dynein (ciliobrevin A),14 or disrupt DNA−Gli
INTRODUCTION
■
interactions (GANT-61).15
The Hedgehog (Hh) signaling pathway plays an important role
In addition to this ‘canonical’ Hh signaling, Hh proteins also
in embryonic development and the overall growth and
promote ‘noncanonical’ signaling that is Gli-independent.16−18
morphology of insects and vertebrates.1,2 Improper Hh
Further complexities are evidenced by the findings that
signaling can result in developmental diseases, such as
different small-molecule inhibitors of Smo can result in
different cellular outcomes. For example, vismodegib prevents
holoprosencephaly.3 Somatic genomic alterations in genes
encoding members of the pathway drive the development
Smo translocation to the primary cilium, while cyclopamine
promotes Smo accumulation in the primary cilium.19,20
and maintenance of several cancers, especially basal cell
carcinoma (BCC) and medulloblastoma.4−7
To enhance our molecular understanding of the pathway, we
The pathway becomes activated when an extracellular
secreted protein from the Hh family, most commonly Sonic
Hedgehog (Shh), binds patched (Ptch), a transmembrane
receptor. In the absence of this binding, Ptch represses the G-
protein coupled transmembrane receptor, smoothened (Smo).
Formation of the Shh/Ptch complex in some still unknown way
derepresses Smo, causing its translocation to the primary cilium
where it influences the state of the transcription regulator Gli.
Smo enables a release of Gli from a repressor complex
comprising Gli and, among others, suppressor of fused (SuFu).
The resulting activated form of Gli translocates to the nucleus
and activates genes involved in cell proliferation and differ-
entiation.4,8,9
Several small-molecule modulators of this complex pathway
have been discovered, with many acting on Smo directly.
Prominent examples are cyclopamine (a natural product found
in Veratrum Californicum) and vismodegib (an FDA-approved
drug for the treatment of BCC).10−12 Other inhibitors have
been reported to act on Shh (robotnikinin),13 modulate the
aimed to discover novel small-molecule probes of Hh signaling.
We first performed a cell-based high-throughput screen for
novel inhibitors of Gli-induced transcription. We discovered a
group of small molecules having compelling stereochemistry-
based structure−activity relationships (SAR), which we
interpret as indirect evidence for a selective interaction with
cellular target(s). Synthetic chemistry to generate analogs
resulted in the elucidation of additional building block-based
SAR and characterization of the novel Shh pathway inhibitors
BRD50837 and BRD9526 with a mechanism-of-action distinct
from cyclopamine.
RESULTS AND DISCUSSION
■
We first screened 21 753 compounds in a cell-based assay using
Shh light II cells. These cells are derived from NIH/3T3 cells
by cotransfection with a Gli-responsive Firefly luciferase
Received: January 2, 2013
Published: May 31, 2013
© 2013 American Chemical Society
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Figure 1. (a) Primary screening data are displayed as percent luciferase activity in Shh light II cells. Each small block displays a heat map associated
with eight stereoisomeric compounds having the same skeleton and appendages. The overall panel A displays a near-complete matrix of multiple
skeletons (only one shown in C) and building blocks used for R2 and R3 (displayed on the left (y-axis) and bottom (x-axis)). In the heat maps, blue
represents inhibition (−100), yellow represents no activity (0), and red represents activation (100) normalized to DMSO control. Empty cells
represent compounds not tested. Values shown are from testing compounds twice in single dose. (b) The highlighted block represents the dose−
response data of eight stereoisomers of a primary subject of this report (BRD50837). (c) Dose−response curve of BRD50837 in C3H10T1/2 cell
differentiation assay and structure of BRD50837 highlighting positions of building block attachment. All values shown are generated from three
independent experiments run in duplicate (values are calculated average SD).
reporter.10,21 All compounds were screened in duplicate at a
single concentration. Screening positives (mean inhibition
≥65%) were retested in dose, and their toxicity was assessed
using CellTiter-Glo to measure cellular adenosine triphosphate
(ATP) levels as a surrogate for viability (Figure S1a-b). A total
of 390 hits were identified and advanced for further
investigation.
activity (Tables 1 and S1). Additionally, to rule out gross
toxicity as source of signal, all compounds were tested in a
viability assay using CellTiter-Glo as a means to estimate
cellular levels of ATP (Table S1). BRD50837 displayed high
potency with an EC50 of 0.09 μM. A PubChem search of other
assays wherein BRD50837 (CID 44499307) was screened
revealed that, as of May 27, 2013, BRD50837 had been tested
in 31 different assays but only scored in our initial screen,
suggesting that it is not broadly active. Compared to other
similarly potent compounds, BRD50837 showed good
phosphate buffered saline (PBS) solubility (64.3 μM) and
was thus chosen as a starting point for further experimentation.
To elucidate additional building block-based SAR, we
synthesized novel analogs, varying the attachments on the
aniline and the extra-annular amine as well as removing the
extra-annular alcohol. BRD50837 (7) and additional novel
analogs 8−19 were synthesized using an abbreviated synthetic
pathway relative to the previously reported solid-phase
synthesis (Schemes 1 and S1). For the synthesis of 7−16,
intermediate 4a was synthesized from 1 and 2a as previously
reported.22,23 Subsequently, the nitrobenzene was reduced to
an aniline that was acylated with acyl chlorides to yield anilides
5. After deprotection of the extra-annular nitrogen with
hydrogen fluoride (HF)/pyridine and addition of a sulfonyl
chloride, the para-methoxybenzyl ether (PMB) group was
removed with 2,3-dichloro-5,6-dicyanobenzo-quinone (DDQ),
resulting in the final compounds.
Both the primary screen and multiple dose-retest data
revealed a striking correlation between activity and stereo-
chemistry of members of a library of the screening collection.
These compounds were initially synthesized using the build/
couple/pair strategy of diversity-oriented synthesis (DOS).22,23
As a consequence, all possible stereoisomers of each structural
type are included in the collection. The compounds in the
library screened include ∼6700 compounds with varying eight-
membered rings that are formed by nucleophilic aromatic
substitution reactions. Based on the primary screening data, two
of the eight stereoisomers of several compounds having the
same eight-membered ring skeleton were active, the RSR and
the SSR isomers, with the sole difference being the
configuration of the extra-annular methyl group (Figure 1).
These initial findings were confirmed in a second biological
assay that measures Hh-induced differentiation of C3H10T1/2
cells into osteoblasts.24 Retesting all eight stereoisomers of
several hit compounds in dose using Shh-conditioned medium-
induced C3H10T1/2 cells confirmed that BRD50837 selec-
tively blocks the Hh pathway in cells (Figures 1b and S1c).
All hits were tested in the secondary differentiation assay
using C3H10T1/2 cells to substantiate their on-pathway
To synthesize analog 18, 2b was used instead of 2a as a
starting material, and the final PMB deprotection step was
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a
Table 1. EC50 in C3H10T1/2 Cells and PBS Solubility of
Analogs of BRD50837
Scheme 1
a
Reagents and conditions: (a) PyBOP, DIPEA, DCM, rt, 52−83%. (b)
borane dimethyl sulfide complex, THF, 65 °C, 88−89%. (c) 2-fluoro-
3-nitrobenzoic acid chloride, NEt3, DCM, rt, 92−100%. (d) CsF,
DMF, 85 °C, 100%. (e) 10% Pd/C, H2, EtOH, 35 °C, 100%. (f) R2Cl,
2,6-lutidine, DCM, rt. (g) NaNO2, NaHSO3, EtOH/AcOH, rt, 48%.
(h) TBSOTf, 2,6-lutidine, DCM, rt, then HF/pyridine, THF, rt. (i)
R3Cl, 2,6-lutidine, DCM, 0 °C, 68% over 3 steps (18). (j) DDQ, pH 7
buffer/DCM, rt, 6−59% over 4 steps (7−16), 49% over 4 steps (19).
(k) TFA, DCM, rt. (l) 4-chlorobenzaldehyde, sodium triacetoxy-
borohydride, DMF/2% AcOH, rt, 10% over 4 steps (17).
trifluoroacetic acid (TFA) and forming the tertiary amine 17
by reductive amination with para-chlorobenzaldehyde. Com-
pound 19 was prepared by treating intermediate 4a with
sodium nitrate and sodium bisulfate, which resulted in the
deaminated product 6. Subsequent deprotection of the Boc
group with HF/pyridine, addition of the sulfonyl chloride and
removal of the PMB group with DDQ yielded 19. All
compounds were purified by column chromatography (30
min, 0−100% ethyl acetate in hexanes) and if necessary by
HPLC purification (SI, synthetic procedures).
Both new and previously synthesized analogs were tested in
C3H10T1/2 cells using Shh-conditioned medium-induced
differentiation as readout of Hh signaling (Tables 1 and S1).
Viability for all compounds was tested in this system as well,
using CellTiter-Glo as a measure of ATP levels (Table S1).
Changes on the aniline moiety of the compound were
tolerated, but activity was optimal for saturated ring systems
(BRD50837, 12, 20). The cyclopropyl derivative (BRD50837)
proved to have better solubility in PBS than the cyclohexyl
derivatives (12, 20) making it the more favorable candidate.
Complete removal of the aniline moiety (19, Table S1) resulted
in a loss of activity. Ureas instead of amides also showed activity
but were less soluble (21, 25, 26, Table S1).
Changing the sulfonamide building block from para-
chlorobenzene sulfonyl chloride to ortho- or meta-chloroben-
zene sulfonyl chloride (13, 14) resulted in reduced activity. An
additional chlorine in the ortho position of the sulfonamide
building block (BRD9526 (15)) did not impact the activity,
suggesting that the chlorine in the para position is interacting
with a putative cellular target, while that in the ortho position is
not. This was also reinforced by the previous observation that
the compound lacking the chlorine entirely had reduced activity
(24). When testing the previously synthesized compounds it
was additionally shown that the chlorine derivative is more
active as compared to the fluorine and methyl derivatives (22,
a
Solubility was measured for compounds that were considered for
subsequent experimentation based on their EC50.
omitted. Analog 17 was synthesized by preparing intermediate
5a as before, removing the PMB group with DDQ,
deprotecting the tert-butoxycarbonyl (Boc) group with
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23). Having an additional methylene in the sulfonyl chloride
also resulted in a decrease of activity (16, Table S1). Preparing
the tertiary amine (17) rather than the sulfonamide resulted in
a decrease of activity, demonstrating a possible electronic or
spatial requirement for the sulfonamide connector. This was
also reflected by additional tertiary amines tested in previous
SAR studies (27, 28, Table S1).
Removing the extra-annular alcohol (18) resulted in a loss of
activity, showing that the alcohol, which originally was used as a
point of attachment in solid-phase synthesis, is necessary.
The original DOS pathway also yielded compounds having
eight-membered rings where the aniline moiety is in the para
instead of the ortho position, and nine-membered rings where
the aniline moiety is also in the para position.22 BRD50837s
analogs having these structural elements were not active (29,
30, Table S1).
SAG-induced differentiation, suggesting a mechanism-of-action
involving modulation of a step in the signaling cascade at or
following Smo signaling. In parallel, we tested the compounds
in Ptch−/− cells, mouse embryonic fibroblasts that contain a β-
galactosidase reporter gene instead of the Ptch gene after the
Ptch promoter.10 Lacking the repressor Ptch, the Hh pathway is
constitutively active in these cells. In this assay cyclopamine
maintained its inhibition of the pathway, but BRD50837 and
BRD9526 had no effect (Figure 2d). When viewing the overall
pathway as a linear set of response nodules, these results are
apparently in contrast to the previous observation; they suggest
that the compounds act at the level of Ptch or a step upstream
of Ptch signaling. However, we also identified compounds from
the original screen with responses similar to cyclopamine in
Ptch−/− cell-based, SAG/C3H10T1/2 cell-based, and BODI-
PY-cyclopamine displacement assays (data not shown), which
gave us confidence that the assays accurately measure
compound/activity profiles.
We prioritized BRD50837 and BRD9526 (15) for further
experimentation as both displayed good EC50s (Figure 2a) and
With these puzzling results in hand, we performed two
additional assays to characterize the compounds. We first tested
the compounds in a competition assay to determine whether
they displace BODIPY-cyclopamine in a cellular assay, thus
suggesting that they bind Smo in the cyclopamine-binding
site.25 Unlike cyclopamine, both BRD50837 and BRD9526 did
not lead to a reduction of BODIPY-cyclopamine binding
(Figures 3a and S3), suggesting that BRD50837 and BRD9526
do not interfere with cyclopamine binding.
We next tested the activity of BRD50837 and BRD9526 in
SuFu−/− cells. These mouse embryonic fibroblasts lack the
pathway repressor SuFu, which leads to constitutively active Hh
signaling.26 It has been reported that Smo antagonists do not
inhibit this signaling while the pathway inhibitor GANT-61
does.15 In our experiments, cyclopamine partially inhibited
downstream Gli1 expression (Figure 3b), perhaps due to an off-
target activity observed at high concentrations.27,28 However,
another more potent Smo inhibitor (vismodegib)11 showed no
suppression of Gli1 expression, consistent with the existing
model of SuFu being downstream of Smo (Figure 3b).
BRD50837 and BRD9526, like cyclopamine, partially lowered
Gli1 expression at concentrations of 2 and 10 μM (Figure 3b).
This partial inhibition may reflect an off-target effect at high
concentrations, but it is also possible that these compounds act
in a way that influences the pathway at the level of or
downstream of SuFu signaling.
The compounds therefore act similar to cyclopamine, a well-
characterized pathway inhibitor, in some aspects (SAG/
C3H10T1/2 cell-based and SuFu−/− cell-based assays) but
seem to have a different mechanism-of-action in other aspects
(Ptch−/− cell-based and BODIPY-cyclopamine displacement
assays). These data suggest that BRD50837 and BRD9526 may
function by mechanisms-of-action that are distinct from
cyclopamine and not easily described by traditional linear
models of the pathway. Consistent with this notion,
BRD50837/BRD9526 repressed Gli1 expression in
C3H10T1/2 cells to a lesser extent than cyclopamine when
the compounds were tested at concentrations that yield similar
responses in Shh-conditioned medium-induced differentiation
of C3H10T1/2 cells (1 μM, and 10 μM, respectively, Figure
S2a).
Figure 2. All values are shown and generated from three independent
experiments run in duplicate (values are calculated average SD). (a)
Inhibition of Shh-conditioned medium (CM) induced differentiation
of C3H10T1/2 cells by BRD50837, BRD9526, and cyclopamine after
48 h. (b) Viability of CM-induced C3H10T1/2 cells in response to 48
h treatment by BRD50837, BRD9526, and cyclopamine. (c) Inhibition
of SAG-induced differentiation of C3H10T1/2 cells by BRD50837,
BRD9526, and cyclopamine after 48 h. (d) β-galactosidase expression
response of Ptch−/− cells to 48 h treatment with BRD50837,
BRD9526, and cyclopamine.
similar PBS solubility (Table 1). Neither showed significant
toxicity based on measuring ATP levels as a surrogate for
growth or viability (Figure 2b). Reduction of Gli1 expression in
C3H10T1/2 cells by 1 μM treatments of these two compounds
was confirmed (Figure S2a).
To understand the mechanisms-of-action of these com-
pounds, we tested the response to BRD50837 and BRD9526 in
several epistasis experiments and a competition assay in
comparison to cyclopamine, a commonly used Smo inhibitor.
We first tested the compounds in C3H10T1/2 cells that were
treated with SAG (a small-molecule activator of Smo) rather
than Shh-conditioned medium to activate the Hh pathway
(Figure 2c).21 Like cyclopamine, the compounds suppressed
CONCLUSION
We report here the discovery of BRD50837 and BRD9526, two
selective small-molecule inhibitors of the Shh pathway. Though
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Figure 3. (a) Effects of cyclopamine, BRD50837, and BRD9526 on BODIPY-cyclopamine (10 nM) binding to exogenously expressed Smo. blue
(Hoechst 33342), red (anti-myc), green (BODIPY-cyclopamine). (b) Gli1 expression in Sufu−/− cells treated with cyclopamine, vismodegib,
GANT61, BRD50837, and BRD9526. All values are shown and generated from three independent experiments run in duplicate (values are
calculated average + SD).
similar in some respects to traditional pathway inhibitors, the
compounds show a distinct pattern of activity in cells perturbed
for components of the pathway. The basis for these differences
is not yet known, but it hints at the complexity of the pathway.
Elucidating the compounds’ mechanism-of-action will help to
realize their full potential as probes and enable the study of this
enigmatic pathway.
Management and Analytical Teams (Broad Institute) for
providing compound plates, purifications, and compound
characterizations, Daniel Smaltz (Harvard University), Mah-
mud Hussain, Andrew Germain, and Zarko Boskovic (all Broad
Institute) for their input and help with compound synthesis,
purification, and characterization, and James Chen (Stanford
University), Marek Nagiec (Broad Institute), Stephan Teglund,
and Rune Toftgard (Karolinska Institute, Sweden) for kindly
̊
EXPERIMENTAL SECTION
For complete experimental information, please refer to the Supporting
Information.
■
providing materials. This research was supported by the
National Institute of General Medical Sciences (GM38627
awarded to S.L.S.).
ASSOCIATED CONTENT
* Supporting Information
Supporting figures, schemes, tables, materials and methods,
assay protocols, synthetic procedures, data analysis, and
characterization data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
stuart_schreiber@harvard.edu
■
Present Address
§Howard Hughes Medical Institute, Department of Devel-
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Nicole Bodycombe, Jaime Cheah, Paul Clemons,
Angela Koehler, Max Majireck, and Michelle Stewart (Broad
Institute) for their input on data analysis and presentation,
Bennett Meier (Harvard University) on his help in the
execution of several experiments presented, Lee Peng
(Massachusetts General Hospital), Joshiawa Paulk (Harvard
University), and Nicola Tolliday (Broad Institute) for initial
support and guidance, James Spoonamore and Thomas Hasaka
(Broad Institute) for help with the setup and execution of the
Shh light II viability and imaging assays, the Compound
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