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JPET#152090
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Title Page
EVIDENCE FOR ALLOSTERIC INTERACTIONS OF ANTAGONIST BINDING TO THE
SMOOTHENED RECEPTOR
Cynthia M. Rominger, Wei-Lin Tiger Bee, Robert A. Copeland, Elizabeth A. Davenport, Aidan Gilmartin,
Richard Gontarek, Keith R. Hornberger, Lorena A. Kallal, Zhihong Lai, Kenneth Lawrie, Quinn Lu,
Lynette McMillan, Maggie Truong, Peter J. Tummino, Brandon Turunen, Matthew Will, William J.
Zuercher and David H. Rominger
Oncology Center for Excellence in Drug Discovery (CMR, AG, RG, ZL, PJT, DHR), Molecular Discovery
Research (WTB, EAD, LAK, QL, LM, MT, BT, MW, WJZ), Oncology Medicinal Chemistry (KRH) and Isotope
Chemistry (KL) GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426
(RAC) EpiZyme, Inc., Cambridge, Massachusetts 02139
JPET Fast Forward. Published on March 20, 2009 as DOI:10.1124/jpet.109.152090
Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.
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Running Title Page
Running title: Smoothened receptor ligand binding mode of inhibition
Corresponding author: David H. Rominger
Current address; 1018 West 8th Avenue Suite AKing of Prussia, PA 19406.
Phone: 610-354-8840 Fax: 610-354-8850
E-mail: drominger@trevenainc.com
Text pages:18
Tables: 2
Figures: 8
References: 27
Abstract: 183
Introduction: 578
Discussion: 1239ABBREVIATIONS: Hh, Hedgehog; Smo, smoothened receptor; Ptch, patched receptor;
purmorphamine, 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine;
SANT-1, N-[(1E)-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-
piperazinamine; SAG-1.3, 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-
pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide; SAG-1.5, 3-chloro-4,7-difluoro-N-[trans-4-
(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide; SANT-
2, N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)benzamide; Compound 1, 6-methyl-
N-{2-[(1,3-thiazol-2-ylmethyl)amino]-2,3-dihydro-1H-inden-5-yl}-4'-(trifluoromethyl)-2-
biphenylcarboxamide; GDC-0449, 2-chloro-N-[4-chloro-3-(2-pyridinyl)phenyl]-4-
(methylsulfonyl)benzamide; Z'''', N-{4-chloro-3-[5-(dimethylamino)-1H-benzimidazol-2-yl]phenyl}-2,3-
dihydro-1,4-benzodioxin-6-carboxamide; HEK, human embryonic kidney; MOI, mode of inhibition
Recommended assignment: Cellular and Molecular
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Abstract
The smoothened receptor (Smo) mediates Hedgehog (Hh) signaling critical for development, cell growth
and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been
implicated in a variety of cancers and small molecule antagonists of Smo have entered human clinical
trials for the treatment of cancer. Here we report the biochemical characterization of allosteric interactions
of agonists and antagonists for Smo. Binding of two radio-ligands, [3H]SAG-1.3 (agonist) and
[3H]cyclopamine (antagonist), was characterized using human Smo expressed in HEK293F membranes.
We observed full displacement of [3H]cyclopamine by all Smo agonist and antagonist ligands examined.
SANT-1, an antagonist, did not fully inhibit the binding of [3H]SAG-1.3. In a functional cell-based β-
lactamase reporter gene assay, SANT-1 and 2 fully inhibited SAG-1.5-induced Hh pathway activation.
Detailed “Schild-type” radio-ligand binding analysis with [3H]SAG-1.3 revealed that two structurally-
distinct Smoothend receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of
allosteric modulation. Our mechanism of action characterization of radio-ligand binding to Smo
combined with functional data provides a better understanding of small molecule interactions with Smo
and their influence on the Hh pathway.
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Introduction
The hedgehog (Hh) signal transduction pathway is critical to development, differentiation, growth and cell
migration. Although Hh signaling is significantly curtailed in adults, it retains functional roles in stem
cell maintenance (Lai, et al., 2003;Machold, et al., 2003;van den Brink, et al., 2004) and tissue repair (Ito,
et al., 1999;Karhadkar, et al., 2004;Miyaji, et al., 2003;Watkins, et al., 2003). Aberrant Hh signaling
pathway activation has been implicated as the cause of certain cancers, including basal cell carcinomas
and medulloblastomas (Dahmane, et al., 1997;Hahn, et al., 1996;Reifenberger, et al., 1998;Xie, et al.,
1998;Zurawel, et al., 2000) and supporting the tumor microenvironment in numerous other cancers
(Berman, et al., 2003;Dierks, et al., 2007;Karhadkar, et al., 2004;Watkins, et al., 2003) via both Hh ligand-
independent and ligand-dependent pathways. Recently, small molecule antagonists of the Smoothened
receptor (Smo) have entered human clinical trials for the treatment of advanced basal cell carcinoma and
metastatic colorectal cancer (Von Hoff, et al., 2008)
Hh polypeptide secretion activates the Hh pathway through two membrane associated receptors, Patched
(Ptch) and Smoothened (Smo). Hh binds to Ptch, the 12-transmembrane spanning receptor, alleviating
suppression of Smo, a 7-transmembrane protein which is structurally similar to G-protein coupled
receptors. Smo activation stimulates the Gli family of transcription factors to induce the expression of
specific genes, including Gli and Ptch, forming both a negative and a positive feedback loop.
Because of the important role the Hh pathway plays in tumorogenesis and stem cell maintenance, small
molecule regulators of the Hh pathway have attracted considerable interest as potential therapeutic agents.
Cyclopamine, a plant-derived steroidal alkaloid, has been shown to directly bind to and inhibit the Smo
receptor (Chen, et al., 2002a). Various screening efforts using cellular Hh pathway or biochemical assays
directed at the Smo receptor have identified potent small molecule agonists and antagonists with
apparently competitive binding behavior for Smo (Chen, et al., 2002b;Frank-Kamenetsky, et al.,
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2002;Williams, et al., 2003). The 12-pass transmembrane protein Ptc is inhibitory to Smo. Under
physiological conditions, activation of the pathway is caused by binding of the endogenous morphogen Hh
to Ptc, which leads to internalization of Ptc, and consequently alleviates the inhibitory affect of Ptc on
Smo. The exact mechanism for Ptc inhibition of Smo has not been elucidated. In addition, no
endogenous “orthosteric” agonist for Smo has been identified. In fact, it has been proposed that Ptc may
translocate an endogenous sterol (like cyclopamine) which inhibits activation and is relieved when Hh
binds Ptc (Bijlsma, et al., 2006).
Here we report the biochemical characterization of allosteric interactions of small molecule
agonists and antagonists for Smo. Binding of two radioligands, [3H]SAG-1.3 (an agonist) and
[3H]cyclopamine (an antagonist), was characterized using human Smo expressed in HEK293F
membranes. “Schild-type” radio-ligand binding analysis with [3H]SAG-1.3 revealed that the Smo
antagonists SANT-1 and SANT-2 bound in a manner consistent with that of allosteric modulation. Both
SANT-1 and 2 appear to bind in an allosteric fashion exhibited by their partial competition of [3H]SAG-
1.3 binding. In contrast, SANT-1 and 2 fully inhibited SAG-1.5-induced Hh pathway activation in a β-
lactamase reporter gene assay in cells. Our results provide biochemical evidence that suggests SANT-1
and SANT-2 are allosteric inhibitors of [3H]SAG-1.3 binding to Smo. The mechanism of action
characterization of radio-ligand binding to Smo combined with functional data supports the idea that
functionally active small molecules interact with Smo in both competitive and allosteric manners. A
deeper understanding of small molecule interactions with Smo can positively influence the search for
Smo-binding, functionally-active chemical starting points.
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Methods
Materials - SAG-1.3 was synthesized for radio-labeling by the Discovery Medicinal Chemistry
Department of GlaxoSmithKline (RTP, NC). The radio-labeling of [3H] SAG-1.3 (specific activity 70
Ci/mmol) was performed by GE-Amersham (Buckinghamshire, UK). [3H]Cyclopamine (specific activity
20 Ci/mmol) was obtained from American Radiochemicals (St Louis, MO). SYTO-63 and Bodipy-
cyclopamine were purchased from Invitrogen (Carlsbad, CA) and Toronto Research Chemicals (North
York, Canada), respectively. The Cell sensor cell line and culture reagents DMEM/GlutaMAX, Opti-
MEM, Newborn Calf Serum, Nonessential amino acids, HEPES and Blasticidin were purchased from
Invitrogen (Carlsbad, CA). Penicillin/Streptomycin was purchased from Cellgro Mediatech, Inc.
(Manassas, VA). Assay plates and compound dilution plates were purchased from Greiner Bio-one
(Monroe, NC). Unlabeled cyclopamine and tomatidine were purchased from Biomol (Plymouth Meeting,
PA), 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine
[purmorphamine], and cyclopamine-KAAD from Calbiochem (San Diego, CA), and N-[(1E)-(3,5-
dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine [SANT-1] from
Tocris (Sunnyvale, CA). 3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-
pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide [SAG-1.3], 3-chloro-4,7-difluoro-N-[trans-4-
(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide [SAG-
1.5], N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)benzamide [SANT-2], 6-methyl-N-
{2-[(1,3-thiazol-2-ylmethyl)amino]-2,3-dihydro-1H-inden-5-yl}-4'-(trifluoromethyl)-2-
biphenylcarboxamide [Compound 1], 2-chloro-N-[4-chloro-3-(2-pyridinyl)phenyl]-4-
(methylsulfonyl)benzamide [GDC-0449] and N-{4-chloro-3-[5-(dimethylamino)-1H-benzimidazol-2-
yl]phenyl}-2,3-dihydro-1,4-benzodioxin-6-carboxamide [Z''''] were synthesized by the Medicinal
Chemistry Research Department of GlaxoSmithKline Research Laboratories, (Durham, NC), and WuXi,
(Shanghai, People's Republic of China). Phenylmethylsulfonyl fluoride (PMSF), pepstatin, Complete
EDTA-free protease tablets and Triton X-100 were purchased from Roche Diagnostics (Indianapolis, IN).
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Leupeptin was purchased from Calbiochem (San Diego, CA). All other components of the membrane
preparation buffers and all other standard reagents were purchased from Sigma (St. Louis, MO).
Generation of recombinant BacMam virus - The Smo BacMam virus was constructed and amplified
based on published methods (Fornwald, et al., 2007). The coding region for the Smo gene, cloned in-
house from human ovary, aligns with NCBI sequence NM_005631.3.
Transduction of HEK293F with Smo BacMam Virus- The cell line used for transductions, HEK293F, was
purchased from Invitrogen (Carlsbad, CA). HEK293F cells were sub-cultured twice weekly in FreeStyle
293 Expression Medium (Invitrogen) and maintained in 3 liter sterile, plastic Erlenmeyer flasks on
shaking platform set at 90 rpm in a humidified 37oC incubator with 5% CO2. Cells were seeded on day 0
at 6x105 cells/ml. The following day the transduction was performed at a cell density of 1.0 – 1.2x106
cells/ml using 40 Smo BacMam virus particles per cell; the culture was supplemented with 2 mM sodium
butyrate. Approximately twenty-four h later, transduced cells were harvested by centrifugation at 400xg
for 30 min at 4oC, washed once with a balanced salt solution, re-pelleted, and the pellet flash frozen in
liquid nitrogen. The cell pellets were stored at -80oC until processed for membranes.
Membrane preparation- HEK293F/Smo_BacMam cell pellets were re-suspended at 4oC in 10 volumes of
ice-cold buffer A (50 mM HEPES pH 7.4, 1 mM EDTA, 25 µg/ml Bacitracin, 100 µM Leupeptin) plus 1
mM PMSF and 2 µM pepstatin per gram of cell pellet, homogenized in a Waring blender for 15 s, placed
on ice for 5 min, and homogenized for an additional 15 s. To remove large particles, a low speed
centrifugation (500xg for 30 min at 4oC) was performed, followed by high-speed centrifugation (48,000xg
for 45 min at 4oC), resuspension in buffer A plus 2 µM pepstatin, and a final high speed centrifugation at
(48,000g for 45 min at 4oC). A dounce homogenizer was used to resuspend the final pellet using ice-cold
buffer A. The membrane suspension was passed through a 23G needle, aliquoted, and stored at -80oC.
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Total protein concentration of the membrane preparation was determined with a Coomassie Plus Reagent
Kit from Pierce Biotechnology (Rockford, IL) using bovine serum albumin as the standard.
[3H]SAG-1.3 and [3H]cyclopamine radioligand binding assays- Radioligand binding assays were
performed under the same conditions, independent of the radioligand used. Membranes were diluted in
buffer B (50 mM HEPES, 3 mM MgCl2 pH 7.2 at 23oC) to a concentration of 0.01-0.02 mg protein/ml.
Assays were initiated by the addition of 200 µl of membrane suspension to 100 µl of radioligand at 1-3
times radioligand Kd and various concentrations of inhibitors in buffer B plus a cocktail of protease
inhibitors (EDTA-free, Roche Diagnostics) and 0.02% BSA to reduce non-specific binding. Binding
assays were performed in duplicate in polypropylene 96 well plates (Costar Corp., Cambridge, MA).
Nonspecific binding was defined in the presence of 1-10 µM cyclopamine ([3H]SAG-1.3 assays) or 10 µM
SANT-2 ([3H]cyclopamine assays). Competition assays were performed at 23°C for 5 h to allow adequate
time for the two ligands to reach equilibrium for binding. The separation of bound from free radioligand
was accomplished by rapid vacuum filtration of the incubation mixture over glass fiber filters (Inotech
Biosystems International, Gaithersburg, MD) presoaked for 2 h in 0.3% polyethylinamine pH 13 using an
Inotech cell harvester. Filters were washed 2 times with 0.3 ml of ice-cold phosphate buffered saline pH
7.0 containing 0.01% Triton X100. Radioactivity on the filters was quantified using a Top Count Liquid
Scintillation Counter, (Perkin Elmer, Waltham, MA). “Hot” saturation experiments were performed with
the addition of labeled [3H]SAG-1.3 over a range of concentrations (0.1-100 nM). Isotopic “cold”
saturation studies were carried out using [3H]cyclopamine or [3H]SAG-1.3 at two fixed concentrations in
the presence of varied concentrations of unlabeled cyclopamine (0.001-10 µM) or SAG-1.3, respectively.
Mode of inhibition (MOI) studies were performed using similar conditions including double titrations of
both radio-ligand and unlabeled Smo ligands.
Bodipy-cyclopamine whole cell binding assay- HEK MSRII cells were transduced at a density of 3X105
cells/mL with 17 SMO BacMam virus particles/cell in Dulbecco’s modified eagle’s medium (DMEM)
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containing 0.5% fetal bovine serum and seeded at 15,000 cells /well in 384-well poly-d-lysine coated
plates followed by overnight (>20 h) incubation at 37°C and 5% CO2. Compound stocks were prepared
in DMSO, except for Bodipy-cyclopamine, which was prepared in 100% ethanol, according to
manufacturer’s instructions. On day 2, DMEM media were removed with a Power Washer 384TM
(TECAN US, Inc., Durham, NC), cells were fixed by adding 3.7% (v/v) formaldehyde (VWR Scientific
Inc., Bridgeport, NJ) to the plates using a Multidrop 384® (Thermo Fisher Scientific, Milford, MA) and
incubating for 20 min. After aspirating the fixation buffer, 2 µM SYTO-63, 25 nM BODIPY-cyclopamine
and indicated concentrations of compounds (all diluted in Hank’s Balanced Salt Solution (HBSS) without
Ca2+/Mg2+) Gibco®Invitrogen (Carlbad, CA), were transferred to the assay plates with a Cybi-well
(Cybio, Jena, Germany). Assay plates were incubated for 2 h, washed several times with buffer, then
imaged on a Perkin Elmer Opera instrument. The fixing and washing protocol was optimized to eliminate
non-specific binding of the Bodipy-cyclopamine probe. On the Opera instrument, 635 nm and 488 nm
excitation lasers were used to excite SYTO-63 and Bodipy, respectively, while 690/50 nm and 525/50 nm
filters were used to collect the fluorescence signals. A primary 405/488/635 nm dichroic filter was used
to filter laser light, a 650 nm long pass filter was used to split Syto63 and Bodipy emission wavelengths,
sending wavelengths longer than 650 nm to the 690/50 nm filter and the remaining light to the 568 nm
dichroic filter. The 568 nm dichroic filter was used to send wavelengths shorter than 568 nm to the
525/50 nm filter for detection of the Bodipy signal. Laser power and exposure times were optimized to
avoid any cross talk between detection channels. The Syto 63 signal was used to identify all cells as
objects, while the Bodipy signal was used to quantify Bodipy-cyclopamine binding to Smo receptors in
cells. Acapella software in the Opera imaging system was used to develop the analysis algorithms for
quantification of the images.
β-lactamase reporter gene assay- CellSensor Gli-bla NIH3T3 cells were maintained at 20 – 80%
confluence in DMEM/GlutaMAX media supplemented with 10% newborn calf serum (NCS), 0.1 mM
nonessential amino acids (NEAA), 25 mM HEPES (pH 7.3), 100 U/mL penicillin, 100 μg/mL
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streptomycin, and 5μg/ml of Blasticidin. Assay medium was Opti-MEM supplemented with 0.5% NCS,
0.1mM NEAA, 1mM sodium pyruvate, 10mM HEPES, 100 U/mL penicillin, and 100 μg/mL
streptomycin. Antagonist and agonist assays were carried out in black-wall, clear bottom, 384-well assay
plates with 10,000 cells/well. For the antagonist assay, cells were plated using a Multidrop 384®
(Thermo Fisher Scientific, Milford, MA) at a volume of 20 μl/well in the presence of an agonist (EC80),
followed by the addition of test compounds with a Cybi well, and incubated for 40-48 h before detection.
For the agonist assay, cells were plated, compounds were added as above, and plates incubated in the
presence of test compounds for 40-48 h prior to detection. Final assay conditions included 1% DMSO
final concentration in a 25 μL total volume assay. After the 40 - 48 h incubation, 5 µl of a 6X LiveBlazer
loading reagent (prepared as instructed by Invitrogen) was added to the assay plates using a Multidrop
384®. Plates were then read using an Envision reader (Perkin Elmer, Waltham, MA) in bottom-read
mode, with an excitation wavelength of 409 nm and emission wavelengths of 460 and 535 nm to measure
the ratio of fluorescence intensity at 460 nm versus 535 nm as specified by Invitrogen.
DATA Analysis - The binding rate constants (k+1 and k-1), apparent equilibrium dissociation constants (Kd)
and the maximum number of binding sites (Bmax), EC50, IC50 and Ki analysis were performed using the
nonlinear iterative curve-fitting computer program GraphPad PRISM (San Diego, CA) (Motulsky and
Christopoulos, 2004). Mode of inhibition (MOI) analysis was performed using GraphPad PRISM as
described in results.
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RESULTS
Characterization of [3H]SAG-1.3 Binding to 293FSmo Cell Membranes- Binding of [3H]SAG-1.3 (2-4
nM) was observed at 23°C to membranes derived from 293FSmo cells and not to those from the 293F
cells transfected with the vector control (Supplemental Figure 1). Incubation of various amounts of
293FSmo derived membrane homogenates with [3H]SAG-1.3 indicated that binding of the radioligand
was linear with protein concentrations up to 12µg/well (Supplemental Figure 2). Based on the protein
titration, a final membrane protein concentration of 2-4 µg/well was used in subsequent assays. Specific
binding measured at this protein concentration was between 1 and 10% of the total amount of radioligand
added, indicating that radioligand depletion was insignificant because of the low receptor concentration
used (i.e., total [L] ~ free ligand). Nonspecific binding was defined in the presence of 1 µM SAG-1.3 as
well as 1-10 µM of cyclopamine or cyclopamine-KAAD. Both yielded a common plateau of maximal
inhibition. Cyclopamine was chosen for subsequent studies since it is structurally distinct from [3H]SAG-
1.3. Under these conditions, [3H]SAG-1.3 binds to Smo containing membranes with 70-80% specificity.
Similar studies were performed to optimize binding of [3H]cyclopamine to 293FSmo membranes (data not
shown).
Kinetics of [3H]SAG-1.3 and [3H]cyclopamine binding; Association/ Dissociation- The time course of
[3H]SAG-1.3 and [3H]cyclopamine binding to 293FSmo cell membranes both exhibited pseudo-first-order
kinetics (Fig. 1A and 1B). At 23°C, equilibrium was reached within 2 h and remained stable up to 5 h.
After an incubation time of 5 min and longer, non-specific binding (defined in the presence of 10 µM
cyclopamine for [3H]SAG-1.3 and 10 µM SANT-2 for [3H]cyclopamine), remained constant. Two
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concentrations of [3H]SAG-1.3 and [3H]cyclopamine were analyzed using global fitting of the association
kinetic model. Using this analysis, we derived a single best-fit estimate for both association (kon) and
dissociation (koff) (Table 1). For [3H]SAG-1.3, we derived an association rate constant of kon = 3.0 x 106
M-1
min-1
and a dissociation k-1 = 8.0 x 10-3
min-1
corresponding to an apparent half-time of 86 min. Non-
linear least squares fitting of this data to a model assuming pseudo first order kinetics best fit the data
compared to a bi-phasic model (F-test , p < 0.05) over the model of one binding site. The equilibrium
dissociation constant (Kd = koff / kon) calculated from the kinetic data was 2.7 nM. The binding of
[3H]cyclopamine, displayed biphasic association kinetics. Fitting of this data to a model assuming the
presence of two binding sites was preferred by the F-test ( p < 0.05) over the model of one binding site.
The fast and slow components of association exhibited half-times of 1.5 and 28 min respectively. The
data fit to pseudo first order kinetics resulted in an association rate constant of kon = 2.4 x 106 M-1
min-1
and a dissociation koff = 2.0 x 10-2
min-1
corresponding to an apparent half-time of 36 min. The
equilibrium dissociation constant (Kd = koff / kon) calculated from the kinetic data was 12 nM.
Saturation of [3H]SAG-1.3 binding to 293FSmo Cell Membranes- Saturation of [3H]SAG-1.3 binding to
293F Smoothened cell membranes is demonstrated in Figure 2. Iterative non-linear analysis indicated
(best fit) a single binding site of high affinity (Kd = 1.7 ± 0.54 nM; Bmax = 34 ± 2.3 pmol/mg protein)
(Fig. 2A). Affinity and Bmax values derived from orthogonal homologous “isotopic” cold saturation
experiments yielded Kd = 5.9 ± 1.7 nM and Bmax = 40 ± 8 pmol/mg protein (Fig. 2B). The limit of
detection for competing compounds utilizing this radioligand was calculated from the density of receptors
(Bmax) expressed in the membrane preparation. Based on the Bmax determined from saturation binding
assays as well as the affinity of [3H]SAG-1.3, the limit of compound affinity which could be measured
using this radioligand was 0.26 to 1.5 nM depending on the final assay volume (1- 0.15 ml).
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Homologous “isotopic-cold” saturation of [3H]cyclopamine binding to 293FSmo Cell Membranes-
Homologous “isotopic-cold” saturation of [3H] cyclopamine specific binding to 293F Smoothened cell
membranes is demonstrated in Figure 3. Iterative non-linear analysis indicated a single binding site (Hill
values 0.9-1.1; Kd = 10 ± 2.5 nM; Bmax = 20 ± 0.9 pmol/mg protein). Based on the density of receptors
determined from saturation binding assays as well as the affinity of [3H]cyclopamine, the limit of
compound affinity which could be measured using this antagonist radio-ligand was 0.4 to 2.6 nM
depending on the final assay volume (1 - 0.15 ml).
Pharmacology of [3H]SAG-1.3 and [3H]cyclopamine Binding to Smoothened Receptors-
The pharmacology of [3H]SAG-1.3 binding to Smo receptors in membranes derived from 293F cells was
examined in competition experiments using small molecule Smo agonists and antagonists. The SAG-1.3
and SAG-1.5 agonists are structurally-related while the purmorphamine agonist is structurally distinct.
Similarly, the cyclopamine, cyclopamine-KAAD, and tomatadine (inactive) antagonists are structurally
similar, while SANT-1, SANT-2, Z'''', Compound 1 (a Smo antagonist which is the racemate of a
representative example #8 structure from Novartis (JAIN, et al., 2007), and GDC-0449, Genentech’s
clinical Smo antagonist (Von Hoff, et al., 2008)(Fig. 4) represent distinct chemotypes. SAG-1.5 was the
most potent agonist inhibitor of [3H]SAG-1.3 binding to Smo (Ki = 0.5 nM). SAG-1.3 bound with high
affinity (Ki = 1 nM) followed by the antagonists GDC-0449, Z'''' (Compound 1), cyclopamine-KAAD,
SANT-2, and cyclopamine (Fig. 5A and B, and summarized in Table 2). Interestingly, GDC-0449
inhibited the binding of [3H]SAG-1.3 in a biphasic manner with an apparent high affinity equal to 1.3 nM
and low affinity of 2.3 µM (Fig 5B). Purmorphamine and tomatidine (an inactive analog of cyclopamine)
were weak inhibitors of binding (inactive up to 10 µM). SANT-1 only exhibited partial inhibition (40%)
of [3H]SAG-1.3 binding with a plateau from 0.04-10 µM, suggesting a possible allosteric interaction
between SANT-1 and SAG-1.3.
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Because of the differential response the agonists and antagonists exhibited against small molecule agonist
[3H]SAG-1.3 binding, we performed competition experiments with the same set of compounds using
[3H]cyclopamine (an antagonist) as the radioligand (Table 2). GDC-0449 possessed the highest affinity
for smoothened receptors labeled by [3H]cyclopamine with a Hill slope of 1 and a four-fold greater affinity
against this ligand compared to the agonist radioligand [3H]SAG-1.3. Agonist compounds, SAG-1.3 and
SAG-1.5, retained similar affinities for Smo defined by competition for [3H]cyclopamine binding to the
Smo receptors with Ki values of 3.7 and 2.3 nM respectively (Fig. 5C and Table 2). Purmorphamine
lacked significant inhibition up to the highest concentration tested. The binding profiles of cyclopamine,
cyclopamine-KAAD, and the inactive analog tomatidine using [3H]cyclopamine were consistent with
affinities measured using the agonist radio-ligand. The antagonist molecules Z'''', SANT-1, and SANT-2
showed high affinities for Smo labeled by [3H]cyclopamine (Fig. 5D and Table 2). In contrast to the
partial inhibition of [3H]SAG-1.3 binding, SANT-1 completely inhibited binding of [3H]cyclopamine with
high affinity (Ki = 2.4 nM).
Mode of Inhibition Studies- The finding that SANT-1 only partially inhibited [3H]SAG-1.3 binding to
Smo in competition assays suggests that various ligands may bind to Smo at distinct sites. To further
investigate the modes of inhibition (MOI) of these ligands, double titrations of both radio-ligand and
compounds were performed for SAG-1.5, cyclopamine, SANT-1, and SANT-2. Compound displacement
(specific binding) and apparent IC50 values were examined in the presence of varying concentrations of
the radioligand probe. This radioligand binding method is comparable to Schild plot analysis (Ehlert and
Authors, 1988) and often referred to as Schild-type plots (Schetz, et al., 1997). Competitive binding
interactions show a linear shift in apparent IC50 in relation to radioligand concentration while allosteric
antagonism can result in an incomplete inhibition of specific binding and or a non-linear hyperbolic plot
of IC50 values. In addition, the data can be quantified by fitting to a modified model based on the Cheng-
Prusoff equation (Kenakin, 2006),
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))/]([1(
)]([1(50
d
dB KA
KAKIC
α+++
= (Eq. 1)
where Kd is the affinity value for the radiolabeled ligand, KB is the Kd for the cold ligand and α is a
cooperativity term. For mutually exclusive ligands i.e. competitive, the value of α in the equation is ∞.
For mixed MOI (including allosteric) α is ≥ 1, but finite.
The Smo agonist SAG-1.5 and antagonist cyclopamine fully inhibited specific binding of [3H]SAG-1.3 to
Smo receptors over the range of radio-ligand concentrations (1-40 x Kd). Apparent IC50 values for SAG-
1.5 shifted linearly with respect to radio-ligand concentration, with α values of 40 and 300 in two different
experiments (Fig. 6A and B). Cyclopamine also behaved in a competitive manner with α >100 in three
experiments (Fig 6C and D). The profile of these two compounds indicated a MOI consistent with
competitive inhibition for binding to Smo labeled with [3H]SAG-1.3. SANT-1 competition resulted in
only partial inhibition (<60%) along with a minor shift in the apparent IC50 values (7-16 nM, α = 4 and
4.5) consistent with hallmarks of an allosteric interaction with [3H]SAG-1 (Fig. 7C and D). The effect of
increasing [3H]SAG-1.3 concentrations on SANT-2 competition binding also revealed a profile consistent
with allosteric inhibition. Incomplete inhibition of [3H]SAG-1.3 binding was observed only at [3H]SAG-
1.3 concentrations above 2 nM. The relationship of apparent IC50 values (17-50 nM) to radio-ligand
concentration was not linear with calculated α values of 5 and 8 (Fig. 7 A and B). In addition, the data
were fit to a more complex ternary model (Kenakin, 2006) to further quantify the interactions (data not
shown). This analysis yielded affinity and α values which were consistent with the previous
determinations indicating a probe-dependent allosteric interaction for SANT-1 and SANT-2 for [3H]SAG-
1.3 binding to Smo.
Whole cell Bodipy-cyclopamine binding assay- The potencies of known smoothened ligands and analogs
for inhibition of fluorescent Bodipy-cyclopamine binding to smoothened receptors are summarized in
Table 2. The rank order and potency of the compounds tested in the Bodipy-cyclopamine assay were
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similar to the affinities determined in both the [3H]SAG-1.3 and [3H]cyclopamine membrane binding
assays. SANT-1 completely competed with Bodipy-cyclopamine, consistent with the complete inhibition
in the [3H]-cyclopamine binding assay.
β-lactamase reporter gene functional assay- To understand the functional consequence of partial
competition of [3H]SAG-1.3 by antagonists like SANT-1 and 2, we also tested these agonists and
antagonists in a β-lactamase reporter gene functional assay (Table 2). The purine derivative agonist
purmorphamine exhibited weak activation of the system, consistent with previous observations in similar
mouse reporter systems (Sinha and Chen, 2006). Both SAG-1.3 and SAG-1.5 displayed nanomolar-range
potency stimulation of Gli-responsive reporter activity, with EC50 values of 0.4 and 7 nM respectively.
Since SAG-1.5 was competitive with [3H]SAG-1.3 for binding to SMO, yet was superior in potency than
the structurally-similar SAG 1.3 in β-lactamase reporter gene functional assay, an EC80 concentration of
SAG 1.5 was utilized as the reporter activator for evaluation of functional inhibitors of Smo The
antagonist ligands SANT-1 and GDC-0449 were the most potent with IC50 values of 20 and 45 nM
respectively. Additional antagonist ligands such as cyclopamine-KAAD, SANT-2, Z'''', and Compound 1,
retained similar rank order pharmacologic potencies to the measured binding affinities determined using
both agonist and antagonist radio-ligands. All of these antagonists, including SANT-1 and 2, exhibited
complete inhibition of the response to the level of background (Fig. 8).
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DISCUSSION
The Hh pathway plays an important role in development, stem cell maintenance, and
tumorigenesis. The pathway activator, Smo, is being pursued as an attractive target for cancer and small
molecule antagonists of Smo have entered clinical trials for the treatment of multiple cancers including
advanced basal cell carcinoma and metastatic colorectal cancer. A better understanding of the interactions
and regulations of antagonists and agonists of Smo will facilitate the design and discovery of the next
generation of small molecule regulators of Smo and the Hh pathway.
Previously, radio-ligand binding of a Smo agonist, [3H]SAG-1.5, has been characterized (Frank-
Kamenetsky, et al., 2002). We have extended their study by examining the binding of both an agonist,
[3H]SAG-1.3, and an antagonist, [3H]cyclopamine to Smo receptors expressed in HEK293F membranes.
Kinetic and equilibrium binding studies revealed that specific binding of both tritium-labeled SAG-1.3
and cyclopamine to human Smo receptors was saturable, reversible, of high affinity, and consistent with
single site binding. Binding kinetic studies revealed that [3H]SAG-1.3 dissociates from Smo receptors
more slowly (t½ = 86 min) compared to cyclopamine (t½ = 36 min). Because of this, we performed
mathematical analysis to show that when the radioligand dissociates more slowly than a competing ligand,
binding is close to equilibrium conditions at t = 3.5/k-1 for the radio-ligand. In the absence of definitive
rate constants for the competing ligands tested, we allowed the binding to equilibrate for 5-7 h, with no
apparent detriment to membrane or ligand integrity. Binding of [3H]cyclopamine to smoothened receptors
displayed a biphasic association (Fig.1 , Table 1) which might indicate a ligand conformational change or
binding site heterogeneity. Saturation binding studies with [3H]SAG-1.3 (classical “hot”) and
[3H]cyclopamine (isotopic-“cold”) demonstrated that each ligand binds to a single site with high affinity,
yielding Kd values of 1.7 ± 0.6 and 10 ± 2.5 nM, respectively (Fig. 2). There was a two fold difference in
the Bmax values determined for [3H]cyclopamine (isotopic) and [3H]SAG-1.3 (“hot) of 20 and 40 pmol/mg
protein respectively (Table 1). Additional assays such as “Schild-type” binding and direct “hot”
[3H]cyclopamine saturation studies are warranted to determine if these ligands bind to the same sites.
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Additional detailed kinetic and direct “hot” saturation analysis with [3H]cyclopamine are required to
further investigate the biphasic association and comparison of binding site number.
A number of Smo agonists and antagonists have been reported previously. The affinity values for
these ligands have been determined using a variety of techniques and often cannot be compared directly.
Using both the agonist and antagonist radioligand probes, we have characterized and compared the
binding affinity of 3 agonists and 8 antagonists of Smo (Table 2). The pharmacology of most compounds
using either [3H]SAG-1.3 or [3H]cyclopamine is similar. SANT-1 shows probe-dependent differences in
competition and will be discussed later. Using [3H]SAG-1.3, the potencies we have determined for SAG-
1.5, SAG-1.3, and cyclopamine-KAAD are in agreement with those determined using [3H]SAG-1.5 to
label murine Smo receptors (Frank-Kamenetsky, et al., 2002). The affinities for most of the other
compounds examined were in agreement with previously reported functional potencies with the exception
of purmorphamine, a hedgehog pathway activator. Purmorphamine has been reported to inhibit BODIPY-
cyclopamine binding to Smo (IC50~ 1µM) and purmorphamine-driven activation of Gli-dependent reporter
was inhibited by cyclopamine (Sinha and Chen, 2006). Interestingly, in our studies, purmorphamine
weakly displaced BODIPY-cyclopamine (IC50 ~ 3.3 µM) binding but did not displace either [3H]SAG-1.3
or [3H]cyclopamine binding up to 10 µM. Two interpretations of this data may be that purmorphamine
activates the Hh pathway indirectly without binding Smo or binds in an allosteric fashion to either
[3H]SAG-1.3 or [3H]cyclopamine consistent with neutral cooperativity. Additional functional studies
employing Schild analysis of the interactions between cyclopamine and agonists such as purmorphamine
and SAG-1.3 would help further the understanding of compound interactions and influence on the
smoothened pathway.
Although the pharmacology of most ligands was similar using either [3H]SAG-1.3 or
[3H]cyclopamine, we observed very different competition patterns for SANT-1, a previously identified
small molecule Smo antagonist. While SANT-1 fully displaced [3H]cyclopamine binding and completely
inhibited SAG-1.5-driven Gli-dependent reporter activity, 10 µM SANT-1 inhibited only 40% of
[3H]SAG-1.3 binding, suggesting potential allosteric interaction between SANT-1 and SAG-1.3. To
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further understand the mechanism of action for SAG-1.5, cyclopamine, SANT-1, and SANT-2 binding to
Smo, we performed a series of double titration (“Schild-type”) experiments with [3H]SAG-1.3 and
[3H]cyclopamine. Analysis of the interactions between [3H]SAG-1.3 and SAG-1.5 or cyclopamine yielded
profiles consistent with competitive binding with α values >>10. In addition, maximal inhibition to the
level of non-specific binding was obtained over the entire range of radio-ligand concentrations tested. It
cannot be fully ruled out that cyclopamine may have a strong allosteric effect, reflected as competitive for
[3H]SAG-1.3 binding. In contrast, our analyses clearly indicated an allosteric interaction for SANT-1 and
SANT-2 towards SAG-1.3. Incomplete inhibition of specific [3H]SAG-1.3 binding (inability to reach
level of non-specific binding in the presence of high concentrations of SANT-1 or SANT-2), a hallmark of
allosteric mode of inhibition, was observed for these antagonists. In addition, the plots for SANT-1 and
SANT-2 each deviate from linearity with calculated α values of <10 for each antagonist. Our data suggest
that the interactions between these two antagonists and SAG-1.3 are not directly competitive. As has been
observed with other receptors, the observed allosteric binding of SANT-1 and SANT-2 appears probe- or
radio-ligand dependent. The fact that SANT-1 and SANT-2 bind smoothened receptors labeled with
[3H]cyclopamine in a manner consistent with competitive antagonism illustrates this point. Further
characterization of the allosteric effects could be performed through sensitive dissociation kinetic studies.
In many cases allosteric inhibitors have been shown to increase the rate of tracer dissociation (Kenakin,
2006). While such assays may be helpful, the particular kinetics of a probe such as [3H]SAG-1.3 should
be considered. The data presented demonstrate SANT-1 and SANT-2 modulate smoothened receptors
labeled with [3H]SAG-1.3 in an allosteric manner.
It has been proposed that SANT-1 may not directly compete for cyclopamine binding since it
failed to directly displace BODIPY-cyclopamine binding. Our competition experiments using both
[3H]cyclopamine (membrane homogenate) and BODIPY-cyclopamine (whole cell) showed complete
displacement by SANT-1. Therefore, we do not have any evidence that SANT-1, SANT-2, and
cyclopamine, have different binding sites. Since SANT-1 and SANT-2 completely inhibit SAG-1.5
induced Hh pathway activation, it seems that partial displacement of SAG-1.3 binding can nevertheless
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produce full functional inhibition. Site-directed mutagenesis studies utilizing both binding and functional
assays would supplement these data and enhance the understanding of smoothened receptor agonist and
antagonist mechanism of action. Our results support the general bias observed in binding assays
compared to functional assays. Because of this, we reemphasize that the choice of ligand used in radio- or
fluorescent-binding studies should be considered, since each probe might specifically identify different
sets of allosteric and competing compounds. The subtle differences in binding of allosteric ligands such
as SANT-1 and SANT-2 may result in unique phenotypes as they may change the way smoothened
receptors interact with accessory proteins. In addition, the specific cellular phenotype can influence the
action of such ligands and is worthy of further investigation.
In conclusion, we have characterized radioligand binding of both an agonist and an antagonist of
Smo. Using both probes, we have compared the binding of multiple antagonists and agonists to Smo.
Our detailed biochemical mechanism of action analyses indicate SANT-1 and SANT-2 are allosteric
inhibitors of [3H]SAG-1.3 binding. Our data have important implications on the understanding of Smo
regulation and discovery of additional small molecule Smo regulators.
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ACKNOWLEDGEMENTS
The authors are grateful to Da-Yuan Wang, for the generation of the Smo BacMam, Kathleen Gallagher
for cloning of the Smo cDNA, and both Terry Kenakin and Thomas Meek for helpful discussions and
suggestions.
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Legends for Figures
Fig.1. A Determination of association and dissociation rates for [3H]SAG-1.3 to 293FSmo cell
membranes. Aliquots of membranes were incubated with 4 and 14 nM [3H]SAG-1.3 at 23°C in the
absence or presence of 10 µM cyclopamine (nonspecific) for varying lengths of time. Data using
two radio-ligand concentrations were fit to the association kinetic model. Non-linear least squares
fitting of this data to a model assuming pseudo first order kinetics best fit the data compared to a bi-
phasic model by the F-test ( p < 0.05) over the model of one binding site. The 95% confidence
bands are shown by thin dashed lines. Individual replicates are plotted. The experiment was
repeated two times with similar results. B Determination of association and dissociation rates for
[3H]cyclopamine to 293FSmo cell membranes. Aliquots of membranes were incubated with 7 and
19 nM [3H]cyclopamine at 23°C in the absence or presence of 10 µM cyclopamine (nonspecific) for
varying lengths of time. Non-linear least squares fitting of this data to a model assuming the
presence of two binding sites was preferred by the F-test ( p < 0.05) over the model of one binding
site. The 95% confidence bands are shown by thin dashed lines. Individual replicates are plotted.
The experiment was repeated two times with similar results.
Fig.2. Saturation assays of [3H]SAG-1.3 binding to HEK 293F cell membranes expressing human
Smoothened receptors. A, direct “hot” saturation binding isotherm showing the total amount of
[3H]SAG-1.3 bound, the amount of [3H]SAG-1.3 bound in the presence of 10 µM cyclopamine
(nonspecific) and the specific (total-nonspecific) binding. The inset shows semilogarithmic
transformation of the specific binding showing that full saturation has been obtained. B,
homologous “isotopic-cold” saturation assay using two concentrations of [3H]SAG-1.3 (2 and 11
nM) in the presence of unlabeled ligand (0.003-10 µM). Saturation analysis best fit a single site
model. Aliquots of 293F cell membranes expressing human Smoothened receptors (1-2 µg/well)
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were incubated for 5 h at 23°C in binding buffer. The data shown are from a representative
experiment. Each value is the average of 2-3 determinations (n=2-7).
Fig.3. Homologous “isotopic-cold” saturation assay using two concentrations of [3H]cyclopamine
(7 and 27 nM) in the presence of unlabeled ligand (0.006-30 µM). Global analysis of both data
sets was performed to derive Kd and Bmax values. Aliquots of 293F cell membranes expressing
human Smoothened receptors (1-2 µg/well) were incubated for 5 h at 23°C in binding buffer. The
data shown are from a representative experiment. Each value is the average of 2-3 determinations
(n=2).
Fig.4. Smoothened receptor agonist and antagonist molecule structures. A, Leiosamine agonist
molecules (SAG-1.3 and SAG-1.5) and a purine derivative purmorphamine. B, Antagonist
chemotypes including cyclopamine, and active derivative cyclopamine-KAAD as well as an
inactive derivative tomatidine. Structurally distinct antagonist chemotypes which inhibit Hh
signaling; SANT-1, SANT-2, Z'''', Compound 1, and the Genentech antagonist GDC-0449.
Fig.5. Characterization of the pharmacological specificity of [3H]SAG-1.3 and [3H]cyclopamine
binding in 293F cells expressing human Smoothened receptor. Membranes prepared as described
under “Materials and Methods”, were incubated with 3 nM [3H]SAG-1.3 (figures A and B) or 7
nM [3H]cyclopamine (figures C and D) and varying concentrations of competing compounds for 5
h at 23°C. The data shown are from representative experiments. Similar curves were obtained
from multiple experiments (n=3-10). The calculated Ki values for the compounds are shown in
table 1.
Fig.6. Schild type plot analysis of SAG-1.5 and cyclopamine inhibition of [3H]SAG-1.3
smoothened receptor binding. A and C: Representative displacement curves of [3H]SAG-1.3
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shown for a range of SAG-1.5 and cyclopamine concentrations. Curves generated in the presence
of varying concentrations of radio-ligand from 1-40 times Kd. B and D: Schild type plot
transformations of IC50 apparent values representing the relative shift in competing compound
potency. Note, α values greater than 10 are consistent with a competitive interaction between
ligands. The data shown are a representative of several experiments (n=2) in duplicate or
triplicate.
Fig.7. Schild type plot analysis of SANT-1 and SANT-2 inhibition of [3H]SAG-1.3 smoothened
receptor binding. A and C: Representative displacement curves of [3H]SAG-1.3 shown for a range
of SANT-1 and SANT-2 concentrations. Curves generated in the presence of varying
concentrations of radio-ligand from 1-40 times Kd. B and D: Schild type plot transformations of
IC50 apparent values representing the relative shift in competing compound potency. Note, α
values less than 10 and incomplete inhibitions are hallmarks of an allosteric binding interaction.
The data shown are a representative of several experiments (n=2) in duplicate or triplicate.
Fig.8. Inhibition by SANT-1 and SANT-2 of the SAG-1.5 induced β-lactamase response.
CellSensor NIH3T3 cells stably expressing the Gli-b-lactamase reporter were exposed to
compounds for 40-48 h in the presence of an EC80 concentration of SAG-1.5. Zero percent and
100% correspond to the basal level of fluorescence and to the SAG-1.5 induced fluorescence level,
respectively. Data are representative of more than three separate experiments all performed in 2-6
replicates.
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Tables TABLE 1
Equilibrium binding of [3H]SAG-1.3 and [3H]cyclopamine to human smoothened receptors in HEK293F cell membranesRadio-ligand Method K d nM B max (pmol/mg protein)
[3H]SAG-1.3 "hot" saturation 1.7 ± 0.5 34 ± 2.3
[3H]SAG-1.3 isotopic- "cold" 5.9 ± 1.7 40 ± 8.0
[3H]cyclopamine isotopic- "cold" 10 ± 2.5 20 ± 0.9
Kinetic parameters of Smo ligand binding to human smoothened receptors in HEK293F cell membranesRadio-ligand k on k off (t ½) k off / k on
x 10 6 M -1 min -1 min -1nM
[3H]SAG-1.3 3.0 ± 0.3 0.008 ± 0.001 2.7 ± 0.6(86 min)
[3H]cyclopaminepseudo-first-order kinetic
determinations2.4 ± 0.6 0.02 ± 0.01 12 ± 8.0
(36 min)biphasic association rate
determinationskobs
kobs fast 0.562 ± 0.126
t ½ fast (1.5 min)
kobs slow 0.023 ± 0.001
t ½ slow (28 min)Data are the mean ± STDEV (n=2-7) K d affinity, B max receptor density and kinetic rate values were determined using non-linear curve fitting as described under Data Analysis
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TABLE 2Binding affinities and functional potencies of various smoothened ligands at wt hSMO receptors
IC50 (nM) XC50 (nM)d
Compound chemotype [3H]SAG-1.3 [3H]cyclopamineWhole cell Bodipy-
cyclopamine binding b-lactamase reporte
SAG1.3 agonist 1.0 ± 0.3 3.7 ± 1.1 13 ± 4.5 7.1 ± 2.9SAG1.5 agonist 0.5 ± 0.2 2.3 ± 0.8 11 ± 3.2 0.39 ± 0.16
purmorphamine agonist >10,000 >10,000 3,300 ± 900 810 ± 250cyclopamine antagonist 13 ± 2.6 17 ± 7 27 ± 5 750 ± 130
cyclopamine-KAAD antagonist 3.9 ± 1.8 9.3 ± 3.5 33 ± 25 60 ± 20tomatidine cyclopamine analogb >10,000 >3,000 N/A 2,310 ± 160
SANT-1 antagonist 40% @ 10µMc 2.4 ± 0.7 5.9 ± 1.9 21 ± 3SANT-2 antagonist 7.8 ± 4.4 8.4 ± 3.9 56 ± 14 135 ± 51
Z'''' antagonist 3.3 ± 0.2 2.8 ± 0.7 11 ± 2.2 96 ± 38Compound 1 antagonist 3.9 ± 2.8 4.8 ± 0.8 50 ± 35 85 ± 10
Genentech GDC-0449 antagonist (H) 1.3 ± 0.5; (L) 2,300 ± 1,200 1.6 ± 0.3 4.9 ± 1.3 45 ± 15aBinding was performed in the presence of 1-4 nM [3H]SAG-1.3 or 7-10 nM [3H]cyclopamineRadioligand binding data are the mean of 3-10 determinations ± SEMbTomatidine is an inactive analog of cyclopaminecSANT-1 gave partial inhibition up to 10µM, Fig 7BdXC50 = EC50 for agonist compounds or IC50 for antagonist compoundseAgonists were examined for stimulation of reporter activity; antagonist activity was determined against a SAG1.5 EC80.
(H) represents high affinity binding (L) represents low affinity bindingReporter assay results represent the mean of 4 - 10 determinations, ± STDEV
Ki (nM)a
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