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.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 20, 2009 as DOI: 10.1124/jpet.109.152090

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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: [email protected]

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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