Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery

Article abstract of DOI:10.1016/j.bmc.2013.04.069

FXR (farnesoid X receptor, NRIH4), a nuclear receptor, plays a major role in the control of cholesterol metabolism. FXR ligands have been investigated in preclinical studies for targeted therapy against metabolic diseases, but have shown limitations. Therefore, there is a need for new agonist or antagonist ligands of FXR, both for potential clinical applications, as well as to further elucidate its biological functions. Here we describe the use of the X-ray crystal structure of FXR complexed with the potent small molecule agonist GW4064 to design and synthesize a novel fluorescent, high-affinity probe (DY246) for time resolved fluorescence resonance energy transfer (TR-FRET) assays. We then used the TR-FRET assay for high throughput screening of a library of over 5000 bioactive compounds. From this library, we identified 13 compounds that act as putative FXR transcriptional antagonists.

Full text of DOI:10.1016/j.bmc.2013.04.069

Bioorganic & Medicinal Chemistry 21 (2013) 4266–4278
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of time resolved fluorescence resonance energy
transfer-based assay for FXR antagonist discovery
a
Donna D. Yu a, , , Wenwei Lin b, , Taosheng Chen b, , Barry M. Forman
⇑
⇑
a Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA
b Department of Chemical Biology & Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
FXR (farnesoid X receptor, NRIH4), a nuclear receptor, plays a major role in the control of cholesterol
metabolism. FXR ligands have been investigated in preclinical studies for targeted therapy against met-
abolic diseases, but have shown limitations. Therefore, there is a need for new agonist or antagonist
ligands of FXR, both for potential clinical applications, as well as to further elucidate its biological func-
tions. Here we describe the use of the X-ray crystal structure of FXR complexed with the potent small
molecule agonist GW4064 to design and synthesize a novel fluorescent, high-affinity probe (DY246)
for time resolved fluorescence resonance energy transfer (TR-FRET) assays. We then used the TR-FRET
assay for high throughput screening of a library of over 5000 bioactive compounds. From this library,
we identified 13 compounds that act as putative FXR transcriptional antagonists.
Received 11 February 2013
Revised 17 April 2013
Accepted 26 April 2013
Available online 7 May 2013
Keywords:
FXR, farnesoid X receptor
NRs, nuclear receptors
HTS, high-throughput screen
LBD, ligand-binding domain
TR-FRET, time resolved fluorescence
resonance energy transfer
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
are well documented in the literature.7 FXR agonists decrease plas-
ma triglyceride levels and increase the synthesis of high-density
Farnesoid X receptor (FXR) is a member of bile acid nuclear hor-
mone receptor superfamily, and is highly expressed in the liver and
intestine.1 FXR is a ligand-dependent transcription factor that reg-
ulates gene networks involved in bile acid homeostasis. Since bile
acids represent the end-product of cholesterol metabolism, FXR
has also a role in regulating lipid and cholesterol homeostasis. Con-
sistent with this role of FXR, bile acids are the primary activating
endogenous ligands of FXR.2 Recent findings suggest that FXR is a
key metabolic regulator in the liver that acts to maintain the
homeostasis of liver metabolites.3 Recent reports also indicate that
FXR has significant effects on vasculature as well.4 Thus, FXR rep-
resents an attractive pharmacological target for the development
of novel therapeutic agents to treat lipid metabolism disorders,
hyperlipidemia and cholestatic disease, as well as atherogenic
disease.5
lipoprotein cholesterol.8 This suggests that highly selective and po-
tent agonists for the FXR might be useful for the treatment of dysl-
ipidemia and cholestasis. However, the preclinical development of
FXR agonists has been limited because activation of FXR leads to
complex responses and potentially undesirable side effects, such
as inhibition of bile acid synthesis by indirectly repressing the
expression of cytochrome 7
of the bile acid synthesis pathway.6
On the other hand, an antagonist of FXR, if selective for Cyp7
a1 (Cyp7a1), the rate-limiting enzyme
a
could be useful therapeutically to increase the conversion of cho-
lesterol to bile acids, resulting in lower low density lipoprotein lev-
els in hyperlipidemic patients. FXR regulates the expression of
small heterodimer partner (SHP).1 SHP attenuates the expression
of CYP7A1 by inhibiting the activity of liver receptor homologue
1 (LRH-1), which is known to augment CYP7A1 expression. FXR
antagonism would be expected to lead to decreased SHP levels
Pharmaceutical control of activity of FXR with synthetic and
natural ligands having agonistic or antagonistic activity is a power-
ful chemical tool for managing various clinical conditions.6 Several
potent and selective FXR agonists have been reported recently and
which, in turn, should increase CYP7a1 activity, enhance
cholesterol metabolism in vivo and reduce serum levels of total
cholesterol. From this point, a FXR antagonist might serve as a use-
ful drug and should be developed.9
Although the search for potent, selective FXR antagonists has
intensified over the past few years, few antagonists have been
identified. Recent efforts involving a coactivator assay have identi-
fied guggulsterone (Fig. 1) as the first example of a direct FXR
antagonist.10 However, use of guggulsterone is limited because it
⇑
Corresponding authors. Tel.: +1 (626) 359 8111x65993; fax: +1 (626) 256 8704
(D.D.Y.); tel.: +1 (901) 595 5937 (T.C.).
E-mail address: dyu@coh.org (D.D. Yu).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.069

D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
4267
is extremely non-specific and targets many other receptors, not
just FXR, and probably targets other pathways as well. Addition-
we developed of a TR-FRET assay suitable for high-throughput
screening (HTS). In this TR-FRET assay, we employed a novel fluo-
rescent FXR ligand (DY246), which we developed based on the
chemical structure of a potent FXR ligand GW4064. The assay
has a high dynamic signal range and Z0 value. We used this assay
to screen for all possible FXR ligands (agonists or antagonists)
within a bioactive library of 5600 compounds, which have been
previously described.22,23 We then characterized for cellular activ-
ity of selected inhibitors identified from the TR-FRET assay. Our re-
sults demonstrated that the TR-FRET assay is suitable for
identifying FXR modulators with cellular activities.
ally, guggulsterone has very low potency (IC50 of 12–25 l
M),10
and its mechanism of antagonism is unclear.11 Thus, guggulsterone
is not a true antagonist of FXR; it is a unique FXR ligand with antag-
onistic activity in coactivator association assays but with the abil-
ity to enhance the action of FXR agonists in vivo.12
Sulfated polyhydroxy sterols isolated from marine invertebrates
were also recently identified as FXR antagonists.13 One of the sul-
fated polyhydroxy sterols, sulfated sterol 8 (Fig. 1), had an antago-
nistic activity toward the expression of a subset of FXR-regulated
genes in liver cells and abrogated the release of nuclear corepressor
from the promoter of these genes. Most recently, theonellasterol
(Fig. 1), a marine FXR antagonist demonstrated as a promising lead
in cholestasis has been reported.14
2. Results and discussion
2.1. Probe design
Nonsteroidal antagonist, exemplified by AGN34 (Fig. 1), antag-
onizes FXR in transient reporter assays but acts in a gene-selective
manner in vivo: it displays agonistic effect on the expression of
To successfully use the TR-FRET assay for HTS to identify novel
FXR ligands, we required a novel fluorescent FXR ligand that had
high affinity and potency. This required that the FXR fluorescent li-
gand contain a potent small molecule ligand that recognizes FXR,
fluorescent tag useful for detection and quantitation (e.g., a fluoro-
phore), and a suitable linker to connect the two (Fig. 2A).
We chose the small molecule GW4064 as the small molecule li-
gand because of its high binding potency for FXR (EC50 = 45 nM) as
assayed by fluorescence resonance energy transfer for recruitment
of the SRC1 peptide to human FXR.24 Similar potency of GW4064
(EC50 value of 90 nM) has also been reported in another study.25
An X-ray co-crystal structure of GW4064 with the ligand bind-
ing domain of FXR and the SRC-1 co-activator peptide has been
solved.26 This structure shed light on the agonist recognition ele-
ments of the FXR ligand binding domain and on the binding modes,
and also further revealed some open space adjacent to the terminal
side-arm of GW4064 that could be exploited in the design of FXR
probe. We used this X-ray crystal structure of the co-complex of
FXR/GW4064 to design a FXR fluorescent probe for FXR.
The high-resolution X-ray structure of the protein-bound com-
plex (FXR–uGW4064) indicated potential side arms of GW4064
could be modified between the carbonyl acid group and isoxazole
hetero atoms, respectively (Fig. 2B). However, because a dichloro-
phenyl oxazole moiety of GW4064 was recognized to provide con-
siderable conformational rigidity to the agonist,27 we kept this
moiety intact in the probe design. It is well established that side-
arm of GW4064 bearing the carboxylate head group is important
CYP7a, antagonistic effect on the expression of the ileal bile acid-
binding protein (IBABP), and is neutral on SHP expression.15 Recent
ligand-based virtual screening of a library of 12480 compounds
identified one nonsteroidal compound, 1,3,4-trisubstituted-pyraz-
olone 12 u as an FXR antagonist (Fig. 1).16 These findings further
confirm that selective FXR modulators can be identified that regu-
late a specific subset of FXR responsive genes in a gene-specific
fashion.
Members of the nuclear receptor family, to which FXR belongs,
is among the most successfully ‘drugged’ targets for the pharma-
ceutical industry, in part because it easily binds and responds to
small, orally bioavailable molecules.17 To date, enormous effort
has been put forth to develop screens for agents that modulate
activity of nuclear receptors. Several robust high-throughput as-
says with high Z’-factor are readily available,18 including fluores-
cence polarization assays for direct ligand binding and FRET/
Alpha-Screen assays for ligand-induced recruitment of coregulator
peptides.19 Z0-factor was routinely used to assess assay
performance. An assay with Z0 >0.5 is considered robust and
reproducible.20
TR-FRET assay has been previously used to screen for modula-
tors of other nuclear receptors with a Z0-factor of >0.75.21 To better
understand the antagonism of FXR and its implication in disease
treatment, with a goal of identifying novel small molecule antago-
nists of FXR activity that might have pharmacological relevance,
O
OH
OH
O
O
O
O
HO
guggulsterone
AGN 34
Lithocholic acid (LCA)
OSO3Na
HO
O
OH
N
O
H3CO
N
O
N
HO
H
NaO3SO
OH
Theonellasterol
1,3,4-Trisubstituted-pyrazolone 12u
sulfated sterol 8
Figure 1. Chemical structures of reported FXR antagonists.

4268
D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
HO
O
OH
(A)
O
O
HO
O
O
S=C=N
FITC
+
OH
O
S
linker
FXR ligand
N
H
Hypothetical binding mode of designed probe
(B)
Figure 2. (A) Design strategy of a fluorescent probe for FXR and hypothetical binding mode of the probe to FXR. (B) Ligand binding domain of the X-ray co-crystal structure of
GW4064 in complex with FXR. Carbon atoms for FXR are shown in cyan. Carbon atoms for GW4064 are shown as green sticks. The semi-transparent gray surface represents
the molecular surface, while hydrogen bonds are depicted as yellow dashed lines.26
for activating FXR by forming a network of hydrogen bonds with
331Arg.28 Further examination revealed that the carboxylic acid
group of GW4064 is co-planar with its phenyl ring, and that the
two oxygen atoms of the carboxylate coordinate with one NH2
acidic function should be incompatible with the formation of the
known ionic interaction to 331Arg, suggesting that there is a ‘hole’
in the structure of the FXR ligand binding pocket that marks an exit
vector for substituents ranging out from the p-position of the ter-
minal aryl passing along the 331Arg that forms an important salt
bridge with the benzoic acid –COO– of the ligand towards the
aqueous surface of the receptor.31 Note that when benzoic acid
of GW4064 was changed into –COOH bioisosteres that has similar
acidity such as a linker acylsulfonamide,31 its FXR agonism and po-
tency correlation is still maintained, suggesting that use of an
amide linker could be suitable for the probe. On the basis of this
information, we placed a linear, rigid linker using hydrazine hy-
drate as an amide bridge that was coupled to the carboxyl moiety
of GW4064.
After designing the linker, we introduced a fluorophore onto the
probe scaffold. We chose fluorescein isothiocyanate (FITC) as the
fluorophore because it is the most widely used fluorophore for
labeling and sensing biomolecules,32 and specifically, it is reactive
towards amino group of the linker connected to the GW4064. The
chemical structure of final probe, which we named DY246, is
shown in Figure 3.
and the e
-NH of the guanidine group of 331Arg. Analysis of the
FXR–GW4064 binding revealed that the electrostatic interaction
mimics the binding mode of the carboxylic acid of the natural bile
acid ligands, such as GCDCA (Glyco-CDCA).29 Furthermore, crystal
analysis of FXR co-complex indicates that the glycine-moiety of
GCDCA already extends outside the receptor such that linkage at
this position via an extended linker cannot interfere with ligand
binding.29 Because the carboxyl-moiety of GW4064 echoes the
binding mode of the carboxylic acid of the natural bile acid ligands,
it could be hypothesized that the flexible guanidine side chain of
331Arg could move to facilitate the interaction with the negatively
charged carboxylic oxygen moiety on GW4064. The potential
binding mode of designed linker for the probe could be twisted
out-of the plane of the aryl ring and extended outside the receptor,
similar to the binding mode of GCDCA, and therefore would be un-
likely to interfere with ligand binding (Fig. 2A). In addition, molec-
ular modeling studies30 suggest that the prolonged distance to the

D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
4269
2.2. Synthesis of DY246 for TR-FRET assay
in the presence of GST–FXR, which brings the Tb-labeled anti-
GST and DY246 into close proximity. The ratio between the donor
and acceptor wavelengths was measured by instruments with TR-
FRET detecting capacity, such as a PHERAstar plate reader.
Our synthetic strategy started with commercially available
GW4064. To prepare the carboxyl hydrazide, the carboxylic acid
group of GW4064 was esterified with methyl bromoacetate to af-
ford compound 1 in a quantitative yield. Compound 1 was subse-
quently treated with excess hydrazine hydrate in methanol to
produce compound 2, which was crystallized from EtoAc at a
48% of yield. The fluorescent moiety (FITC) was added onto 2 in
THF/EtOH, followed by purification with flash chromatography,
producing the fluorescein adduct compound 3 as a yellowish-or-
ange solid at a 31% yield. Compound 3 (DY246) was fully character-
ized by 1H NMR, 13C NMR, and HRMS. Thus, a synthetic procedure
( Fig. 3) to produce the FXR fluorescent probe DY246 has been
achieved in a three-step process starting from GW4064 and led
to 14% overall yield.
2.4. Pre-screening characterization of DY246 as an FXR
fluorescent ligand for TR-FRET binding assays
The new fluorescein labeled ligand DY246 had an EC50 value of
550 nM in a cell-based transfection assay. To assess the utility of
DY246 as an hFXR fluorescent probe for TR-FRET binding assays,
we determined the binding interactions between 10 nM DY246,
10 nM GST–hFXR–LBD, and 1.5 nM Tb-anti-GST in the presence
or absence of various concentrations of GW4064 at 15, 20, and
30 min. Both total binding (with DMSO) and non-specific binding
(with 5
20 min and slightly decreased at 30 min. Correspondingly, the sig-
nal/background ratios (DMSO/5 M GW4064 ratios, for which the
M GW4064 signals were normalized to 1) had similar trends
lM GW4064) were relatively stable (Fig. 4A) at 15 and
2.3. Components of the DY246-based TR-FRET assay
l
5
l
We used DY246 as a fluorescent probe (acceptor moleculae) in
the TR-FRET assay, in which energy transfer can be detected by an
increase in the fluorescence emission of the acceptor molecule and
a decrease in the fluorescence emission of the donor molecule. In
our TR-FRET assay, the components are Terbium (Tb) labeled-
anti-GST antibody, DY246 and GST–hFXR–LBD. The established
TR-FRET assay from Invitrogen uses a fluorescein labeled coactiva-
tor (fluorescein-SRC 2–2) as a fluorescent acceptor. The disadvan-
tage of screening by established Invitrogen assay is that it
narrows the range of ligands that can be identified. However, use
of DY246 can overcome this disadvantage. For testing DY246, Tb
in the Tb labeled anti-GST antibody was the donor species, and
FITC in DY246 was the acceptor species. Excitation of the Tb-la-
beled anti-GST antibody by an energy source would trigger an en-
ergy transfer to DY246, which would then emit light at 520 nm.
This energy transfer is distance dependent, and could only occur
to the total binding and non-specific binding signals (Fig. 4B), with
values of 9.52, 9.28, and 8.33 for 15, 20 and 30 min, respectively.
The Z0-factor remained constant at 15, 20, and 30 min, with corre-
sponding values of 0.75, 0.70, and 0.78 (Fig. 4C). An acceptable as-
say typically has a Z0-factor value of more than 0.5.20 The EC50
values for GW4064 were constant for 15 and 20 min (317 and
373 nM, respectively) and then increased to 753 nM at 30 min
(Fig. 4D). The DY246-based hFXR TR-FRET assay data for Figure 4
was from assays using 20
lL/well, but assays using 30 lL/well
had similar results (data not shown).
DY246 exhibited spectroscopic properties that were well-suited
for TR-FRET. The constant EC50 values for GW4064 in the DY246-
based FXR TR-FRET assays suggest that a short incubation time,
such as 15 or 20 min, is preferred, which is also compatible with
HTS because a shorter incubation time contributes to higher
throughput. In an automated large-scale screen, which would use
O
N
Cl
O
N
O
Cl
BrCH2COOCH3
O
O
Cl
O
O
OH
Cl
Cl
O
COOCH3
GW4064
Cl
1
NH2NH2
O
HO
C
N
O
OH
Cl
O
O
FITC
Cl
O
NHNH2
S
N
Cl
2
THF/EtOH
O
HO
O
O
N
OH
O
Cl
O
S
O
Cl
N
H
NH
N
H
Cl
3 (DY246)
Figure 3. Synthesis of the novel fluorescent probe DY246 (compound 3) for the TR-FRET assay.

4270
D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
DMSO
DMSO
8
6
4
2
0
15
5 µM GW4064
5 µM GW4064
A
B
10
5
0
15 min
20 min
Incubation Time
30 min
15 min
20 min
30 min
Incubation Time
8
6
4
2
0
15 min
1.0
0.8
0.6
0.4
0.2
0.0
C
D
20 min
30 min
15 min
20 min
30 min
-10
-9
-8
-7
-6
-5
Incubation Time
Log[GW4064] M
Figure 4. Longitudinal signal stability of the interaction of DY246 with GST–FXR–LBD and Tb-anti-GST. (A) Interaction of 10 nM DY246 with 10 nM GST–FXR–LBD and 1.5 nM
Tb-anti-GST at the indicated time points in the presence of DMSO or 5
M GW4064. (B) Signal-to-background ratio of the interaction of the data shown in A. (C) Z0-factor
values of the interaction of the data shown in A and B. The Z0-factor was calculated from the total binding signal (DMSO) and background signal (5
M GW4064) by using Eq. 2
l
l
(see Section 4). (D) GW4064 dose–response curves in the presence of 10 nM DY246 with 10 nM GST–FXR–LBD and 1.5 nM Tb-anti-GST at the indicated time points.
scheduling software, the signal from each plate would be mea-
sured exactly at a pre-set incubation time, and therefore there
should not be variation on incubation time between plates. We
chose to use a 20 min incubation time for further HTS because this
incubation time best fits the automation system. For the same rea-
son, we used a 20
lL/well assay volume for the primary HTS and a
30 L/well assay volume for the following high throughput dose
l
response screening. Another FXR agonist fexaramine was used in-
stead of GW4064 to evaluate the DY246-based TR-FRET assay. Sim-
ilar high Z0 value (0.82) was obtained (Fig. 5). Taken together, these
results indicate that the DY246-based TR-FRET assay is suitable for
HTS to evaluate a variety of FXR ligands that have differing
affinities.
2.5. HTS to identify FXR ligands
Figure 5. TR-FRET assay for identification of FXR ligands. The assay was performed
with the FXR probe ligand DY246 (GW-FL) alone giving high signal output.
However, signal was lost in the presence of unlabeled competitor ligand Fexar-
amine. The assay was repeated 22 times yielding a calculated Z0-factor of 0.82
indicating an excellent assay.
Current screening strategies for FXR (and many other nuclear
receptors) use TR-FRET, AlphaScreen or other homogeneous assays
to detect ligand-induced interactions between receptors and small
coregulator peptides. A ‘hit’ is identified when the test compound
promotes or disrupts the receptor–peptide interaction. Antagonists
can be identified when the assay, typically a cell-based assay, is set
under antagonistic mode in which the assay system includes a
putative agonist such that the assay readout will be high in the ab-
sence of an antagonist and low in the presence of an antagonist.
Co-activator recruitment assays might have the advantage of
detecting more ligands, but cannot distinguish whether the ligands
directly bind to the LBD. Although these assays are useful, they are
biased in that they rely on interactions with a single coregulator
peptide. Therefore, by design, this strategy is inherently limited
as it excludes the detection of selective FXR modulators that fail
to recruit the particular peptide used in the assay. To overcome this
obstacle we developed a TR-FRET assay that directly detects ligand
binding without a bias for any downstream conformation change
or biochemical activity. We subsequently used the DY246-based
TR-FRET assay in a 384-well format to screen a collection of 5600
bioactive compounds (3200 unique compounds) representing
drugs, drug candidates, and other molecules with characterized
biological activities from a variety of sources, including Micro-
Source, Prestwick, and Sigma as previously described.22,23 For this
screen, we used the putative hFXR agonist GW4064 (5
DMSO as positive and negative controls, respectively. The com-
pounds were screened at 15 M. To exclude false-positive com-
lM) and
l
pounds that non-specifically interfered with the assay, including
compounds that self-absorb fluorescence, we counter-screened
by replacing GST–FXR+DY246 with a GST–FXR–fluorescein cova-
lent conjugate.
We arbitrarily selected 50% inhibition as the hit criterion be-
cause we are interested in further characterizing relatively potent
compounds (the screening concentration was 15 lM). Using this

D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
4271
criterion, the screen identified 249 chemicals (a hit rate of 249/
5600 = 4.44%), of which 193 were unique chemical entities (an ac-
tual hit rate of 193/3200 = 6.03%). The 249 compounds were then
tested in a dose-responsive analysis with the TR-FRET assay, using
acid showed moderate activity (27% and 19% maximum activation,
respectively), which is consistent with previous observations.34,35
The 17 compounds identified by the TR-FRET displayed varying
PXR agonistic activity, ranging from inactive (e.g., ivermectin) to
very potent (e.g., 120% maximum activation and an EC50 of
compound concentrations ranging from 1.2 nM–23.3 lM, in tripli-
cate. Only compounds that displayed dose–responsive inhibitory
activity were selected for further characterization. Among the
249 chemicals tested in the dose–responsive analysis, we selected
17 compounds for further follow-up (Table 1).
6.1 lM for flutrimazole). Flutrimazole is a close analog of clotrimz-
ole, and had not been previously reported as a PXR agonist. Clo-
trimazole, a known PXR agonist37 showed moderate PXR activity
(36% maximum activation). Nimodipine and felodipine are known
PXR agonists with potency comparable to rifampicin.38,39 We
showed that felodipine had a maximum activation of 31%, and
2.6. Characterization of hits from HTS
nimodipine had a maximum activation of 126% (EC50 of 0.4 lM).
The 17 selected compounds were then examined for their abil-
ity to act as antagonists of ligand activated FXR in cell-based tran-
scription reporter assays (Fig. 6). However, two of the 17 selected
compounds, C16-carnitine and 6-OH-L-DOPA, are poorly perme-
able in cells that lack specific trans-membrane transporters.33
C16-carnitine (palmitoyl-DL-carnitine) was excluded from follow-
up because of its poor cell permeability, while 6-OH-L-DOPA was
included as an additional negative control. We also excluded
(R,R)-cis-diethyl tetrahydro-2,8-chrysenediol) because it is toxic
at high concentrations. The remaining compounds were tested
The other 13 compounds, thyoxine, fulvestrant, sulconazole,
raloxifene, loratadine, 6-HO-DL-DOPA, JWH-015, ivermectin, NBI
27914, (R,R)-cis-diethyl tetrahydro-2,8-chrysenediol, GW7647,
palmitoyl-DL-carnitine, and bio, have not been previously evaluated
for their ability to activate PXR. These results are in agreement
with previous observations for other FXR antagonists34–36 and also
suggest that a PXR transactivation assay can be used to facilitate
the selection of more specific FXR antagonists for further
characterization.
Although the above findings demonstrate that the current assay
and hit selection criteria are appropriate for large-scale screening,
we have identified some areas that can be further improved to re-
duce the false positive rate. Specifically, we found that some com-
pounds non-specifically inhibit TR-FRET to a certain degree when
for their ability to inhibit transactivation mediated by 8
Compounds were tested at concentrations ranging from 3 to
30 M, a range that reflects toxicity-limiting concentrations for
lM CDCA.
l
certain compounds. Of the remaining 14 compounds, 13 inhibited
transactivation by >50%, and 9 of these inhibited transactivation by
75–100% (Fig. 6). These compounds also displayed potent activity
in the TR-FRET assay. For example, ivermectin revealed an IC50 of
ꢀ300 nM in the TR-FRET assay (Fig. 7). The compounds that failed
to inhibit transactivation by 50% or more in the cell-based reporter
assay (T4, or thyroxine) only showed 54% inhibition in the TR-FRET
assay. Taken together, these findings strongly suggest that use of
the TR-FRET assay with a selection criterion of >50% inhibition
can accurately predict true biological activity.
tested at 10 lM or higher. This can be seen when comparing inhi-
bition of non-specific FRET (GST–fluorescein–TbAb, Fig. 9) with
inhibition of specific FRET (DY246-FXR-TbAb, Fig. 10). For example,
of the 17 compounds tested for non-specific inhibition, three pro-
duced ꢀ50% inhibition [Bio, (R,R)-cis-diethyl tetrahydro-2,8-chry-
senediol, and 6-OH-L-DOPA] ( Fig. 9). Initial dose response
analysis indicated that, as expected, these non-specific effects were
not seen at lower doses (e.g., 2 M; data not shown). Nonetheless,
l
it must be noted that when compared with specific inhibition of
FXR, the non-specific inhibition was much less significant (Figs. 9
and 10).
The selected compounds were also tested for agonist activity
relative to 100
porter assays (Fig. 8). Compounds were tested at concentrations
ranging from 3 to 30 M, a range that reflects toxicity-limiting
lM CDCA in similar cell-based transcription re-
These findings suggest the following:
l
concentrations for certain compounds. Only one compound,
(1) Although the screen is acceptable as is, screening com-
nimodipine, had significant agonist activity. Because nimodipine
pound at 10–15 lM concentrations may produce weak
also inhibited activation by 8
l
M CDCA (Fig. 6), this compound
non-specific inhibition for a minority of compounds. The
extent of this inhibition can be as high as 50%, the cutoff
used to define a hit. Therefore, it may be useful to screen
is best characterized as a weak or partial agonist. We noticed
that although the vast majority of selected compounds acted
as antagonists, only one acted as weak or partial agonist. The
high hit rate of 6.03% for antagonists might be due to the selec-
tion of compound library. In contrast the hit rate for agonists
was 0.4%, which is in line with the hit rate expected for screen-
ing nuclear receptors.18 These findings further suggest that our
TR-FRET assay is appropriate for large-scale screening as it can
successfully identify all ligands, regardless of whether they are
agonists or antagonists.
at slightly lower concentration (5–7.5 lM) to further min-
imize non-specific inhibition. A follow-up dose–response
study should be helpful in establishing the optimal
screening concentration.
(2) It may also be useful to increase the selection criteria to >60
or 70% inhibition. This level of inhibition appears to be
higher than any non-specific inhibition observed for any
compound. The cutoff used to define a hit can be selected
after the large-scale screen is completed, that is, the larger
the number of hits, the more stringent the selection criteria
can be. Selection of the proper screening concentration and
use of proper selection criteria will help minimize false-pos-
itives and false-negatives.
(3) If any questions do arise as to the validity of certain com-
pounds, the GST–fluorescein counter screen provides a rapid
and accurate approach to filter out false-positives, including
compounds that self-absorb fluorescence. Although this
option may not be needed, it is useful to know that a rapid
and affordable counter screen exists to eliminate false-posi-
tives. Comparison of FXR inhibition versus GST–fluorescein
appears to be as accurate and predictive of true activity as
the cell-based transcription reporter assay.
2.7. A PXR transactivation assay
Pregnane X receptor (PXR) is a xenobiotic receptor that binds
promiscuously to structurally diverse chemicals, including ligands
for other nuclear receptors23 Several FXR antagonists, including Z-
guggulsterone and lithocholic acid, could be endowed with PXR
agonistic activity.34–36 We used a PXR transactivation assay de-
scribed previously23 to evaluate the 17 compounds identified by
the TR-FRET assay, in addition to Z-guggulsterone and lithocholic
acid, for their ability to activate PXR. In the assay, DMSO (0% acti-
vation) and 10 lM rifampicin (100% activation; rifampicin is a
known PXR agonist37 was used as negative and positive control,
respectively. As shown in Table 1, Z-guggulsterone and lithocholic

Table 1
Summary of compounds identified by TR-FRET
Compound
Name
Structure
clogPa
FXR–TR–FRET activity
PXR transactivation activity
% Inhibitionb
IC50
(lM)
% Activationd
EC50
(lM)
c
e
O
N
O
Cl
Cl
1
Felodipine
5.29
58.2
4.96 1.4
30.6 1.3 (40
lM)
NA
OH
O
I
I
O
I
2
Thyoxine
3.51
58.2
7.86 1.7
13.9 0.4 (40
lM)
NA
HO
I
OH
H2N
O
OH
Me
HO
3
Fulvestrant
8.5
62.1
0.79 0.28
102.8 3.9 (20
l
M)
1.95 0.01
7
S
O
F
F
F
F
F
F
F
4
Flutrimazole
4.47
66.7
13.8 4.9
120.4 2.1 (40
l
M)
6.10 0.16
N
N
NO2
O
O
N
OH
5
Nimodipine
3.01
67.5
8.96 1.95
125.9 4.1 (2.5
l
M)
0.39 0.01
O
O

Cl
Cl
6
7
Sulconazole
Raloxifene
5.66
6.86
71.1
71.4
6.88 1.59
50.0 1.6 (10
l
M)
M)
NA
N
N
S
Cl
OH
HO
S
11.56 2.99
81.5 0.2 (20
l
11.71 0.33
N
O
O
Cl
N
8
Loratadine
5.05
72.5
3.07 0.76
57.4 1.7 (10
l
M)
3.00 0.05
N
O
O
OH
HO
NH2
9
6-HO-DL-DOPA
-3.53
6.55
73.0
75.5
7.92 2.34
Inactive
NA
O
OH
N
OH
10
JWH-015
3.22 0.85
125.0 1.1 (40
l
M)
2.64 0.12
O
HO
O
O
O
O
O
O
O
O
O
11
Ivermectin
5.96
77.4
0.26 0.02
Inactive
NA
O
OH
O
OH
(continued on next page)

Table 1 (continued)
Compound
Name
Structure
clogPa
FXR–TR–FRET activity
PXR transactivation activity
% Inhibitionb
IC50
(lM)
% Activationd
EC50
(lM)
c
e
Cl
Cl
N
N
12
13
NBI 27914
7.65
77.7
2.77 1.00
114.8 1.2 (20
l
M)
4.95 0.46
N
H
N
Cl
Cl
Cl
Clotrimazole
5.25
6.11
81.4
81.9
3.24 0.87
35.6 1.9 (10
l
M)
M)
NA
N
N
OH
14
(R,R)-cis-Diethyl tetrahydro-2,8-chrysenediol
1.31 0.31
53.1 2.8 (20
l
8.60 0.98
HO
HN
O
N
15
GW7647
7.08
84.8
4.91 0.92
25.9 1.4 (40
l
l
M)
NA
S
OH
O
Cl-
N
O
O
16
17
Palmitoyl-DL-Carnitine
0.53
4.27
93.6
96.3
9.75 1.86
40.7 0.1 (40
M)
NA
NA
OH
14
Br
H
N
OH
NO
Bio
3.4 0.58
42.6 1.5 (1.25 lM)
HN

D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
4275
(4) The non-specific activity that we observed against GST–fluo-
rescein is likely to be a general problem for all assays that rely
on Tb-fluorescein-based-TR-FRET. As time and resources per-
mit, it may be useful to screen the compound library
intended to be used for TR-FRET assay against GST–fluores-
cein. The resulting database may be helpful in minimizing
false-negatives in a broad array of related screening projects.
3. Conclusions
FXR plays a critical role in the control of cholesterol, lipid and
glucose metabolism, and has become a challenging drug target
for the treatment of hyperlipidemia and cholestatic disease as well
as atherosclerotic heart diseases. FXR antagonists raise the exciting
possibility that they may be useful as therapeutic agents for these
COOH
A
OH
HO
CDCA
B
Figure 6. Abilities of compounds identified throught TR-FRET assay act as
antagonists of ligand-activated FXR in cell-based reporter transcription assays.
(A). Chemical structure of CDCA (chenodeoxycholic acid). (B). Inhibition of FXR/
CDCA-mediated transactivation by the indicated compounds identified in the TR-
FRET assay.
110
90
70
50
30
10
-10
-10
-9
-8
-7
-6
-5
-4
Log[Ivermectin] M
Figure 7. Dose response studies for ivermectin in the TR-FRET assay.

4276
D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
Figure 8. Agonist activity of compounds in transactivation assay.
Figure 10. Specific effects of compounds (10 lM) on FXR-mediated TR-FRET by
DY246.
4. Experimental section
4.1. Chemistry
4.1.1. General procedures
Organic reagents were purchased from commercial suppliers
unless otherwise noted and were used without further purification.
All solvents were analytical or reagent grade. All reactions were
carried out in flame-dried glassware under argon or nitrogen. Melt-
ing points were determined and reported automatically by an
optoelectronic sensor in open capillary tubes and were uncor-
rected. 1H NMR and 13C NMR spectra were measured at 500 and
125 MHz, respectively, using CDCl3 or CD3OD as the solvents and
tetramethylsilane (Me4Si) as the internal standard. Flash column
chromatography was performed using Sigma–Aldrich silica gel 60
(200–400 mesh), carried out under moderate pressure with col-
umns of an appropriate size packed and eluted with appropriate
eluents. All reactions were monitored by thin layer chromatogra-
phy (TLC) on precoated plates (silica gel HLF). TLC spots were visu-
alized either by exposure to iodine vapors or by irradiation with UV
light. Organic solvents were removed under vacuum by a rotary
evaporator.
Figure 9. Non-specific effect of compounds (10 lM) on non-specific TR-FRET (GST–
fluorescein).
4.1.2. (E)-2-Methoxy-2-oxoethyl 3-(2-chloro-4-((3-(2,6-dichloro
phenyl)-5-isopropylisoxazol-4-yl)methoxy)styryl)benzoate (1)
(E)-3-(2-Chloro-4-((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-
4-yl)methoxy)styryl)benzoic acid (GW4064) (25 mg, 0.046 mmol)
was dissolved in DMF (2 mL) and treated with methyl bromoace-
tate (15 mg, 0.1 mmol) and powder potassium carbonate (13 mg,
0.1 mmol). The resulting mixture was heated at 75 °C overnight.
After cooling to room temperature, the reaction mixture was
poured into 20 mL of saturated ammonium chloride solution
and then extracted with EtOAc (3 ꢁ 20 mL). The organic extracts
were washed with H2O (15 mL), followed by brine (2 ꢁ 15 mL),
dried over Na2SO4, and concentrated under vacuum to give the
desired ester as an oil (25 mg) that was used for the next step
without further purification. 1H NMR (CDCl3) d 7.58 (m, 1H),
7.52 (d, 2H), 7.39 (m, 4H), 7.33 (m, 2H), 6.92 (d, 1H), 6.78 (s,
1H), 6.77 (d, 1H), 4.88 (s, 2H), 4.72 (s, 2H), 3.77 (s, 3H), 3.35
(m, 1H), 1.44 (d, 6H).
diseases. To facilitate the search for modulators of FXR activity, we
developed an FXR binding assay based on TR-FRET. We developed
and synthesized the novel fluorescently-labeled FXR probe DY246,
and showed that it selectively bound to the ligand binding domain
of FXR. This suggests it is a promising fluorescent FXR ligand suit-
able for TR-FRET assays directed at the discovery of novel FXR li-
gands, including antagonists. We demonstrated that the TR-FRET
assay we reported here using DY246 offers an efficient primary
screening tool for identifying FXR modulators. Use of DY246 in
TR-FRET is expected to provide further insights into ligand binding
mechanisms and to improve the specificity of therapeutics target-
ing FXR biological functions. Studies aimed at further in vitro pro-
filing of FXR antagonists identified in our initial HTS and expanding
our knowledge of their structure–activity relationships (SAR), with
an ultimate goal of developing orally active FXR antagonists in
vivo, are under way.

D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
4277
GW4064) group. The 520 nm/490 nm ratios for dilutions of
GW4064 were fit into a one-site competitive binding equation in
a dose-dependent manner to derive the EC50 value for GW4064.
Experiments were repeated in triplicate.
4.1.3. (E)-3-(2-Chloro-4-((3-(2,6-dichlorophenyl)-5-isopropyliso
xazol-4-yl)methoxy) styryl)benzohydrazide (2)
To
a solution of (E)-2-methoxy-2-oxoethyl 3-(2-chloro-4-
((3-(2,6-dichlorophenyl)-5-isopropylisoxazol-4-yl)methoxy)styryl)
benzoate (1) (25 mg, 0.04 mmol) in methanol (2 mL), hydrazine
monohydrate (50 mg, 1 mmol) was added. The reaction mixture
was stirred at room temperature for 36 h. Then organic solvent
and excess hydrazine were removed in vacuo. The residue was
poured into 10 mL cold water and stirred for 10 min. The formed
solid was washed with cold water several times and dried under
vacuum. Crystallization of the crude solid from warm EtOAc gener-
ated product 2 (10 mg, 48%) as a beige solid.
4.2.2. TR-FRET for detection of FXR ligand-binding activity
A TR-FRET kit (LanthaScreen™) from Invitorgen was used as per
the vendor’s instructions. Terbium chelate (GST–FXR + ter-
bium ꢂ anti-GST antibody) donor species was mixed with a fluo-
rescein-tagged SRC2 derived peptide (LKEKHKILHRLLQDSSSPV)
acceptor species. The TR-FRET value was determined as a ratio of
the FRET-specific signal measured with a 520 nm filter to that of
the signal measured with the terbium-specific 495 nm filter. The
fluorescent signals were read in a time-resolved manner to reduce
assay interference and increase data quality. The dose–response
1H NMR (CDCl3) d 7.64 (m, 3H), 7.33 (m, 2H), 7.30 (m, 4H), 6.94
(d, 1H), 6.77 (s, 1H), 6.57 (dd, 1H), 4.73 (s, 2H), 3.46 (m, 1H), 1.43
(d, 6H); 13C NMR (CDCl3) d 176.4, 159.7, 159.0, 157.1, 142.2, 135.7,
134.2, 132.0, 131.4, 130.9, 128.7, 128.1, 127.7, 127.1, 124.4, 115.7,
113.7, 109.1, 59.6, 27.1, 25.5, 25.0.
curve yielded an EC50 of ꢀ4
lM for CDCA which is consistent with
measurements form other assays.
4.3. Cell-based co-transfection reporter assay1
4.1.4. (E)-5-(2-(3-(2-Chloro-4-((3-(2,6-dichlorophenyl)-5-isopro
pylisoxazol-4-yl)methoxy) styryl)benzoyl)hydrazinecarbothioa
mido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoate (3)
5 Fluorescein isothiocyanate, isomer I (16 mg, 0.042 mmol) was
added to a solution of product 2 (10 mg, 0.010 mmol) in EtOH/THF
(3:2, 2 mL). The resulting orange reaction mixture was stirred at
room temperature in the dark for 24 h. The reaction mixture was
then concentrated under vacuum in the dark to give an orange
semi-solid that was purified by flash chromatography (95:3:2
CH2Cl2/MeOH/AcOH, v/v) to generate the product 3 (5 mg, 30%)
as a yellowish-orange solid. 1H NMR (MeOD) d 8.31 (s, 1H), 7.91
(m, 1H), 7.82 (d, 1H), 7.77 (s, 1H), 7.47 (m, 2H), 7.42 (m, 2H),
7.35 (d, 2H), 7.12 (m, 2H), 6.96 (d, 1H), 6.68 (s, 1H), 6.63 (m, 6H),
6.56 (m, 5H), 4.75 (s, 2H), 3.35 (m, 1H), 1.38 (d, 6H); 13C NMR
(MeOD) d 188.5, 176.5, 174.6, 169.9, 160.7, 159.5, 157.3, 153.5,
145.9, 143.1, 140.6, 135.8, 134.0, 131.4, 128.6, 127.3, 125.8,
124.8, 123.9, 112.8, 110.3, 102.4, 59.3, 27.1, 19.5. HRMS (ESI);
Calcd for C49H35Cl3N4O8S (M+1): 944.1242. Found: 944.1259.
CV-1 cells were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% resin charcoal–stripped fetal bovine ser-
um, 50 U/mL penicillin G, and 50 lg/mL streptomycin sulfate
(DMEM-FBS) at 37 °C in 5% CO2. One day prior to transfection, cells
were plated to 50–80% confluence using phenol red free DMEM-
FBS. Cells were transiently transfected as described.1 Reporter con-
structs (300 ng/105 cells) and cytomegalovirus-driven expression
vectors (20–50 ng/105 cells) were added as indicated along with
CMX-b-gal (500 ng/105 cells) as an internal control. After 2 h, the
liposomes were removed and cells were treated for approximately
45 h with phenol red free DMEM-FBS containing either vehicle
control (DMSO or the indicated compounds. After exposure to
ligandindicated compound, the cells were harvested and assayed
for luciferase and b-galactosidase activity. Results are expressed
as the mean standard deviation of at least three independent
experiments, and error bars indicate the standard deviation.
4.2.5. TR-FRET-based hFXR HTS
4.2. Biology
In the primary TR-FRET hFXR HTS, 30 nl of 10 mM testing chem-
ical was transferred with a pintool into 15
hFXR and Tb-anti-GST in a 384-well black plate, and then 5
40 nM DY246 was dispensed to give a final volume of 20 L/well
with 10 nM GST–hFXR, 1.5 nM Tb-anti-GST, 15 M testing chemi-
cal and 10 nM DY246. In addition, selected wells containing 5
l
L of a mixture of GST–
4.2.1. Pre-screening characterization of DY246 as FXR fluoresce
nt ligand in a TR-FRET binding assay
l
L of
l
In a black 384-well plate (Corning Incorporated, Corning, NY),
l
DMSO, GW4064 (5
lM) or serial dilutions of GW4064 (Tocris Bio-
lM
scicnce, Minneapolis, MN) (5
lM to 2.54 nM, 1:3 titration at 10
GW4064 or DMSO were used as positive and negative controls,
respectively. The final DMSO concentration was 0.15% in all wells.
The plates were then spun down after a brief shake and incubated
for 20 min at room temperature. The TR-FRET signal was then col-
lected for each well with an Envison plate reader using an excita-
tion wavelength of 340 nm and emission wavelengths of 520 nm
and 490 nm. The 520/490 nm ratio from each well was calculated
and employed for the % inhibition calculation. The % inhibition
for each well was calculated using the following equation:
concentration levels) were mixed with 10 nM GST–hFXR–LBD,
1.5 nM Tb-anti-GST antibody (Invitrogen, Carlsbad, CA), 10 nM
DY246, and TR-FRET assay buffer (Invitrogen, Carlsbad, CA) in a
reaction volume of 20
lL/well. The final DMSO concentration was
0.1% for all wells. The DMSO group and GW4064 (5
lM) group
served as negative and positive controls, respectively. The plate
was spun down after a brief shake and incubated under room tem-
perature. The TR-FRET signal for each well was then collected,
using an Envision plate reader with excitation wavelength of
340 nm and emission wavelengths of 520 nm and 490 nm at 15,
20 and 30 min. The 520 nm/490 nm ratio from each well was cal-
culated. The Signal/Background ratio was defined as the ratio of
negative control group (DMSO) against positive control group
DMSO520 nm=490 nm ꢂ Chemical520 nm=490 nm
%Inhibition ¼ 100% ꢁ
DMSO520 nm=490 nm ꢂ GW4064520 nm=490 nm
(5
following equation:20
l
M GW4064). The Z0-factor values were calculated using the
In the dose–response TR-FRET hFXR HTS, the general protocol
used for the primary screening was followed with minor modifi-
cation. Briefly, 70 nl of titrated testing chemical was transferred
3
rþ þ 3rꢂ
with a pintool into 20 lL of a mixture of GST–hFXR and Tb-anti-
Z0 ¼ 1 ꢂ
Meanþ ꢂ Meanꢂ
GST in a 384-well black plate, and then 10
lL of 30 nM DY246
where r+ is the standard deviation of the negative control (DMSO)
was dispensed to give a final volume of 30
lL/well with 10 nM
group; rꢂ is the standard deviation of the positive control (5
GW4064) group; Mean+ is the mean of the negative control (DMSO)
group; and Mean– is the mean of the positive control (5
l
M
GST–hFXR, 1.5 nM Tb-anti-GST, 1-to-3 titrated testing chemical
from 23.3
taining 5
l
M to 1.2 nM and 10 nM DY246. Selected wells con-
lM GW4064 or DMSO were again used as positive
lM

4278
D. D. Yu et al. / Bioorg. Med. Chem. 21 (2013) 4266–4278
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lM rifampi-
cin signal ꢂ DMSO signal)]. For compound with maximum %
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PRISM 6 (GraphPad Software, La Jolla, CA).
DMSO, rifampicin, nimodipine, L-thyroxine, JWH-015, clotrima-
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palmitoyl-DL-carnitine chloride and lithocholic acid were obtained
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Acknowledgments
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We gratefully thank Dr. Arthur D. Riggs for helpful discussion,
support, and encouragement. We also thank Ramani Ravirala for
technical assistance and Keely Walker for editing the manuscript.
This work was supported by the American Lebanese Syrian Asso-
ciated Charities (ALSAC), St. Jude Children’s Research Hospital
(St. Jude), National Institutes of Health National Institute of Gen-
eral Medical Sciences [Grant GM086415], and National Institutes
of Health National Cancer Institute [Grant P30-CA21765].
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