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Introduction
Nuclear receptors (NRs) are a superfamily of ligand activated transcription
factors that modulate specific gene expression. To date, more than
100 NRs have been identified including class I (ligand-dependent),
class II (ligand-independent), and orphan receptors. A common feature
of NRs is that they all contain a DNA binding domain that interacts
with respective target genes to exert physiological functions[1].
Peroxisome proliferator-activated receptors (PPARs) with three isoforms
(α, β, and γ) regulate gene transcription in response to small,
lipophilic ligands[2-5]. PPARα is present in the liver,
kidney, and heart, PPARβ (also known as PPARδ) is expressed ubiquitously,
and PPARγ is mainly found in the adipose tissue and muscle. Upon
ligand binding, PPARs release relevant co-repressors and form heterodimers
with retinoid X receptors (RXRs)[6]. The heterodimers
bind to peroxisome proliferator response elements (PPREs)[7,8]
and recruit co-activators to initiate transcription of target
genes. It is known that PPARγ is activated by fatty acids and prostaglandin
J2 derivatives, although the identities of its physiologically
relevant activators are not certain[9,10].
Because PPARγ activation can cause insulin sensitization, its synthetic
agonists have been used in the treatment of type 2 diabetes[11,12].
Recently discovered liabilities of such therapy, namely, weight
gain and edema, led to regulatory concerns on the long-term administration
of drugs acting through PPARγ[13,14]. The elimination
of such adverse effects may depend on the discovery of novel compounds
with improved tissue selectivity while retaining insulin-sensiti-zing
property.
Conventional methods to study and characterize NRs include non-homogeneous
hydroxyapatite (HA) and gel shift assays which require a laborious
separation procedure and thus, are not suitable for high-throughput
screening (HTS). Scintillation proximity assay (SPA)[15]
technology, howerer, provides a homogeneous screening approach that
does not involve post-reaction liquid handling steps and is well-suited
to automation and HTS. In the SPA system, an isotope (eg, [3H])
is brought very close to a scintillant-impregnated microbead or
FlashPlate by binding to its surface. Because the emitted β particles
or augur electrons can only travel short distances in the bulk solution,
the microbead or FlashPlate preferentially captures electrons from
the bound radiolabeled ligand. Therefore, the amount of light emitted
from the scintillant in the microbead or FlashPlate is directly
proportional to the amount of bound radiolabeled ligand (Figure
1). Several SPA-based NR competitive binding assays were developed
and applied to HTS using biotinylated receptor ligand binding domains
(LBDs; 'ABC' method)[16,17]. In this paper, we describe
a more complex SPA-based assay system which includes the full-length
PPARγ and RXRα, biotinylated PPRE, [3H]BRL49653 and streptavidin-coated
FlashPlate or microbead in a homogeneous setting. This 'ABCDE' approach
was fully validated and applied to HTS of a sizable compound library.
A series of structurally diversified 'hits' were found, and subsequent
characterization led to the discovery of two novel PPARγ binders
with sub-micromolar potency and high specificity.
Materials and methods
Reagents Potassium chloride, sodium phosphate monobasic
anhydrous, and magnesium chloride haxahydrate were purchased from
Shanghai Chemical Co, Ltd. Edetic acid was purchased from Sigma-Aldrich
(USA). BRL49653 and troglitazone were purchased from Cayman Chemical
Co (USA). 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate
(CHAPS) was purchased from Boehringer Mannheim GmbH (Germany). Dithiothreitol
(DTT) was purchased from BioBasic Inc (Canada) and hydroxyapatite
was obtained from Bio-Rad Laboratories (USA). Aprotinin and leupeptin
were purchased from Merck KGaA (Germany). [3H]BRL49653
(53 Ci/mmol) was obtained from American Radiolabeled Chemicals,
Inc (USA), FlashPlate and flat-bottom IsoplateTM was
obtained from PerkinElmer, Inc (USA), and streptavidin-coated microbead
was obtained from Amersham Biosciences UK Ltd (England). The plasmids
of human NRs used in this study were from Dr Shen X of Shanghai
Institute of Materia Medica, Chinese Academy of Sciences and Dr
Chen SJ of Shanghai Institute of Hematology. Full-length PPARγ,
RARα, β, γ, and RXRα, β, γ were produced with a baculovirus expression
system using IPLB-Sf-21 cells[18]. The stock solutions
for PPARγ, RARα, β, γ and RXRα, β, γ extract proteins were at 7,
12-15, and 7-13 g/L, respectively. Double-strand 5´-biotinylated-PPRE
(CCTT-TGACCTATTGAACTATTACCT) was synthesized by Shanghai Sangon
Biological Engineering Technology & Service Co, Ltd.
HA assay The assay buffer consists of 10% glycerol (v/v),
NaH2PO4 25 mmol/L, MgCl2 0.5 mmol/L,
DTT 1 mmol/L, edetic acid 1 mmol/L, CHAPS 5 mmol/L, aprotinin 2
mg/L and leupeptin 100 µmol/L. PPARγ 1 µL extract protein
(70 mg/L) was loaded into each well of IsoplateTM containing
the assay buffer, followed by [3H]BRL49653 (1.2 µL,
10 nmol/L) and various concentrations of BRL49653 or troglita-zone
(2.5 µL), to give a final volume of 100 µL per well. The
plates were sealed and incubated overnight at 4 oC. HA
(25%, v/v) 25 mL was added to each well the next morning and the
plates were gently agitated twice for 5 min each. Following centrifugation
at 1200×g for 3 min, the supernatant was decanted and
100 µL assay buffer was added to each well. This washing procedure
was repeated twice before adding 150 µL scintillation liquid
(PerkinElmer), the plates were gently agitated to resuspend HA and
counting was measured by a MicroBeta counter (PerkinElmer).
FlashPlate based SPA assay Biotinylated-PPRE (4 µL
from a stock solution of 10 g/L) was mixed with the above assay
buffer (20 mL) containing fish sperm DNA (Sangon; 20 µL from
a 10 g/L stock solution), loaded to streptavidin-coated FlashPlate
(200 µL/well) and incubated overnight at 4 oC.
It was then washed three times with the assay buffer, 200 µL
reaction solution containing 14 µg PPARγ extract protein (70
mg/L), 0.94 mg RXRα extract protein (4.7 mg/L), 10 nmol/L [3H]BRL49653
and various concentrations of BRL49653 or troglitazone were added
to each well. Following incubation at 4 oC for 4 h, the
plates were counted by the MicroBeta counter. For validation purpose,
various concentrations of PPARγ and RXRα extract proteins, as well
as different reaction time lengths, were studied to determine an
optimal assay condition.
Microbead based SPA assay Biotinylated-PPRE (2 µL
from a stock solution of 10 g/L) was mixed with the assay buffer
(10 mL) containing fish sperm DNA (Sangon; 10 µL from a 10
g/L stock solution) and 4 mg streptavidin-coated microbead in
a conical polypropylene centrifuge tube (Corning Inc,
USA) and incubated overnight at 4 oC. The mixture was
centrifuged for 10 min at 1500×g. The supernatant was
then removed and washed three times with the 10-mL assay buffer.
Reaction solution 10 mL containing 700 µg PPARγ extract protein
(70 mg/L), 47 µg RXRα extract protein (4.7 mg/L), 10 nmol/L
[3H]BRL49653 and various concentrations of BRL49653 or
troglitazone were distributed to each well of a IsoplateTM
(100 µL/well) and incubated at 4 oC for 4 h before
counting by the MicroBeta counter. For validation purposes, various
amounts of microbead were used to determine an optimal assay condi-tion.
HTS studies The compound library used for screening consists
of 16 000 pure synthetic compounds and extracts of natural products.
A 10-compound pool per well mix was applied to the primary screening
(microbead based SPA assay), with an average concentration of 7
µmol/L for each compound dissolved in 100% Me2SO
solution. This matrix system maximizes the advantage of HTS and
allows duplicate screening of each compound[19] . In
each 96-well IsoplateTM, 16 wells were used as positive
control (BRL-49653) and samples showing greater than 70% inhibition
were considered 'hits'. Positive compounds were re-screened with
FlashPlate based SPA assay and confirmed 'hits' studied for their
binding cross-reactivities with RARα, β, γ and RXRα, β, γ using
respective HA assay.
Results
Assay validation In the present study, we first assessed
the kinetics of the signal strength generated by FlashPlate assay.
Time-course experiment suggested that the equilibrium reached after
3.5 h of incubation at 4 oC and prolongation of the reaction
time did not improve the assay efficiency (Figure 2A). Various concentrations
of PPARγ and RXRα were used to establish the optimal assay condition.
In the absence of unlabeled BRL49653, a maximum signal was detected
with a combination of 140 mg/L (1:50 of the stock solution) PPARγ
extract protein and 7 mg/L (1:1000 of the stock solution) RXRα (Figure
2B). The fully optimized assay possessed a signal to background
ratio of 5. Thus, optimal receptor concentrations were determined
for PPARγ (70 mg/L; 1:100) and RXRα (4.7 mg/L; 1:1500), respectively.
Under this assay condition, IC50 values for the two PPARγ
agonists, BRL49653 and troglitazone were measured (Figure 2C), and
found to be comparable to those calculated from the HA assay (Figure
2D). Since the principle of microbead- based SPA assay is similar
to that of FlashPlate, identical receptor concentrations were used
with a reduced assay volume (100 µL/well). When different concentrations
of microbead were used, a saturation reached between 2 and 4 g/L
(Figure 3A) with a signal-to-background ratio equal to 5. The IC50
values for BRL49653 and troglitazone determined by this assay were
within the range described earlier (Figure 3B).
Assay parameters In order to apply the microbead-based
SPA assay to HTS, both non-specific binding (NSB; using 22.5 mmol/L
BRL49653) and maximum binding (MB; using 0 mmol/L BRL49653) were
studied. Coefficient of variation (CV) values were 8.5% for
NSB and 6.2% for MB, respectively (Figure 3C). The Z' factor, which
estimates the suitability to HTS[20], was calculated
to be 0.71.
High-throughput screening Of the 16 000 samples
initially screened, 178 'hits' (1.11%)
showing greater than 70% competitive inhibition on BRL49653
binding to PPARγ were discovered (all synthetic compounds; Figure
4A). Secondary (single compound per well) screening confirmed that
24 of the above 'hits' displayed consistent inhibitory effects with
IC50 values between 0.2 and 28.5 mmol/L. Cross-reactivity
studies with RARα, β, γ and RXRα, β, γ revealed that 12 of these
compounds possess specific PPARγ binding properties including 2
with IC50 values less than 0.5 mmol/L (Table 1). In this
HTS campaign, the signal-to-noise ratio (10-to-15-fold), CV
(5%-8%) (Figure 4B) and Z' factor (0.66-0.75) are of high quality
nature.
Discussion
Three receptor binding assays were employed and compared side-by-side
in the present study to measure specific binding properties of two
known PPARγ agonists, namely, BRL49653 and troglitazone. Conventional
HA assay is a non-homogeneous method widely used to assess competitive
interaction between a testing agent and receptor in the presence
of radiolabeled ligand. For PPARγ, such interaction involves additional
components such as RXRα and PPRE. Therefore, the 'ABCDE' method
utilizing either FlashPlate or microbead based on SPA technology
was developed and validated to include both RXRα and PPRE in the
assay system. The final readout is fully dependent upon specific
binding of biotinylated PPRE to streptavidin-coated FlashPlate or
microbead as addition of free biotin was able to block this interaction
completely[16]. Although the IC50 values of
BRL49653 and troglitazone generated by these three approaches were
very similar and comparable to those reported previously with a
relatively simple 'ABC' SPA method (BRL49653: Kd=26
nmol/L, IC50=36 nmol/L; troglitazone: Kd=310
nmol/L, IC50=320 nmol/L)[16], the 'ABCDE'
model is obviously superior in terms of physiologic mimicry, easy
to use, robustness and efficiency, largely due to its inclusiveness
and homogenous nature.
We found that both FlashPlate and microbead-based SPA
methods could be readily adapted to automated HTS. However, the
cost of FlashPlate, including assay volume (200 µL), associated
reagent consumption, and compound depletion, etc, is approximately
5 times greater than that of microbead (100 µL). This may constitute
a major concern when implementing a large HTS campaign. All the
key assay parameters, such as CV and Z' factor, obtained
from our microbead-based SPA validation experiments, indicate that
it is well suited to HTS[20]. Indeed, when employed in
HTS of potential PPARγ modulators, this assay system demonstrated
a consistently high quality in terms of the signal-to-noise ratio,
CV and Z' factor in all the 40 pooled compound matrix plates.
One implication of this is that microbead-based SPA technology may
be expanded to other NRs that form heterodimers upon activation.
Following the initial screening of 16 000 samples, a "hit"
rate of 1.11% was achieved, of which, only 13.5% of the 'hits' could
be confirmed by secondary screening. They were further tested for
cross-reactivities with both RARs and RXRs (defined as IC50
less than 70 µmol/L), and 12 compounds showed high specificity
for PPARγ with IC50 values ranging from 0.2 to 28.5 µmol/L.
The IC50 for one thiazolidinedione-like compound is 0.2
µmol/L, better than several currently marketed PPARγ agonists.
The other compound with an IC50 of 0.49 mmol/L belongs
to the imide class. Both of them possess novel chemical structures
(data not shown). If their activities could be demonstrated by cell-based
functional assays, bioassay-guided structure modification and optimization
may lead to the discovery of some entirely new PPARγ modulators.
Acknowledgement
We thank Dr Dale E MAIS, Xin XIE, Na LI, and Cheng-he JIN for their
valuable discussions.
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