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Recent advances in automation and in the identification of new molecular targets for therapeutic intervention, and the
increasing number of compounds available for screening are the driving forces behind the development of new drug
discovery systems. High-throughput screening (HTS) has become increasingly important in the process of drug
development[1]. Thereinto, measurement techniques are particularly important with respect to the quality of HTS. During the past 7 years,
fluorescence polarization (FP) assays have gained popularity in HTS because the method is homo-geneous, highly sensitive,
and easily miniaturizable, has a rapid readout, and is neither radioactive nor labor
intensive[2].
FP is a versatile technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. FP
assays are homogenous in that they do not require a separation step and do not require attachment to an immobilized phase.
Polarization values can be measured repeatedly and after the addition of reagents, because measuring the polarization is rapid
and does not destroy the sample. Generally, this technique can be used to measure the polarization values of fluorophores
from low picomolar to micromolar levels. Polarization does not change with fluorescence intensity; therefore a 1 pmol/L and
a 1 nmol/L solution of fluorescein should have the same polarization
values[3].
For HTS assays, FP can be used to directly measure the binding and dissociation between two molecules if the binding
partners are small and fluorescently tagged. The binding can be simply monitored by reading the polarization value of the
small fluorescent molecule before and after it binds to a large molecule. Developing competitive binding assays that can be
used to measure analyte concentrations directly in solution is
straightforward[4,5].
In the present study, we developed a novel high-throughput assay based on competitive FP to screen the ligands of
lectin-like oxidized low-density lipoprotein receptor-1
(LOX-1), a membrane receptor that is expressed on the surface of
endothelial cells, and has roles in endothelial injury and
atherosclerosis[6,7]. The basic principle of this assay is that cloned,
expressed, and purified LOX-1 is labeled by fluorescein isothiocyanate (FITC), and allowed to bind its natural ligand, oxidized
low-density lipoprotein (oxLDL), in a competitive displacement assay in a homogeneous solution to find compounds that are
active with respect to LOX-1.
Materials and methods
Reagent preparation Human low-density lipoprotein (LDL; density 1.019-1.063 g/mL) was isolated by sequential
ultracentrifugation at 4 ¡ãC from normol lipidemic fasting volunteers and oxidized to oxLDL by
CuSO4[8,9]. oxLDL was labeled with
1,1¡¯-dioctadecyl-3,3,3¡¯,3¡¯-tetramethylindo-carbocyanine perchlorate (DiI; Molecular Probe, Sigma, St Louis, USA) according to
the method of Zouhair and Yurachek[10].
Construction, expression and purification of recombinant hLOX-1
The human LOX-1 gene was obtained by RT-PCR from THP-1 cells stimulated with 50 µmol/L histamine. The purified hLOX-1 gene was cloned into a pMD 18-T (TaKaRa,
Dalian, China) vector, amplified in Escherichia
coli strain DH5a, and the positive plasmid-harboring hLOX-1 cDNA was
digested with EcoRI and NotI. hLOX-1 cDNA was subcloned into pPIC9K (Invitrogen, Carlsbad, CA, USA), and digested
with the same restriction enzymes, resulting in the recombinant plasmid pPIC9K-His-hLOX-1.
The plasmid pPIC9K-His-hLOX-1 was linearized with the restriction enzyme
SacI and transformed into Pichia
pastoris GS115 (Invitrogen) with PEG1000 as recommended in the instructions of the
Pichia expression kit (Version F; Invitrogen).
The yeast strain was cultured on YEPD medium (1% yeast extract, 2% peptone, 2% glucose). MD medium (1.34% yeast
nitrogen base, 0.04% biotin, 2% glucose and 1.5% agar) was used for the selection and maintenance of
His+ (His 4 genotype) transformants. Transformants were incubated for 18 h in buffered minimal glycerol medium, then the cultures were
centrifuged and the cell pellet was resuspended in buffered minimal methanol medium. Cells were cultured in a baffled shake flask
for 5 d at 30 ºC. Methanol was added to maintain a concentration of 1%
(v/v) every day. Recombinant hLOX-1 was purified
using HiTrap ChelatingHP (Amersham Biotech, Piscataway, USA) according to the manufacturer¡¯s
instructions[11,12].
Validation of hLOX-1 Western blot analysis was used to validate the recombinant hLOX-1 receptor. The purified
hLOX-1 receptor (10 and 20 µg) was separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel
and transferred to polyvinylidene difluoride membrane (PVDF). After incubation in blocking solution (5% nonfat milk;
Sigma), the membrane was incubated with a 1:150 dilution of the anti-hLOX-1 monoclonal primary antibody (JTX92; a gift
from Prof T SAWAMURA, Osaka, Japan)[13] overnight at 4 ¡ãC. The membrane was washed and then incubated with a 1:2000
dilution of the second antibody (Amersham) for 1 h, and fragments binding to the membrane were detected with the Amersham
enhanced chemiluminescence (ECL) kit.
Three concentrations of purified hLOX-1 (0.4, 0.2, and
0.1 mg/mL) plus bovine serum albumin (BSA; 1 mg/mL) were used for
printing on poly-L-lysine-coated slides by robotic printers equipped with split pins (diameter 300 µm). The slides were
incubated for 5 min at 4 ¡ãC. Ten microliters of DiI-oxLDL (5 ng/mL) was added to each slide, then slides were covered with a
slip, incubated in a humidified chamber at room temperature for approximately 30 min, and then rinsed with a gentle stream of
phosphate-buffered saline (pH 7.4) for 3 min. Slides were air-dried and then used in the microarray scanner (ScanArray
Express; PerkinElmer, Boston, USA).
Fluorescent labeling Purified hLOX-1 receptor was labeled with FITC after it was dialyzed overnight against 0.01 mol/L
carbonate buffer (pH 9.5). A stock solution of FITC (Sigma) was prepared by dissolving 1 mg FITC in 1 mL of dimethyl
sulfoxide (Me2SO). The FITC solution was added to the hLOX-1 solution drop by drop, and the final ratio of FITC to
hLOX-1 is 50 µg to 1 mg. After this mixture was incubated for 16 h at 4 ¡ãC in the dark with a magnetic stirrer, FITC-hLOX-1 was
collected by using a Sephadex G25 column. Absorbance at 495 and 280 nm was measured in order to calculate the labeling
efficiency as follows:
F/P=(2.87¡ÁA495)/(A280
-0.35¡ÁA495). Only FITC-hLOX-1 in which the value of F/P was between 2 and 4 was
used for the assay.
Optimization of the FP assay Optimization of the FP assay was achieved by titrating to determine the optimal oxLDL and
FITC-hLOX-1 concentrations[14]. Three FITC-hLOX-1 concentrations (100, 200, and 400 nmol/L) were selected to betitrated
by oxLDL at concentrations ranging from 0.05 nmol/L to 100 µmol/L.
FP measurements were carried out using a SpectraMaxM5
(Molecular Devices, California USA) and black Costar
96-well plates (Corning Incorporated, Corning, NY, USA). Polarization was measured by using an excitation filter of 485 nm and an
emission filter of 520 nm, each well was flashed 50 times, and average values were obtained. Binding experiments were
performed in 96-well microplates of 150 µL total volume per well, and both FITC-hLOX-1 and oxLDL were diluted with 20
nmol/L phosphate-buffered saline (pH 7.4), which contained 0.24 nmol/L ethylenediamine tetraacetic acid (EDTA).
Me2SO tolerance and stability
Me2SO was necessary to maintain the solubility of the chemical library components. To
assess the effect of Me2SO on polarization in all experiments, 0%, 1%, 3%, or 5% final
Me2SO concentrations were used in the displacement assay buffer. To determine the optimal time of incubation and the stability of the signal, FITC-hLOX-1 with
oxLDL was incubated for 15 min, 30 min, 1 h, or 2 h before the polarization value was measured.
FP competition assay Assay was carried out according to optimal condition, and the maximal bound oxLDL
concentrations and optimal concentrations of FITC-hLOX-1 were determined. To assess the competition assay, the displacement
reaction was investigated by adding unlabeled
hLOX-1, after the binding of FITC-hLOX-1 and oxLDL reached a maximum. The
titration concentration of unlabeled hLOX-1 spanned from 5 nmol/L to 1 mmol/L.
High-throughput screening The optimized FP assay was used to screen
12 700 single chemicals. In homogenous solution, a polarization value was obtained when only fully displacement FITC-hLOX-1 was present. When oxLDL was
added and binding reached a maximum, the sample (final concentration 2.5 mg/mL) was added to competitively displace
oxLDL and bind hLOX-1. Three polarization values were used to determine the binding affinity of the sample with hLOX-1,
and unlabeled hLOX-1 was used as a positive control. The reaction system and assay conditions were all as described earlier
for the optimized assay.
Data analysis All polarization values are expressed in millipolarization units (mP). The polarization values of the FP assay
were calculated by using Analyst software based on the
equation[15]: mP =1000¡Á[(I||
-G*I^)/(I||
+G*I^)], where I||
is the fluorescence intensity measured when the excitation and emission polarizers are parallel,
I^ is the fluorescence intensity measured
when the excitation and emission polarizers are perpendicular, and G is the grating factor that corrects for instrument bias.
The G factor was calculated for each experiment by using the basal polarization value determined with the FITC-only wells.
To assess the robustness of the HTS of FP, the Z¡¯ value was calculated by using the
equation[15]: Z¡¯=1-[(3*s
free+3*sbound)/(mPbound
-mPfree)], where s is the standard deviation in the signal, the subscript ¡°bound¡± corresponds to the mean signal
obtained in the absence of a displacing substance, and the subscript ¡°free¡± corresponds to a completely displaced
tracer. Data from 96 positive and negative controls were used to calculate the Z¡¯ factor. Data are presented as mean¡ÀSD.
Results
Validation of hLOX-1 To validate the purity of the
hLOX-1, the purified hLOX-1 protein was blotted with anti-hLOX-1
monoclonal antibody. Purified hLOX-1 and anti-hLOX-1
monoclonal antibody had a high binding at 43 kDa (Figure
1). Incubation of purified hLOX-1 (at 3 concentrations: 0.4, 0.2, and 0.1 mg/mL) and BSA (1 mg/mL) with DiI-oxLDL showed that
binding of hLOX-1-oxLDL occurred in a concentration-dependent manner, and that BSA did not bind with DiI-oxLDL (Figure
2). This demonstrated the binding activity of
hLOX-1 with oxLDL.
FP assay optimization The oxLDL and fluorescently labeled-hLOX-1 titration curves are shown in Figure 3.
Labeled-hLOX-1 concentrations of 100, 200, and 400 nmol/L were tested and used to determine the optimal concentration of oxLDL.
With increasing oxLDL concentration, the polarization value increased and reached a plateau at an oxLDL concentration of
30 µmol/L. Once this plateau reached, the polarization value barely changed with further increases in the concentration of
oxLDL. A system with a total volume of 150 µL containing 200 nmol/L FITC-hLOX-1 and 50 µmol/L oxLDL was selected to use
in the actual assay.
FP Me2SO tolerance and assay stability
For the HTS chemical library, all compounds needed to be dissolved in
Me2SO. To obtain a tolerance value for
Me2SO in the FP assay, 0%, 1%, 3%, and 5%
Me2SO was used to assess the effect on the
polarization value. As shown in Figure 4, the more
Me2SO was added to each well, the lower the polarization value became.
However, for up to 5% Me2SO, the Z¡¯ factor was not significantly different for the different concentrations of
Me2SO.
Different durations for the receptor-ligand reaction were tested to determine the stability of the signal. FITC-LOX-1 at a
concentration of 200 nmol/L was titrated with oxLDL, and the reaction was incubated for varying periods of time before
measurement. There was little difference in the polarization values after 15 min, 30 min, 1 h, and 2 h incubation.
An incubation time of 20 min was chosen for the actual FP HTS assay (Figure 5).
Robustness of the FP assay for HTS The ultimate aim of the HTS assay is to identify active compounds, so the validation
and design of assays for robust HTS is critically important. The
Z¡¯ factor is widely used to measure the quality of an assay.
The average polarization values were approximately 106.0¡À7.6 and 232¡À6.8 mP in negative controls (free) and positive
controls (maximal binding), respectively (Figure 6). The
Z¡¯ factor for this assay was 0.66, which indicates that it should be a
robust HTS assay.
FP competition assay Given fixed concentrations of FITC-hLOX-1 and oxLDL, when the binding of receptor and ligand
reached an equilibrium, unlabeled hLOX-1 was titrated to assess the competition effect (Figure 7). At a concentration of 100
nmol/L of unlabeled hLOX-1, the polarization value began to reduce and the FITC-labeled hLOX-1-oxLDL began to
dissociate and be displaced by unlabeled hLOX-1. At a titrating concentration of 300 µmol/L unlabeled hLOX-1, the polarization
value was unchanged and FITC-hLOX-1 was almost displaced by unlabeled hLOX-1.
High-throughput screening results A total of
12 700 single chemicals were screened using the optimized FP-based HTS
assay system. After the primary screening, 28 chemicals were retested in duplicate. The distribution of the duplicate test
results was very consistent. During retesting with 5 multiple diluted concentrations, 3 chemicals were confirmed to have
activity with respect to LOX-1, and their
IC50 values are shown in Table 1.
Discussion
Membrane receptors are large receptors that are expressed on the cell surface, one of the most important classes of
therapeutic targets for drug discovery. In the present study, we selected hLOX-1 as a target, and developed a competitive
FP-based HTS assay. LOX-1 receptor is critically related to the progression of atherosclerosis and endothelial cell injury, so
the identification of antagonists of LOX-1 will have potential significance for diseases induced by endothelial activation,
dysfunction, and injury[16].
FP provides information on the rotational mobility of a fluorescent molecule. The rotational speed of a molecule is
dependent on the size of the molecule, and the temperature and viscosity of the solution. Therefore, if viscosity and
temperature are held constant, polarization is directly proportional to molecular
volume[17]. The basic theory of FP is that
mobilized small molecules have low polarization values and that large molecules have high polarization values. In a
homogeneous solution, the labeled compound is small and rotates rapidly (low polarization); when the labeled compound binds to
the larger molecule, its rotation slows down considerably (polarization changes from low to
high)[18,19]. Because the mobility of a fluorescent molecule will change if it binds to another molecule, FP can be used to study and quantify biomolecular
interactions. Therefore FP is a powerful technique for the study of biomolecular interactions in solution. It has been used to
investigate DNA-protein interactions, protein-protein interactions, and small molecule-protein
interactions[20,21].
For FP assay, the change in polarization value comes from the change of molecular volume, so fluorophores generally
were labelled to a small volume molecule, and were obtained high polarization values when small labeled-tracers with low
polarization values bind to large molecules in solution. In an FP-based receptor-ligand reactive system, the fluorophore is
always labeled onto ligands, because the ligands are almost smaller than the correlated
receptors[17]. In our FP-based competition assay, the molecular weight of hLOX-1 was 43 kDa, even though it is a membrane receptor, and that of its ligand,
oxLDL, was 3000 kDa. According to the theory of FP-based assay, FITC was used to label
hLOX-1. The compounds of chemical libraries are almost all small molecules, and far smaller than oxLDL. If the natural ligands competitively displace oxLDL from
hLOX-1, the polarization value will be greatly decreased relative to that of hLOX-1-oxLDL. Therefore it was feasible to use the
designed FP assay for screening hLOX-1 ligands.
In FP-based assays, the concentration of the fluorophore in the assay mixture is a major factor determining interference
of the compounds. A high labeled-tracer concentration in the screening process would result in reduced sensitivity for weak
inhibitors, because it is difficult for the weak inhibitors to compete with the
ligand[22]. However, a low labeled-tracer
concentration would produce low fluorescence counts, decreasing the signal-to-noise ratio. In the present study,
3 labeled-tracer concentrations were selected to determine the optimal concentration, and a concentration of 200 nmol/L for FITC-hLOX-1
was selected for use in the actual assay as the final concentration. At a concentration of 30 µmol/L oxLDL, the binding
plateaued, and the fixed 200 nmol/L concentration of FITC-hLOX-1 was titrated by oxLDL. In actual FP-based HTS, it is
possible that free tracer binding to compounds could conceal some displacement, so an excess oxLDL concentration (50
µmol/L) was used to prevent free FITC-hLOX-1 occurring in the reaction solution and reducing the background signal.
For HTS, identification of an active compound is known as a ¡°hit¡±, and the sensitivity of an assay is the critical
determinant of the hit ratio. The ability of a particular HTS assay to identify hits depends largely on the suitability or quality of the
assay used in the screening. The Z¡¯ factor, a characteristic parameter for the quality of the assay itself, was used to evaluate
the quality of the competitive FP-based HTS assay developed in the present study. The closer the Z¡¯ factor is to 1.0, the more
robust the assay for HTS is, and if Z¡¯ is =0.5, it indicates that the HTS assay is
robust[15]. The Z¡¯ value for our assay was 0.66,
indicating that it is robust and well suited for HTS.
In summary, an FP assay for the binding of oxLDL to FITC-hLOX-1 was optimized and miniaturized for HTS. The assay
performance was excellent, with a Z¡¯ value of 0.66. The distributions of the duplicates of the retest were very consistent,
indicating the reliability and robustness of the assay. The competitive FP assays described here for the detection of ligands
of LOX-1 are sensitive, robust, and suitable for adaptation to the HTS format.
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