Development of a cell-based high-throughput assay to screen for inhibitors of organic anion transporting polypeptides 1B1 and 1B3.

Abstract

The two organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are expressed at the sinusoidal membrane of hepatocytes. They have a broad and overlapping substrate specificity and transport many endobiotics and drugs. Specific inhibitors are required to determine the contribution of each OATP to the hepatocellular uptake of common substrates. We have developed a cell-based high-throughput assay to screen chemical libraries in order to identify such inhibitors for OATP1B1 and OATP1B3. We have used OATP1B1- or OATP1B3-expressing Chinese Hamster Ovary cells on 96-well plates and determined uptake of fluorescein-methotrexate (FMTX). We validated the assay with known inhibitors and screened the well characterized Prestwick library of 1120 drugs. Along with several known OATP inhibitors including rifampicin, cyclosporine A and mifepristone we identified some new inhibitors. For inhibitors that seemed to be able to distinguish between OATP1B1- and OATP1B3-mediated FMTX uptake IC(50) values were determined. Estropipate (estrone-3-sulfate stabilized with piperazine) was the most selective OATP1B1 inhibitor (IC(50) = 0.06 microM vs. 19.3 microM for OATP1B3). Ursolic acid was the most selective OATP1B3 inhibitor (IC(50) = 2.3 microM vs. 12.5 microM for OATP1B1). In conclusion, this cell-based assay should allow us to identify even more specific inhibitors by screening larger chemical libraries.

Full text

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Current Chemical Genomics, 2010, 4, 1-8 1

1875-3973/10 2010 Bentham Open
Open Access
Development of a Cell-Based High-Throughput Assay to Screen for
Inhibitors of Organic Anion Transporting Polypeptides 1B1 and 1B3
Chunshan Gui1, Amanda Obaidat1, Rathnam Chaguturu2,3 and Bruno Hagenbuch*,1,2
1Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City,
Kansas 66160, USA
2The University of Kansas Cancer Center, Kansas City, Kansas 66160, USA
3The High Throughput Screening Laboratory, The University of Kansas, Structural Biology Center, Lawrence, Kansas
66045, USA
Abstract: The two organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are expressed at the sinusoidal mem-
brane of hepatocytes. They have a broad and overlapping substrate specificity and transport many endobiotics and drugs.
Specific inhibitors are required to determine the contribution of each OATP to the hepatocellular uptake of common sub-
strates. We have developed a cell-based high-throughput assay to screen chemical libraries in order to identify such in-
hibitors for OATP1B1 and OATP1B3. We have used OATP1B1- or OATP1B3-expressing Chinese Hamster Ovary cells
on 96-well plates and determined uptake of fluorescein-methotrexate (FMTX). We validated the assay with known inhibi-
tors and screened the well characterized Prestwick library of 1120 drugs. Along with several known OATP inhibitors in-
cluding rifampicin, cyclosporine A and mifepristone we identified some new inhibitors. For inhibitors that seemed to be
able to distinguish between OATP1B1- and OATP1B3-mediated FMTX uptake IC50 values were determined. Estropipate
(estrone-3-sulfate stabilized with piperazine) was the most selective OATP1B1 inhibitor (IC50 = 0.06 μM vs. 19.3 μM for
OATP1B3). Ursolic acid was the most selective OATP1B3 inhibitor (IC50 = 2.3 μM vs. 12.5 μM for OATP1B1). In
conclusion, this cell-based assay should allow us to identify even more specific inhibitors by screening larger chemical
libraries.
Keywords: OATP1B1, OATP1B3, fluorescein-methotrexate, cell based assay, high-throughput screening.
INTRODUCTION

OATPs; rodents: Oatps) form a large gene superfamily of
transport proteins expressed in multiple organs [1, 2]. Many
of their members are multispecific transporters that mediate
the uptake of numerous endo- and xeno-biotics. Human
OATP1B1 and OATP1B3 are expressed in the sinusoidal
membrane of hepatocytes and are thought to be liver-specific
under normal physiological conditions. They are responsible
for the uptake of numerous drugs, including statins, endo-
thelin receptor antagonists,
methotrexate and paclitaxel, as well as the antibiotic rifam-
picin [1]. Besides specific substrates like prostaglandin E2
for OATP1B1 and CCK-8 for OATP1B3 [3, 4], OATP1B1
and OATP1B3 have a number of common drug substrates
including bosentan [5], fluvastatin [6], methotrexate [7, 8],
olmesartan [9], pitavastatin [10], rifampicin [11], rosuvasta-
tin [12], and valsartan [13]. Given that both OATPs are ex-
pressed in human hepatocytes, it will be almost impossible
without specific inhibitors to elucidate which of the two pro-
teins is more important for the transport of a certain drug.
Furthermore, these transporters can affect the bioavailability

Organic anion transporting polypeptides (humans:
anticancer drugs like

*Address correspondence to this author at the Department of Pharmacology,
Toxicology and Therapeutics, The University of Kansas Medical Center,
3901 Rainbow Blvd, Kansas City, KS 66160, USA; Tel: +1-913-588-0028;
Fax: +1-913-588-7501; E-mail: bhagenbuch@kumc.edu
of drugs. In 1996 we demonstrated that the peptide-based
thrombin inhibitor CRC 220 is a substrate of rat Oatp1a1
[14]. This explained the very high hepatic first pass elimina-
tion of CRC 220 which was partially responsible for its low
bioavailability. Specific OATP1B1 or OATP1B3 inhibitors
could be co-applied with a drug which is efficiently cleared
by the liver and this could increase its bioavailability.

OATP1B1 and OATP1B3, we decided to develop an assay
for high-throughput screening. Fluorescent substrates are
preferred for high-throughput screening because of the sensi-
tivity and dynamic range that the fluorescence signal affords.
So far only fluorescent bile acids [15] and Fluo-3, a fluores-
cent calcium indicator [16, 17] have been demonstrated to be
substrates for OATP1B3 while fluorescein methotrexate
(FMTX) has been reported as a substrate for OATP1B1 [18].
However, methotrexate has been shown to be a substrate for
both OATP1B1 and OATP1B3 [8] and is commercially
available as fluorescein methotrexate (FMTX) and Alexa
Fluor® 488 methotrexate (AMTX). Preliminary data from
our laboratory also demonstrated that the anticancer drug
paclitaxel is a substrate for both OATPs. A fluorescent ver-
sion of paclitaxel is also available commercially as Oregon
Green® 488 Taxol (Flutax-2). Therefore, we compared these
different fluorescent compounds along with fluorescein as
substrates for OATP1B1 and OATP1B3. We further charac-
terized FMTX transport by both OATPs and established an
assay on 96-well plates for high-throughput screening. We
In order to identify potentially specific inhibitors for

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2 Current Chemical Genomics, 2010, Volume 4 Gui et al.
used this assay to screen the Prestwick library for modulators
(inhibitors or stimulators) and confirmed several hits by
measuring IC50 or EC50 values for inhibition and stimulation
of OATP1B1- and OATP1B3-mediated FMTX transport.
MATERIALS AND METHODS
Materials

Alexa Fluor® 488 methotrexate (AMTX), and Oregon
Green® 488 Taxol (Flutax-2) were purchased from Invitro-
gen (Carlsbad, CA). Fetal bovine serum was from Hyclone
(Logan, UT). The Prestwick library was obtained from the
University of Kansas High Throughput Screening Labora-
tory. Individual chemicals for the confirmatory experiments
were either from Prestwick Chemical or from Sigma and
stock solutions were prepared in DMSO.
Cell culture reagents, fluorescein-methotrexate (FMTX),
Cell Culture and Transport Assay on 24-well Plates

OATP1B1 and OATP1B3 were described previously [19]
and were grown at 37 °C in a humidified 5% CO2 atmos-
phere in Dulbecco’s Modified Eagle Medium, containing 1
g/l D-glucose, 2 mM L-glutamine, 25 mM Hepes buffer and
110 mg/l sodium pyruvate, supplemented with 10% FBS
(Hyclone, Logan, UT), 50 ?g/ml L-proline, 100 U/ml peni-
cillin and 100 ?g/ml streptomycin, and 500 μg/ml G-418.
Cells were passaged twice a week and used up to passage 55.
Protein concentrations were very consistent, both on 24-well
plates (0.150 ± 0.005 mg/well) as well as on 96-well plates
(0.045 ± 0.002 mg/well). Transport assays on 24-well plates
were performed as described [19] with the following modifi-
cations: cells were washed three times with 1 ml of pre-
warmed (37 °C) uptake buffer (116.4 mM NaCl, 5.3 mM
KCl, 1 mM NaH2PO4, 0.8 mM MgSO4, 5.5 mM D-glucose
and 20 mM Hepes, pH adjusted to 7.4 with Trizma base) and
uptake was started by adding 200 ?l of uptake buffer con-
taining the indicated concentrations of fluorescent com-
pound. After the indicated time period at 37 °C, uptake was
stopped by removing the uptake solution and washing the
cells four times with 1 ml of ice-cold uptake buffer. The cell
membranes were then disrupted with 300 ?l of 1% Triton X-
100 (dissolved in PBS) and fluorescence was measured in a
Bio-Tek Synergy HT microplate reader (Winooski, VT) at
an excitation wavelength of 485 nm and an emission wave-
length of 528 nm.
Chinese Hamster Ovary (CHO) cells stably expressing
High-Throughput Uptake Experiments in 96-well Plates

expressing CHO cells were plated at 12,000 cells per well on
96-well plates and 48 hours later medium was replaced with
medium containing 5 mM Na-butyrate to induce nonspecific
gene expression. Previous experiments with OATP express-
ing CHO cells have demonstrated that after treatment of
CHO cells with Na-butyrate transporter function was in-
creased due to an increase in the maximal transport rate [20].
After another 24 hours in culture, the cells were used for
uptake experiments. Cells were washed three times with 200
?l of room temperature uptake buffer (116.4 mM NaCl, 5.3
mM KCl, 1 mM NaH2PO4, 0.8 mM MgSO4, 5.5 mM D-
glucose and 20 mM Hepes, pH adjusted to 7.4 with Trizma
base) and uptake was started by adding 70 ?l of uptake
buffer containing 5 ?M substrate in the presence or absence
For high-throughput uptake experiments, OATP-
of inhibitors. After 30 min culture at room temperature, up-
take was stopped by removing the uptake solution and wash-
ing the cells four times with 200 μl of room temperature up-
take buffer. The cells were then solubilized with 100 ?l of
1% Triton X-100 (dissolved in PBS). Fluorescence was
measured in a Bio-Tek Synergy HT microplate reader (Wi-
nooski, VT) at an excitation wavelength of 485 nm and an
emission wavelength of 528 nm.
Data Analysis

cording to NIH assay guidance manual [21]. The data were
analyzed using Sigmaplot and Graphpad prism. Statistical
significance was determined using the two-tailed unpaired
Student’s t test.
Z’ factor calculation and assay validation were done ac-
RESULTS
Characterization of Fluorescent Substrate Transport by
OATP1B1 and OATP1B3

four new fluorescent compounds, fluorescein-methotrexate
(FMTX), Alexa Fluor® 488 methotrexate (AMTX), Oregon
Green® 488 Taxol (Flutax-2) and fluorescein to the uptake
of the known OATP substrate Fluo-3. As can be seen in Fig.
(1), four of the five compounds namely Fluo-3, FMTX, Flu-
tax-2 and fluorescein are substrates for OATP1B3, while
AMTX was not transported. OATP1B1 on the other hand
only transported FMTX and fluorescein. Given that radiola-
beled methotrexate has previously been documented as a
substrate for both OATP1B1 and OATP1B3 [8] and that the
uptake signal for both OATP1B3 (about 4.5 fold) and
OATP1B1 (about 3 fold) was highest for FMTX, we decided
to further characterize it. As the results in Fig. (2) demon-
strate, uptake of 10 μM FMTX was linear over a 5 min pe-
riod. Thus, we used the 2 min time point to determine the
kinetic parameters of OATP-mediated FMTX transport. Fig.
(3) shows one of three independent experiments and demon-
In the first set of experiments, we compared the uptake of










Fig. (1). Comparison of OATP1B1- and OATP1B3-mediated trans-
port of fluorescent substrates. Uptake was measured with 1 μM of
the indicated fluorescent compounds on 24-well plates for 30 min.
Values are means ± SE of two combined independent experiments
each performed with triplicate determinations. FMTX: fluorescein
methotrexate; AMTX: Alexa Fluor® 488 methotrexate; Flutax-2:
Oregon Green® 488 Taxol; WT: wild-type CHO cells; OATP1B1:
CHO cells expressing OATP1B1; OATP1B3: CHO cells
expressing OATP1B3.
4000
t units)
e (fluorescent
uptake
3000
WT
OATP1B1
OATP1B3
2000
1000
0
Fluo-3FMTXAMTX Flutax-2 Fluorescein

Page 3

High-Throughput Assay for OATP1B1 and OATP1B3 Transporters Current Chemical Genomics, 2010, Volume 4 3
strates that OATP1B3 (closed circles) has a much higher
capacity than OATP1B1 (open circles) to transport FMTX.
Based on all three experiments we calculated for OATP1B1
an apparent Km value of 3.8 ± 0.7 μM and a Vmax value of
961 ± 198 fluorescent units/mg protein * min and for
OATP1B3 an apparent Km value of 7.9 ± 2.0 μM and a Vmax
value of 4772 ± 709 fluorescent units/mg protein * min.












Fig. (2). Time dependent uptake of FMTX into CHO cells express-
ing OATP1B1 and OATP1B3. Uptake of 10 μM FMTX was de-
termined at 37 °C with wild-type (closed triangles), OATP1B1
(open circles) or OATP1B3 (closed circles) expressing CHO cells
on 24-well plates. Values are means ± SE of quadruplicate determi-
nations in a single experiment.












Fig. (3). Kinetics of OATP1B1- and OATP1B3-mediated FMTX
uptake. Uptake of increasing concentrations of FMTX was meas-
ured at 37 °C for 2 min on 24-well plates with CHO wild-type and
OATP1B1 or OATP1B3 expressing cells. Net OATP1B1 (open
circles) or OATP1B3 (closed circles) uptake was calculated by
subtracting uptake into wild-type cells from uptake into OATP-
expressing cells. The values (means ± SE) were fitted to the Micha-
elis-Menten equation using non-linear regression analysis.

Establishing a High-Throughput Assay on 96-well Plates

on 96-well plates instead of on 24-well plates, we deter-
mined uptake of 5 μM FMTX in the absence or presence of
In order to test whether this cell-based assay could be run
known OATP substrates or inhibitors for 30 min at room
temperature on 96-well plates. We could detect FMTX up-
take mediated by both OATPs. A typical uptake resulted in
4,400 ± 80 fluorescent units for wild-type CHO cells, 15,060
± 1,540 fluorescent units for OATP1B1-expressing cells and
33,880 ± 3,645 fluorescent units for OATP1B3-expressing
cells. Thus, signal-to-basal ratios of 3.4 for OATP1B1 and of
7.7 for OATP1B3 were obtained. As can be seen from Fig.
(4), uptake by OATP1B1 and OATP1B3 was inhibited by all
tested compounds. In additional experiments (data not
shown), we determined that uptake at room temperature was
linear over at least 30 min. Furthermore, we calculated the Z’
factors for the assays (0.26 for OATP1B1 and 0.46 for
OATP1B3) in the absence (negative control) and presence
(positive control) of 10 μM rifampicin with three independ-
ent 96-well plates. Validation was performed following the
recommendations of the NIH assay guidance manual [21].
The coefficient of variance for all maximal signals, unnor-
malized middle signals and minimal signals was less than
20%. Furthermore, all minimal signal standard deviations
were smaller than both, the standard deviations of the middle
and the maximal signals.





















Fig. (4). Inhibition of OATP-mediated FMTX uptake by known
inhibitors/substrates. Uptake of 5 μM FMTX was measured for 30
min at room temperature on 96-well plates with OATP-expressing
and wild-type CHO cells in the absence or presence of the indicated
compounds. Values obtained with wild-type CHO cells were sub-
tracted from values obtained with OATP-expressing cells and are
given as percent of control. Means ± SE of two combined inde-
pendent experiments each performed with quadruplicate determina-
tions are shown. * p < 0.05; E3S: estrone-3-sulfate; E17?G: estra-
diol-17?-glucuronide.
otein)
103 / mg pro
cent units x
ake (fluoresc
upta
50 50
60
wild-type
OATP1B1
OATP1B3 OATP1B3
40
30
10
20
010203040
0
time [min]
x min)
mg protein
ent units / m
e (fluoresce
uptake
6
OATP1B1
OATP1B3
4
2
00 20 204040606080 80 100100
0
Fluorescein-methotrexate [M]
OATP1B1
Control
10 M Rifampicin
50 M Pravastatin
50 M E3S 50 M E3S
50 M E17G
50 M Mifepristone
2.5 M Paclitaxel

50 M Clotrimazole
*
*
*
*
*
**
*
020406080100120
Control
10 M Rifampicin
50 M P50 M Pravastatin
50 M E3S
50 M E17G
50 M Mifepristone

2.5 M Paclitaxel
50 M Clotrimazole
t ti
OATP1B3
*
*
*
020 4060 80100120
*
*
% of control

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4 Current Chemical Genomics, 2010, Volume 4 Gui et al.
Screening of Prestwick Library

we included 8 wells of negative (DMSO controls) and 8
wells of positive (10 μM rifampicin in DMSO) controls in
each plate. As can be seen from the screening statistics
shown in Table 1, we have developed a useful assay to
screen for OATP1B3 inhibitors but the assay needs to be
improved to be used with OATP1B1. Z’ values for the
OATP1B1 part were close to zero or even negative, while
the values for the OATP1B3 part were between 0.41 and
0.79. Nevertheless, we identify inhibitors for both OATP1B1
and OATP1B3 mediated uptake and a stimulator for
OATP1B3. Among the strongest inhibitors found were
known OATP substrates and/or inhibitors like cyclosporine
A, rifampicin, saquinavir and several statins. In addition to
these compounds, several new substances were found to in-
hibit FMTX transport mediated by either OATP1B1 or
OATP1B3 or by both.
To screen the 1120 compounds in the Prestwick library,

mined IC50 values for several compounds that preferentially
inhibited either OATP1B1 or OATP1B3 or seemed to stimu-
late one or the other OATP. Among the tested compounds
we identified five compounds that inhibited OATP1B1 more
potently than OATP1B3 and three that preferentially inhib-
ited OATP1B3 mediated FMTX transport (Table 2, Fig. 5).
To confirm these hits of the screening results, we deter-
Estropipate, which is estrone-sulfate stabilized with
piperazine, was the strongest OATP1B1 inhibitor with an
IC50 of 0.06 ± 0.01 μM, while it hardly inhibited OATP1B3
at concentrations up to 1 μM (IC50 of 19.3 ± 6.3 μM) (Table
2). In addition to estropipate, we found that bromocryptine,
ergocryptine, fipexide, niflumic acid and repaglinide prefer-
entially inhibited OATP1B1 (Table 2, Fig. 5), while
beclamethasone, moricizine and ursolic acid preferentially
inhibited OATP1B3 (Table 2, Fig. 5). Among the potential
stimulators we could confirm that progesterone stimulated
OATP1B3-mediated FMTX uptake with an EC50 of 0.2 μM,
while it inhibited OATP1B1-mediated FMTX uptake with an
IC50 of 5.4 ± 1.9 μM (Fig. 6).
DISCUSSION

say for OATP1B3 (Z’ factor > 0.4) that allows screening for
inhibitors and stimulators of OATP1B3. The assay estab-
lished for OATP1B1 needs more optimization perhaps using
a different substrate to be as valuable as the one for
OATP1B3. Initially we tested several fluorescent com-
pounds as potential substrates for OATP1B1 and OATP1B3
and compared them to the known OATP1B1 and OATP1B3
substrate Fluo-3 [16, 17, 19]. Because fluorescein-
methotrexate (FMTX) resulted in the highest signal for both
OATP1B1 and OATP1B3 and because radiolabeled
In this study, we established a good high-throughput as-
Table 1. Statistics of Prestwick Library Screening
OATP1B1
Plate number 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Mean (neg
control)
5,170 5,906 5,428 7,400 6,166 4,984 4,866 6,113 5,670 6,713 6,390 6,054 6,042 9,159
STDEV (neg
control)
390 1,107 807 924 579 645 451 721 634 1,150 425 605 444 921
Mean (pos
control)
9,411 10,044 9,499 14,100 10,904 9,080 8,950 11,267 10,641 11,879 12,990 12,183 14,967 16,572
STDEV (pos
control)
871 715 753 1,520 1,267 803 537 1,042 935 1,663 954 1,226 1,249 1,760
Sample mean 9,654 10,381 9,101 13,350 9,857 8,448 8,967 11,136 10,397 11,423 12,343 11,735 12,785 16,997
Z' factor 0.11 -0.32 -0.15 -0.09 -0.17 -0.06 0.27 -0.03 0.05 -0.63 0.37 0.10 0.43 -0.08
OATP1B3
Plate number 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Mean (neg
control)
7,161 8,034 7,766 10,591 7,901 7,506 7,734 9,639 8,273 8,231 8,418 9,093 9,047 9,856
STDEV (neg
control)
760 1,315 1,037 1,814 523 834 1,194 1,495 761 332 370 472 570 1,026
Mean (pos
control)
26,768 31,694 27,097 31,987 28,351 25,272 30,369 26,319 26,882 30,781 31,653 34,132 34,603 32,986
STDEV (pos
control)
1,704 2,889 1,778 1,212 1,789 2,271 2,257 1,792 1,171 4,063 1,281 3,003 3,917 3,014
Sample mean 26,308 26,850 23,819 33,715 26,838 25,368 25,738 29,565 26,661 29,799 29,365 33,188 31,714 36,503
Z' factor 0.62 0.47 0.56 0.58 0.66 0.48 0.54 0.41 0.69 0.42 0.79 0.58 0.47 0.48

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High-Throughput Assay for OATP1B1 and OATP1B3 Transporters Current Chemical Genomics, 2010, Volume 4 5
Table 2.
pounds
Inhibition of OATP1B1- and OATP1B3-Mediated Fluorescein-Methotrexate (FMTX) Uptake by Prestwick Library Com-
Compound IC50 on OATP1B1 (μ μM) IC50 on OATP1B3 (μ μM)
2-aminobenzenesulfonamide
> 100 > 100
Aconitine
> 100 No inhibition
Beclomethasone
6.7 ± 1.2 1.4 ± 0.4
Bromocryptine
0.7 ± 0.08 1.8 ± 0.3
Captopril
No inhibition No inhibition
Ergocryptine
0.8 ± 0.2 2.2 ± 1.2
Estropipate
0.06 ± 0.01 19.3 ± 6.3
Etomidate
> 100 > 100
Fipexide
34.5 ± 6.8 > 100
Metronidazole
> 100 > 100
Moricizine
8.1 ± 1.9 2.7 ± 0.4
Niflumic acid
3.7 ± 1.4 22.0 ± 6.8
Ramipril
4.0 ± 0.5 3.3 ± 0.3
Repaglinide
1.1 ± 0.2 4.8 ± 0.7
Resveratrol
11.2 ± 2.0 23.7 ± 2.5
Rifampicin
1.3 ± 0.2 1.5 ± 0.5
Trifluridine
No inhibition No inhibition
Ursolic acid
12.5 ± 1.9 2.3 ± 0.03
Results are means ± SE of at least three independent experiments.



















Fig. (5). Inhibition of OATP-mediated FMTX uptake by selected Prestwick compounds. Concentration dependent inhibition of 5 μM FMTX
by indicated compounds was measured for 30 min at room temperature on 96-well plates with wild-type and OATP-expressing CHO cells.
Values obtained with wild-type CHO cells were subtracted from values obtained with OATP-expressing cells and are given as percent of
control. Means ± SE of the means of three independent experiments are shown and were used to calculate IC50 values.