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

Gui C, Obaidat A, Chaguturu R, Hagenbuch B - Curr Chem Genomics (2010)

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

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.

No MeSH data available.


Time dependent uptake of FMTX into CHO cells expressing OATP1B1 and OATP1B3. Uptake of 10 µM FMTX was determined 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 determinations in a single experiment.
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Figure 2: Time dependent uptake of FMTX into CHO cells expressing OATP1B1 and OATP1B3. Uptake of 10 µM FMTX was determined 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 determinations in a single experiment.

Mentions: In the first set of experiments, we compared the uptake of 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, Flutax-2 and fluorescein are substrates for OATP1B3, while AMTX was not transported. OATP1B1 on the other hand only transported FMTX and fluorescein. Given that radiolabeled 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) demonstrate, uptake of 10 μM FMTX was linear over a 5 min period. 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 demonstrates 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.


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

Gui C, Obaidat A, Chaguturu R, Hagenbuch B - Curr Chem Genomics (2010)

Time dependent uptake of FMTX into CHO cells expressing OATP1B1 and OATP1B3. Uptake of 10 µM FMTX was determined 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 determinations in a single experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864424&req=5

Figure 2: Time dependent uptake of FMTX into CHO cells expressing OATP1B1 and OATP1B3. Uptake of 10 µM FMTX was determined 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 determinations in a single experiment.
Mentions: In the first set of experiments, we compared the uptake of 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, Flutax-2 and fluorescein are substrates for OATP1B3, while AMTX was not transported. OATP1B1 on the other hand only transported FMTX and fluorescein. Given that radiolabeled 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) demonstrate, uptake of 10 μM FMTX was linear over a 5 min period. 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 demonstrates 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.

Bottom Line: 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).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, Kansas 66160, USA.

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.

No MeSH data available.