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, Bruno Hagenbuch*, 1, 2
1 Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, Kansas 66160, USA
2 The University of Kansas Cancer Center, Kansas City, Kansas 66160, USA
3 The High Throughput Screening Laboratory, The University of Kansas, Structural Biology Center, Lawrence, Kansas 66045, USA

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© Gui et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

* 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:


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 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.