A cell‐based high‐throughput assay to identify inhibitors of Organic Anion Transporting Polypeptides 1B1 and 1B3

Abstract

Organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are expressed at the basolateral membrane of hepatocytes and exhibit broad and overlapping substrate specificity for many endobiotics and drugs. Without specific inhibitors it is almost impossible to determine the contribution of the individual transporters to hepatocellular uptake of shared substrates (drugs). In order to identify such inhibitors of OATP1B1 and OATP1B3, we developed a cell‐based high throughput assay to screen chemical libraries. We used Chinese Hamster Ovary cells that stably express human OATP1B1 or OATP1B3 on 96‐well plates and measured uptake of fluorescein‐methotrexate (F‐MTX). After validating the assay with known inhibitors we screened the pharmacologically well characterized Prestwick library of 1120 compounds. Among the numerous hits were known OATP inhibitors including rifampicin, cyclosporine A and mifepristone. For inhibitors that seemed to be able to distinguish between OATP1B1 and OATP1B3 mediated F‐MTX uptake we determined IC50 values. Estropipate (estrone‐3‐sulfate stabilized with piperazine) turned out to be a most distinctive OATP1B1 inhibitor (IC50 = 0.07 μM vs. 11.2 μM for OATP1B3) while ursolic acid was the most distinctive OATP1B3 inhibitor (IC50 = 3.4 μM vs. 29.6 μM for OATP1B1). In conclusion, this cell‐based assay should allow us to screen larger libraries and identify even more specific inhibitors.