Figure 1

[³H]TC transport and [³H]preS1 binding screening assay. NTCP-HEK293 cells were seeded onto 96-well plates and were incubated with tetracycline in order to induce expression of the human NTCP protein. Cells without tetracycline treatment were used as 0% controls of [³H]TC uptake and [³H]preS1 binding, respectively. Cells were subjected to bile acid transport experiments with 1 µM [³H]TC and myr-preS1_2-48 lipopeptide binding experiments with 5 nM [³H]preS1, each over 10 min at 37 °C. Experiments were performed with solvent alone (set to 100%) and with increasing concentrations of the indicated inhibitors. The mean of the 0% control was subtracted to calculate net [³H]TC transport rates (shown in blue) as well as net [³H]preS1 binding rates (shown in red), which are depicted in the diagrams (expressed as % of control at the y-axis). Half maximal inhibitory concentrations (IC₅₀) were calculated by nonlinear regression analysis using the equation log(inhibitor) versus response (GraphPad Prism). In order to validate the assay, (a) TC and (b) myr-preS1_2-48 lipopeptide were used as inhibitors. Furthermore, the already established NTCP inhibitors (c) ciglitazone, (d) troglitazone, (e) cyclosporine A, and (f) rapamycin were analyzed for [³H]TC transport inhibition and [³H]preS1 binding inhibition. Data represent means ± SD of quadruplicate determinations of representative experiments.