Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1
Section snippets
Materials
[1-14C]tetraethylammonium bromide (14C-TEA; 2.4 mCi/mmol), [6,7-3H]estradiol-17β-d-glucuronide (3H-E17βG; 45 Ci/mmol), and [6,7-3H]estrone-3-sulfate (3H-E3S; 57.3 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). N-[methyl-3H]-4-phenylpyridinium acetate (3H-MPP; 80 Ci/mmol) was from Biotrend Chemikalien GmbH (Koeln, Germany). Unlabeled estrone-3-sulfate, ibuprofen and furosemide were obtained from Sigma–Aldrich (Schnelldorf, Germany). Cytostar-T scintillating microplates were
Applicability of the SPA for detecting hOCT1-, hOATP1B1- and hOAT3-mediated transport of various 14C- and 3H-labeled substrates
HEK-hOCT1 (clone mix) and HEK control cells were incubated with the hOCT1 substrate, 14C-tetraethylammonium, in 96-well Cytostar-T plates, and scintillation signals were recorded. Fig. 1A shows time- and concentration-dependent uptake of 14C-TEA into hOCT1-transfected cells detected by scintillation signals significantly increasing over time, whereas no uptake of 14C-TEA was seen in the control cells, where the signals remained unchanged over time (Fig. 1B). These data clearly demonstrate the
Conclusion
This study demonstrates the applicability of a scintillation proximity assay for detecting compound interactions with human SLC transporters in real time. The assay is high throughput and amenable to automation and allows for determination of Km values, for selection of clonal cell lines by their level of transporter function, and for identification of SLC transporter inhibitors in a convenient and reliable fashion, suggesting its successful use in future drug discovery.
Acknowledgments
We thank Stefan Hallén and Björn Johansson for excellent technical advice on multiplate scintillation counting.
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