Elsevier

Analytical Biochemistry

Volume 366, Issue 2, 15 July 2007, Pages 117-125
Analytical Biochemistry

Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1

https://doi.org/10.1016/j.ab.2007.04.038Get rights and content

Abstract

Increasing evidence suggests a key role of transport proteins in the pharmacokinetics of drugs. Within the solute carrier (SLC) family, various organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs) that interact with drug molecules have been identified. Traditionally, cellular uptake assays require multiple steps and provide low experimental throughput. We here demonstrate the use of a scintillation proximity approach to detect substrate uptake by human drug transporters in real time. HEK293 cells stably transfected with hOCT1, hOATP1B1, or hOAT3 were grown directly in Cytostar-T scintillating microplates. Confluent cell monolayers were incubated with 14C- or 3H-labeled transporter substrates. Cellular uptake brings the radioisotopes into proximity with the scintillation plate base. The resulting light emission signals were recorded on-line in a microplate scintillation counter. Results show time- and concentration-dependent uptake of 14C-tetraethylammonium, 3H-methylphenylpyridinium (HEK-hOCT1), 3H-estradiol-17β-d-glucuronide (HEK-hOATP1B1), and 3H-estrone-3-sulfate (HEK-hOAT3), while no respective uptake was detected in empty vector-transfected cells. Km of 14C-tetraethylammonium and 3H-estrone-3-sulfate uptake and hOAT3 inhibition by ibuprofen and furosemide were similar to conventional dish uptake studies. The scintillation proximity approach is high throughput, amenable to automation and allows for identification of SLC transporter substrates and inhibitors in a convenient and reliable fashion, suggesting its broad applicability in drug discovery.

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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