Rapid detection of ABC transporter interaction: potential utility in pharmacology.
BACKGROUND
The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are known to transport a wide range of structurally diverse compounds. Their high level of expression at the blood-brain, maternal-fetal, and blood-testis barriers as well as their purported roles in oral absorption suggests that ABC transporters play important pharmacologic roles.


METHODS
We have developed a method to characterize the function and inhibition of ABC transporters using an automated cell counter with fluorescence detection capability. The assay was performed using stably-transfected HEK293 cells expressing P-gp, MRP1, or ABCG2 and examining transport of fluorescent substrates in the presence or absence of known inhibitors and compared to results obtained with a flow cytometer. Fold increase in intracellular fluorescence was then calculated for cells incubated with fluorescent substrate in the absence of inhibitor versus in the presence of inhibitor.


RESULTS
Fold increase values obtained either with the cell counter or flow cytometer were comparable for cells expressing either MRP1 or ABCG2; slightly higher fold increase values were observed when cells expressing P-gp were read on a flow cytometer compared to the cell counter.


CONCLUSIONS
The assay described provides an inexpensive detection method to aid in the development of novel ABC transporter inhibitors or to characterize potential drug-drug interactions.
Medical Oncology Branch, Center for Cancer Research, National Institutes of Health, Bethesda, MD 20892, USA. robeyr@mail.nih.gov
Robert W
RW
Robey
Bo
B
Lin
Jean
J
Qiu
Leo Li-Ying
LL
Chan
Susan E
SE
Bates
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D003524:Cyclosporins / Q000494:pharmacology; D055808:Drug Discovery / Q000379:methods*; D004347:Drug Interactions; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection
C513055:ABCB1 protein, human; C116614:ABCG2 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D003524:Cyclosporins; D005456:Fluorescent Dyes; D007211:Indoles; D009363:Neoplasm Proteins; D011422:Propionates; D011804:Quinolines; C059141:verlukast; C010522:tryptoquivaline; C070272:valspodar