High-Throughput Flow Cytometry to Detect Selective Inhibitors of ABCB1, ABCC1, and ABCG2 Transporters

Abstract

Up-regulation of pump (transporter) expression and selection of resistant cancer cells result in cancer multidrug resistance to diverse substrates of these transporters. While more than 48 members of the ATP binding cassette (ABC) transporter superfamily have been identified, up to now only three human ABC transporters-ABCB1, ABCC1, and ABCG2-have unambiguously been shown to contribute to cancer multidrug resistance. The use of low-toxicity and high-specificity agents as a targeted transporter inhibition strategy is necessary to effectively overcome multiple drug resistance. An objective of the present studies was to develop and validate HyperCyt (IntelliCyt, Albuquerque, NM) flow cytometry high-throughput screeening assays to assess the specificity of test compounds that inhibited transporters as an integral part of the screen. Two separate duplex assays were constructed: one in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide as substrate, and the other in which ABCB1 and ABCC1 transporters were evaluated in parallel using fluorescent calcein acetoxymethyl ester as substrate. ABCB1-expressing cells were color-coded to allow their distinction from cells expressing the alternate transporter. The assays were validated in a screen of the Prestwick Chemical Library (Illkirch, France). Three novel selective inhibitors of the ABCC1 transporter were identified in the screen, and the activity of each was confirmed in follow-up chemosensitivity shift and reversal studies. This high-throughput screening assay provides an efficient approach for identifying selective inhibitors of individual ABC transporters, promising as probes of transporter function and therapeutic tools for treating chemotherapy-resistant cancers.

Nature and uses of fluorescent dyes for drug transporter studies

“…SLC transporter inhibition % = (DF Control -DF +Compound X ) Â 100/(DF Control -DF +Reference inhibitor ) Because dye transport assays can generally be adapted to high-throughput screening [159], libraries of compounds can be easily tested towards dye transport activity, permitting thus to study structure--activity relationships and thereby to identify critical physical and chemical parameters required for inhibition. By this way, structure--activity relationships have been notably analyzed for inhibition of BCRP, OCT2, MATE1, OATP1B1, OATP1B3, OAT1 and OAT3 [95,99,106,[160][161][162].…”

A High-Content Assay Strategy for the Identification and Profiling of ABCG2 Modulators in Live Cells

“…For this reason, we investigated the use of the JC-1 dye as an alternative fluorescent ABCG2 substrate. 9,12 In contrast to Hoechst, the significant difference in JC-1 intracellular accumulation between U87MG and U87MG-ABCG2 (three-to eightfold) remains constant for *22 h (Fig. 3B-D), thus being more amenable to our assay and automated screening.…”

“…Despite significant efforts, suitable ABCG2 inhibitors are still lacking. Several in vitro assays have been established for the identification of new ABCG2 modulators, such as drug-efflux activity using FACS, [8][9][10][11][12] transport assays measuring the net flux across the monolayer, 13 bioluminescence imaging, 14 and ATPase assays. 15 All these assays measure only one parameter and provide hits based on a single criterion: the ABCG2 function, as measured by the efflux of a fluorescent substrate.…”

The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation

“…verapamil for ABCG2) should be avoided. [26,27] The first functional identification of tissue-derived cells with stem cell-like properties was achieved by a lower level 285 accumulation of the Hoechst 33 342 dye in such cells, forming a 'side population'. [28,29] It has been documented that the active extrusion of this dye by the ABCG2 protein is responsible for this phenotype, [30] while other ABC transporters may also be relevant in Hoechst 33 342 extrusion.…”