Identification of Inhibitors of ABCG2 by a Bioluminescence Imaging–Based High-Throughput Assay

Abstract

ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)-based assay was developed to allow highthroughput compound screening. This assay exploits our finding that D-luciferin, the substrate of firefly luciferase ( fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of D-luciferin and enhance bioluminescence signal by increasing intracellular D-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty-seven compounds showed BLI enhancement, a measure of anti-ABCG2 activity, of z5-fold, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g., mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from a FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing.

Labeling of Luciferase/eGFP-Expressing Bone Marrow-Derived Stromal Cells with Fluorescent Micron-Sized Iron Oxide Particles Improves Quantitative and Qualitative Multimodal Imaging of Cellular Grafts In Vivo

“…It was recently shown by Zhang and colleagues that ABCG2/ BCRP transporters on the cell membrane play a crucial role in D-luciferin retention in the cell. Moreover, they demonstrated that the level of BLI signal from firefly luciferasepositive cells can be influenced by using inhibitors of these transporters [42,43]. Although our data demonstrate that a particle should be intracellular in order to achieve BLI amplification ((i) in Fig.…”

Cisplatin Selects for Multidrug-Resistant CD133+ Cells in Lung Adenocarcinoma by Activating Notch Signaling

“…In vitro study showed that ABCG2-overexpressing human NSCLC cell lines were more resistant to chemotherapy (44,45). Inhibition of ABCG2 and/or ABCB1 by selective inhibitors sensitized NSCLCs to chemotherapy (46,47).…”

Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

“…To test the relative effectiveness of reported ABCG2 inhibitors in vitro, we measured the bioluminescence signal of HEK cells transfected with fLuc and ABCG2 (23) in the presence of the ABCG2 inhibitors Ko143 (19), tariquidar (24), elacridar (25), gefitinib, or nilotinib (26). The ABCB1 inhibitor (2R)-anti-5-f3-[4-(10,11-dichloromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxygquinoline trihydrochloride(DCPQ) (27,28) and HEK cells without ABCG2 (HEK/empty/fLuc) were used as controls.…”