Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

Abstract

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [125I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.

The microfluidic multitrap nanophysiometer for hematologic cancer cell characterization reveals temporal sensitivity of the calcein-AM efflux assay

“…Our data suggest a calcein-AM trapping effect that only becomes apparent when the calcein-AM efflux pump, MRP-1, is sufficiently inhibited. This effect seems at odds with the currently held belief that calcein-AM rapidly diffuses across the cell membrane25. Passive diffusion down its concentration gradient would imply that calcein-AM diffuses out of the cell at a similar rate as that with which it is taken up after extracellular calcein-AM is removed.…”

Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors

“…Therefore, Calcein efflux assay is considered a functional indicator of MDR activity. 55–57 We observed that the encapsulated MCF-7R cells could be divided into three distinct subtypes based on high, medium and minimal levels of Calcein AM fluorescence (Fig. 4A).…”

Human ABCB1 (P-glycoprotein) and ABCG2 mediate resistance to BI 2536, a potent and selective inhibitor of Polo-like kinase 1

“…In contrast, BI 2536 appeared not to interact with ABCC1 at all. Interestingly, in a recent study aimed to develop a novel high-throughput assay identifying ABCB1 interacting compounds, BI 2536 was identified as a positive hit from a library 193 compounds [44]. Next, we measured the effect of BI 2536 on the vanadate-sensitive ATPase activity of ABCB1 and ABCG2 (Fig.…”