Abstract 5635: Development of a high-throughput cell and fluorescent image-based ABCB1-mediated efflux assay for screening inhibitors of ABCB1.

Abstract

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 the IncuCyteFLR technology. Time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells was recorded using the IncuCyteFLR. 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. A kinase inhibitor library and other compounds with known therapeutic targets were screened. The assay is highly reproducible and inhibitors for ABCB1-mediated efflux were identified from the kinase inhibitor library. Among compounds with known targets, BEZ235, BI 2536, IKK 16, and Ispinesib (SB-715992) inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry based efflux assay. BEZ235, BI 2536, and IKK 16 also successfully competed with radiolabeled photoaffinity substrate [125I]iodoarylazidoprazosin (IAAP) for binding to ABCB1. Inhibition of ABCB1 lowered the IC50 value of BI 2536 treated ABCB1-overexpressing cancer cells. Phase and fluorescent images taken by the IncuCyteFLR provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. This high-throughput assay provides an efficient, reliable, and simple technique for screening libraries of natural and synthetic compounds to identify ABCB1 inhibitors. The same approach may be applied to screen inhibitors of other ABC transporters when suitable cell lines and fluorescent substrates are used. Citation Format: Megan R. Ansbro, Suneet Shukla, Suresh V. Ambudkar, Stuart H. Yuspa, Luowei Li. Development of a high-throughput cell and fluorescent image-based ABCB1-mediated efflux assay for screening inhibitors of ABCB1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5635. doi:10.1158/1538-7445.AM2013-5635