Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
PLoS One. 2013 Apr 10;8(4):e60334. doi: 10.1371/journal.pone.0060334. Print 2013.
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 [(125)I]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.
ABCB1 也被称为 P-糖蛋白（P-gp）或多药耐药蛋白 1（MDR1），是 ATP 结合盒（ABC）转运体家族的一种膜相关的多药转运体。它是研究最多的能够使癌细胞产生耐药性的转运体之一。能够识别与 ABCB1 相互作用的化合物的可靠高通量测定法对于开发新的治疗药物至关重要。使用荧光和相差活细胞成像系统开发了一种用于测量 ABCB1 介导的 calcein AM 外排的高通量测定法。该测定法证明了 ABCB1 过表达 KB-V1 细胞中荧光 calcein 的时间和剂量依赖性积累。用已知的 ABCB1 抑制剂 XR9576、维拉帕米和环孢菌素 A 对测定法进行了验证，所有这些抑制剂在该测定法中均显示出剂量依赖性抑制 ABCB1 介导的 calcein AM 外排。成像系统拍摄的相差和荧光图像为评估具有细胞毒性或产生假阳性信号的化合物提供了更多机会。对具有已知治疗靶点和激酶抑制剂文库的化合物进行了筛选。该测定法鉴定出多种作为 ABCB1 介导的外排抑制剂的化合物，且重现性高。在所鉴定的作为 ABCB1 抑制剂的化合物中，BEZ235、BI 2536、IKK 16 和伊匹单抗被进一步评估。这四种化合物以剂量依赖性方式抑制 calcein AM 外排，并且在基于流式细胞术的 calcein AM 外排测定法中也具有活性。BEZ235、BI 2536 和 IKK 16 还成功抑制了放射性标记的光亲和底物 [125I]碘代芳基叠氮 praxosin 与 ABCB1 的标记。用 XR9576 和环孢菌素 A 抑制 ABCB1 增强了 BI 2536 对 ABCB1 过表达癌细胞 HCT-15-Pgp 的细胞毒性，并使 BI 2536 的 IC50 值降低了几个数量级。这种高效、可靠且简单的高通量测定法已经鉴定出可能影响药物效力或药物相互作用并预测临床治疗中多药耐药性的 ABCB1 底物/抑制剂。
