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A High-Throughput Cell-Based Assay for Inhibitors of ABCG2 ActivityBasic Research Program, SAIC-Frederick, Inc., NCI Frederick, Frederick, MD, Molecular Targets Development Program, NCI Frederick, Frederick, MD
Basic Research Program, SAIC-Frederick, Inc., NCI Frederick, Frederick, MD, Molecular Targets Development Program, NCI Frederick, Frederick, MD
Laboratory of Genomic Diversity, NCI Frederick, Frederick, MD
Cancer Therapeutics Branch, National Cancer Institute, Bethesda, MD
Cancer Therapeutics Branch, National Cancer Institute, Bethesda, MD
Molecular Targets Development Program, NCI Frederick, Frederick, MD, Data Management Services, Inc., NCI Frederick, Frederick, MD
Molecular Targets Development Program, NCI Frederick, Frederick, MD
Molecular Targets Development Program, NCI Frederick, Frederick, MD
ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 µM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z' 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (
Key Words: ABCG2 • pheophorbide a • drug resistance
This version was published on March
1, 2006 Journal of Biomolecular Screening, Vol. 11, No. 2,
176-183 (2006) This article has been cited by other articles:
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50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 µM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity. 
