A high-throughput cell-based assay for inhibitors of ABCG2 activity.
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 microM 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 (> or = 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 microM) 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.
Henrich
Curtis J
CJ
Basic Research Program, SAIC-Frederick, Inc., NCI Frederick, Frederick, MD 21702, USA. henrichc@ncifcrf.gov
Bokesch
Heidi R
HR
Dean
Michael
M
Bates
Susan E
SE
Robey
Robert W
RW
Goncharova
Ekaterina I
EI
Wilson
Jennifer A
JA
McMahon
James B
JB
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000037:antagonists & inhibitors*; D045744:Cell Line, Tumor; D002734:Chlorophyll / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D006801:Humans; D007192:Indenes / Q000494:pharmacology*; D008942:Mitoxantrone / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000032:analysis / Q000037:antagonists & inhibitors*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D007192:Indenes; D009363:Neoplasm Proteins; D011838:Radiation-Sensitizing Agents; D002734:Chlorophyll; D008942:Mitoxantrone; C032623:pheophorbide a; C024743:Fumitremorgin A