title: A high-content assay strategy for the identification and profiling of ABCG2 modulators in live cells.
abstract: ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which has been implicated in resistance to various chemotherapeutic agents. Though a number of cell-based assays to screen for inhibitors have been reported, they do not provide a content-rich platform to discriminate toxic and autofluorescent compounds. To fill this gap, we developed a live high-content cell-based assay to identify inhibitors of ABCG2-mediated transport and, at the same time, assess their cytotoxic effect and potential optical interference. We used a pair of isogenic U87MG human glioblastoma cell lines, with one stably overexpressing the ABCG2 transporter. JC-1 (J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine iodide) was selected as the optimal reporter substrate for ABCG2 activity, and the resulting assay was characterized by a Z' value of 0.50 and a signal-to-noise (S/N) ratio of 14 in a pilot screen of ∼ 7,000 diverse chemicals. The screen led to the identification of 64 unique nontoxic positives, yielding an initial hit rate of 1%, with 58 of them being confirmed activity. In addition, treatment with two selected confirmed positives suppressed the side population of U87MG-ABCG2 cells that was able to efflux the Hoechst dye as measured by flow cytometry, confirming that they constitute potent new ABCG2 transporter inhibitors. Our results demonstrate that our live cell and content-rich platform enables the rapid identification and profiling of ABCG2 modulators, and this new strategy opens the door to the discovery of compounds targeting the expression and/or trafficking of ABC transporters as an alternative to functional inhibitors that failed in the clinic.
authors.affiliation: 1 High-Throughput Screening Core Facility, Memorial Sloan-Kettering Cancer Center , New York, New York.
authors.forename: Christophe
authors.initials: C
authors.lastname: Antczak
authors.forename: Boyoung
authors.initials: B
authors.lastname: Wee
authors.forename: Constantin
authors.initials: C
authors.lastname: Radu
authors.forename: Bhavneet
authors.initials: B
authors.lastname: Bhinder
authors.forename: Eric C
authors.initials: EC
authors.lastname: Holland
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay / Q000379:methods; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins