title: Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters.
abstract: ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.
authors.affiliation: Cytometry, Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.
authors.forename: J Jacob
authors.initials: JJ
authors.lastname: Strouse
authors.forename: Irena
authors.initials: I
authors.lastname: Ivnitski-Steele
authors.forename: Anna
authors.initials: A
authors.lastname: Waller
authors.forename: Susan M
authors.initials: SM
authors.lastname: Young
authors.forename: Dominique
authors.initials: D
authors.lastname: Perez
authors.forename: Annette M
authors.initials: AM
authors.lastname: Evangelisti
authors.forename: Oleg
authors.initials: O
authors.lastname: Ursu
authors.forename: Cristian G
authors.initials: CG
authors.lastname: Bologa
authors.forename: Mark B
authors.initials: MB
authors.lastname: Carter
authors.forename: Virginia M
authors.initials: VM
authors.lastname: Salas
authors.forename: George
authors.initials: G
authors.lastname: Tegos
authors.forename: Richard S
authors.initials: RS
authors.lastname: Larson
authors.forename: Tudor I
authors.initials: TI
authors.lastname: Oprea
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Edwards
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; C513055:ABCB1 protein, human; C116614:ABCG2 protein, human; C421109:multidrug resistance-associated protein 1