title: Antibody binding shift assay for rapid screening of drug interactions with the human ABCG2 multidrug transporter.
abstract: The ABCG2 multidrug transporter protein has been identified as a key player in cancer drug resistance and xenobiotic elimination, as its actively transported substrates include anticancer drugs, intermediates of heme metabolism, xenobiotics, and also drug conjugates. Several transported substrates at higher concentrations, and some anticancer agents even at low concentrations directly inhibit the ABCG2 transporter, thus it is difficult to provide estimation for pharmacologically important ABCG2-dependent interactions. In addition, as documented here, in mutant variants of the transporter, inhibitors of the wild-type ABCG2 may become actively transported substrates. In this paper we describe a rapid in vitro assay to identify transport modulation by measuring the cell surface interaction of a conformation sensitive monoclonal antibody (5D3) with ABCG2 in intact cells. As documented, in conjunction with membrane ATPase, transport and cytotoxicity measurements, this assay provides a reliable estimate of concentration-dependent modulation of ABCG2 by newly emerging pharmacophores. A high-throughput, 96-well plate assay platform is also provided.
authors: Membrane Research Group of Hungarian Academy of Sciences, Department of Biophysics, Semmelweis University and National Blood Center, Diószegi u 64, H-1113 Budapest, Hungary.
authors: Ágnes
authors: Á
authors: Telbisz
authors: Csilla
authors: C
authors: Hegedüs
authors: Csilla
authors: C
authors: Özvegy-Laczka
authors: Katalin
authors: K
authors: Goda
authors: György
authors: G
authors: Várady
authors: Zoltán
authors: Z
authors: Takáts
authors: Eszter
authors: E
authors: Szabó
authors: Brian P
authors: BP
authors: Sorrentino
authors: András
authors: A
authors: Váradi
authors: Balázs
authors: B
authors: Sarkadi
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000937:Antigen-Antibody Reactions / Q000187:drug effects*; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D055808:Drug Discovery / Q000379:methods*; D019008:Drug Resistance, Neoplasm; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D049990:Membrane Transport Modulators / Q000494:pharmacology; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D011994:Recombinant 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; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D008024:Ligands; D049990:Membrane Transport Modulators; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins