title: A high-throughput screening microplate test for the interaction of drugs with P-glycoprotein.
abstract: P-glycoprotein (P-gp) is a multidrug transporter responsible for resistance to anticancer chemotherapy and physiologically involved in absorption, distribution, and excretion of a large number of hydrophobic xenobiotics. P-gp exhibits both an ATPase activity correlated with its drug transport function and a basal ATPase activity in the absence of any drug. We have developed a high-throughput screening test to detect specific interactions between drugs and P-gp. We took into account the existence of multiple binding sites on P-gp to propose and validate an optimized strategy, based on the modulation of the basal ATPase activity of P-gp and of the ATPase activity stimulated by three reference substrates (verapamil, vinblastine, and progesterone). The ATPase activity measurements were performed on P-gp-containing membrane vesicles from actinomycin-D-selected hamster DC-3F lung fibroblasts by a spectrophotometric method based on continuous monitoring of ADP formation, regenerated in ATP by a coupled enzyme system. This assay may be performed on 96- or 384-well microtiter plates. When applying this ATPase assay to 41 compounds known from the literature for their interaction with P-gp, 95% of them were found to be positive, whereas only 78% were positive when considering solely the modulation of the basal activity.
authors.affiliation: CEA, DBCM, Section de Biophysique des Protéines et des Membranes, LRA 17V Université Paris-Sud, F-91191 Gif-sur-Yvette, France.
authors.forename: Alexia
authors.initials: A
authors.lastname: Garrigues
authors.forename: Jérôme
authors.initials: J
authors.lastname: Nugier
authors.forename: Stéphane
authors.initials: S
authors.lastname: Orlowski
authors.forename: Eric
authors.initials: E
authors.lastname: Ezan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D006224:Cricetinae; D003412:Cricetulus; D003609:Dactinomycin / Q000037:antagonists & inhibitors; D018432:Drug Resistance, Multiple / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism; D006868:Hydrolysis; D008168:Lung / Q000166:cytology; D009243:NAD / Q000378:metabolism; D011374:Progesterone / Q000494:pharmacology*; D011485:Protein Binding; D012015:Reference Standards; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000379:methods*; D014700:Verapamil / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009243:NAD; D003609:Dactinomycin; D011374:Progesterone; D014747:Vinblastine; D014700:Verapamil; D000251:Adenosine Triphosphatases