A high-throughput screening microplate test for the interaction of drugs with P-glycoprotein

被引:61
作者
Garrigues, A
Nugier, J
Orlowski, S
Ezan, E [1 ]
机构
[1] CEA, DBCM, Sect Biophys Prot & Membranes, F-91191 Gif Sur Yvette, France
[2] Univ Paris Sud, CNRS, URA 2096, LRA V 17, F-91191 Gif Sur Yvette, France
[3] CEA, DRM, Serv Pharmacol & Immunol, F-91191 Gif Sur Yvette, France
关键词
P-glycoprotein; ATPase; drug; HTS;
D O I
10.1006/abio.2002.5650
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2002 Elsevier Science (USA).
引用
收藏
页码:106 / 114
页数:9
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