Garrigues Alexia, Nugier Jérôme, Orlowski Stéphane, Ezan Eric
CEA, DBCM, Section de Biophysique des Protéines et des Membranes, LRA 17V Université Paris-Sud, F-91191 Gif-sur-Yvette, France.
Anal Biochem. 2002 Jun 1;305(1):106-14. doi: 10.1006/abio.2002.5650.
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.
P-糖蛋白（P-gp）是一种多药转运蛋白，负责对抗癌化疗产生耐药性，并且在生理上参与大量疏水性外源性物质的吸收、分布和排泄。P-gp既表现出与其药物转运功能相关的ATP酶活性，又在没有任何药物的情况下表现出基础ATP酶活性。我们开发了一种高通量筛选试验，以检测药物与P-gp之间的特异性相互作用。我们考虑到P-gp上存在多个结合位点，基于对P-gp基础ATP酶活性以及由三种参考底物（维拉帕米、长春碱和孕酮）刺激的ATP酶活性的调节，提出并验证了一种优化策略。通过基于连续监测ADP形成的分光光度法，利用偶联酶系统将ADP再生为ATP，对来自放线菌素-D筛选的仓鼠DC-3F肺成纤维细胞的含P-gp膜囊泡进行ATP酶活性测量。该测定可在96孔或384孔微量滴定板上进行。当将这种ATP酶测定应用于文献中已知的41种与P-gp相互作用的化合物时，发现其中95%呈阳性，而仅考虑基础活性的调节时，只有78%呈阳性。
