title: Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase--further experimental evidence for a multisite model.
abstract: P-glycoprotein, a plasma membrane protein overexpressed in multidrug-resistant (MDR) cells, exhibits in vitro an ATPase activity and is responsible for the energy-dependent efflux of structurally unrelated cytotoxic drugs (like vinblastine) and various MDR-reversing agents (like verapamil and progesterone) from these MDR cells. To investigate the mechanism of P-glycoprotein interaction with various compounds, we measured the P-glycoprotein ATPase activity on membrane vesicles prepared from the MDR cell line DC-3F/ADX, and we studied the effects of vinblastine, verapamil and progesterone on this ATPase activity. The basal P-glycoprotein ATPase activity is increased by verapamil and progesterone, with respective half-maximal activating concentrations of approximately 1.5 microM and approximately 25 microM, and activation factors of approximately 1.7 and approximately 2.2. Vinblastine inhibits the activation of P-glycoprotein ATPase induced by verapamil or progesterone with an inhibition constant approximately 0.5 microM in both cases. This demonstrates that vinblastine has a specific modulating site on P-glycoprotein. The combined modulation of P-glycoprotein ATPase by vinblastine and verapamil reveals that these two drugs are mutually exclusive. Since these two molecules have different effects both on the basal P-glycoprotein ATPase activity and on the MgATP concentration dependence of P-glycoprotein ATPase activity, they could bind P-glycoprotein either on different and overlapping sites, or on distant but interacting sites. In contrast, the combined modulation of P-glycoprotein ATPase by vinblastine and progesterone reveals a non-competitive relationship between these two drugs, and hence shows that they can independently and simultaneously bind P-glycoprotein on distinct sites. Since verapamil and progesterone are mutual inhibitors of P-glycoprotein ATPase stimulation in a non-competitive manner, these two molecules can also bind independently P-glycoprotein on separated sites. This is confirmed here by the observation of a synergistic effect when mixtures of verapamil and progesterone are tested for the modulation of P-glycoprotein ATPase. Three MDR-related molecules, taken as models for interaction with P-glycoprotein, appear thus to bind on at least two different separated specific sites. These results favor a multisite model rather than a universal site model to describe the broad substrate specificity characterizing P-glycoprotein function.
authors.affiliation: Section de Biophysique des Protéines et des Membranes, DBCM and URA 2096 CNRS, CEA Saclay, Gif-sur-Yvette, France.
authors.forename: M
authors.initials: M
authors.lastname: Garrigos
authors.forename: L M
authors.initials: LM
authors.lastname: Mir
authors.forename: S
authors.initials: S
authors.lastname: Orlowski
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D006224:Cricetinae; D018432:Drug Resistance, Multiple; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D014700:Verapamil / Q000378:metabolism / Q000494:pharmacology; D014747:Vinblastine / Q000378:metabolism / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004791:Enzyme Inhibitors; D011374:Progesterone; D014747:Vinblastine; D014700:Verapamil; D000251:Adenosine Triphosphatases