Competitive and Non‐Competitive Inhibition of the Multidrug‐Resistance‐Associated P‐glycoprotein ATPase

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 ≈1.5 μM and ≈25 μM, and activation factors of ≈1.7 and ≈2.2. Vinblastine inhibits the activation of P‐glycoprotein ATPase induced by verapamil or progesterone with an inhibition constant ≈0.5 μM 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.