Understanding polyspecificity within the substrate-binding cavity of the human multidrug resistance P-glycoprotein

Human P-glycoprotein (P-gp) controls drugs bioavailability by pumping structurally unrelated drugs out of cells. The X-ray structure of the mouse P-gp ortholog has been solved, with two SSS enantiomers or one RRR enantiomer of the selenohexapeptide inhibitor QZ59, found within the putative drug-binding pocket (Aller SG, Yu J, Ward A, Weng Y, Chittaboina S, Zhuo R, Harrell PM, Trinh YT, Zhang Q, Urbatsch IL et al. (2009). Science 323, 1718-1722). This offered the first opportunity to localize the well-known H and R drug-binding sites with respect to the QZ59 inhibition mechanisms of Hoechst 33342 and daunorubicin transports, characterized here in cellulo. We found that QZ59-SSS competes efficiently with both substrates, with K-I,K- app values of 0.15 and 0.3 mu M, which are 13 and 2 times lower, respectively, than the corresponding K-m,K- app values. In contrast, QZ59-RRR non-competitively inhibited daunorubicin transport with moderate efficacy (K-I,K- app = 1.9 mu M); it also displayed a mixed-type inhibition of the Hoechst 33342 transport, resulting from a main non-competitive tendency (K-i2,K- app = 1.6 mu M) and a limited competitive tendency (K-i1,K- (app) = 5 mu M). These results suggest a positional overlap of QZ59 and drugs binding sites: full for the SSS enantiomer and partial for the RRR enantiomer. Crystal structure analysis suggests that the H site overlaps both QZ59-SSS locations while the R site overlaps the most embedded location.