The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein

1 The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [H-3]-XR9576 w ith the multidrug resistance P-glycoprotein (P-gp). XR9576 displayed specif ic high-affinity binding to P-gp (B-max=275 pmol mg(-1), K-d=5.1 nM). The t ransport substrates [H-3]-vinblastine and [H-3]-paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P-gp. The duration of action o f XR9576 with P-gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate. 2 The relative affinities of several modulators and transport substrates to interact with P-gp were determined from displacement drug equilibrium bind ing assays. Vinblastine and paclitaxel could only fractionally displace [H- 3]-XR9576 binding, displaying K-i values significantly different from their measured K-d values. This suggests a non-competitive interaction between X R9576 and the P-gp substrates vinblastine and paclitaxel. 3 XR9576 was shown to be a potent modulator of P-gp mediated [H-3]-vinblast ine and [H-3]- paclitaxel transport as it increased the steady-state accumu lation of these cytotoxics in CH(r)B30 cells to levels observed in non-P-gp -expressing AuxB1 cells (EC50= 487+/-50 nM). This inhibition of drug transp ort is not mediated through competition for transport since [H-3]-XR9576 ac cumulation was not influenced by P-gp expression or function. 4 These results demonstrate that the P-gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this t ransporter than any other reported modulators. Several lines of evidence su ggest that XR9576 inhibits P-gp function by binding at a site which is dist inct from the site of interaction of transport substrates. The two sites ma y be classified as serving modulatory or transport functions.