P-glycoprotein, a member of the ATP-binding cassette transporter family, is able to confer resistance on tumors against a large number of functionally and chemically distinct cytotoxic compounds. Several recent investigations suggest that P-glycoprotein contains multiple drug binding sites rather than a single site of broad substrate specificity. In the present study, radioligand-binding techniques were used to directly characterize drug interaction sites on P-glycoprotein and how these multiple sites interact. The drugs used were classified as either 1) substrates, which are known to be transported by P-glycoprotein (e.g., vinblastine) or 2) modulators, which alter P-glycoprotein function but are not themselves transported by the protein (e.g., XR9576). Drug interactions with P-glycoprotein were either competitive, at a common site, or noncompetitive, and therefore at distinct sites. Based on these data, we can assign a minimum of four drug binding sites on P-glycoprotein. These sites fall into two categories: transport, at which translocation of drug across the membrane can occur, and regulatory sites, which modify P-glycoprotein function. Intriguingly, however, some modulators interact with P-glycoprotein at a transport site rather than a regulatory site. The pharmacological data also demonstrate that both transport and regulatory sites are able to switch between high- and low-affinity conformations. The multiple sites on P-glycoprotein display complex allosteric interactions through which interaction of drug at one site switches other sites between high- or low-affinity conformations. The data are discussed in terms of a model for the mechanism of transport by P-glycoprotein.
Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2 ؊/؊ knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649 -15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 M of the cyclin-dependent kinase inhibitor UCN-01 or 1 M of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 M tariquidar, and ABCG2-transfected cells were 6 -7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.
1 The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [ 3 H]-XR9576 with the multidrug resistance Pglycoprotein (P-gp). XR9576 displayed speci®c high-anity binding to P-gp (B max =275 pmol mg 71 , 4 These results demonstrate that the P-gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P-gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classi®ed as serving modulatory or transport functions.
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