title: The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein.
abstract: 1 The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]-XR9576 with the multidrug resistance P-glycoprotein (P-gp). XR9576 displayed specific high-affinity binding to P-gp (Bmax = 275 pmol mg-1, Kd = 5.1 nM). The transport substrates [3H]-vinblastine and [3H]-paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P-gp. The duration of action of 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 binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]-XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non-competitive interaction between XR9576 and the P-gp substrates vinblastine and paclitaxel. 3 XR9576 was shown to be a potent modulator of P-gp mediated [3H]-vinblastine and [3H]-paclitaxel transport as it increased the steady-state accumulation of these cytotoxics in CHrB30 cells to levels observed in non-P-gp-expressing AuxB1 cells (EC50 = 487+/-50 nM). This inhibition of drug transport is not mediated through competition for transport since [3H]-XR9576 accumulation 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 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 classified as serving modulatory or transport functions.
authors.affiliation: Nuffield Department of Clinical Biochemistry & Cellular Science, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU.
authors.forename: C
authors.initials: C
authors.lastname: Martin
authors.forename: G
authors.initials: G
authors.lastname: Berridge
authors.forename: P
authors.initials: P
authors.lastname: Mistry
authors.forename: C
authors.initials: C
authors.lastname: Higgins
authors.forename: P
authors.initials: P
authors.lastname: Charlton
authors.forename: R
authors.initials: R
authors.lastname: Callaghan
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000187:drug effects*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D006224:Cricetinae; D019008:Drug Resistance, Neoplasm; D007700:Kinetics; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding; D011804:Quinolines / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000972:Antineoplastic Agents, Phytogenic; D011804:Quinolines; D014747:Vinblastine; D000255:Adenosine Triphosphate; C402343:tariquidar; D017239:Paclitaxel