The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction.
1. The interaction of the indolizin sulfone SR33557 with the multidrug resistance P-glycoprotein (P-gp), was used to explore the nature of drug binding site(s) on this transporter. The steady-state accumulation of [3H]-vinblastine in P-gp expressing CHrB30 cells was increased by SR33557 with greater potency than verapamil. Furthermore, SR33557 potentiated the affinity of verapamil to modulate vinblastine transport when added simultaneously. 2. Verapamil elicited a 1.5 to 2.5 fold stimulation of basal ATPase activity in CHrB30 membranes, whereas SR33557 and vinblastine inhibited activity, but only at relatively high concentrations. However, SR33557 and vinblastine decreased the Vmax but not the Km for verapamil stimulation of ATPase activity. This is indicative of a non-competitive interaction, most likely at distinct sites. 3. The specific [3H]-vinblastine binding to P-gp in CHrB30 cell membranes was displaced by SR33557 with an IC50 of 8.3 +/- 4.5 nM. Moreover, SR33557 caused a 3 fold increase in the dissociation rate of vinblastine binding to P-gp indicating a negative allosteric effect on the vinca alkaloid acceptor site. 4. These results demonstrate that SR33557 interacts with a site on P-gp which is distinct from, but allosterically linked to the vinca alkaloid site. The apparent broad substrate specificity displayed by P-gp may be explained by a multiple drug binding site model.
Martin
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Nuffield Department of Clinical Biochemistry, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital.
Berridge
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Higgins
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Callaghan
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation; D007212:Indolizines / Q000494:pharmacology*; D010627:Phenethylamines / Q000494:pharmacology*; D014316:Tritium; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007212:Indolizines; D010627:Phenethylamines; D014316:Tritium; D014747:Vinblastine; D014700:Verapamil; D000251:Adenosine Triphosphatases; C059901:fantofarone