REVERSIBLE LABELING OF A CHEMOSENSITIZER BINDING DOMAIN OF P-GLYCOPROTEIN WITH A NOVEL 1,4-DIHYDROPYRIDINE DRUG TRANSPORT INHIBITOR

A photoreactive dihydropyridine (DHP), BZDC-DHP ethyl)phenyl)-1,4-dihy dropyridine-3,5-dicarboxylic acid {2-[3-(4-benzoylphenyl)propionylamin o]ethyl} ester ethyl ester), and its tritiated derivative were synthes ized as novel probes for human p-glycoprotein (p-gp). (-)-[H-3]BZDC-DI B specifically photolabeled p-gp in membranes of multidrug-resistant C CRF-ADR5000 cells. In reversible labeling experiments a saturable, vin blastine-sensitive and high-affinity (K-d = 16.3 nM, B-max = 58 pmol/m g of protein, k(+1) = 0.031 nM(-1) min(-1), k(-1) = 0.172 min(-1)) bin ding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and k nown chemosensitizers with a p-gp characteristic pharmacological profi le. For eight chemosensitizers tested, the potency for binding inhibit ion correlated (r > 0.94) with the potency for drug transport inhibiti on (measured using rhodamine 123 accumulation). The DHP niguldipine an d a structurally related pyrimidine stereoselectively stimulated rever sible (-)-[H-3]BZDC-DHP binding, suggesting that more than one DHP mol ecule can bind to p-gp at the same time. Our data demonstrate that DHP s label multiple chemosensitizer domains on p-gp, distinct from the vi nblastine interaction site. (-)-[H-3]BZDC-DHP represents a valuable to ol to characterize the molecular organization of chemosensitizer bindi ng domains on p-gp by both reversible binding and photoinduced covalen t modification. It provides a novel simple screening assay for p-gp ac tive drugs.