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 (2,6-dimethyl-4-(2-(trifluoromethyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid {2-[3-(4-benzoylphenyl)propionylamino]ethyl} ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (−)-[3H]BZDC−DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine-sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k+1 = 0.031 nM-1 min-1, k-1 = 0.172 min-1) binding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally relate...