B9209-005, AN AZIDO DERIVATIVE OF THE CHEMOSENSITIZER DEXNIGULDIPINE-HCL, PHOTOLABELS P-GLYCOPROTEIN

P-glycoprotein is an energy-dependent drug extrusion pump for a variet y of anticancer drugs and is involved in the development of multidrug resistance in cancer. Dexniguldipine-HCl is a potent chemosensitizer f or P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway. To investigate the mechanisms of chem osensitization and to identify the binding sites for dexniguldipine-HC l on target proteins involved in chemosensitization, [H-3]B9209-005, a n azido derivative of dexniguldipine-HCl, was synthesized and used as a photoaffinity ligand. In two models of multidrug resistance reversal , i.e., sensitization to vincristine and modulation of rhodamine-123 u ptake, B9209-005 and dexniguldipine-HCl showed identical biological ac tivities. Photoaffinity labeling experiments with [H-3]B9209-005 in ce ll membranes from multidrug-resistant CCRF ADR-5000 cells, in comparis on with labeling experiments with [H-3]azidopine (an established photo affinity ligand for P-glycoprotein), showed that [H-3]B9209-005 labele d two proteins, with apparent molecular masses of 170 and 95 kDa. The pharmacological specificity of labeling was demonstrated by inhibition of photoincorporation by several cytostatic drugs transported by P-gl ycoprotein, as well as by chemosensitizers. Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C219 and with a site-directed polyclonal antibody to the amino-termin al sequence of P-glycoprotein (amino acids 389-406) identified these p roteins as intact P-glycoprotein and the amino-terminal fragment there of. No specific labeling was obtained in the drug-sensitive parent cel l line CCRF-CEM, which is devoid of significant P-glycoprotein express ion. Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude me mbrane protein was obtained. The affinity of [H-3]B9209-005 for bindin g to and photoincorporation into P-glycoprotein was 5-fold greater tha n that of [H-3]azidopine, and photoincorporation of [H-3]B9209-005 sho wed a different photoincorporation pattern, compared with [H-3]azidopi ne, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein. In contrast to [ H-3]azidopine, no specific labeling of this fragment was obtained with [H-3]B9209-005, indicating different binding sites for or different p hotoincorporation of the two dihydropyridine ligands. Because B9209-00 5 carries the photoreactive azido group in the dihydropyridine moiety, whereas the azido group of azidopine is located in the side chain, th ese results suggest that the dihydropyridine moiety of the two compoun ds probably interacts with the amino-terminal part of P-glycoprotein, whereas the side chains react preferentially with the carboxyl-termina l 55-kDa fragment. The data clearly show that the chemosensitizing pot ency of dexniguldipine-HCl is due to direct interaction of dexniguldip ine-HCl with and inhibition of P-glycoprotein. Furthermore, [H-3]B9209 -005 is a valuable ligand for the identification, at the molecular lev el, of the dexniguldipine-HCl binding sites on P-glycoprotein.