BIBW22 BS, POTENT MULTIDRUG RESISTANCE-REVERSING AGENT, BINDS DIRECTLY TO P-GLYCOPROTEIN AND ACCUMULATES IN DRUG-RESISTANT CELLS

The expression of P-glycoprotein (P-gp) in tumor cells causes a multid rug resistance (MDR) phenotype. P-gp has been shown to mediate the tra nsport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a ph enylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW 22 BS s(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the p hotoaffinity labeling of a 170-kDa protein in drug-resistant cells imm unoprecipitated with P-gp-specific monoclonal antibodies. The photolab eling of P-gp by [H-3]azido-BIBW22 BS was specific and saturable. Furt hermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, in hibited the photolabeling oi P-gp by [H-3]azido-BIBW22 BS. Drug bindin g studies showed that membranes from MDR cells bound more BIBW22 BS th an parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug trans port studies demonstrated that BIBW22 BS is not a substrate for P-gp e fflux pump. Interestingly, BIBW22 BS was shown to accumulate more in r esistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -re sistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [H-3] vinblastine seen in the same MDR cells. A comparison of [H-3]azido-BIB W22 BS or [H-3]azidopine photolabeled P-gp by Cleveland mapping with S taphylococcus aureus V8 protease showed differences in the photolabele d peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.