5-((3-azidophenethyl)methylamino)-2-(3-4-5-trimethoxyphenyl)-2-isopropylvaleronitrile and Leukemia

Verapamil enhances anticancer drug cytotoxicity in multidrug resistant (MDR) cells, apparently by competing with these agents for binding to P-glycoprotein (Pgp). In this study, we provide direct evidence for this competition. We studied the binding of an optically pure photoaffinity analogue of verapamil, (S)-5-[(3-azidophenylethyl)-[N-methyl-3H]- methylamino]-2-(3,4,5-trimethoxyphenyl)-2-isopropylvaleronitrile (LU-49888), to Pgp from MDR cell lines. LU-49888 specifically labeled a single Mr 170,0000 protein that was identified as Pgp on Western blots and also by specific immunoprecipitation with monoclonal antibody C-219. A 200-fold molar excess of vinblastine or vincristine specifically inhibited this binding by greater than 98%. LU-49888 labeling of Pgp was also inhibited by actinomycin D (45%), podophyllotoxin (47%), and amsacrine (82%), marginally by doxorubicin (25%), colchicine (22%), daunorubicin (18%), and etoposide (14%), but not by teniposide. Modulators of Pgp-MDR also compete with LU-49888 for binding to Pgp: verapamil (82%), diltiazem (73%), quinidine (91%), reserpine (91%), rescinnamine (88%), and trimethoxybenzoylyohimbine (89%). Chloroquine was moderately inhibitory (25%), whereas chlorpromazine and yohimbine, which are not modulators in our MDR cell lines, did not inhibit the binding of LU-49888 to Pgp. LU-49888 labeling of Pgp was also completely inhibited by (R)-, (S)-, and racemic desmethoxyverapamil, all with the same efficiency. Our results demonstrate that the verapamil analogue LU-49888 specifically binds to Pgp and suggest that verapamil and some MDR modulators exert their effects by interacting with Pgp.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Drug Resistance; Humans; Leukemia; Membrane Glycoproteins; Neoplasm Proteins; Tumor Cells, Cultured; Verapamil