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Binding of an Optically Pure Photoaffinity Analogue of Verapamil, LU-49888, to P-Glycoprotein from Multidrug-resistant Human Leukemic Cell Lines¹

Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

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 M_r 170,000 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 >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.

¹ Supported in part by research Grant CA-40570 and Cancer Center Support (CORE) Grant CA 21765 from the National Cancer Institute, Department of Health and Human Services, Bethesda, MD, and in part by American Lebanese Syrian Associated Charities.

² To whom requests for reprints should be addressed, at Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38101.

Received 4/18/89. Revised 10/23/89. Accepted 11/ 6/89.

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