B9209-005, an azido derivative of the chemosensitizer dexniguldipine-HCl, photolabels P-glycoprotein.
P-glycoprotein is an energy-dependent drug extrusion pump for a variety of anticancer drugs and is involved in the development of multidrug resistance in cancer. Dexniguldipine-HCl is a potent chemosensitizer for P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway. To investigate the mechanisms of chemosensitization and to identify the binding sites for dexniguldipine-HCl on target proteins involved in chemosensitization, [3H]B9209-005, an 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 uptake, B9209-005 and dexniguldipine-HCl showed identical biological activities. Photoaffinity labeling experiments with [3H]B9209-005 in cell membranes from multidrug-resistant CCRF ADR-5000 cells, in comparison with labeling experiments with [3H]azidopine (an established photoaffinity ligand for P-glycoprotein), showed that [3H]B9209-005 labeled 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-glycoprotein, as well as by chemosensitizers. Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C 219 and with a site-directed polyclonal antibody to the amino-terminal sequence of P-glycoprotein (amino acids 389-406) identified these proteins as intact P-glycoprotein and the amino-terminal fragment thereof. No specific labeling was obtained in the drug-sensitive parent cell line CCRF-CEM, which is devoid of significant P-glycoprotein expression. Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude membrane protein was obtained. The affinity of [3H]B9209-005 for binding to and photoincorporation into P-glycoprotein was 5-fold greater than that of [3H]azidopine, and photoincorporation of [3H]B9209-005 showed a different photoincorporation pattern, compared with [3H]azidopine, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein. In contrast to [3H]azidopine, no specific labeling of this fragment was obtained with [3H]B9209-005, indicating different binding sites for or different photoincorporation of the two dihydropyridine ligands. Because B9209-005 carries the photoreactive azido group in the dihydropyridine moiety, whereas the azido group of azidopine is located in the side chain, these results suggest that the dihydropyridine moiety of the two compounds probably interacts with the amino-terminal part of P-glycoprotein, whereas the side chains react preferentially with the carboxyl-terminal 55-kDa fragment.(ABSTRACT TRUNCATED AT 400 WORDS)
Borchers
C
C
Fakultät für Chemie, Universität Konstanz, Germany.
Ulrich
W R
WR
Klemm
K
K
Ise
W
W
Gekeler
V
V
Haas
S
S
Schödl
A
A
Conrad
J
J
Przybylski
M
M
Boer
R
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D000345:Affinity Labels; D001386:Azides / Q000737:chemistry* / Q000494:pharmacology; D002460:Cell Line; D004095:Dihydropyridines / Q000737:chemistry* / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D004357:Drug Synergism; D006801:Humans; D010777:Photochemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; C094804:B 9209-005; D004095:Dihydropyridines; C054074:niguldipine