Reversible labeling of a chemosensitizer binding domain of p-glycoprotein with a novel 1,4-dihydropyridine drug transport inhibitor.
A photoreactive dihydropyridine (DHP), BZDC-DHP (2,6-dimethyl-4-(2-(trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5- dicarboxylic acid (2-[3-(4-benzoylphenyl)propionylamino]ethyl) ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (-)-[3H]BZDC-DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine-sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k(+1) = 0.031 nM-1 min-1, k(-1) = 0.172 min-1) binding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally related pyrimidine stereoselectively stimulated reversible (-)-[3H]BZDC-DHP binding, suggesting that more than one DHP molecule can bind to p-gp at the same time. Our data demonstrate that DHPs label multiple chemosensitizer domains on p-gp, distinct from the vinblastine interaction site. (-)-[3H]BZDC-DHP represents a valuable tool to characterize the molecular organization of chemosensitizer binding domains on p-gp by both reversible binding and photoinduced covalent modification. It provides a novel simple screening assay for p-gp active drugs.
Boer
R
R
Institut für Biochemische Pharmakologie, Innsbruck, Austria.
Dichtl
M
M
Borchers
C
C
Ulrich
W R
WR
Marecek
J F
JF
Prestwich
G D
GD
Glossmann
H
H
Striessnig
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D001577:Benzophenones / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D015220:Calcium Channels / Q000378:metabolism; D004095:Dihydropyridines / Q000378:metabolism*; D018432:Drug Resistance, Multiple / Q000502:physiology*; D006801:Humans; D008954:Models, Biological; D015335:Molecular Probes / Q000378:metabolism; D017319:Photosensitizing Agents / Q000378:metabolism; D014747:Vinblastine / Q000378:metabolism
C099532:2,6-dimethyl-4-(2-(trifluoromethyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid (2-(3-(4-benzoylphenyl)propionylamino)ethyl) ester ethyl ester; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001577:Benzophenones; D015220:Calcium Channels; D004095:Dihydropyridines; D015335:Molecular Probes; D017319:Photosensitizing Agents; D014747:Vinblastine; C054074:niguldipine