Reversible labeling of a chemosensitizer binding domain of p-glycoprotein with a novel 1,4-dihydropyridine drug transport inhibitor

R. Boer, M. Dichtl, C. Borchers, W. R. Ulrich, J. F. Marecek, G. D. Prestwich, H. Glossmann, J. Striessnig

Research output: Contribution to journalArticlepeer-review

39 Citations (Scopus)

Abstract

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 (K(d) = 16.3 nM, B(max) = 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.

Original languageEnglish
Pages (from-to)1387-1396
Number of pages10
JournalBiochemistry
Volume35
Issue number5
DOIs
Publication statusPublished - 6 Feb 1996
Externally publishedYes

Fingerprint

Dive into the research topics of 'Reversible labeling of a chemosensitizer binding domain of p-glycoprotein with a novel 1,4-dihydropyridine drug transport inhibitor'. Together they form a unique fingerprint.

Cite this