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

Biochemistry. 1996 Feb 6;35(5):1387-96. doi: 10.1021/bi951912u.

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 (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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • Benzophenones / metabolism*
  • Binding Sites
  • Biological Transport / drug effects
  • Calcium Channels / metabolism
  • Dihydropyridines / metabolism*
  • Drug Resistance, Multiple / physiology*
  • Humans
  • Models, Biological
  • Molecular Probes / metabolism
  • Photosensitizing Agents / metabolism
  • Vinblastine / metabolism

Substances

  • 2,6-dimethyl-4-(2-(trifluoromethyl)phenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid (2-(3-(4-benzoylphenyl)propionylamino)ethyl) ester ethyl ester
  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Benzophenones
  • Calcium Channels
  • Dihydropyridines
  • Molecular Probes
  • Photosensitizing Agents
  • Vinblastine
  • niguldipine