title: Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity.
abstract: The aim of the present study was to demonstrate that the modulation of P-glycoprotein (Pgp) ATPase activity by peptides, drugs, and chemosensitizers takes place on a common drug pharmacophore. To this end, a highly emetine-resistant Chinese hamster ovary cell line was established, in which Pgp constituted 18% of plasma membrane protein. Reconstituted proteoliposomes, the Pgp content of which was up to 40%, displayed a basal activity of 2.6 +/- 0.45 micromol of Pi/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 micromol of Pi/min/mg of protein) by valinomycin and various Pgp substrates, whereas, to our surprise, gramicidin D, an established Pgp substrate, was inhibitory. Taking advantage of this novel inhibition of Pgp ATPase activity by gramicidin D, a drug competition assay was devised in which gramicidin D-inhibited Pgp ATPase was coincubated with increasing concentrations of various substrates that stimulate its ATPase activity. Gramicidin D inhibition of Pgp ATPase was reversed by Pgp substrates, including various cytotoxic agents and chemosensitizers. The inhibition of the basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We conclude that the various MDR type substrates and chemosensitizers compete on a common drug binding site present in Pgp.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: M J
authors.initials: MJ
authors.lastname: Borgnia
authors.forename: G D
authors.initials: GD
authors.lastname: Eytan
authors.forename: Y G
authors.initials: YG
authors.lastname: Assaraf
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000633:toxicity*; D001667:Binding, Competitive; D016466:CHO Cells; D002999:Clone Cells; D003198:Computer Simulation; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple; D004640:Emetine / Q000633:toxicity; D006096:Gramicidin / Q000378:metabolism / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology*; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D007476:Ionophores; D009842:Oligopeptides; D006096:Gramicidin; D014634:Valinomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000251:Adenosine Triphosphatases; D004640:Emetine