title: Binding site(s) on P-glycoprotein for a newly synthesized photoaffinity analog of agosterol A.
abstract: Agosterol A (AG-A) is a novel agent that reverses P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1)-meditated multidrug resistance (MDR). We have synthesized [125I]11-azidophenyl agosterol A (azidoAG-A), a photoaffinity analog of AG-A, and characterized its binding to P-gp in membrane vesicles prepared from multidrug-resistant P-gp-overexpressing KB-C2 cells. The photoanalog photolabeled intact P-gp and both the N- and C-terminal fragments of P-gp. [125I]AzidoAG-A is transported by P-gp and the intracellular accumulation of both [125I]azidoAG-A and [3H]AG-A in KB-C2 cells was lower than that in the parental drug-sensitive KB-3-1 cells. [125I]AzidoAG-A bound to the drug binding site(s) on P-gp because photoaffinity labeling of P-gp was inhibited by a variety of known P-gp substrates, including anticancer, reversing, and anti-human immunodeficiency virus (HIV) agents. The binding of [125I]azidoAG-A to P-gp differs from the binding of other photolabeled probes such as iodoaryl-azidoprazosin (IAAP) to P-gp and from the binding of [125I]azidoAG-A to MRP1 based on the differing effects of flupentixol and glutathione (GSH) on their binding. Thus, [125I]azidoAG-A will be a useful tool to elucidate the structure and function of P-gp because it directly binds to the drug binding site(s) on P-gp, is transported by P-gp, and exhibits different P-gp binding characteristics than IAAP.
authors.affiliation: Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Mitsuo
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Noguchi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Nakajima
authors.forename: Shunji
authors.initials: S
authors.lastname: Aoki
authors.forename: Xiao-Qin
authors.initials: XQ
authors.lastname: Ren
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Sumizawa
authors.forename: Misako
authors.initials: M
authors.lastname: Haraguchi
authors.forename: Motomasa
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Masanori
authors.initials: M
authors.lastname: Baba
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Nagata
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Akiyama
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Furukawa
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D018432:Drug Resistance, Multiple; D006801:Humans; D019920:Photoaffinity Labels; D011233:Precipitin Tests; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D019920:Photoaffinity Labels; D013261:Sterols; C429345:agosterol A