Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P‐glycoprotein

Abstract

P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P-glycoproteincontaining plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125 I-labeled peptides were separated by tricine/ PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248±312 [transmembrane segment (TM)4 to TM5], 758±800 (beyond TM7 to beyond TM8) and 1160±1218 (after the Walker A motif of the second nucleotide-binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.Keywords: ABC transporter; multidrug resistance; P-glycoprotein; photoaffinity labeling.Chemotherapy is one of the three major options for the treatment of cancer. However, after the initial treatment with cytostatic drugs, tumor cells can become resistant to a broad range of structurally different drugs. Such resistance during chemotherapy is known as multidrug resistance (MDR). MDR is mainly based on overexpression of P-glycoprotein, an ATP-binding cassette (ABC) transporter. P-glycoprotein actively transports many amphiphilic cytostatic drugs out of cancer cells (causing resistance against these drugs) [1±4].The large 1276 amino-acid plasma membrane protein P-glycoprotein is made up of 12 transmembrane segments (TMs) and two nucleotide-binding domains (NBDs) [3]. The major issue of how P-glycoprotein can handle so many substances has been mainly approached by photoaffinity labeling and mutagenesis studies [5,6]. Active mutagenesis identified numerous motifs and positions in the transmembrane domains and both nucleotide-binding folds which are directly or indirectly involved in drug binding [7]. These data demonstrate the complexity of the 3D structure of the substrate-binding pocket of P-glycoprotein. Moreover, binding and transport of the drug were found to be coupled to ATP hydrolysis [8] Epitope mapping revealed two major photobinding sites for the prazosin derivative [ 125 I]iodoarylazidoprazosin in each half of the P-glycoprotein (TM6 and TM12) and one minor photobinding site from TM4 up to, but not including, TM6 [15]. The minor photolabeling site overlaps that found for its photoreactive P-glycoprotein substrates iodipine and iodomycin [10,11], the second cytoplasmic loop between TM4 and TM5. Drug-transport studies ...

Evidence for the Locations of Distinct Steroid and Vinca Alkaloid Interaction Domains within the Murine mdr1b P-Glycoprotein

“…These results are consistent with the bipartite P-gp structure seen by Rosenberg et al (2001). Iodoprazosin may have separate binding sites within both halves of the protein (Dey et al, 1997;Isenberg et al, 2001) and the azidopine binding site may involve elements from both halves of the protein (Bruggemann et al, 1992;Morris et al, 1995). Several other approaches have yielded evidence of distinct drug binding sites but were not designed to indicate the location of the sites within the protein (Tamai and Safa, 1991;Spoelstra et al, 1994;Ayesh et al, 1996;Pascaud et al, 1998;Shapiro et al, 1999).…”

Sipholenol A, a marine‐derived sipholane triterpene, potently reverses P‐glycoprotein (ABCB1)‐mediated multidrug resistance in cancer cells

“…(32) In human P-gp, the IAAP binding was localized to residues 1134 -1169, which correspond to TM11 and the sixth extracellular loop. (33) In the authors' studies, sipholenol A inhibited the photoaffinity labeling of […”

Identification of Ligand-Binding Regions of P-Glycoprotein by Activated-Pharmacophore Photoaffinity Labeling and Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry

“…In the C-terminal half, ligand-binding regions have been reported to extend from the C terminus of TM11 to the N terminus of the Walker A motif of NBD2 (Greenberger, 1998). Isenberg et al (2001) …”