Identification and characterization of the binding sites of P-glycoprotein for multidrug resistance-related drugs and modulators - PubMed (original) (raw)

Review

Ahmad R Safa. Curr Med Chem Anticancer Agents. 2004 Jan.

Abstract

A major problem in cancer treatment is the development of resistance to multiple chemotherapeutic agents in tumor cells. A major mechanism of this multidrug resistance (MDR) is overexpression of the MDR1 product P-glycoprotein, known to bind to and transport a wide variety of agents. This review concentrates on the progress made toward understanding the role of this protein in MDR, identifying and characterizing the drug binding sites of P-glycoprotein, and modulating MDR by P-glycoprotein-specific inhibitors. Since our initial discovery that P-glycoprotein binds to vinblastine photoaffinity analogs, many P-glycoprotein-specific photoaffinity analogs have been developed and used to identify the particular domains of P-glycoprotein capable of interacting with these analogs and other P-glycoprotein substrates. Furthermore, significant advances have been made in delineating the drug binding sites of this protein by studying mutant P-glycoprotein. Photoaffinity labeling experiments and the use of site-directed antibodies to several domains of this protein have allowed the localization of the general binding domains of some of the cytotoxic agents and MDR modulators on P-glycoprotein. Moreover, site-directed mutagenesis studies have identified the amino acids critical for the binding of some of these agents to P-glycoprotein. Furthermore, equilibrium binding assays using plasma membranes from MDR cells and radioactive drugs have aided our understanding of the modes of drug interactions with P-glycoprotein. Based on the available data, a topological model of P-glycoprotein and the approximate locations of its drug binding sites, as well as a proposed classification of multiple drug binding sites of this protein, is presented in this review.

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Figures

Fig. (1)

Fig. (1)

Structures of photoaffinity analogs of MDR-related drugs known to covalently label P-glycoprotein: (I) [125I]NASV, (2) [125I]NASC, (3) [3H]NAB-DNR, (4) [125I]NAS-DNR, (5) [125I]ASA-Rh123, (6) [125I]ASA-BZ, and (7) [3H]BzDC-paclitaxel.

Fig. (2)

Fig. (2)

(A) Autoradiogram of [125I]NASV photolabeled plasma membranes (20 μg protein) from CEM, CEM/VBL100, CEM/VBL1000 and CEM/VBL5000 cells in the absence (lanes 1, 2, 4 and 6) or presence of 10 μM nonradioactive vinblastine (lanes 3, 5 and 7). (B) Autoradiogram of [125I]NASV photoaffinity labeled KB-3-1 (lane 1) and KB-GRC1 transfectants (KB-3-1 cells transfected with the _MDR_1 gene) (lanes 2–7), KB-GRC1 transfectants (8 × 106 cells) were photoaffinity labeled with 50 nM [125I]NASV (50–60 Ci/mMole) in the absence (lane 2) or presence of 100 μM vinblastine, actinomycin D, doxorubicin, colchicine, or methotrexate, respectively (lanes 3–7), and after subjecting to SDS-PAGE, the samples were processed for autoradiography.

Fig. (3)

Fig. (3)

Structures of photoaffinity analogs of MDR modulators known to covalently label P-glycoprotein: (1) [125I]NAS-VP, (2) LU-49888, (3)[3H]azidopme, (4) [3H]B92009-005, (5) [125I]AAP and (6) [125I]NAPS.

Fig. (4)

Fig. (4)

Structures of photoaffinity analogs of MDR modulators known to covalently label P-glycoprotein: (1) cyclosporin aziridine, (2) synthetic isoprenoid, (3) _N_-solanesyl-N', N'-bis(3, 4-dimethoxybenzoyl)ethylenediamine, (4) forskolin analogs, (5) estramustine photoaffinity analog, and (6) VF-13.

Fig. (5)

Fig. (5)

Topological model of P-glycoprotein and the approximate locations of its drug binding sites. The figure shows the structure of P-glycoprotein with twelve transmembrane domains (TMDs) predicted by hydrophobicity plot. Domains of the protein that are involved in the binding of iodomycin (IDM), [125I]NASV, [125I]NAS-VP, [125I]NAST, [3H]azidopine (AZP), 6-o-[[2-[3-(4-azido-3-[125I]iodophenyl) propionamido]ethyl]-carbamyl)forskolin (AIPPF), [125I]iodoarylazidoprazosin (IAAP), [3H]-3'-BzDC-paclitaxel, [3H]-7-BzDC-paclitaxel (dashed lines, amino acid residues 985–1008 are marked). For details, see the text.

Fig. (6)

Fig. (6)

Photoaffinity labeling of P-glycoprotein from SH-SY5Y/VCR cells with [125I]AAP (lane 1) and the effects of 100 μM trifluoperazine, chlorpromazine, thioridazine, perphenazine, W-7, cis-flupentixol, trans-flupentixol, fluphenazine and fluphenazine N-mustard (lanes 2–10), respectively.

Fig. (7)

Fig. (7)

Proposed classification of multiple drug binding sites of P-glycoprotein. Based on the kinetic analysis of drug binding to P-glycoprotein under equilibrium conditions and photoaffinity labeling using various photoaffinity analogs of the cytotoxic agents and MDR modulators as well as competition experiments described in the text, seven binding sites for P-glycoprotein are proposed. Arrows indicate positive or negative communication between these sites.

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