Photoaffinity Labeling of P-Glycoprotein

Peer, Michael; Csaszar, Edina; Vorlaufer, Elisabeth; Kopp, Stephan; Chiba, Peter

doi:10.2174/1389557053402738

Cited by 20 publications

(23 citation statements)

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“…Protons from each CH 2 chain segment have the same chemical shift but could contribute differently to each cross-peak volume due to different contacts with the substrate. A better spatial resolution is offered by other NMR approaches such as 2 H NMR on selectively chain deuterated lipids or 13 C MAS NMR, as demonstrated for the antidepressants desipramine and imipramine (also PGP substrates). They have been found to show contacts down to the sixth of 14 carbons on the lipid acyl chain (26,47).…”

Section: Discussionmentioning

confidence: 99%

“…So far, insight into drug location and interaction with membranes has relied on calorimetry (24), molecular modeling (25), 1 H, 14 N, and 31 P NMR spectroscopy (26,27), 1 H- 13 C dipolar coupling constants (26,28), 2 H order parameters (29,30), and fluorescence measurements (29,31). MAS-NOESY (magic angle spinning assisted nuclear Overhauser enhancement spectroscopy) has been shown by Gawrisch and co-workers to be a powerful tool in studying the distribution of the substrate within model lipid bilayers in multilamellar dispersions (32)(33)(34).…”

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confidence: 99%

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Localization of Multidrug Transporter Substrates within Model Membranes

2006

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Active extrusion of drugs from the cell interior by primary and secondary efflux pumps is an essential mechanism underlying the phenomenon of multidrug resistance. The first discovered and best characterized primary efflux pump found in humans is the ABC transporter P-glycoprotein (PGP), which shows very broad substrate specificity. Many of these molecules are lipophilic, and binding most likely takes place within the membrane. PGP could either translocate them from the inner to the outer leaflet (flippase) or extrude them from the membrane into the extracellular environment (hydrophobic vacuum cleaner). Recognition and binding of such a diverse set of substrates must be associated with a preferred membrane location, determined by molecular properties and lipid interactions. Therefore, a systematic study of the interaction among seven PGP substrates (phenazine, doxorubicin, cephalexin, ampicillin, chloramphenicol, penicillin G, and quercetin) and two modulators (quinidine and nicardipine) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membranes is reported here. The location profile of these molecules across the membrane was determined by (1)H NOESY MAS NMR based on (1)H-(1)H cross-peaks between their aromatic fingerprint region and lipid resonances. Although structurally rather diverse, all tested substances are found to have their highest concentration between the phosphate of the lipid headgroup and the upper segments of the lipid hydrocarbon chains. Our findings are consistent with PGP substrate and modulator binding from the membrane interface region.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Localization of Multidrug Transporter Substrates within Model Membranes

2006

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“…Furthermore, MsbA is also in the ABC family of multi-drug resistance protein and confers drug resistance in bacteria and MsbA and P-gp share many common substrates. Thus, MsbA and its homology model may be used to explain the transport activities of the ABC transporter family in general [56], especially because the two transmembrane domains of both contain six α-helical segments that consist of the substrate transporting channel [55,56,59].…”

Section: Anthracycline Drug Resistance Is Mediated By Multi-drug Resimentioning

confidence: 99%

Anthracyclines as effective anticancer drugs

Nadas¹,

Sun

2006

Expert Opinion on Drug Discovery

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Anthracyclines have received significant attention due to their effectiveness and extensive use as anticancer agents. At present, the clinical use of these drugs is offset by drug resistance in tumours and cardiotoxicity. Therefore, a relentless search for the 'better anthracycline' has been ongoing since the inception of these drugs > 30 years ago. This review focuses on the most recent pharmacology and medicinal chemistry developments on the design and use of anthracyclines. Based on their crystal structures as well as molecular modelling, a more detailed mechanism of topoisomerase poisoning by these new anthracyclines has emerged. Chemical modifications of anthracyclines have been found to possibly change the target selectivity among various topoisomerases and, thus, vary their anticancer activity. Additionally, recent sugar modifications of anthracyclines have also been found to overcome P-glycoprotein-mediated drug resistance in cancer therapy. The continued improvement of anthracycline clinical applications so far and the clinical trials of the 'third generation' of anthracyclines (such as sabarubicin) are also discussed. To finally find the 'better' anthracycline, further areas of research still need to be explored such as: the elucidation of the topoisomerase and P-glycoprotein crystal structures, molecular modelling based on crystal structure in order to design the next generation of better anthracycline drugs, the continued modifications of the anthracycline sugar moieties, and the further improvement of anthracycline drug delivery methods.

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“…Nevertheless, an atomic detail structure of P-gp or of any other ATPdependent multidrug transporter is not available to date. Photoaffinity labeling studies have identified regions that are accessible for substrates (Peer et al, 2004). Atomic details of the substrate binding domain and of the dynamics of the transport process, however, still remain elusive.…”

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confidence: 99%

P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

Pleban¹,

Kopp²,

Csaszar³

et al. 2004

Mol Pharmacol

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121

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P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization timeof-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino-and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.Multidrug resistance represents a serious obstacle to successful cancer chemotherapy. Although multifactorial in etiology, one type of multidrug resistance is associated with the overexpression of energy-dependent membrane-bound pumps, which intercept and efflux drugs before they reach their intracellular target structures. P-glycoprotein (ABCB1) represents a paradigm ATP-dependent efflux pump expressed in human cancer cells. In addition to its expression in cancer cells, P-gp is also physiologically expressed in a number of tissues such as intestinal epithelial cells, at the brush border of renal tubule epithelial cells, the canalicular side of hepatocytes, and in capillary endothelial cells forming the blood-brain barrier. It thus interferes with oral drug absorption and drug delivery to the brain, and it enhances renal and biliary excretion. P-gp has therefore attracted considerable attention as a nontarget in the field of drug development, because for a large number of active compounds, interaction with P-glycoprotein might compromise their future development into a drug. Considerable energy has therefore been devoted to the characterization of molecular features that make compounds P-gp substrates and to the definition of the molecular mechanism of drug transport by P-gp. A number of studies have dealt with the kinetics and thermodynamics of the transport process

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Photoaffinity Labeling of P-Glycoprotein

Cited by 20 publications

References 0 publications

Localization of Multidrug Transporter Substrates within Model Membranes

Localization of Multidrug Transporter Substrates within Model Membranes

Anthracyclines as effective anticancer drugs

P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

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