Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The organosulfur compounds (OSCs), present in garlic, are studied for their protective effect against human cancers. P-glycoprotein (P-gp) and multidrug resistance protein 2 (Mrp2) are two transporters involved in the defense of cells and in the development of multidrug resistance. Whereas OSCs increase glutathione S-transferase activity (GST), Mrp2 plays a role in the transport of glutathione (GSH)-conjugates. In this study, we have investigated the effect of two OSCs, diallyl disulfide (DADS) and S-allyl cysteine (SAC), on P-gp and Mrp2 expression in renal brush-border membranes. By Western blot analysis, our results show that DADS induces Mrp2 expression (by 7-fold), which correlates with the rise of GST activity and GSH levels. Surprisingly, a co-administration of OSC with cisplatin, an anticancer drug, significantly increased Mrp2 gene and protein expression (by 30-fold), suggesting that DADS could potentiate the effects of cisplatin. Interestingly, SAC and cisplatin in co-treatment decreased P-gp protein expression and mdr1b isoform mRNA levels. In addition, modulation of the mdr1b isoform and Mrp2 by cisplatin was completely abolished by a glutathione precursor, N-acetyl cysteine. These results indicate that OSCs present in a garlic-rich diet might alter chemotherapeutic treatments using P-gp or Mrp2 substrates.
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PMID:Diallyl disulfide, a chemopreventive agent in garlic, induces multidrug resistance-associated protein 2 expression. 1547 18

Overexpression of P-glycoprotein (P-gp; ABCB1) can cause multidrug resistance during cancer and AIDS chemotherapy because of its ability to transport a broad range of structurally unrelated compounds from the cell. P-gp is a member of the ABC family of proteins. It is a single polypeptide containing four domains--two transmembrane (TM) domains each of which contains six TM segments, and two nucleotide-binding domains. Chemical modification and cross-linking studies of cysteine mutants of P-gp indicate that the common drug-binding pocket is at the interface between the TM domains. It has been postulated that drug substrates enter the lipid bilayer, are extracted by P-gp and transported to the extracellular medium. It is not clear how drug substrates enter the drug-binding pocket. Here, we propose that drug-substrates diffuse from the lipid bilayer into the drug-binding pocket through "gates" formed by TM segments at either end of the drug-binding pocket.
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PMID:Do drug substrates enter the common drug-binding pocket of P-glycoprotein through "gates"? 1573 3

The human multidrug resistance P-glycoprotein (P-gp) uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. The relatively high expression of P-gp in organs such as the intestine, kidney, blood-brain/testes barrier and in some tumor cells can compromise chemotherapy treatments for patients with cancer or AIDS/HIV. It has been difficult to inhibit P-gp during chemotherapy with noncovalent inhibitors because the relatively high levels of inhibitors have severe side effects. An alternative approach to inhibit P-gp would be to covalently modify cysteine residues within the NBDs. In this study, we tested whether metabolites of disulfiram, a drug currently used to treat chronic alcoholism, could inhibit P-gp. We show that the disulfiram metabolites, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the verapamil-stimulated ATPase activity of P-gp with IC50 values (concentrations that result in 50% inhibition of activity) of 9 and 4.8 microM, respectively. Similarly, S-methyl N,N-diethylthiocarbamate sulfoxide and S-methyl N,N-diethylthiocarbamate sulfone inhibited the activity of aldehyde dehydrogenase with IC50 values of 3.2 and 1.7 microM, respectively. Inhibition of P-gp by the metabolites was not reversed by addition of the reducing compound, dithiothreitol. We then determined which endogenous cysteine residue was responsible for inhibiting P-gp activity after exposure to the disulfiram metabolites. Treatment of P-gp mutants containing a single cysteine residue showed that inactivation was primarily due to modification of Cys1074 in NBD2. These results indicate that metabolites of disulfiram can covalently inactivate P-gp. Covalent modification of drug transporters could be a useful approach for inhibiting their activities during chemotherapy.
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PMID:Disulfiram metabolites permanently inactivate the human multidrug resistance P-glycoprotein. 1602 54

P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.
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PMID:ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein. 1604 2

Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.
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PMID:Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy. 1622 90

P-glycoprotein is a membrane protein involved in the phenomenon of multidrug resistance. Its activity and transport function have been largely characterized by various biochemical studies and a low-resolution image has been obtained by electron microscopy. Obtaining a high-resolution structure is, however, still remote due to the inherent difficulties in the experimental determination of membrane protein structures. We present here a three-dimensional (3D) atomic model of P-glycoprotein in absence of ATP. This model was obtained using a combination of computational techniques including comparative modeling and rigid body dynamics simulations that embody all available cysteine disulfide crosslinking data characterizing the whole protein in absence of ATP. The model features rather well most of the experimental interresidue distances derived both in the transmembrane domains and in the nucleotide binding domains. The model is also in good agreement with electron microscopy data, particularly in terms of size and topology. It features a large cavity detected in the protein core into which seven ligands were successfully docked. Their predicted affinity correlates well with experimental values. Locations of docked ligands compare favorably with those suggested by cysteine-scanning data. The finding of different positions both for a single ligand and for different ligands corroborates the experimental evidence indicating the existence of multiple drug binding sites. The interactions identified between P-glycoprotein and the docked ligands reveal that different types of interactions such as H-bonds, pi-pi and cation-pi interactions occur in agreement with a recently proposed pharmacophore model of P-glycoprotein ligands. Furthermore, the model also displays a lateral opening located in the transmembrane domains connecting the lipid bilayer to the central cavity. This feature supports rather well the commonly admitted mechanism of substrate uptake from the lipid bilayer. We propose that this 3D model may be an important tool to understand the structure-function relationship of P-glycoprotein.
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PMID:Predicting the three-dimensional structure of human P-glycoprotein in absence of ATP by computational techniques embodying crosslinking data: insight into the mechanism of ligand migration and binding sites. 1646 78

P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.
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PMID:Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket. 1649 38

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.
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PMID:Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket. 1681 63

Our previous study identified a vincristine-selected multidrug resistance (MDR) cell line, HOB1/VCR, derived from a lymphoblastoma HOB1. The HOB1/VCR cells are resistant to typical MDR drugs and are cross-resistant to P-glycoprotein-independent drugs such as cisplatin (cis-diamminedichloroplatinum [II]). The mechanism of this atypical MDR phenotype is uncertain. The present study provides evidence regarding the contribution of reactive oxygen species (ROS) to the resistance of cells in response to treatments (vincristine, cisplatin and H2O2). Notably, the HOB1/VCR cells were cross-resistant to H2O2. High levels of ROS formed in both sensitive and HOB1/VCR cells by H2O2, and moderate levels of ROS were generated by treatment with cisplatin and vincristine. The ROS level in HOB1/VCR cells was lower than that in sensitive cells following treatments. The ROS level was reduced markedly by a non-toxic concentration of N-acetyl-L-cysteine, a ROS scavenger, in drug-treated cells, and was correlated with reduced cytotoxicity. Furthermore, concentrations of glutathione and glutathione peroxidase, but not superoxide dismutase and catalase, increased in HOB/VCR cells. The DL-buthionine-[S,R]-sulfoximine inhibited formation of glutathione and sensitized both cell types to treatments. Therefore, overexpression of an H2O2-reducing system, glutathione-glutathione peroxidase, has a role in resistance. Experimental results further demonstrate that ROS is likely a primary signal in the acquisition of the MDR phenotype and therefore a potential target when designing drugs for chemoresistance.
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PMID:Involvement of reactive oxygen species in multidrug resistance of a vincristine-selected lymphoblastoma. 1751 54

The recently reported structures of the bacterial multidrug exporter Sav1866 suggest a domain architecture in which both nucleotide-binding domains (NBDs) of this ATP binding cassette (ABC) transporter contact both transmembrane domains (TMDs). Such a domain arrangement is particularly unexpected because it is not found in the structures of three solute importers BtuCD, HI1470/1, and ModBC from the same protein family. There is also no precedent for such an arrangement from biochemical studies with any ABC transporter. Sav1866 is homologous with the clinically relevant human P-glycoprotein (ABCB1). If the structure proposed for Sav1866 is physiologically relevant, the long intracellular loops of P-glycoprotein TMD2 should contact NBD1. We have tested this by using cysteine mutagenesis and chemical cross-linking to verify proximal relationships of the introduced sulfhydryls across the proposed interdomain interface. We report the first biochemical evidence in support of the domain arrangement proposed for the multidrug resistance class of ABC transporters. With a domain arrangement distinctly different from the three solute importers it seems likely that the TMDs of ABC importers and exporters have evolved different mechanisms to couple to common conformational changes at conserved NBDs.
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PMID:Evidence for a Sav1866-like architecture for the human multidrug transporter P-glycoprotein. 1762 29


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