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)

P-glycoprotein (Pgp), the ATP-binding cassette multidrug transporter, exhibits a drug (substrate)-stimulatable ATPase activity, and vanadate (Vi) inhibits this activity by stably trapping the nucleoside diphosphate in the Pgp.ADP.Vi conformation. We recently demonstrated that Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp in the absence of substrate occurs both in the presence of 8-azido-[alpha-(32)P]ATP (following 8-azido-ATP hydrolysis) or 8-azido-[alpha-(32)P]ADP (without hydrolysis) and, the transition state intermediates generated under either condition are functionally indistinguishable. In this study, we compare the effect of substrates on Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp under both non-hydrolysis and hydrolysis conditions. We demonstrate that whereas substrates stimulate the Vi-induced trapping of 8-azido-[alpha-(32)P]ADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of nucleoside diphosphate in the absence of Vi, indicating that the substrate-induced conformation exerts its effect at a step distinct from nucleoside diphosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition state, Pgp x ADP x P(i) (Vi), can be generated both via the hydrolysis of ATP or by directly providing ADP to the system, in the presence of substrate the reaction is driven in the forward direction, i.e. hydrolysis of ATP. These data suggest that substrate-stimulated ATP hydrolysis by Pgp is a vectorial process.
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PMID:Evidence for the vectorial nature of drug (substrate)-stimulated ATP hydrolysis by human P-glycoprotein. 1145 43

The absorption of many drugs is affected by their interaction with ATP-binding cassette (ABC) transporters. The most extensively studied of these ABC transporters is the proein product of MDR1 (multidrug resistance) that encodes a 170-kDa integral plasma membrane phosphorylated glycoprotein known as P-glycoprotein (P-gp). The purpose of this study was to determine, using two different methods, whether the nonsedating antihistamine loratadine (L) and its active metabolite desloratadine (DL) interact with P-gp. MDR cells presenting human P-gp were incubated with the fluorescent P-gp substrate daunorubicin with or without L, DL, and several positive controls. The IC(50) of loratadine (approximately 11 microM) was approximately 160 times the maximum observed plasma concentration (C(max)) following a dose of 10 mg. The IC(50) of desloratadine (approximately 43 microM) was approximately 880 times the C(max) following a dose of 5 mg. The positive control, cyclosporin A, had an IC(50) of approximately 1 microM. ATP hydrolysis activity was measured in the membrane fraction prepared from MDR cells presenting P-gp, which were exposed to various concentrations of test compounds. Known substrates of P-gp demonstrated clear, repeatable, concentration-dependent increases in ATP hydrolysis activity. L caused an increase in ATPase activity above basal levels. L had a V(max) about 200% basal activity and K(m) of approximately 3 microM for P-gp. In contrast, DL had no significant effect on baseline ATP hydrolysis. L inhibited human P-gp much less than verapamil or cyclosporin A. DL inhibited human P-gp significantly less than L (4 times). DL therefore is not a significant inhibitor of P-gp and should not cause clinical drug interactions with agents that are P-gp substrates.
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PMID:Evaluation of the interaction of loratadine and desloratadine with P-glycoprotein. 1145 24

P-glycoprotein is a member of the ATP-binding cassette (ABC) transport superfamily. It plays an important role in the development of multidrug resistance in cancers by effluxing a wide variety of anticancer drugs. A large amount of information on the structure and function of P-glycoprotein has been accumulated over recent years from studies using molecular, biochemical, and biophysical approaches. It remains unclear, however, how this protein folds in membranes and how it transports such a wide variety of hydrophobic compounds. This paper highlights the recent progress in the structural and biogenesis aspects of P-glycoprotein. A model mechanism of P-glycoprotein action is proposed as a hypothesis that is based on recent progress in studying the topological folding of P-glycoprotein.
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PMID:The multi-structural feature of the multidrug resistance gene product P-glycoprotein: implications for its mechanism of action (hypothesis). 1146 6

Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy. One important mechanism of MDR involves the multidrug transporter, P-glycoprotein (Pgp), which confers upon cancer cells the ability to resist lethal doses of certain cytotoxic drugs by pumping the drugs out of the cells and thus reducing their cytotoxicity. Pgp belongs to the ATP-binding cassette (ABC) family of transporter molecules which require hydrolysis of ATP to run the transport mechanism. The substrates of Pgp may be endogenous (steroid hormones, cytokines) or exogenous (cytostatic drugs). A number of studies have demonstrated a negative correlation between Pgp expression levels and chemosensitivity or survival in a range of human malignancies. In principle, Pgp mediated drug resistance can be circumvented by treatment regimens that either exclude Pgp substrate drugs or include Pgp inhibitory agents. Experimental studies have demonstrated that certain structural modifications of anthracyclines confer the ability to escape Pgp transport. The therapeutic benefit of Pgp inhibitors as chemosensitizers is currently being explored in phase III clinical trials, and the first promising results have already been reported. Another therapeutic option for Pgp inhibitors has recently evolved as several Pgp inhibitors, many of which are generally low-toxic substances, by themselves constrain proliferation and cause cell death by apoptosis in certain MDR cancer cell lines. The dual effect of Pgp inhibitors, targeting MDR cancer cells selectively, may translate into improved efficacy of cancer chemotherapy and perhaps new and less toxic drug treatment strategies in human MDR cancer.
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PMID:P-glycoprotein as a drug target in the treatment of multidrug resistant cancer. 1147 37

The human multidrug resistance P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of transporters, is frequently responsible for the failure of chemotherapy by virtue of its ability to export hydrophobic cytotoxic drugs from cells. Elucidating the inter- and intramolecular interactions of this protein is critical to understanding its cellular function and mechanism of action. Toward this end, we have used both biochemical and genetic techniques to probe potential oligomerization interactions of P-gp. Differentially epitope-tagged P-gp molecules did not co-immunoprecipitate when co-expressed in HEK293 cells or when co-translated in vitro, demonstrating that P-gp is monomeric in both the presence and absence of detergents. The two cytoplasmic domains of P-gp did not interact with each other in vivo when co-expressed as gene fusions in yeast. In contrast, the homologous domains of the transporter associated with antigen processing (TAP), which reside on separate polypeptides and must form a heterodimeric transporter (TAP1/TAP2), did interact in this system, suggesting a role for these domains in TAP dimerization. Implications for understanding the subunit organization of ABC transporters are discussed.
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PMID:The multidrug resistance P-glycoprotein. Oligomeric state and intramolecular interactions. 1149 94

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C(5)-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter.
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PMID:Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator. 1150 16

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.
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PMID:Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding. 1151 17

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.
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PMID:Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle. 1159 5

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) drug efflux pump, has been recently shown to play an important role in the physiology of Langherans cells, a subtype of dendritic cells (DC) found in the skin. The present study was designed to investigate expression and activity of P-gp and of multidrug resistance-associated protein (MRP), another ABC efflux pump sharing numerous substrates with P-gp, in human monocyte-derived DC. Immunolabeling experiments and dye efflux assays indicated that such cells displayed elevated levels of MRP activity and expression when compared to those present in parental monocytes. Generation of DC from monocytes in the presence of the MRP inhibitor indomethacin did not, however, alter the capacity of DC to stimulate allogeneic T cells proliferation in mixed lymphocyte reaction. In addition, indomethacin did not inhibit the up-regulation of the CD1a, a marker occurring during the differentiation of monocytes into DC. In contrast to that of MRP, functional expression of P-gp was not detected in monocyte-derived DC. Such antigen presenting cells that constitute a promising tool for antitumor vaccinal therapy therefore display differential expression of the efflux pumps P-gp and MRP.
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PMID:Differential expression of the efflux pumps P-glycoprotein and multidrug resistance-associated protein in human monocyte-derived dendritic cells. 1160 Feb 13

P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is an important mediator of multidrug resistance in cancer. Pgp exhibits a very broad specificity for substrates. These substrates share a common feature of being amphipathic and can orient into either leaflet of the membrane bilayer. Current evidence suggests that Pgp recognizes and extracts substrates from the membrane bilayer, but from which leaflet is unresolved. To directly test whether Pgp can decrease substrate concentration in the extracellular leaflet of the plasma membrane in living cells, we used the fluorescent lipid analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH). TMA-DPH in the extracellular solution rapidly partitions into the extracellular leaflet of the plasma membrane and exhibits slow transbilayer flipping into the cytoplasmic leaflet. Because TMA-DPH fluorescence is confined to the extracellular leaflet in early time points after addition but labels intracellular membranes after longer incubation, we can assess the effect of Pgp on TMA-DPH concentration from both extracellular leaflet and intracellular membranes. Transient transfection with a Pgp and the green fluorescence protein (GFP) fusion protein generated cells with heterogeneous expression levels of Pgp-GFP. Compared with nonexpressing cells, cells expressing Pgp-GFP showed decreased accumulation of TMA-DPH in intracellular membranes but similar levels of accumulation in the extracellular leaflet of the plasma membrane. Additionally, in drug-selected MCF7/Adr cells, which constitutively express high levels of Pgp, inhibition of Pgp by cyclosporin A resulted in significantly increased accumulation of TMA-DPH in intracellular membranes but no difference in its accumulation in the extracellular leaflet of the plasma membrane. These data indicate that whereas Pgp can extract TMA-DPH from the cytoplasmic leaflet of the membrane, any activity Pgp may possess in the extracellular leaflet is insufficient to decrease TMA-DPH concentration there and, therefore, does not contribute to lowering the cellular levels. Pgp is the prototype of an increasing number of clinically important ATP-binding cassette transporters of amphipathic drugs and lipids. These results may help decipher a common mechanism of these transporters.
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PMID:P-glycoprotein does not reduce substrate concentration from the extracellular leaflet of the plasma membrane in living cells. 1169 90


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