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 (P-gp) is a membranous ATPase responsible for the multidrug resistance (MDR) phenotype. Using membrane vesicles prepared from the highly resistant cell line DC-3F/ADX we studied the influence of P-gp ATPase activity of four progesterone derivatives which specifically bind to P-gp and reverse MDR. Progesterone and desoxycorticosterone stimulate P-gp ATPase activity with, respectively, apparent concentrations giving half-maximal activation of 20-25 microM and 40-50 microM, and activation factors of 2.3 (at 100 microM progesterone) and 1.8 (at 170 microM desoxycorticosterone). Hydrocortisone above 100 microM stimulates P-gp ATPase activity while corticosterone has no apparent stimulating effect. Our data are consistent with the location of the binding sites for the progesterone derivatives on the P-gp membranous domain. The effects of these steroids on verapamil-stimulated P-gp ATPase activity support a non-competitive mechanism, i.e. the binding sites for verapamil and steroids are mutually non-exclusive for P-gp ATPase modulation. A similar non-competitive inhibition of progesterone-stimulated P-gp ATPase activity by desoxycorticosterone or by corticosterone leads to the conclusion that these steroids, although sharing related structures, have distinct modulating sites on P-gp. As expected from their mutually non-exclusive interactions on P-gp, progesterone and verapamil when mixed induce a synergistic modulation of P-gp ATPase activity. Since drug transport by P-gp is believed to be coupled to its ATPase activity, a corresponding synergistic effect of these two modulators for the inhibition of P-gp-mediated drug resistance can be expected.
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PMID:Effects of steroids and verapamil on P-glycoprotein ATPase activity: progesterone, desoxycorticosterone, corticosterone and verapamil are mutually non-exclusive modulators. 871 80

Among the mechanisms by which cancer cells evade chemotherapy, multidrug resistance (MDR) is certainly the best known. MDR is characterised by cross-resistance between numerous natural products used in cancer treatment, especially antibiotics and plant alkaloids. MDR results from a defect in cell accumulation of the drugs, which are actively effluxed from cells by a plasma membrane pump, which is a high molecular weight glycoprotein termed P-glycoprotein. This protein is encoded by a gene called mdr1, and can be inhibited by a variety of pharmacological compounds. The activation of the mdr1 gene can occur via numerous types of stimulation, especially anticancer drugs themselves, which can induce mdr1 gene transcription. P-glycoprotein is an ATPase transporter which is believed to extrude xenobiotics from the plasma membrane rather than from cytoplasm. Although potential sites of interaction of P-glycoprotein with its various ligands have been identified, especially at the level of putative transmembrane domains, the exact mechanism for drug pumping has never been elucidated. Reversal of MDR in vitro is easy to obtain and to characterise. An important development aims at identifying substances able to reverse MDR in the clinical setting, that are devoid of any pharmacological properties other than interaction with P-glycoprotein. Other targets can be postulated for these MDR modulators, whose combination could well lead to a synergistic reversal of drug resistance.
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PMID:[Multidrug resistance and its reversal. General review of fundamental aspects]. 873 88

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 microM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 microM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7ADR cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.
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PMID:Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells. 875 33

P-Glycoprotein is a member of the ABC superfamily of membrane transporters, and functions as an ATP-driven active efflux pump for natural products and chemotherapeutic drugs. Overexpression of P-glycoprotein is a major cause of multidrug resistance in human cancers. Sulfhydryl modification agents are known to inactivate both P-glycoprotein ATPase activity and transport function. In the present study, P-glycoprotein purified from CHRB30 cells was covalently labeled at two conserved Cys residues, one within each of the nucleotide binding domains, using 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS). MIANS modification inactivated P-glycoprotein ATPase function, in a concentration-dependent fashion. Increasing concentrations of ATP blocked MIANS labeling with an IC50 of 0.37 mM (similar to the KM for ATP hydrolysis), which suggests that the label is located close to the site of ATP binding within the nucleotide binding domain. A blue shift in the fluorescence spectrum of MIANS bound to P-glycoprotein indicated that the labeled Cys residues are situated in a nonpolar environment. MIANS-labeled P-glycoprotein was still able to bind ATP, as demonstrated by quenching of the fluorescence, with a Kd of 0.46 mM. Addition of a variety of drugs and chemosensitizers to MIANS-labeled P-glycoprotein led to substantial quenching of the probe fluorescence within the nucleotide binding domains. Dissociation constants for drug binding measured by fluorescence quenching were in the range of 0.77 microM for vinblastine to 158 microM for colchicine. Quenching by ATP and drugs was independent and additive, suggesting that each produces a defined change in the protein. The rate of MIANS labeling of Pgp was reduced in the presence of drugs and chemosensitizers, implying that a long-range conformational change arises from drug binding which alters the accessibility of the nucleotide binding domains to MIANS. These results suggest that there is conformational communication between the drug binding site(s) of P-glycoprotein and the ATPase catalytic sites within the nucleotide binding domains.
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PMID:Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains. 879 69

The structure of purified P-glycoprotein functionally reconstituted into liposomes was investigated by attenuated total reflection Fourier transform infrared spectroscopy. A quantitative evaluation of the secondary structure and a kinetic of 2H/H exchange of the P-glycoprotein were performed both in the presence and in the absence of MgATP, MgATP-verapamil, and MgADP. This approach was previously shown to be a useful tool to detect tertiary structure changes resulting from the interaction between a protein and its specific ligands, as established for the Neurospora crassa H+-ATPase. 2H/H exchange measurements provided evidence that a large fraction of the P-glycoprotein is poorly accessible to the aqueous medium. Addition of MgATP induced an increased accessibility to the solvent of a population of amino acids, while addition of MgATP-verapamil resulted in a subtraction of a part of the protein from access to the aqueous solvent. No significant changes were observed upon addition of MgADP or verapamil alone. The secondary structure of P-glycoprotein was not affected by addition of ligands. The variations observed in the 2H/H exchange rate when P-glycoprotein interacted with the above ligands therefore represented tertiary structure changes. Fluorescence quenching experiments confirmed that MgATP-induced changes are to be found in the tertiary structure of the enzyme.
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PMID:Secondary and tertiary structure changes of reconstituted P-glycoprotein. A Fourier transform attenuated total reflection infrared spectroscopy analysis. 879 27

The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer. Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation. The laboratory of Dr. Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype. Since that time, a similar correlation has been observed in many other MDR cell lines. Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-alpha isoenzyme. Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn. More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions. To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms. However, these areas are still under active investigation and not fully answered. Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function. This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.
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PMID:P-glycoprotein, multidrug resistance and protein kinase C. 882 Sep 51

Cytochalasins are a family of structurally related natural product cytotoxins that selectively depolymerize microfilaments. In this study, the interaction between several cytochalasins and the drug transporter P-glycoprotein was investigated. Dihydrocytochalasin B and cytochalasin E consistently sensitized P-glycoprotein-overexpressing human breast carcinoma cells (MCF-7/ADR) to daunomycin, vinblastine, and actinomycin D without affecting the cytotoxicity of cisplatin. These compounds did not affect the sensitivities of the parental MCF-7 cells to anticancer drugs, indicating that their effects are due to P-glycoprotein inhibition. Effects of dihydrocytochalasin B and cytochalasin E were observed at concentrations as low as 2.5 and 5 microM, respectively. In contrast, cytochalasins A, B, C, D, H, and J did not sensitize MCF-7/ADR cells to any of the drugs. The accumulation of [3H]-vinblastine by MCF-7/ADR cells and by drug-resistant human ovarian carcinoma cells (SKVLB1) was increased to the greatest extent by verapamil, followed by dihydrocytochalasin B > cytochalasin E > cytochalasin B, whereas cytochalasins A, C, D, H, and J did not alter intracellular accumulation of the drug. Similarly to verapamil, dihydrocytochalasin B significantly stimulated the ATPase activity of P-glycoprotein, while other cytochalasins were ineffective. These results demonstrate that very closely related compounds can differentially interact with P-glycoprotein. For example, the only difference between cytochalasin B and dihydrocytochalasin B is the saturation of a carbon-carbon double bond in dihydrocytochalasin B. These structural differences may provide important insight into chemical determinants for drug interaction with P-glycoprotein.
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PMID:Differential interactions of cytochalasins with P-glycoprotein. 883 87

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.
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PMID:Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system. 889 56

Each homologous half of P-glycoprotein consists of a transmembrane domain with six potential transmembrane segments and an ATP-binding domain. Labeling studies with photoactive drug analogs show that labeling occurs within or close to predicted transmembrane segments (TM) 6 (residues 331-351) and TM12 (residues 974-994). To test if these segments are in near-proximity we generated 42 different P-glycoprotein mutants in which we re-introduced a pair of cysteine residues into a Cys-less P-glycoprotein, one within TM6 (residues 332-338) and one within TM12 (residues 975-980) and assayed for cross-linking between the cysteines. All the mutants retained verapamil-stimulated ATPase activity. We found that only the mutant containing Cys-332 and Cys-975 was cross-linked in the presence of oxidant as judged by its decreased mobility on SDS gels. Similar results were obtained when the same mutations were introduced into Cys-less NH2-terminal and COOH-terminal half-molecules of P-glycoprotein followed by coexpression and treatment with oxidant. Cross-linking between Cys-332 and Cys-975, however, was inhibited by verapamil or vinblastine but not by colchicine. These results suggest that residues Cys-332 and Cys-975, which occupy equivalent positions when TM6 and TM12 are aligned, are close to each other in the tertiary structure of P-glycoprotein.
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PMID:Inhibition of oxidative cross-linking between engineered cysteine residues at positions 332 in predicted transmembrane segments (TM) 6 and 975 in predicted TM12 of human P-glycoprotein by drug substrates. 891 Mar 31

Several studies have demonstrated the presence of oligomers of P-glycoprotein in multidrug-resistant cells. The minimum functional unit of P-glycoprotein, however, is not known. In order to determine whether the functional unit is an oligomer, we tested for associations between P-glycoproteins containing either a histidine tag or the epitope tag for monoclonal antibody A52 at the COOH-terminal end of the molecule. Both tagged molecules were active and had indistinguishable drug resistance profiles. The tagged P-glycoproteins were expressed contemporaneously in HEK 293 cells, purified by nickel-chelate chromatography followed by immunoblot analysis. We found that P-glycoprotein-A52 did not copurify with functionally active P-glycoprotein-(His)10, even when the former was overexpressed relative to the histidine-tagged protein. Similar results were obtained with phosphorylation-deficient mutants of P-glycoprotein. By contrast, we could purify and reconstitute drug-stimulated ATPase activity when the half-molecules NH2-terminal half-(His)10/COOH-terminal half-A52 or NH2-terminal half-A52/COOH-terminal half-(His)10 were coexpressed in HEK 293 cells. These results suggest that nickel-chelate chromatography may be a suitable method for studying protein-protein interactions in membrane proteins and that the minimal functional unit of P-glycoprotein is likely to be a monomer.
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PMID:The minimum functional unit of human P-glycoprotein appears to be a monomer. 891 Mar 32


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