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 human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket.
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PMID:Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein. 1290 21

Delivery of drugs to the brain is impeded by the activity of efflux pumps expressed by endothelial cells of brain vasculature. The ATP binding cassette (ABC) transporters, among which ABCB1/MDR1 P-glycoprotein and ABCC1/multidrug resistance-associated protein 1 are expressed in brain endothelial cells, participate in drug efflux properties of the blood-brain barrier (BBB). Searches of the EST (expressed sequence tags) database with the conserved ABC domain, conducted to identify other ABC transporters expressed in the BBB, recovered 15 ABC transporter sequences expressed in human brain cDNA libraries. One of these sequences, identical to ABCG2, was highly expressed in cultured human cerebromicrovascular endothelial cells and human brain tissue at both mRNA and protein levels. Overexpression of human ABCG2 in immortalized rat brain endothelial cells resulted in enhanced polarized abluminal to luminal transport of various substrates tested in the in vitro BBB model. Brain vessels extracted from tissue sections of nonmalignant human brain and glioblastoma tumors by laser capture microdissection microscopy and analyzed by real-time polymerase chain reaction showed higher expression of ABCG2 relative to ABCB1/MDR1 and ABCC1/MRP1. ABCG2 was up-regulated in both glioblastoma vessels and parenchymal tissue. These studies suggest a role for brain endothelial ABCG2 transporter in modulating drug delivery to the brain and in conferring drug resistance to glioblastomas.
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PMID:The expression and functional characterization of ABCG2 in brain endothelial cells and vessels. 1295 61

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.
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PMID:Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites. 1452 74

ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.
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PMID:Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein. 1455 Dec 17

A noncoding C3435T mutation in exon 26 of the ABCB1 gene was found to be often associated with a G2677T(A) mutation in exon 21 encoding an Ala893Ser P-glycoprotein and with a noncoding C1236T mutation in exon 12. We developed a Pyrosequencing screening method that simultaneously detects all three mutations. After separate PCRs for each exon, the sequences around the potentially mutated nucleotide positions were simultaneously analyzed in a multiplex assay. The method was tested with DNA from 100 volunteers. Allele frequencies of the T1236, T2677, and T3435 alleles were 44, 42, and 50%, respectively. A mutation at position 3435 occurred together with a mutation at position 2677 or 1236 in 64 and 65% of the subjects, respectively. The most frequent haplotype, with 44.4%, was not mutated at all three positions, i.e., C1236, G2677, C3435. The second most frequent haplotype, with 37.1%, was mutated at all three positions, i.e., T1236, T2677, T3435. The most frequent genotype, with 36%, was heterozygously mutated at all three positions, i.e., C/T1236, G/T2677, C/T3435. The next most frequent genotypes were a homozygous nonmutated genotype, with 20%, and a homozygous mutated genotype, with 13%.
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PMID:Simultaneous screening for three mutations in the ABCB1 gene. 1455 7

Transplantation has transformed the treatment of patients with organ failure in a number of clinical settings, and immunosuppressive drug therapy is fundamental to its success. However, all the drugs in current use have a narrow therapeutic index. Under-dosing can lead to rejection, while over-dosing increases the risks of infection, malignant disease, and serious drug-specific adverse effects, including diabetes mellitus, nephrotoxicity, hypertension, and hyperlipidemia. Heterogeneity in the pharmacokinetics of these drugs makes initial dose determination difficult, as there is a poor correlation between dose and blood concentration. This results in difficulties in achieving target blood concentrations early after transplantation, which are important for reducing the rate of immunological rejection. This problem is compounded by the observation that neither drug dose nor drug blood concentration accurately predict clinical efficacy or toxicity. The main determinant of heterogeneity in dose requirements is intestinal absorption of the active drug. The oxidative enzymes, cytochrome P450 (CYP) 3A4 and CYP3A5, and the drug efflux pump P-glycoprotein (P-gp) in enterocytes regulate this process. Most substrates for the P-gp pump are also substrates for the CYP3A enzymes. An efficient barrier to xenobiotic absorption is formed by the CYP enzymes and P-gp, and by the two systems working synergistically. Genetic polymorphisms have been reported for the genes associated with the expression of the CYP3A enzymes and P-gp. Genotyping patients for CYP3A genes has the potential to aid the establishment of optimal dosage regimens for transplant patients. Genetic polymorphism of the multiple drug resistance gene-1 (MDR1, also known as ABCB1) [3435C/T] and the CYP3A5 genes (CYP3A5*1, CYP3AP1*1) have the greatest potential to influence the pharmacokinetics of immunosuppressants. Homozygosity of the T allele of the MDR1 3435C/T polymorphism has been associated with reduced enterocyte expression of P-gp resulting in increased drug absorption. The presence of the CYP3A5*1 allele is necessary for the production of a fully catalytic CYP3A5 protein, and also influences the ratio of CYP3A4 : CYP3A5 as well as the overall CYP3A catalytic activity. The CYP3A4 : CYP3A5 ratio may, in turn, influence the pattern of drug metabolites formed. Heterogeneity in the production of active and inactive metabolites has implications for both the pharmacokinetics and pharmacodynamics of these drugs.Gene frequencies and drug dose requirements differ between ethnic groups. Ethnic differences in dose requirements for immunosuppressants have been discussed widely. However, ethnicity is a rather crude marker for genotype. Pharmacogenetic typing offers the possibility of significant improvement in the individualization of immunosuppressive drug prescribing with reduced rates of rejection and toxicity.
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PMID:The pharmacogenetics of immunosuppression for organ transplantation: a route to individualization of drug administration. 1457 18

Tumor cells may become resistant to conventional anticancer drugs through the occurrence of transmembrane transporter proteins such as P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), or members of the multidrug resistance-associated protein family (MRP1-MRP5; ABCC1-ABCC5). In this report, we studied whether tumor cells that are cytostatic drug resistant because of overexpression of one of the above mentioned proteins are sensitive to a new anticancer agent, interleukin-4 toxin (IL-4 toxin). IL-4 toxin is a fusion protein composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE) [IL-4(38-37)-PE38KDEL]. Ninety-six-h cytotoxicity assays and 10-day clonogenic assays showed that drug-selected multidrug resistant (MDR) tumor cells that overexpress P-glycoprotein or breast cancer resistance proteins are still sensitive to IL-4 toxin. Also, tumor cells transfected with cDNA for MRP2-5 showed no resistance, or marginal resistance, only to the toxin as compared with the parent cells. In contrast, MRP1-overexpressing cells, both drug selected and MRP1 transfected, are clearly resistant to IL-4 toxin with resistance factors of 4.3 to 8.4. MRP1-overexpressing cells were not resistant to PE itself. IL-4 toxin resistance in MRP1-overexpressing cells could be reversed by the MRP1 inhibitors probenecid or MK571 and were not affected by glutathione depletion by DL-buthionine-S,R-sulfoximine. In a transport assay using plasma membrane vesicles prepared from MRP1-overexpressing cells, IL-4 toxin and IL-4, but not PE, inhibited the translocation of the known MRP1 substrate 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). These data suggest that MRP1-overexpressing cells are resistant to IL-4 toxin because of extrusion of this agent by MRP1. Still, the results of this study demonstrate that IL-4 toxin effectively kills most MDR tumor cells and, therefore, represents a promising anticancer drug.
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PMID:Multidrug-resistant tumor cells remain sensitive to a recombinant interleukin-4-Pseudomonas exotoxin, except when overexpressing the multidrug resistance protein MRP1. 1458 76

Advances in transplantation technology have brought about great benefits to patients suffering from organ failure, but the problem still remains of complications induced by steroids used for post-transplant immunosuppression. Among the side-effects caused by steroids, non-traumatic osteonecrosis of the femoral head (ONF) constitutes a serious problem. The same protocol for steroid administration induces ONF in some patients, but not in others, indicating the presence of individual difference in steroid sensitivity. We hypothesized that this difference might be mediated by the drug-transport protein, P-glycoprotein (P-gp), and investigated the relationship between single nucleotide polymorphisms in the multidrug resistance gene 1 (ABCB1, MDR1) encoding P-gp and ONF. Subjects comprised 136 patients receiving kidney transplantation. Thirty patients developed post-transplant ONF. Genomic DNA was extracted from peripheral blood, and genotypes of ABCB1 C3435T (exon 26) and G2677T/A (exon 21) were determined by direct sequencing. Multivariate analyses based on clinical information were performed to determine the relationship between ABCB1 genotypes and ONF. The dose/concentration (D/C) ratios of tacrolimus were also determined to estimate the activity of P-gp in patients with different genotypes of ABCB1 C3435T (CC, CT, TT), and in those who did and did not develop ONF. The ABCB1 3435TT genotype showed a significantly lower incidence of ONF (adjusted odds ratio = 0.10, P = 0.034). The D/C ratio in the 3435TT genotype was significantly higher than that in the 3435CC genotype. The D/C ratio in patients developing ONF was significantly higher than in those patients who did not develop ONF. The results suggest increased activity of P-gp in patients with the 3435TT genotype and in those who did not develop ONF. The ABCB1 2677 homozygous variant type also showed a lower incidence of ONF (adjusted odds ratio = 0.26, P = 0.056). The 3435T and 3435C alleles were in linkage disequilibrium with the 2677T and the 2677G alleles, respectively, in the study population. An assessment of C3435T and G2677T/A polymorphisms preceding steroid treatment could be useful for predicting the resistance to ONF development.
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PMID:ABCB1 C3435T and G2677T/A polymorphism decreased the risk for steroid-induced osteonecrosis of the femoral head after kidney transplantation. 1458 80

Active transport across biological membranes has become a noticeable factor in the absorption, distribution, and excretion of an increasing number of drugs. Different transmembrane transport systems including organic anion transporters (OATP, solute carrier family SLC21A), organic cation transporters (OCT, SLC22A), dipeptide transporters (PEPT, SLC15A), nucleoside transporters (CNT, SLC28A), monocarboxylate carriers (MCT, SLC2A), and members of the large ATP-binding cassette family (ABC, SLC3A) are involved in drug disposition. Genetic polymorphisms in transport proteins frequently occur and contribute to interindividual differences in the efficacy and safety of pharmatherapy. Currently, the most advanced research has been done on P-glycoprotein (ABCB1, SLC3A1.201.1). Knowledge of this transporter indicates that haplotype analysis rather than association with single nucleotide polymorphisms (SNPs) provides the most appropriate interpretation of pharmacogenetic data from drug transporters. This review gives an overview and update on the pharmacological impact of genetic variants in transmembrane transporters.
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PMID:Impact of genetic polymorphisms in transmembrane carrier-systems on drug and xenobiotic distribution. 1459 19

Idarubicin (IDA) is an anthracycline anticancer drug utilized in the treatment of acute leukemias. There are conflicting data published with regard to the cross-resistance of IDA in multidrug-resistant (MDR) cells expressing P-glycoprotein (P-gp). We evaluated the cytotoxicity and cellular accumulation of IDA in a panel of anthracycline-selected MDR cell lines. Leukemia K562/R7 cells and sarcoma MES-SA/Dx5 cells expressing high levels of the MDR1 (ABCB1) gene were resistant to IDA (42-fold and 150-fold, respectively). In both of these cell lines, resistance to IDA was equivalent to that for doxorubicin, the drug used to select for the MDR variants. The P-gp inhibitor PSC 833 (valspodar) at 2 microM completely restored sensitivity to IDA. IDA accumulation was decreased 12-fold in MES-SA/Dx5 cells vs parental cell line, and drug uptake was restored to control levels by PSC 833. Reduced intracellular IDA was correlated with P-gp content by flow cytometry. Experiments in NIH3T3 murine cells transfected with the human MDR1 gene substantiated the findings of cross-resistance to IDA and reversal of resistance by PSC 833. Our data indicate that IDA is a high-affinity substrate for P-gp.
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PMID:Modulation of resistance to idarubicin by the cyclosporin PSC 833 (valspodar) in multidrug-resistant cells. 1464 19


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