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)

Mutagenesis was used to investigate the functional role of six pairs of aspartate and glutamate residues (D450/D1093, E482/E1125, E552/E1197, D558/D1203, D592/D1237, and E604/E1249) that are highly conserved in the nucleotide binding sites of P-glycoprotein (Mdr3) and of other ABC transporters. Removal of the charge in E552Q/E1197Q and D558N/D1203N produced proteins with severely impaired biological activity when the proteins were analyzed in yeast cells for cellular resistance to FK506 and restoration of mating in a ste6Delta mutant. Mutations at other acidic residues had no apparent effect in the same assays. These four mutants were expressed in Pichia pastoris, purified to homogeneity, and biochemically characterized with respect to ATPase activity. Studies with purified proteins showed that mutants D558N and D1203N retained 14 and 30% of the drug-stimulated ATPase activity of wild-type (WT) Mdr3, respectively, and vanadate trapping of 8-azido[alpha-(32)P]nucleotide confirmed slower basal and drug-stimulated 8-azido-ATP hydrolysis compared to that for WT Mdr3. The E552Q and E1197Q mutants showed no drug-stimulated ATPase activity. Surprisingly, drugs did stimulate vanadate trapping of 8-azido[alpha-(32)P]nucleotide in E552Q and E1197Q at a level similar to that of WT Mdr3. This suggests that formation of the catalytic transition state can occur in these mutants, and that the bond between the beta- and gamma-phosphates is hydrolyzed. In addition, photolabeling by 8-azido[alpha-(32)P]nucleotide in the presence or absence of drug was also detected in the absence of vanadate in these mutants. These results suggest that steps after the transition state, possibly involved in release of MgADP, are severely impaired in these mutant enzymes.
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PMID:Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein. 1108 62

Cyclosporin A (CsA) treatment of HEK 293 cells expressing the rat heart RHE-1 (NCX1.1, EMBL accession number ) or the rat brain RBE-2 (NCX1.5, GenBank(TM) accession number ) Na(+)-Ca(2+) exchanger inhibited their transport activity in a concentration-dependent manner. The inhibition was detectable at 2 microm CsA, and exposure of the cells to 20 microm CsA resulted in a decrease of the Na(+)-dependent Ca(2+) uptake to about 20% relative to that of untreated cells. Determination of the surface expression of the exchanger protein revealed a parallel concentration-dependent reduction in the amount of the immunoreactive protein. No reduction was detected in the amount of total immunoreactive exchanger protein in CsA-treated cells relative to untreated ones. Among the different drugs tested, only PSC833, an analog of cyclosporin D, mimicked the effects of CsA. Exposure of the transfected cells to the chemically related cyclosporin D and macrolide drugs (FK506 or rapamycin) had no effect on the transport activity or the surface expression of the Na(+)-Ca(2+) exchanger. Co-expression of the human multidrug transporter P-glycoprotein (of which both drugs are modulators) with the cloned Na(+)-Ca(2+) exchanger revealed that transport activity and surface expression of each transporter in the co-transfected system were similar to those of each transporter alone in both the presence and absence of CsA or PSC833. CsA and PSC833 inhibited the surface expression of the NCX1 protein but did not alter the surface expression of P-glycoprotein. Unlike some P-glycoprotein endoplasmic reticulum-retained mutants (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 709-712), CsA did not rescue RBE-2/F913-->Stop, an endoplasmic reticulum-retained function-competent mutant of the Na(+)-Ca(2+) exchanger (Kasir, J., Ren, X., Furman, I., and Rahamimoff, H. (1999) J. Biol. Chem. 274, 24873-24880) and did not induce its kinesis to the surface membrane, further demonstrating molecular differences between P-glycoprotein and NCX1 mutants for interaction with CsA.
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PMID:Transport activity and surface expression of the Na+-Ca2+ exchanger NCX1 are inhibited by the immunosuppressive agent cyclosporin A and by the nonimmunosuppressive agent PSC833. 1170 Mar 17

SNF4435C and D, novel immunosuppressants produced by a strain of Streptomyces spectabilis, were examined for their reversing effects in vitro on various multidrug-resistant (MDR) tumor cells overexpressing P-glycoprotein. These two compounds in the range of 3-10 microM completely reversed the resistance of MDR variant cells, mouse leukemia P388 cells [vincristine (VCR)-resistant P388/VCR and adriamycin (ADM)-resistant P388/ADM], human myelogenous leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM) and human ovarian cancer A2780 cells (ADM-resistant AD(10)), against VCR. Both compounds moderately potentiated the sensitivity of the MDR cells to ADM but the reversal was not complete. SNF4435C and D significantly increased the intracellular accumulation of VCR in AD(10) cells as potently as verapamil, cyclosporin A (CysA) and FK506, whereas the compounds exerted no effect on the accumulation of VCR in the drug-sensitive parent cells. Moreover, SNF4435C improved the chemotherapeutic efficacy of VCR in the treatment of P388/VCR-bearing mice. When 10 mg/kg SNF4435C was administered intraperitoneally to the mice concurrently with 0.2 mg/kg VCR for every 5 days, a treated/control (T/C) value of 143% was obtained. These results suggest that the compounds are useful candidates or tools for MDR modification in cancer chemotherapy.
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PMID:Reversal of multidrug resistance by novel nitrophenyl pyrones, SNF4435C and D. 1171 49

Clinically important resistance of fungal pathogens to azole antifungal drugs is most frequently caused by the over-expression of energy-dependent drug efflux pumps. These pumps usually belong to either the ATP-binding cassette (ABC) family or the major facilitator superfamily (MFS) class of membrane transporter. Little is known about how these pumps work and there is urgent need to develop pump antagonists that circumvent resistance. The expression system is based on an S. cerevisiae AD1-8u- strain deleted in seven major ABC transporters which has reduced background and endogenous efflux activity. Plasmid pABC3 was engineered to allow functional hyper-expression of foreign proteins in this host. The main advantages of our system are its cloning efficiency: the use of homologous recombination to stably integrate single copy constructs into the host genome under the control of a highly active transcriptional regulator. The expression system has been used to clone and express genes encoding drug efflux pumps from several pathogenic fungi. Furthermore, functional over-expression of human P-glycoprotein was also demonstrated. The protein hyper-expression system will be useful for the screening of pump inhibitors and the study of membrane protein pumping mechanisms. This system has been used to screen chemicals for pump inhibitors. It was found that FK506 and milbemycins chemosensitized pump-expressing and fluconazole-resistant strains and inhibited pump ATPase activity.
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PMID:[An efficient system for functional hyper-expression of multidrug efflux pumps in Saccharomyces cerevisiae]. 1511 61

This study aimed to determine the impact of maintenance immunosuppressive therapy with cyclosporin A (CsA), tacrolimus (FK506) and sirolimus (Rapa) on the in vivo activity of both intestinal and hepatic cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (PGP) in renal transplant patients. The activity of these four elimination pathways was measured by the recently validated intravenous (iv) and per oral (po)14C erythromycin breath and urine test. In addition, overall hepatic P450 activity was measured by the (13)C aminopyrin breath test. Three groups of stable renal transplant patients on maintenance therapy with corticosteroids (CS) and mycophenolate mofetil (MMF) plus either CsA or FK506 or Rapa were examined. A significant increase in intestinal CYP3A4 activity and a significant decrease in hepatic and intestinal PGP activity was seen in patients on CsA in comparison with those on FK506 or Rapa (p < 0.01). A similar analysis in six healthy volunteers at baseline and after intake of CsA, FK506 and Rapa confirmed the results seen in the patients. There was no difference in CYP3A4 and PGP activity in the patients taking either FK506 or Rapa and healthy controls. These data suggest that a different pattern of drug interactions might be expected in patients treated with CsA vs. FK506/Rapa.
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PMID:CYP3A4 and P-glycoprotein activity in healthy controls and transplant patients on cyclosporin vs. tacrolimus vs. sirolimus. 1530 40

Ischemia-reperfusion injury is an unavoidable problem for organ transplantation including small bowel transplantation, and causes a large intra-individual variation of tacrolimus (FK506) pharmacokinetics. Little information is available about the regulation of the intestinal P-glycoprotein expression during tissue regeneration. In the present study, we have examined the molecular and functional variations of ileum P-glycoprotein using rats after ischemia-reperfusion treatment. Morphological study revealed a rapid regeneration of the intestinal wall during 24 h after reperfusion. A reverse transcription-coupled competitive PCR and Western blot analysis revealed that the intestinal expression of P-glycoprotein recovered with time after reperfusion. At 24 h after reperfusion, the ileum P-glycoprotein level was transiently increased to two-fold, and the absorption rate of dihydro-[(3)H]FK506 from in situ ileum loop into portal vein was markedly low in comparison with the control. P-glycoprotein was detected in the crypt area as well as in villous cells at 6 h after reperfusion, and then localized to the apical surface at 24 h consistent with the cell proliferation and differentiation. However, the P-glycoprotein level returned to normal at 48 h. The intra-individual variation in the absorptive rate of tacrolimus was suggested to be regulated by the morphological status of the intestinal epithelium and enterocyte expression level of P-glycoprotein. Therefore, the monitoring of the enterocyte P-glycoprotein level would provide useful information for determining the dosage of tacrolimus immediately after small bowl transplantation.
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PMID:Transient up-regulation of P-glycoprotein reduces tacrolimus absorption after ischemia-reperfusion injury in rat ileum. 1567 May 75

Pdr5p in Saccharomyces cerevisiae is a functional homologue of mammalian P-glycoprotein implicated in multidrug resistance (MDR). In order to obtain useful inhibitors to overcome MDR in clinical tumors, screening of Pdr5p inhibitors has been carried out. We isolated a fungal strain producing Pdr5p inhibitors using our original assay system, and it was classified as Trichoderma sp. P24-3. The purified inhibitor was identified as isonitrile, 3-(3'-isocyano-cyclopent-2'-enylidene)-propionic acid, a compound whose carboxyl residue is essential for the inhibitory activity. A non-toxic concentration of the isonitrile (41.5 microg/ml, 255 microM) inhibited Pdr5p-mediated efflux of cycloheximide or cerulenin in Pdr5p-overexpressing cells. In addition, addition of the isonitrile led to accumulation of rhodamine 6G, a substrate of Pdr5p, in the Pdr5p-overexpressing cells. The inhibitory profiles of the isonitrile against S1360 mutants (S1360A and S1360F) of Pdr5p were different from those of FK506 and enniatin. The isonitrile did not influence PDR5 gene expression and the amount of Pdr5 protein, nor did it inhibit the function of Snq2p, a homologue of Pdr5p. Interestingly, the isonitrile inhibited the function of Cdr1p and Cdr2p, Pdr5p homologues in pathogenic yeast Candida albicans. Thus, it was found that the isonitrile shows a different inhibitory spectrum from that of FK506 and enniatin as a potent inhibitor for Pdr5p, Cdr1p, and Cdr2p.
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PMID:A new function of isonitrile as an inhibitor of the Pdr5p multidrug ABC transporter in Saccharomyces cerevisiae. 1579 29

At present, the two calcineurin inhibitors-cyclosporine (CsA) and tacrolimus (FK506)-are among the most frequently used immunosuppressants in clinical transplantation. Both drugs share variable oral bioavailability, which necessitates intense drug monitoring. This variability is attributed to large interindividual differences in drug catabolism by cytochrome P450 3A4/5 (CYP3A4/5) and drug efflux by P-glycoprotein (PGP). In addition, the activity of both CYP3A4 and PGP can vary substantially within the same individual due to environmental factors such as concomitant intake of inducing/inhibiting medications (eg, rifampicin/sporanox) or food substances (eg, grapefruit juice). More recently, an inducing effect of methylprednisolone on intestinal and hepatic CYP3A4 has been shown. Also, an influence of gender on CYP3A4 activity (being higher in women) has been reported. Once CsA and FK506 are absorbed and reach the bloodstream, both drugs are avidly bound to erythrocytes (up to 95% for FK506 and 50% for CsA) and plasma proteins, leaving only a small fraction of circulating active drug. This phenomenon also limits further hepatic catabolism and hence clearance of drug, which is influenced by hematocrit and levels of plasma proteins such as albumin. The aim of the present study was to compare the influence of changing steroid doses, hematocrit, and albumin on trough and dose levels of FK506 versus CsA during the first year after transplantation. In addition, the evolution of trough and dose levels of FK506 versus CsA was stratified according to gender.
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PMID:Different evolution of trough and dose levels during the first year after transplantation for tacrolimus versus cyclosporine. 1596 36

Calcineurin inhibitors, tacrolimus (FK506) and cyclosporine (ciclosporin A), are the primary immunosuppressive agents used on recipients of organ transplantations. The hepatic metabolism of these drugs by cytochrome P450 IIIA (CYP3A) subfamilies is considered a major eliminating process. The intestinal efflux-pump P-glycoprotein (Pgp) (multidrug resistance 1 [MDR1], ATP-binding cassette B1 [ABCB1]) and CYP3A4 have been demonstrated as important for the bioavailability of drugs, so called "absorptive barriers". Recently, an important role for CYP3A5 in the intestine for the oral clearance of drugs has been identified. Both tacrolimus and cyclosporine are substrates of Pgp, CYP3A4 and CYP3A5, and therefore, these molecules are potential pharmacokinetic factors with which to establish personalized dosage regimens for these drugs. Although the effect of single nucleotide polymorphisms in the MDR1/ABCB1 and CYP3A5 genes on the pharmacokinetics of immunosuppressant has been widely examined, some contradictions have been emerged. In living-donor liver transplant (LDLT) patients, the intestinal mRNA expression level of MDR1 and CYP3A5 genotyping both in the native intestine and in the grafted liver are suggested to be potential pharmacokinetic factors for adjusting initial dosage and predicting post-operative variation in the pharmacokinetics of tacrolimus. We review the pharmacokinetic and pharmacodynamic characteristics of these drugs including the large pharmacokinetic variation and potential individualized dosage adjustments based on the genomic information of transporters and metabolic enzymes as well as classical pharmacokinetic analyses based on therapeutic drug monitoring (TDM).
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PMID:An up-date review on individualized dosage adjustment of calcineurin inhibitors in organ transplant patients. 1675 7

Tacrolimus hydrate (FK506) reduces the symptoms of myasthenia gravis (MG) due to its immunosuppressive properties. A drug efflux pump P-glycoprotein (P-gp) actively transports FK506 out of target cells, thereby reducing their efficacy. We investigated the influence of FK506 therapy on the P-gp function of peripheral-blood mononuclear cells (PBMCs) in MG patients. Six MG patients treated with FK506 (MG(FK+)), four MG patients treated without FK506 administration (MG(FK-)), and 18 healthy subjects were included in this study. P-gp function was estimated by transporter activity that was inferred from a decrease in fluorescent P-gp substrate Rhodamine 123 (Rh123) and its inhibition by cyclosporine A (CsA). The P-gp efflux function in MG (FK+) patients assessed by the Kolmogorov-Smirnov (KS) statistic D was lower than in the healthy subjects (p=0.0084). However, PBMC sensitivity to FK506 in MG (FK+) patients was significantly higher compared to that of the healthy subjects (p=0.02). There was a significant correlation between the Rh123 efflux activity and PBMC sensitivity to FK506 in vitro (p=0.011). The data raise the possibility that FK506 treatment attenuated P-gp function in the PBMCs of the MG patients.
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PMID:P-glycoprotein function in peripheral blood mononuclear cells of myasthenia gravis patients treated with tacrolimus. 1726 68


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