Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We identified a thiol-reactive substrate, Tris-(2-maleimidoethyl)amine (TMEA), to explore the contribution of the TM segments 6 and 12 of the human multidrug resistance P-glycoprotein (P-gp) during transport. TMEA is a trifunctional maleimide and stimulated the ATPase activity of Cys-less P-gp about 7-fold. Cysteine-scanning mutagenesis of TM12 showed that the activity of mutant V982C was inhibited by TMEA. P-gp mutants containing V982C (TM12) and another cysteine in TM6 were constructed and tested for cross-linking with TMEA. A cross-linked product was observed in SDS-polyacrylamide gel electrophoresis for mutant L339C(TM6)/V982C(TM12). Cross-linking by TMEA also inhibited the ATPase activity of the mutant protein. Substrates such as cyclosporin A, vinblastine, colchicine, or verapamil inhibited cross-linking by TMEA. In the presence of ATP at 37 degrees C, cross-linking of mutant L339C/V982C was decreased. In contrast, there was enhanced cross-linking of mutant F343C(TM6)/V982C(TM12) in the presence of ATP. These results show that cross-linking must be within the drug-binding domain, that residues L339C(TM6)/V982C(TM12) must be at least 10 A apart, and that ATP hydrolysis promotes rotation of one or both TM helices.
 ...
PMID:Cross-linking of human multidrug resistance P-glycoprotein by the substrate, tris-(2-maleimidoethyl)amine, is altered by ATP hydrolysis. Evidence for rotation of a transmembrane helix. 1142 7

1. Increasing the lipophilicity is a strategy often used to improve a compound's cellular uptake and retention but this may also convert it into a substrate for an ATP-dependent transporter such as P-glycoprotein or the multidrug resistance-associated protein (MRP1), which are involved in cellular efflux of drugs. Tris-Lipidation of compounds is a convenient way of modifying drug lipophilicity and generating an array of derivatives with diverse properties. 2. To determine the effect of Tris-Lipidation on a drug's cytoxicity in multidrug resistant cells, various glycyl-Tris-mono- (GTP1), di- (GTP2) and tri-palmitate (GTP3) derivatives were prepared of the cancer chemotherapeutic drugs chlorambucil and methotrexate, and of the anti-HIV drug AZT. The cytotoxicity of these derivatives and their parent compounds was determined in the CEM/VLB(100) cells with increased P-glycoprotein expression, the CEM/E1000 cells that overexpress MRP1 and the parent, drug-sensitive CCRF-CEM cells. 3. Increasing the lipophilicity of AZT increased its cytotoxicity in the sensitive CCRF-CEM parental cell line while decreased cytotoxicity was observed for the methotrexate derivatives. For the chlorambucil derivatives, both increased (GTP1) and decreased (GTP2) cytotoxicity occurred in the CCRF-CEM cells. With the exception of AZT-GTP1, all GTP1 and GTP2 derivatives of chlorambucil, methotrexate and AZT had decreased cytotoxicity in the P-glycoprotein-expressing CEM/VLB(100) cells while chlorambucil-GTP1, methotrexate-GTP2 and methotrexate-GTP3 were the only compounds with decreased cytotoxicity in the MRP1-overexpressing CEM/E1000 cells. 4. The number of palmitate residues, the position of derivatisation and the type of linkage all may affect the P-glycoprotein and MRP1 substrate properties. 5. Tris-Lipidation may therefore provide a useful way of manipulating the pharmacokinetic properties of drugs.
 ...
PMID:The use of Tris-lipidation to modify drug cytotoxicity in multidrug resistant cells expressing P-glycoprotein or MRP1. 1246 37

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.
 ...
PMID:Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein. 1290 21

Triclabendazole (TCBZ) and albendazole (ABZ) are flukicidal benzimidazole compounds extensively used in veterinary medicine. Although TCBZ has excellent activity against mature and immature stages of the liver fluke, Fasciola hepatica, ABZ action is restricted to flukes older than 12 wk. The intensive use of TCBZ has resulted in the development of resistance. To gain insight into the mechanisms of resistance to TCBZ, the ex vivo diffusion of TCBZ, TCBZ sulfoxide (TCBZSO, the active metabolite of TCBZ), and ABZ into TCBZ-susceptible and -resistant adult flukes was compared. TCBZ-susceptible (Cullompton) and -resistant (Sligo) flukes were incubated in Krebs-Ringer Tris buffer with either TCBZ, TCBZSO, or ABZ (5 nmol/ ml) for 90 min. Drug/metabolite concentrations were quantified by high-performance liquid chromatography. All the assayed molecules penetrated through the tegument of both susceptible and resistant flukes. However, significantly lower concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes. In contrast, ABZ entrance into the susceptible and resistant flukes was equivalent. The influx/efflux balance for TCBZ, TCBZSO, and ABZ in susceptible and resistant flukes in the presence or absence of a substrate (ivermectin) of the drug transporter P-glycoprotein was assessed. The ivermectin-induced modulation of P-glycoprotein activity decreased TCBZ efflux from the resistant flukes. Higher concentrations of TCBZ and TCBZSO were recovered from the resistant liver flukes in the presence of ivermectin. Thus, an altered influx/efflux mechanism may account for the development of resistance to TCBZ in F. hepatica.
 ...
PMID:Resistance-induced changes in triclabendazole transport in Fasciola hepatica: ivermectin reversal effect. 1730 20

Membrane and membrane-associated proteins are rich in known or potential pharmaceutical drug targets for carcinogenesis. In order to systemically analyze membrane proteins of human breast cancer, we isolated membrane from MCF-7 cells by sequential extraction by washing with three different buffers, namely, phosphate buffer (5 mM, pH 8.0), Tris (40 mM, pH 9.5), and sodium carbonate (100 mM pH 11). The extracted proteins were separated by two-dimensional gel electrophoresis (2-DE) using cup-loading and were then analyzed by peptide mass fingerprinting (PMF). A total of 137 spots from the gels of the three procedures were successfully identified. They corresponded to 79 distinct proteins. Among them, 22 exclusive proteins belonging to each washing procedure were also found, including P-glycoprotein, endoplasmin, Stress-70 protein, ADAM 10, protein disulfide isomerase, and glutamate receptor. These results indicate phosphate buffer to be the most beneficial for enrichment of peripheral membrane proteins, and sodium carbonate is beneficial for the presentation of integral membrane proteins but usually with poor resolution. The reference maps and identified proteins will serve as a basis for the further investigation of breast cancer, especially the proteomic comparison among different cell types of breast cancer, or among the different stages in the drug interfering process of the MCF-7 cell line.
 ...
PMID:Membrane protein analysis of human breast cancer cell line MCF-7 by different membrane washing methods. 1870 47

Dogs with a 4-bp deletion in the MDR1 (or ABCB1) gene show intolerance to certain drugs routinely used in veterinary medicine, such as ivermectin, vincristine, and doxorubicin. The mutation leads to a dysfunctional P-glycoprotein drug transporter, which results in drug accumulation in the brain and severe neurotoxicity. A rapid and accurate in-house test to determine the genotype of patients in cases of acute neurotoxic signs or in tumor patients is desirable. We describe a cost-effective detection method with simple technical equipment for veterinary practice. Two allele-specific methods are presented, which allow discrimination of all genotypes, require little hands-on time, and show the results within ~1 h after DNA sampling. DNA from buccal swabs of 115 dogs with known genotype (no mutation, n = 54; heterozygous for the mutation, n = 37; homozygous for the mutation, n = 24) was extracted either by using a column-based extraction kit or by heating swabs in a simple NaOH-Tris buffer. Amplification was performed either by allele-specific fast polymerase chain reaction or by allele-specific loop-mediated isothermal amplification (LAMP). Analysis was done either on agarose gels, by simple endpoint visualization using ultraviolet light, or by measuring the increase of fluorescence and time to threshold crossing. Commercial master mixes reduced the preparation time and minimized sources of error in both methods. Both methods allowed the discrimination of all 3 genotypes, and the results of the new methods matched the results of the previous genotyping. The presented methods could be used for fast individual MDR1/ ABCB1 genotyping with less equipment than existing methods.
 ...
PMID:Fast and simple detection methods for the 4-base pair deletion of canine MDR1/ ABCB1 gene by PCR and isothermal amplification. 2806 49