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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The multidrug efflux pump P-glycoprotein (Pgp) couples drug transport to ATP hydrolysis. Previously, using a synthetic library of tetramethylrosamine ( TMR) analogues, we observed significant variation in ATPase stimulation ( V m (D)). Concentrations required for half-maximal ATPase stimulation ( K m (D)) correlated with ATP hydrolysis transition-state stabilization and ATP occlusion (EC 50 (D)) at a single site. Herein, we characterize several TMR analogues that elicit modest turnover ( k cat <or= 1-2 s (-1)) compared to verapamil (VER) ( k cat approximately 10 s (-1)). Apparent ATPase activities manifest as nearly equivalent to basal values. In some cases, K m (D) parameters for drug stimulation of ATPase could not be accurately determined, yet these same TMR analogues promoted ATP occlusion at relatively low concentrations ( approximately 0.4-40 microM). Moreover, the TMR analogues competitively inhibited VER-dependent ATPase activity at concentrations similar to those required for ATP occlusion. Finally, the TMR analogues facilitated uptake of calcein-AM into CR1R12 and MDCK-MDR1 cells and are actively transported by Pgp in monolayers of MDCK-MDR1 cells at similarly low concentrations ( approximately 1-20 microM). ADP.V i release kinetics were identical in the presence of the TMR derivatives, VER, or in the absence of drug, suggesting that slow turnover is not likely due to slow release of the ATP hydrolysis products ADP and P i. These data support the partition model in which drug site occupancy converts residual basal ATPase activity to a drug-dependent mechanism even in cases where stimulation appears to be exactly compensatory to basal values. It is noteworthy that when compared to previously reported TMR analogues, subtle modification of the TMR scaffold can confer large differences in ATP turnover.
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PMID:ATP occlusion by P-glycoprotein as a surrogate measure for drug coupling. 1827 55

The objective was to examine the influence of Pluronic block-copolymers on the interaction between the drug efflux transporter, P-glycoprotein and HIV-1 protease inhibitors (PIs). The ATPase assay determined the effect of various Pluronics on PI-stimulated P-gp ATPase activity. Cellular accumulation studies were conducted using MDCKII and LLC-PK1 cells transfected with human MDR1 to assess Pluronic modulation of PI efflux. Pluronic P85 inhibited both basal and nelfinavir-stimulated P-gp ATPase activity, while Pluronic F127 had no effect. In cell accumulation studies, Pluronic P85 restored the accumulation of nelfinavir in MDCKII-MDR1 cells while Pluronic F127 and F88 had no effect. Pluronic P85 increased saquinavir accumulation in wild-type and MDR1-transfected cells in both the MDCKII and LLC-PK1 cell models, suggesting inhibition of multiple transporters, including MRPs. In conclusion, this study provides evidence that a block-copolymer, Pluronic P85, effectively inhibits the interaction of P-gp with nelfinavir and saquinavir. These data indicate that effective inhibition of HIV-1 PI efflux by Pluronic P85 may influence the distribution of antiretroviral agents to sites protected by efflux mechanisms, such as the blood-brain barrier, and possibly increase the brain exposure of these drugs resulting in suppression of viral replication and reduction in the incidence of drug resistant mutants.
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PMID:Interactions of pluronic block copolymers on P-gp efflux activity: experience with HIV-1 protease inhibitors. 1839 90

Chloroacetanilide herbicides are among the most commonly used herbicides in agriculture. Several studies have demonstrated a number of them to be carcinogenic. ATP binding cassette (ABC) transporters are efflux pumps expressed in cell membranes, which form an important wall of defense against xenobiotics from different sources. We tested the interaction of the herbicides acetochlor, alachlor, dimetachlor, metazachlor, metolachlor, propachlor and prynachlor with human multidrug resistance transporters MDR1, MRP1, MRP2 and BCRP. A number of metabolites were studied for interaction with MRP1, MRP2 and MRP3. Transporter interactions were studied by measuring ATPase activity, inhibition of fluorescent dye efflux and vesicular transport. Also inhibition of MDR1 was monitored by measuring digoxin transport on Caco-2 monolayers and paclitaxel toxicity on K562-MDR cells. Acetochlor, alachlor, metolachlor and metazachlor showed specific interactions with MDR1. Digoxin permeability and paclitaxel cytotoxicity studies revealed that these herbicides are potent inhibitors of MDR1 that can modulate drug absorption and cause chemosensitization of cells. MRP1 was demonstrated to transport an important intermediate of the acetochlor detoxification pathway. Several specific interactions were shown when studying the interaction of chloroacetanilides with human transporter proteins. This study suggests an important role for transporter proteins in hazard prediction of agrochemicals and demonstrates how transporter interactions can be easily detected using in vitro screening methods.
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PMID:Specific interactions of chloroacetanilide herbicides with human ABC transporter proteins. 1843 74

P-glycoprotein (ABCB1) prevents absorption (e.g., blood-brain barrier) or enhances excretion (e.g., kidney) by moving substrates from the cytosolic to the extracellular membrane leaflet at the expense of ATP hydrolysis. It translocates various drugs and functions in membranes exhibiting different lateral packing densities. To gain more functional insight, we measured the temperature dependence of the P-glycoprotein ATPase activity in NIH-MDR1-G185 cell membranes in the absence and presence of three drugs (promazine, verapamil, and PSC833), exhibiting significantly different transporter affinities. Activation enthalpies (Delta H(++)) and entropies ( TDelta S(++)) were derived from Eyring plots. In the absence of drugs, the activation enthalpy and the free energy of activation for P-glycoprotein ATPase activity was determined as Delta H(++) = 92.6 +/- 4.2 kJ/mol and Delta G(++) = 73.1 +/- 7.2 kJ/mol, respectively. Increasing the drug concentration reduced the activation enthalpy, whereby the drug with the highest transporter affinity had the strongest effect (DeltaDelta H(++) = -21%). The free energy of activation decreased for activating (DeltaDelta G(++) = approximately -3.8%) and increased for inhibitory compounds (DeltaDelta G(++) = approximately +0.7%). The drug-specific changes of the free energy of activation are thus barely above thermal energy. A comparison with literature data revealed that a decrease of the lateral membrane packing density reduces the enthalpic and the entropic contribution to the free energy of activation. Although the P-glycoprotein ATPase activity increases only slightly with decreasing lateral membrane packing density, the mode of action changes from strongly entropy-driven at high, to essentially enthalpy-driven at low packing densities. This suggests that the transporter and the membrane form a functional entity.
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PMID:P-glycoprotein senses its substrates and the lateral membrane packing density: consequences for the catalytic cycle. 1875 52

A series of chalcogenopyrylium dyes were evaluated as modulators/inhibitors of P-glycoprotein (Pgp). Their ability to inhibit verapamil (VER)-dependent ATPase activity (IC(50) values) in lipid-activated, mouse Cys-less mdr3 Pgp was determined. Their ability to promote calcein-AM (CAM) uptake in MDCKII-MDR1 cells and their capacity to be transported by Pgp in monolayers of MDCKII-MDR1 cells were also evaluated. The chalcogenopyrylium dyes promoted CAM uptake with values of EC(50) between 5 x 10(-6) and 3.5 x 10(-5)M and 7 of the 9 dyes examined in transport studies were substrates for Pgp with efflux ratios (P(BA/AB)) between 14 and 390. Binding of three compounds (1-S, 3-S, and 4-S) to Pgp was also assessed by fluorescence. These three thiopyrylium dyes showed increased fluorescence upon binding to Pgp, giving apparent binding constants, K(app), on the order of 10(-7) to 10(-6)M. Compound 8-Te was particularly intriguing since it appeared to influence Pgp at low micromolar concentrations as evidenced by its influence on VER-stimulated ATPase activity (IC(50) of 1.2 x 10(-6)M), CAM uptake (EC(50) of 5.4 x 10(-6)M), as well as [(3)H]-vinblastine transport by Pgp in cells (IC(50) of 4.3 x 10(-6)M) and within inside-out membrane vesicles (IC(50) of 9.6 x 10(-6)M). Yet, Pgp did not influence the distribution of 8-Te in MDCKII-MDR1 monolayers suggesting that 8-Te may bind to an allosteric site.
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PMID:Chalcogenopyrylium dyes as inhibitors/modulators of P-glycoprotein in multidrug-resistant cells. 1884 67

Inherent or acquired resistance of tumor cells to cytotoxic drugs represents a major limitation to the successful chemotherapeutic treatment of cancer. During the past three decades dramatic progress has been made in the understanding of the molecular basis of this phenomenon. Analyses of drug-selected tumor cells which exhibit simultaneous resistance to structurally unrelated anti-cancer drugs have led to the discovery of the human MDR1 gene product, P-glycoprotein, as one of the mechanisms responsible for multidrug resistance. Overexpression of this 170 kDa N-glycosylated plasma membrane protein in mammalian cells has been associated with ATP-dependent reduced drug accumulation, suggesting that P-glycoprotein may act as an energy-dependent drug efflux pump. P-glycoprotein consists of two highly homologous halves each of which contains a transmembrane domain and an ATP binding fold. This overall architecture is characteristic for members of the ATP-binding cassette or ABC superfamily of transporters. Cell biological, molecular genetic and biochemical approaches have been used for structure-function studies of P-glycoprotein and analysis of its mechanism of action. This review summarizes the current status of knowledge on the domain organization, topology and higher order structure of P-glycoprotein, the location of drug- and ATP binding sites within P-glycoprotein, its ATPase and drug transport activities, its possible functions as an ion channel, ATP channel and lipid transporter, its potential role in cholesterol biosynthesis, and the effects of phosphorylation on P-glycoprotein activity.
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PMID:Molecular analysis of the multidrug transporter, P-glycoprotein. 1900 82

Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3 is a MDM2 inhibitor under preclinical investigation as nongenotoxic activator of the p53 pathway for cancer therapy. Here, nutlin-3 was evaluated for its activity alone or in combination with established chemotherapeutic drugs for antitumor action in chemosensitive and chemoresistant neuroblastoma and rhabdomyosarcoma cell lines. Effects of nutlin-3 single treatment were much more pronounced in p53 wild-type cell lines (IC(50)s <3 micromol/L) than in p53-mutated cell lines (IC(50)s >17 micromol/L). In sharp contrast to the expectations, nutlin-3 concentrations that did not affect viability of p53-mutated cell lines strongly increased the efficacy of vincristine in p53-mutated, P-glycoprotein (P-gp)-overexpressing cell lines (decrease in IC(50)s 92- to 3,434-fold). Similar results were obtained for other P-gp substrates. Moreover, nutlin-3 reduced efflux of rhodamine 123 and other fluorescence dyes that are effluxed by P-gp. Investigation of Madin-Darby canine kidney (MDCK) II cells stably transfected with plasmids encoding for P-gp (MDCKII MDR1) or multidrug resistance protein 1 (MRP-1, MDCKII MRP1) revealed that nutlin-3 not only interferes with P-gp but also affects MRP-1-mediated efflux. Kinetic studies and investigation of P-gp-ATPase activity showed that nutlin-3 is likely to act as a P-gp transport substrate. Examination of the nutlin-3 enantiomers nutlin-3a and nutlin-3b revealed that, in contrast to MDM2-inhibitory activity that is limited to nutlin-3a, both enantiomers similarly interfere with P-gp-mediated drug efflux. In conclusion, nutlin-3-induced inhibition of P-gp and MRP-1 was discovered as a novel anticancer mechanism of the substance in this report.
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PMID:Reversal of P-glycoprotein-mediated multidrug resistance by the murine double minute 2 antagonist nutlin-3. 1914 53

A literature review revealed that mefloquine neurotoxicity has been demonstrated at both the preclinical and clinical levels, with nausea, dizziness, sleep disturbances, anxiety and psychosis, amongst other adverse neuropsychiatric events, reported in users. Females and individuals of low body mass index (BMI) are at apparent greater risk. Mechanisms of possible neurotoxicity may include binding to neuroreceptors and cholinesterases, inhibition of sarcoendoplasmic reticulum ATPase (SERCA) and interference with cellular Ca(2+) homeostasis, accumulation in the CNS, and reductions in CNS efflux in individuals possessing certain MDR1 polymorphisms. It may be prudent to avoid mefloquine in females and low BMI individuals, and in combination with other potentially neurotoxic agents such as the artemisinin antimalarials.
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PMID:Mefloquine neurotoxicity: a literature review. 1917 93

Multidrug resistance protein MDR1 (P-glycoprotein/ABCB1) is an ATP-dependent efflux pump for various cytotoxic agents, and is implicated in the resistance of human tumors to chemotherapeutic drugs. To achieve the three-dimensional structural analysis for its mechanistic implications, large amounts of high-quality and homogeneous MDR1 protein are essential. Here we report a cost-effective method for large-scale expression of human MDR1 using a baculovirus/insect expressSF+ cell system and an alterative purification method to maintain MDR1 in a monodispersed state. After extensively optimizing the detergent, pH, and additives, a high yield (2.8 mg/L) of purified MDR1 was obtained by immobilized metal chelate affinity and size-exclusion chromatographies with 49% recovery. The purified MDR1 exhibited specific ATP hydrolase activity (1.7 micromol/min/mg) in the presence of a substrate, verapamil. This value was 14-fold greater than the basal activity without the drug. Size-exclusion chromatography analysis of purified MDR1 showed a monodispersed elution profile. The present purification method provides suitable material for structural and functional studies on human MDR1.
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PMID:Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells. 1923 88

The extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we have investigated the role of P-glycoprotein (P-gp) in the transport of tanshinone I (TSI), a major active constituent of S. miltiorrhiza. The TSI transport across Caco-2 monolayers was pH-, energy-, and temperature-dependent, but not sodium-dependent. TSI exhibited a polarized transport in Caco-2 monolayers which was attenuated by P-gp inhibitors. The permeability (P(app)) values of TSI in the basolateral to apical direction were significantly higher in MDCK-II cells over-expressing MDR1, as compared to the wild-type control cells. Furthermore, TSI significantly inhibited the transport of digoxin in Caco-2 cells with an IC(50) value of 0.53 +/- 0.09 microM. TSI also moderately stimulated P-gp ATPase activity with K(m) and V(max) values of 31.70 +/- 7.09 microM and 57.71 +/- 5.26 nmol/min/mg protein, respectively. Our findings indicate that TSI is a substrate and inhibitor of P-gp, which has important clinical and toxicological implications.
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PMID:Role of P-glycoprotein in the transport of tanshinone I, one active triterpenoid from Salvia miltiorrhiza. 1935 97


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