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Journal of Pharmacological and... Mar 2012P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either...
INTRODUCTION
P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches.
METHODS
Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology.
RESULTS
Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2=0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans.
DISCUSSION
This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 min, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Biological Transport, Active; Cricetinae; Cyclosporine; Drug Interactions; Humans; Inhibitory Concentration 50; LLC-PK1 Cells; Liposomes; Pharmaceutical Preparations; Protein Binding; Recombinant Proteins; Swine
PubMed: 22394995
DOI: 10.1016/j.vascn.2012.02.002 -
Journal of Neurochemistry May 2004We investigated the cellular/subcellular localization and functional expression of P-glycoprotein, an ATP-dependent membrane-associated efflux transporter, in...
We investigated the cellular/subcellular localization and functional expression of P-glycoprotein, an ATP-dependent membrane-associated efflux transporter, in astrocytes, a brain parenchyma compartment that is poorly characterized for the expression of membrane drug transporters. Analyses were carried out on primary cultures of astrocytes isolated from the cerebral cortex of neonatal Wistar rats and CTX TNA2, an immortalized rat astrocyte cell line. Both cell cultures display morphological features typical of type I astrocytes. RT-PCR analysis revealed mdr1a and mdr1b mRNA in primary cultures of astrocytes and in CTX TNA2 cells. Western blot analysis using the P-glycoprotein monoclonal C219 antibody detected a single band of appropriate size in both cell systems. Immunocytochemical analysis using the monoclonal antibodies C219 and MRK16 labeled P-glycoprotein along the plasma membrane, caveolae, coated vesicles and nuclear envelope. Immunoprecipitation studies using the caveolin-1 polyclonal H-97 antibody demonstrated that P-glycoprotein is physically associated with caveolin-1 in both cell culture systems. The accumulation of [(3)H]digoxin (an established P-glycoprotein substrate) by the astrocyte cultures was significantly enhanced in the presence of standard P-glycoprotein inhibitors and an ATP depleting agent. These results demonstrate the cellular/subcellular location and functional expression of P-glycoprotein in rat astrocytes and suggest that this glial compartment may play an important role in the regulation of drug transport in the CNS.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Astrocytes; Biological Transport; Blotting, Western; Caveolin 1; Caveolins; Cells, Cultured; Digoxin; Enzyme Inhibitors; Immunohistochemistry; Precipitin Tests; RNA, Messenger; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Subcellular Fractions
PubMed: 15086534
DOI: 10.1111/j.1471-4159.2004.02417.x -
PloS One 2021P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the...
P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer's disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer's associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphate; Alzheimer Disease; Amyloid beta-Peptides; Biocatalysis; Blood-Brain Barrier; Cell Line, Tumor; Disease Progression; Humans; Hydrolysis; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Peptide Fragments; Protein Binding; Protein Conformation, beta-Strand; Protein Domains; Protein Transport; Quinolines; Signal Transduction; Substrate Specificity
PubMed: 33901197
DOI: 10.1371/journal.pone.0250371 -
Cells Mar 2021Zebrafish has emerged as a powerful model in studies dealing with pigment development and pathobiology of pigment diseases. Due to its conserved pigment pattern with...
Zebrafish has emerged as a powerful model in studies dealing with pigment development and pathobiology of pigment diseases. Due to its conserved pigment pattern with established genetic background, the zebrafish is used for screening of active compounds influencing melanophore, iridophore, and xanthophore development and differentiation. In our study, zebrafish embryos and larvae were used to investigate the influence of third-generation noncompetitive P-glycoprotein inhibitor, tariquidar (TQR), on pigmentation, including phenotype effects and changes in gene expression of chosen chromatophore differentiation markers. Five-day exposure to increasing TQR concentrations (1 µM, 10 µM, and 50 µM) resulted in a dose-dependent augmentation of the area covered with melanophores but a reduction in the area covered by iridophores. The observations were performed in three distinct regions-the eye, dorsal head, and tail. Moreover, TQR enhanced melanophore renewal after depigmentation caused by 0.2 mM 1-phenyl-2-thiourea (PTU) treatment. qPCR analysis performed in 56-h post-fertilization (hpf) embryos demonstrated differential expression patterns of genes related to pigment development and differentiation. The most substantial findings include those indicating that TQR had no significant influence on leukocyte tyrosine kinase, GTP cyclohydrolase 2, tyrosinase-related protein 1, and forkhead box D3, however, markedly upregulated tyrosinase, dopachrome tautomerase and melanocyte inducing transcription factor, and downregulated purine nucleoside phosphorylase 4a. The present study suggests that TQR is an agent with multidirectional properties toward pigment cell formation and distribution in the zebrafish larvae and therefore points to the involvement of P-glycoprotein in this process.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Cell Differentiation; Gene Expression Profiling; Gene Expression Regulation; Gene Expression Regulation, Developmental; Larva; Melanins; Melanophores; Pigmentation; Quinolines; RNA, Messenger; Zebrafish; Zebrafish Proteins
PubMed: 33804686
DOI: 10.3390/cells10030690 -
Amino Acids Jul 2020Elevated plasma concentrations of the uremic toxin asymmetric dimethylarginine (ADMA) and low plasma concentrations of L-homoarginine are independently associated with...
Vectorial transport of the arginine derivatives asymmetric dimethylarginine (ADMA) and L-homoarginine by OATP4C1 and P-glycoprotein studied in double-transfected MDCK cells.
Elevated plasma concentrations of the uremic toxin asymmetric dimethylarginine (ADMA) and low plasma concentrations of L-homoarginine are independently associated with cardiovascular events and mortality. Key enzymes involved in the homeostasis of both arginine derivatives are expressed in proximal tubule cells of the kidney. To get access to these enzymes, transport proteins are important. One of the transporters mediating the transport of ADMA and L-homoarginine is the solute carrier superfamily (SLC) member OATP4C1, located in the basolateral membrane of proximal tubule cells. To gain insights into the role of export pumps in the transport of both substances, we established a double-transfected MDCK cell line expressing OATP4C1 and the export pump P-glycoprotein (P-gp). Using MDCK cell monolayers, we demonstrated in time-dependent and concentration-dependent vectorial transport experiments that ADMA and L-homoarginine are transported from the basolateral to the apical compartment of MDCK-OATP4C1-P-gp cells with significantly higher transport rates compared to single-transfected MDCK-OATP4C1, MDCK-P-gp and MDCK-VC (control) cells (e.g. transport ratio MDCK-OATP4C1-P-gp/MDCK-VC: for 50 µM ADMA = 2.0-fold, for 50 µM L-homoarginine = 3.4-fold). These results indicate that both OATP4C1 and P-gp transport the arginine derivatives ADMA and L-homoarginine and are, therefore, important for the homoeostasis of both substances.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Arginine; Dogs; Homoarginine; Humans; Madin Darby Canine Kidney Cells; Organic Anion Transporters; Transcytosis; Transfection
PubMed: 32642843
DOI: 10.1007/s00726-020-02867-8 -
Protein Science : a Publication of the... Jan 2014The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible...
The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpretations in the latter model. To address this concern, we generated an experimental electron density map from single-wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Å resolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug-free structure was refined to 3.8 Å resolution with a 9.4 and 8.1% decrease in R(work) and R(free), respectively, (R(work) = 21.2%, R(free) = 26.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ∼95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was corrected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addition of three N-terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted >6 Å from their original positions. The drug translocation pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Amino Acid Motifs; Amino Acid Sequence; Amino Acids, Aromatic; Animals; Biological Transport; Crystallography, X-Ray; Fourier Analysis; Humans; Mice; Models, Molecular; Protein Conformation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Sequence Homology, Amino Acid
PubMed: 24155053
DOI: 10.1002/pro.2387 -
Drug News & Perspectives Sep 2009Epilepsy affects more than 60 million people worldwide. While most patients can be treated with antiepileptic drugs, up to 40% of patients respond poorly to... (Review)
Review
Epilepsy affects more than 60 million people worldwide. While most patients can be treated with antiepileptic drugs, up to 40% of patients respond poorly to pharmacotherapy. This drug resistance is not well understood and presents a major clinical problem. In this short review we provide background information on one potential cause of antiepileptic drug resistance, namely, upregulation of the drug efflux transporter P-glycoprotein at the blood-brain barrier. We summarize recent findings that connect antiepileptic drug resistance with P-glycoprotein upregulation and show a mechanistic link between seizures and upregulation of this transporter. We provide an overview of results demonstrating that glutamate released during seizures signals through N-methyl-Daspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) to increase P-glycoprotein. In this context we discuss the NMDA receptor and COX-2 as potential therapeutic targets and provide information on current clinical trials on drugresistant epilepsy involving blood-brain barrier efflux transporters. Finally, we provide a perspective on future research that could help improve the treatment of drug-resistant epilepsy.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Anticonvulsants; Drug Resistance; Epilepsy; Humans; Signal Transduction; Up-Regulation
PubMed: 19890496
DOI: 10.1358/dnp.2009.22.7.1401354 -
Cells Apr 2020Doxorubicin is a strong inducer of immunogenic cell death (ICD), but it is ineffective in P-glycoprotein (Pgp)-expressing cells. Indeed, Pgp effluxes doxorubicin and...
Doxorubicin is a strong inducer of immunogenic cell death (ICD), but it is ineffective in P-glycoprotein (Pgp)-expressing cells. Indeed, Pgp effluxes doxorubicin and impairs the immunesensitizing functions of calreticulin (CRT), an "eat-me" signal mediating ICD. It is unknown if classical Pgp inhibitors, designed to reverse chemoresistance, may restore ICD. We addressed this question by using Pgp-expressing cancer cells, treated with Tariquidar, a clinically approved Pgp inhibitor, and -3 compound, a ,-bis(alkanol)amine aryl ester derivative with the same potency of Tariquidar as Pgp inhibitor. In Pgp-expressing/doxorubicin-resistant cells, Tariquidar and -3 increased doxorubicin accumulation and toxicity, reduced Pgp activity, and increased CRT translocation and ATP and HMGB1 release. Unexpectedly, only -3 promoted phagocytosis by dendritic cells and activation of antitumor CD8T-lymphocytes. Although Tariquidar did not alter the amount of Pgp present on cell surface, -3 promoted Pgp internalization and ubiquitination, disrupting its interaction with CRT. Pgp knock-out restores doxorubicin-induced ICD in MDA-MB-231/DX cells that recapitulated the phenotype of -3-treated cells. Our work demonstrates that plasma membrane-associated Pgp prevents a complete ICD notwithstanding the release of ATP and HMGB1, and the exposure of CRT. Pharmacological compounds reducing Pgp activity and amount may act as promising chemo- and immunesensitizing agents.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Apoptosis; Calreticulin; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Endocytosis; Esters; Humans; Immunogenic Cell Death; Kinetics; Proteolysis; Quinolines; Ubiquitination
PubMed: 32331368
DOI: 10.3390/cells9041033 -
The Journal of Biological Chemistry Sep 1995P-glycoprotein consists of two homologous halves, each composed of a transmembrane domain and a nucleotide-binding domain. In order to understand how the domains...
P-glycoprotein consists of two homologous halves, each composed of a transmembrane domain and a nucleotide-binding domain. In order to understand how the domains interact in P-glycoprotein, we expressed each domain as a separate polypeptide and tested for associations using coimmunoprecipitation assays. We found that the interactions between the two halves of P-glycoprotein were mediated through associations between the two transmembrane domains as well as through the nucleotide-binding domains. In addition, the nucleotide-binding domain also associated with the transmembrane domain in each half of the molecule. By contrast, we could not detect any association either between the first nucleotide-binding domain and the second transmembrane domain, or between the second nucleotide-binding domain and the first transmembrane domain. We then tested whether individual domains associated with molecular chaperones, since biogenesis of P-glycoprotein appears to involve the chaperones calnexin and Hsc70. We found that calnexin associated only with the transmembrane domains, while Hsc70 associated only with the nucleotide-binding domains. These results suggest that noncovalent interaction between the domains of P-glycoprotein can contribute to structure and function of P-glycoprotein and that chaperones may participate in the folding of each domain.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Amino Acid Sequence; Antibodies, Monoclonal; Binding Sites; Calcium-Binding Proteins; Calnexin; Carrier Proteins; Cell Line; Cell Membrane; Epitopes; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Models, Structural; Molecular Chaperones; Molecular Sequence Data; Protein Folding; Protein Structure, Secondary; Recombinant Proteins; Transfection
PubMed: 7545169
DOI: 10.1074/jbc.270.37.21839 -
Current Topics in Medicinal Chemistry 2010The issue of pharmacoresistance in epilepsy has received considerable attention in recent years, and a number of plausible hypotheses have been proposed. Of these, the... (Review)
Review
The issue of pharmacoresistance in epilepsy has received considerable attention in recent years, and a number of plausible hypotheses have been proposed. Of these, the so-called transporter hypothesis is the most extensively researched and documented. This hypothesis assumes that refractory epilepsy is associated with a localised over-expression of drug transporter proteins such as P-glycoprotein (Pgp) in the region of the epileptic focus, which actively extrudes antiepileptic drugs (AEDs) from their intended site of action. However, although this hypothesis has biological plausibility, there is no clinical evidence to support the assertion that AEDs are sufficiently strong substrates for transporter-mediated extrusion from the brain. The use of modern brain imaging techniques to determine Pgp function in patients with refractory epilepsy has started only recently, and may ultimately determine whether increased expression and function of Pgp or other efflux transporters are involved in AED resistance.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Anticonvulsants; Drug Resistance, Multiple; Epilepsy; Humans; Positron-Emission Tomography
PubMed: 20645916
DOI: 10.2174/156802610792928095