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Biochemical Pharmacology Oct 2018The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range...
The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed "small" and "large" protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these "small" and "large" protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 Å AFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the "small" and "large" protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Lipid Bilayers; Liposomes; Mice; Microscopy, Atomic Force; Protein Conformation
PubMed: 30121251
DOI: 10.1016/j.bcp.2018.08.017 -
BioMed Research International 2015The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system... (Review)
Review
The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Antipsychotic Agents; Blood-Brain Barrier; Drug Resistance; Humans; Schizophrenia
PubMed: 26491671
DOI: 10.1155/2015/484963 -
The International Journal of... Sep 2010Antipsychotic drugs are the mainstay pharmacotherapy for schizophrenia and related psychiatric disorders. While the metabolic pathways of antipsychotic drugs have been... (Comparative Study)
Comparative Study
Antipsychotic drugs are the mainstay pharmacotherapy for schizophrenia and related psychiatric disorders. While the metabolic pathways of antipsychotic drugs have been well defined, the role of drug transporters in the disposition and effects of antipsychotic drugs has not been systematically explored. P-glycoprotein has ubiquitous expression in brain endothelial cells and plays a protective role by effluxing substrates for elimination and by limiting their accumulation in the central nervous system. Risperidone and several other antipsychotic drugs are substrates of P-glycoprotein. Increased antipsychotic drug entry into the brain via blockade of the P-glycoprotein transporter may facilitate the amount of available drug to its targets, particularly dopamine receptors. By increasing available antipsychotic drug concentrations, P-glycoprotein inhibition offers a novel means of enhanced drug delivery. This study evaluated whether selective P-glycoprotein transporter inhibition would increase the effects of risperidone on relevant indices of behaviour (catalepsy and locomotion) and neurochemistry (dopamine release and metabolism as measured by in-vivo microdialysis). We administered the P-glycoprotein inhibitor, PSC 833 (100 mg/kg p.o.), to rats prior to administration of risperidone at varying doses (0.01-4.0 mg/kg s.c.). P-glycoprotein inhibition significantly increased risperidone-induced cataleptic effects, blockade of amphetamine-induced locomotion, and effects on dopamine turnover as seen by increased striatal dopamine metabolite levels. These results provide functional evidence concordant with prior data for increased brain levels of risperidone following PSC 833 treatment.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Catalepsy; Corpus Striatum; Cyclosporins; Dopamine; Dose-Response Relationship, Drug; Drug Synergism; Male; Motor Activity; Rats; Rats, Sprague-Dawley; Risperidone
PubMed: 19835667
DOI: 10.1017/S1461145709990782 -
FEBS Letters Dec 1995P-glycoprotein is a plasma-membrane glycoprotein which confers multidrug-resistance on cells and displays ATP-driven drug-pumping in vitro. It contains two... (Review)
Review
P-glycoprotein is a plasma-membrane glycoprotein which confers multidrug-resistance on cells and displays ATP-driven drug-pumping in vitro. It contains two nucleotide-binding domains, and its structure places it in the 'ABC transporter' family. We review recent evidence that both nucleotide-sites bind and hydrolyse Mg-ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high-energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Adenosine Triphosphatases; Adenosine Triphosphate; Binding Sites; Biological Transport; Drug Resistance, Multiple
PubMed: 8549739
DOI: 10.1016/0014-5793(95)01345-8 -
Advances in Pharmacology (San Diego,... 2014The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances... (Review)
Review
The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Blood-Brain Barrier; Endothelial Cells; Humans; Pharmaceutical Preparations; Protein Transport
PubMed: 25307213
DOI: 10.1016/bs.apha.2014.06.009 -
Acta Pharmacologica Sinica Jun 2012To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.
AIM
To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.
METHODS
Caco-2 cells were exposed to NO donors for designated times. P-gp function and expression were assessed using Rhodamine123 uptake assay and Western blotting, respectively. Intracellular reactive oxygen species (iROS) and intracellular reactive nitrogen species (iRNS) levels were measured using ROS and RNS assay kits, respectively.
RESULTS
Exposure of Caco-2 cells to 0.1 or 2 mmol/L of sodium nitroprusside (SNP) affected the function and expression of P-gp in concentration- and time-dependent manners. A short-term (4 h) exposure reduced P-gp function and expression accompanied with significantly increased levels of iROS and iRNS. In contrast, a long-term (24 h) exposure stimulated the P-gp function and expression. The stimulatory effects of 2 mmol/L SNP was less profound as compared to those caused by 0.1 mmol/L SNP. The other NO donors SIN-1 and SNAP showed similar effects. Neither the NO scavenger PTIO (2 mmol/L) nor soluble guanylate cyclase inhibitor ODQ (50 μmol/L) reversed the SNP-induced alteration of P-gp function. On the other hand, free radical scavengers ascorbate, glutathione and uric acid (2 mmol/L for each), PKC inhibitor chelerythrine (5 μmol/L), PI3K/Akt inhibitor wortmannin (1 μmol/L) and p38 MAPK inhibitor SB203580 (10 μmol/L) reversed the upregulation of P-gp function by the long-term exposure to SNP, but these agents had no effect on the impaired P-gp function following the short-term exposure to SNP.
CONCLUSION
NO donors time-dependently regulate P-gp function and expression in Caco-2 cells: short-term exposure impairs P-gp function and expression, whereas long-term exposure stimulates P-gp function and expression. The regulation occurs via a NO-independent mechanism.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Caco-2 Cells; Free Radical Scavengers; Gene Expression Regulation; Humans; Nitric Oxide Donors; Nitroprusside; Reactive Nitrogen Species; Reactive Oxygen Species; Signal Transduction
PubMed: 22543702
DOI: 10.1038/aps.2012.25 -
European Journal of Biochemistry Sep 2000The ABC superfamily of membrane transporters is one of the largest classes of proteins across all species and one of the most intensely researched. ABC proteins are... (Review)
Review
The ABC superfamily of membrane transporters is one of the largest classes of proteins across all species and one of the most intensely researched. ABC proteins are involved in the trafficking of a diverse variety of biological molecules across cell membranes, with some members implicated in medical syndromes such as cystic fibrosis and multidrug resistance to anti-cancer drugs. In the absence of X-ray crystallographic data, structural information has come from spectroscopy, electron microscopy, secondary structure prediction algorithms and residue substitution, epitope labelling and cysteine cross-linking studies. These have generally supported a model for the topology of the transmembrane domains of ABC transporters in which a single aqueous pore is formed by a toroidal ring of 12 alpha helices, deployed in two arcs of six helices each. Although this so-called 6 + 6 helix model can be arranged in either mirror or rotational symmetry configurations, experimental data supports the former. In this review, we put forward arguments against both configurations of this 6 + 6 helix model, based on what is known generally about symmetry relationships in proteins. We relate these arguments to P-glycoprotein, in particular, and discuss alternative models for the structure of ABC transporters in the light of the most recent research.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Animals; Models, Molecular; Protein Conformation
PubMed: 10951188
DOI: 10.1046/j.1432-1327.2000.01628.x -
Biochimica Et Biophysica Acta.... Apr 2018ABC (ATP binding cassette) transporters, ubiquitous in all kingdoms of life, carry out essential substrate transport reactions across cell membranes. Their transmembrane... (Comparative Study)
Comparative Study Review
ABC (ATP binding cassette) transporters, ubiquitous in all kingdoms of life, carry out essential substrate transport reactions across cell membranes. Their transmembrane domains bind and translocate substrates and are connected to a pair of nucleotide binding domains, which bind and hydrolyze ATP to energize import or export of substrates. Over four decades of investigations into ABC transporters have revealed numerous details from atomic-level structural insights to their functional and physiological roles. Despite all these advances, a comprehensive understanding of the mechanistic principles of ABC transporter function remains elusive. The human multidrug resistance transporter ABCB1, also referred to as P-glycoprotein (P-gp), is one of the most intensively studied ABC exporters. Using ABCB1 as the reference point, we aim to compare the dominating mechanistic models of substrate transport and ATP hydrolysis for ABC exporters and to highlight the experimental and computational evidence in their support. In particular, we point out in silico studies that enhance and complement available biochemical data. "This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain."
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Adenosine Triphosphate; Animals; Biological Transport; Humans; Models, Biological; Molecular Dynamics Simulation; Protein Binding; Protein Conformation
PubMed: 29097275
DOI: 10.1016/j.bbamem.2017.10.028 -
Molecules (Basel, Switzerland) Nov 2017Seven medicinal plants popularly used for treating malaria in West Africa were selected to assess herb-drug interaction potential through a series of in vitro methods....
Seven medicinal plants popularly used for treating malaria in West Africa were selected to assess herb-drug interaction potential through a series of in vitro methods. Fluorescent cytochrome P450 (CYP) assays were conducted using the recombinant CYP enzymes for CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 to assess the effect of the methanolic extracts on the metabolic activity of CYPs. Secondly, the inhibitory effect of the extracts was evaluated on P-glycoproteins (P-gp) using calcein-AM, a fluorescent substrate, in MDCK-II and hMDR1-MDCK-II cells. The inhibition of P-gp activity was determined as a reflection of increase in calcein-AM uptake. Additionally, the enzyme induction potential of the extracts was assessed through the modulation of PXR activity in HepG2 cells transiently transfected with pSG5-PXR and PCR5 plasmid DNA. Significant inhibition of CYP activity ( < 10 µg/mL) was observed with the following herbs: [CYP2C9, 3A4 and CYP2D6]; [CYP2C9]; [CYP2C9 and CYP2C19]; [CYP2C19, CYP2C9 and CYP3A4]; [CYP2C19 and CYP3A4]. Extracts of four herbs (, , and ) exhibited significant inhibition of P-gp with values (µg/mL) of 17 ± 1, 16 ± 0.4, 26 ± 1, and 24 ± 1, respectively. In addition, four herbs (, , and ) showed a >two-fold increase in induction in PXR activity. These findings suggest that these herbs may be capable of eliciting herb-drug interactions if consumed in high quantities with concomitant use of conventional therapies.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Antimalarials; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Enzyme Activation; Herb-Drug Interactions; Humans; Plant Extracts; Pregnane X Receptor; Receptors, Steroid
PubMed: 29168799
DOI: 10.3390/molecules22122049 -
Journal of Pharmacological Sciences 2010This study was conducted to determine the rate of P-glycoprotein (P-gp)-mediated efflux of digoxin analogues and metabolites and to assess the effects of macrolide...
This study was conducted to determine the rate of P-glycoprotein (P-gp)-mediated efflux of digoxin analogues and metabolites and to assess the effects of macrolide antibiotics on this efflux. Bidirectional transport studies were conducted using our Caco-2 sub clone with high P-gp expression (CLEFF9). HPLC methods were employed to measure drug transport. All digoxin metabolites were P-gp substrates, although digoxin had the greatest efflux ratio. Erythromycin had no effect on the transport of digoxin, maintaining a basolateral to apical efflux ratio of 14.8, although it did reduce the efflux ratio of dihydrodigoxin and digoxigenin by 34% and 43%, respectively. Azithromycin also had little effect on the transport of digoxin or any of its metabolites. In contrast, clarithromycin and roxithromycin almost eliminated basolateral targeted efflux. Using paclitaxel as a known P-gp substrate, erythromycin demonstrated only partial P-gp inhibitory capacity, maintaining an efflux ratio over 100. In contrast, clarithromycin and roxithromycin were 10-fold greater P-gp inhibitors. Clarithromycin and roxithromycin are likely to exhibit drug interactions with digoxin via inhibition of efflux mechanisms. Azithromycin appears to have little influence on P-gp-mediated digoxin absorption or excretion and would be the safest macrolide to use concurrently with oral digoxin.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Anti-Bacterial Agents; Caco-2 Cells; Chromatography, High Pressure Liquid; Digoxin; Humans; Limit of Detection; Macrolides
PubMed: 20724802
DOI: 10.1254/jphs.10109fp