P-glycoprotein (ABCB1) interacts directly with lipid-based anti-cancer drugs and platelet-activating factorsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 1022-1033 ◽  
Author(s):  
Paul D.W. Eckford ◽  
Frances J. Sharom
Keyword(s):  
Atpase Activity ◽  
Lipid A ◽  
Atp Hydrolysis ◽  
Membrane Lipids ◽  
Binding Pocket ◽  
Drug Binding ◽  
Cancer Drugs ◽  
Outer Leaflet ◽  
P Glycoprotein ◽  
Anti Cancer

The P-glycoprotein multidrug transporter (Pgp; ABCB1) is an ATP-binding cassette (ABC) protein that has been implicated in the multidrug resistance of human cancers. Pgp couples ATP hydrolysis to active extrusion from the cell of a broad array of amphipathic compounds via an ill-defined mechanism. Substrates are believed to interact with Pgp within the membrane. Reconstituted Pgp functions as an ATP-dependent flippase for a variety of fluorescently labelled membrane lipids. The protein may also function as a drug ‘flippase’, moving its substrates from the inner to the outer leaflet of the bilayer. We show that lipid-based anti-cancer drugs, such as miltefosine, and signaling molecules, such as platelet-activating factors, bind saturably to Pgp with Kd values in the low micromolar range, and modulate its ATPase activity. These compounds also inhibit Pgp-mediated flipping of fluorescent lipids and transport of Hoechst 33342 and tetramethylrosamine, which occupy different subsites in the drug-binding pocket. Bacterial lipid A modulates Pgp ATPase activity, and glycolipid flipping is inhibited by unlabelled glucosylceramide, suggesting that these lipids also interact with the transporter. These results indicate that Pgp treats a variety of lipid-based molecules as substrates, and likely interacts with lipids and drugs in the same manner.

scholarly journals Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket

2006 ◽  
Vol 396 (3) ◽  
pp. 537-545 ◽  
Author(s):  
Tip W. Loo ◽  
M. Claire Bartlett ◽  
David M. Clarke
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Covalent Attachment ◽  
Acetoxymethyl Ester ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.

scholarly journals Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein

2020 ◽  
Vol 117 (47) ◽  
pp. 29609-29617
Author(s):  
Andaleeb Sajid ◽  
Sabrina Lusvarghi ◽  
Megumi Murakami ◽  
Eduardo E. Chufan ◽  
Biebele Abel ◽  
...  
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cancer Drug ◽  
Binding Domains ◽  
Cancer Drug Resistance ◽  
P Glycoprotein ◽  
Cell Transforming

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

scholarly journals Voruciclib, a Potent CDK4/6 Inhibitor, Antagonizes ABCB1 and ABCG2-Mediated Multi-Drug Resistance in Cancer Cells

2018 ◽  
Vol 45 (4) ◽  
pp. 1515-1528 ◽  
Author(s):  
Pranav Gupta ◽  
Yun-Kai Zhang ◽  
Xiao-Yu Zhang ◽  
Yi-Jun Wang ◽  
Kimberly W. Lu ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Cellular Localization ◽  
Intracellular Localization ◽  
Scintillation Counter ◽  
Flow Cytometric Analysis ◽  
Multi Drug Resistance ◽  
Dependent Manner ◽  
Cancer Drugs ◽  
Intracellular Accumulation ◽  
Anti Cancer

Background/Aims: The overexpression of ATP-Binding Cassette (ABC) transporters has known to be one of the major obstacles impeding the success of chemotherapy in drug resistant cancers. In this study, we evaluated voruciclib, a CDK 4/6 inhibitor, for its chemo-sensitizing activity in ABCB1- and ABCG2- overexpressing cells. Methods: Cytotoxicity and reversal effect of voruciclib was determined by MTT assay. The intracellular accumulation and efflux of ABCB1 and ABCG2 substrates were measured by scintillation counter. The effects on expression and intracellular localization of ABCB1 and ABCG2 proteins were determined by Western blotting and immunofluorescence, respectively. Vanadate-sensitive ATPase assay was done to determine the effect of voruciclib on the ATPase activity of ABCB1 and ABCG2. Flow cytometric analysis was done to determine the effect of voruciclib on apoptosis of ABCB1 and ABCG2-overexpressing cells and docking analysis was done to determine the interaction of voruciclib with ABCB1 and ACBG2 protein. Results: Voruciclib significantly potentiated the effect of paclitaxel and doxorubicin in ABCB1-overexpressing cells, as well as mitoxantrone and SN-38 in ABCG2-overexpressing cells. Voruciclib moderately sensitized ABCC10- overexpressing cells to paclitaxel, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, voruciclib increased the intracellular accumulation and decreased the efflux of substrate anti-cancer drugs from ABCB1- or ABCG2-overexpressing cells. However, voruciclib did not alter the expression or the sub-cellular localization of ABCB1 or ABCG2. Voruciclib stimulated the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner. Lastly, voruciclib exhibited a drug-induced apoptotic effect in ABCB1- or ABCG2- overexpressing cells. Conclusion: Voruciclib is currently a phase I clinical trial drug. Our findings strongly support its potential use in combination with conventional anti-cancer drugs for cancer chemotherapy.

scholarly journals Substrate Specificity of Aglaia loheri Active Isolate towards P-glycoprotein in Multidrug-Resistant Cancer Cells

2016 ◽  
Vol 11 (11) ◽  
pp. 1934578X1601101
Author(s):  
Else Dapat ◽  
Sonia Jacinto ◽  
Thomas Efferth
Keyword(s):  
Substrate Specificity ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Multidrug Resistant ◽  
Contributory Factor ◽  
Acetoxymethyl Ester ◽  
P Glycoprotein ◽  
Drug Concentrations

Multidrug resistance (MDR) is a major contributory factor in the failure of chemotherapy. Concrete interpretation of P-glycoprotein (P-gp) substrate specificity, whether a substance is a substrate or an inhibitor, represents an important feature of a compound's pharmaceutical profiling in drug design and development. In this work, the P-gp substrate specificity of Maldi 531.2[M+H]+, a phenol ester from Aglaia loheri Blanco leaves was investigated. This study focuses on the effect of Maldi 531.2[M+H]+ on P-gp ATPase activity, which was examined by measuring the amount of inorganic phosphates (Pi) released as a result of ATP hydrolysis. To test the effects of Maldi 531.2[M+H]+ on MDR activity, an attempt to combine Maldi 531.2[M+H]+ with a potent P-gp substrate such as verapamil was performed. As a result of this combination treatment, two distinct patterns of interaction with P-gp activity were determined by a calcein-acetoxymethyl ester (AM) assay. Depending on the concentratgion, both stimulation and inhibition of MDR activity were observed at certain drug concentrations suggesting biphasic reactions, which can be understood as cooperative stimulation and competitive inhibition, respectively. Verapamil is a strong substrate to P-gp. Substrate specificity of Maldi 531.2[M+H]+ may be less than the substrate specificity of verapamil, but it acts additively together with low concentrations of verapamil in stimulating ATPase activity. On the one hand, verapamil and Maldi 531.2[M+H]+ exerted cooperative stimulation on P-gp. On the other hand, Maldi 531.2[M+H]+ acts as competitive inhibitor at higher concentrations.

scholarly journals Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

2016 ◽  
Vol 36 (2) ◽  
Author(s):  
Kaitlyn V. Ledwitch ◽  
Robert W. Barnes ◽  
Arthur G. Roberts
Keyword(s):  
Drug Interactions ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Mechanistic Model ◽  
Cardiovascular Drugs ◽  
Drug Binding ◽  
P Glycoprotein ◽  
Complex Drug

P-glycoprotein (Pgp) plays a major role in promoting drug–drug interactions (DDIs) with verapamil and digoxin. In the present study, we present a comprehensive molecular and mechanistic model of Pgp DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes.

scholarly journals Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

2006 ◽  
Vol 401 (2) ◽  
pp. 597-605 ◽  
Author(s):  
Yasuhisa Kimura ◽  
Noriyuki Kioka ◽  
Hiroaki Kato ◽  
Michinori Matsuo ◽  
Kazumitsu Ueda
Keyword(s):  
Atpase Activity ◽  
Molecular Mass ◽  
Binding Site ◽  
Drug Binding ◽  
Soluble Form ◽  
Strongly Correlated ◽  
Drug Binding Site ◽  
Hydrophobic Compounds ◽  
P Glycoprotein ◽  
Human Mdr1

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug–MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on Km values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). Vmax values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

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