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 ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

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 In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

scholarly journals Dissection of drug-binding-induced conformational changes in P-glycoprotein

1998 ◽  
Vol 255 (2) ◽  
pp. 383-390 ◽  
Author(s):  
Guichun Wang ◽  
Roxana Pincheira ◽  
Jian-Ting Zhang
Keyword(s):  
Conformational Changes ◽  
Drug Binding ◽  
P Glycoprotein

scholarly journals Computational Pharmacogenetics of P-Glycoprotein Mediated Antiepileptic Drug Resistance

2016 ◽  
Author(s):  
Ashok Palaniappan ◽  
Sindhu Varghese
Keyword(s):  
Drug Resistance ◽  
Treatment Failure ◽  
Conformational Changes ◽  
Point Mutations ◽  
Active Role ◽  
Docking Studies ◽  
Drug Binding ◽  
Experimental Investigations ◽  
Drug Bioavailability ◽  
P Glycoprotein

AbstractThe treatment of epilepsy using antiepileptogenic drugs is complicated by drug re-sistance, resulting in treatment failure in more than one-third of cases. Human P-glycoprotein (hPGP; MDR1) is a known epileptogenic mediator. Given that experimental investigations have suggested a role for pharmacogenetics in this treatment failure, it would be of interest to study hPGP polymorphisms that might contribute to the emergence of drug resistance. Changes in protein functional activity could result from point mutations as well as altered abundance. Bioinformatics approaches were used to assess and rank the functional impact of 20 missense MDR1 polymorphisms and the top five were selected. The structures of the wildtype and mutant hPGP were modelled based on the mouse PGP structure. Docking studies of the wildtype and mutant hPGP with four standard anti-epileptic drugs were carried out. Our results revealed that the drug binding site with respect to the wildtype protein was uniform. However the mutant hPGP proteins displayed a repertoire of binding sites with stronger binding affinities towards the drug. Our studies indicated that specific polymorphisms in MDR1 could drive conformational changes of PGP structure, facilitating altered contacts with drug-substrates and resulting in drug extrusion. This suggests that MDR1 polymorphisms could play an active role in modifying drug bioavailability, leading to pharmacoresistance in antiepileptic chemotherapy.

scholarly journals Computational Pharmacogenetics of P-Glycoprotein Mediated Antiepileptic Drug Resistance

2018 ◽  
Vol 11 (1) ◽  
pp. 197-207 ◽  
Author(s):  
Sindhu Varghese ◽  
Ashok Palaniappan
Keyword(s):  
Drug Resistance ◽  
Treatment Failure ◽  
Conformational Changes ◽  
Binding Sites ◽  
Docking Studies ◽  
Drug Binding ◽  
Experimental Investigations ◽  
Binding Affinities ◽  
Drug Binding Site ◽  
P Glycoprotein

Background:The treatment of epilepsy using antiepileptogenic drugs is complicated by drug resistance, resulting in treatment failure in more than one-third of cases. Human P-glycoprotein (hPGP;MDR1) is a known epileptogenic mediator.Methods:Given that experimental investigations have suggested a role for pharmacogenetics in this treatment failure, it would be of interest to study hPGP polymorphisms that might contribute to the emergence of drug resistance. Changes in protein functional activity could result from mutations as well as altered abundance. Bioinformatics approaches were used to assess and rank the functional impact of 20 missenseMDR1polymorphisms and the top five were selected. The structures of the wildtype and variant hPGP were modelled based on the mouse PGP structure. Docking studies of the wildtype and variant hPGP with four standard anti-epileptic drugs were carried out.Results:Our results revealed that the drug binding site with respect to the wildtype protein was uniform. However, the variant hPGP proteins displayed a repertoire of binding sites with stronger binding affinities towards the drug.Conclusion:Our studies indicated that specific polymorphisms inMDR1could drive conformational changes of PGP structure, facilitating altered contacts with drug-substrates and thus modifying their bioavailability. This suggests thatMDR1polymorphisms could actively contribute to the emergence of pharmaco-resistance in antiepileptic therapy.

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 Computational Insights into Allosteric Conformational Modulation of P-Glycoprotein by Substrate and Inhibitor Binding

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6006
Author(s):  
Juan Xing ◽  
Shuheng Huang ◽  
Yu Heng ◽  
Hu Mei ◽  
Xianchao Pan
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Molecular Design ◽  
Conformational Dynamics ◽  
Water Environment ◽  
Inhibitor Binding ◽  
Binding Domains ◽  
Allosteric Communication ◽  
P Glycoprotein ◽  
Dynamics Simulations

The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a physiologically essential membrane protein that protects many tissues against xenobiotic molecules, but limits the access of chemotherapeutics into tumor cells, thus contributing to multidrug resistance. The atomic-level mechanism of how substrates and inhibitors differentially affect the ATP hydrolysis by P-gp remains to be elucidated. In this work, atomistic molecular dynamics simulations in an explicit membrane/water environment were performed to explore the effects of substrate and inhibitor binding on the conformational dynamics of P-gp. Distinct differences in conformational changes that mainly occurred in the nucleotide-binding domains (NBDs) were observed from the substrate- and inhibitor-bound simulations. The binding of rhodamine-123 can increase the probability of the formation of an intermediate conformation, in which the NBDs were closer and better aligned, suggesting that substrate binding may prime the transporter for ATP hydrolysis. By contrast, the inhibitor QZ-Leu stabilized NBDs in a much more separated and misaligned conformation, which may result in the deficiency of ATP hydrolysis. The significant differences in conformational modulation of P-gp by substrate and inhibitor binding provided a molecular explanation of how these small molecules exert opposite effects on the ATPase activity. A further structural analysis suggested that the allosteric communication between transmembrane domains (TMDs) and NBDs was primarily mediated by two intracellular coupling helices. Our computational simulations provide not only valuable insights into the transport mechanism of P-gp substrates, but also for the molecular design of P-gp inhibitors.

scholarly journals Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter

2006 ◽  
Vol 399 (2) ◽  
pp. 315-323 ◽  
Author(s):  
Paula L. Russell ◽  
Frances J. Sharom
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Functional Characterization ◽  
Tryptophan Fluorescence ◽  
Fold Increase ◽  
Nucleotide Binding ◽  
Forward Direction ◽  
Multidrug Transporter ◽  
Bivalent Cation ◽  
P Glycoprotein

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (Vi) and fluoroaluminate (AlFx) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp–M2+–ADP–X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeFx) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and Vi, BeFx or AlFx using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with Vi and ATP led to a 6-fold increase in the acrylamide quenching constant, KSV, suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with Vi and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)–ATP/TNP–ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeFx-trapped complex was much more polar than that of the Vi and AlFx complexes. Functionally, all the trapped complexes were able to bind drugs and TNP–nucleotides with unchanged affinity compared with native Pgp.

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