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David M Clarke - One of the best experts on this subject based on the ideXlab platform.
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Transmembrane segment 7 of human P-glycoprotein forms part of the Drug-Binding pocket.
Biochemical Journal, 2006Co-Authors: M. Claire Bartlett, David M ClarkeAbstract:P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the Drug-Binding pocket is important for understanding the mechanism of transport. The common Drug-Binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537–545] that the first transmembrane segment (TM1) contributed to the Drug-Binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS–verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to Drug Binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the Drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS–verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS–verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the Drug-Binding pocket. Disulfide cross-linking was inhibited by some Drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the Drug-Binding pocket of P-gp.
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Transmembrane segment 1 of human P-glycoprotein contributes to the Drug-Binding pocket
Biochemical Journal, 2006Co-Authors: M. Claire Bartlett, David M ClarkeAbstract: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.
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Do Drug substrates enter the common Drug-Binding pocket of P-glycoprotein through “gates”?
Biochemical and Biophysical Research Communications, 2005Co-Authors: David M ClarkeAbstract:Abstract Overexpression of P-glycoprotein (P-gp; ABCB1) can cause multiDrug resistance during cancer and AIDS chemotherapy because of its ability to transport a broad range of structurally unrelated compounds from the cell. P-gp is a member of the ABC family of proteins. It is a single polypeptide containing four domains—two transmembrane (TM) domains each of which contains six TM segments and two nucleotide-Binding domains. Chemical modification and cross-linking studies of cysteine mutants of P-gp indicate that the common Drug-Binding pocket is at the interface between the TM domains. It has been postulated that Drug substrates enter the lipid bilayer, are extracted by P-gp and transported to the extracellular medium. It is not clear how Drug substrates enter the Drug-Binding pocket. Here, we propose that Drug-substrates diffuse from the lipid bilayer into the Drug-Binding pocket through “gates” formed by TM segments at either end of the Drug-Binding pocket.
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The Drug-Binding pocket of the human multiDrug resistance P-glycoprotein is accessible to the aqueous medium.
Biochemistry, 2004Co-Authors: M. Claire Bartlett, David M ClarkeAbstract:: P-Glycoprotein (P-gp) is an ATP-dependent Drug pump that transports a broad range of compounds out of the cell. Cross-linking studies have shown that the Drug-Binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different Drug substrates. Here, we determined whether cysteine residues within the Drug-Binding pocket were accessible to the aqueous medium. Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and [2-(trimethylammonium)ethyl)]methanethiosulfonate (MTSET). Residue Ile-306(TM5) is close to the verapamil-Binding site. It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity. Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil. We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid. Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine. MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the Drug-Binding pocket. For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10). Inhibition was enhanced by ATP hydrolysis. By contrast, cross-linking of cysteine residues located outside the Drug-Binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET. These results indicate that the Drug-Binding pocket is accessible to water.
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Permanent activation of the human P-glycoprotein by covalent modification of a residue in the Drug-Binding site.
Journal of Biological Chemistry, 2003Co-Authors: M. Claire Bartlett, David M ClarkeAbstract:Abstract The human multiDrug resistance P-glycoprotein (ABCB1) transports a broad range of structurally diverse compounds out of the cell. The transport cycle involves coupling of Drug Binding in the transmembrane domains with ATP hydrolysis. Compounds such as verapamil stimulate ATPase activity. We used cysteine-scanning mutagenesis of the transmembrane segments and reaction with the thiol-reactive substrate analog of verapamil, methanethiosulfonate (MTS)-verapamil, to test whether it caused permanent activation of ATP hydrolysis. Here we report that one mutant, I306C(TM5) showed increased ATPase activity (8-fold higher than untreated) when treated with MTS-verapamil and isolated by nickel-chelate chromatography. Drug substrates that either enhance (calcein acetoxymethyl ester, demecolcine, and vinblastine) or inhibit (cyclosporin A and trans-(E)-flupentixol) ATPase activity of Cys-less or untreated mutant I306C P-glycoprotein did not affect the activity of MTS-verapamil-treated mutant I306C. Addition of dithiothreitol released the covalently attached verapamil, and ATPase activity returned to basal levels. Pretreatment with substrates such as cyclosporin A, demecolcine, verapamil, vinblastine, or colchicine prevented activation of mutant I306C by MTS-verapamil. The results suggest that MTS-verapamil reacts with I306C in a common Drug-Binding site. Covalent modification of I306C affects the long range linkage between the Drug-Binding site and the distal ATP-Binding sites. This results in the permanent activation of ATP hydrolysis in the absence of transport. Trapping mutant I306C in a permanently activated state indicates that Ile-306 may be part of the signal to switch on ATP hydrolysis when the Drug-Binding site is occupied.
J Zhao - One of the best experts on this subject based on the ideXlab platform.
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screening of type i and ii Drug Binding to human cytochrome p450 3a4 in nanodiscs by localized surface plasmon resonance spectroscopy
Analytical Chemistry, 2009Co-Authors: J Zhao, George C Schatz, Stephen G Sligar, Richard P Van DuyneAbstract:A prototype nanoparticle biosensor based on localized surface plasmon resonance (LSPR) spectroscopy was developed to detect Drug Binding to human membrane-bound cytochrome P450 3A4 (CYP3A4). CYP3A4 is one of the most important enzymes in Drug and xenobiotic metabolism in the human body. Because of the inherent propensity of CYP3A4 to aggregate, it is difficult to study Drug Binding to this protein in solution and on surfaces. In this paper, we use a soluble nanometer scale membrane bilayer disk (Nanodisk) to functionally stabilize monomeric CYP3A4 on Ag nanoparticle surfaces fabricated by nanosphere lithography. CYP3A4-Nanodiscs have absorption bands in the visible wavelength region, which upon Binding certain Drugs shift to either shorter (type I) or longer wavelengths (type II). On the basis of the coupling between the LSPR of the Ag nanoparticles and the electronic resonances of the heme chromophore in CYP3A4-Nanodiscs, LSPR spectroscopy is used to detect Drug Binding with high sensitivity. This paper ...
Stephen Curry - One of the best experts on this subject based on the ideXlab platform.
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structural basis of the Drug Binding specificity of human serum albumin
Journal of Molecular Biology, 2005Co-Authors: J Ghuman, Masaki Otagiri, P A Zunszain, I Petitpas, A A Bhattacharya, Stephen CurryAbstract:Human serum albumin (HSA) is an abundant plasma protein that binds a remarkably wide range of Drugs, thereby restricting their free, active concentrations. The problem of overcoming the Binding affinity of lead compounds for HSA represents a major challenge in Drug development. Crystallographic analysis of 17 different complexes of HSA with a wide variety of Drugs and small-molecule toxins reveals the precise architecture of the two primary Drug-Binding sites on the protein, identifying residues that are key determinants of Binding specificity and illuminating the capacity of both pockets for flexible accommodation. Numerous secondary Binding sites for Drugs distributed across the protein have also been identified. The Binding of fatty acids, the primary physiological ligand for the protein, is shown to alter the polarity and increase the volume of Drug site 1. These results clarify the interpretation of accumulated Drug Binding data and provide a valuable template for design efforts to modulate the interaction with HSA.
Ralph Weissleder - One of the best experts on this subject based on the ideXlab platform.
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abstract 1968 nanoparticle mediated measurement of target Drug Binding in cancer cells
Cancer Research, 2012Co-Authors: Adeeti V Ullal, Thomas Reiner, Katherine S Yang, Rostic Gorbatov, Changwook Min, David Issadore, Hakho Lee, Ralph WeisslederAbstract:Responses to molecularly targeted therapies can be highly variable and depend on mutations, fluctuations in target protein levels in individual cells and Drug delivery. The ability to rapidly quantitate Drug response in cells harvested from patients in a point-of-care setting would have far reaching implications. Capitalizing on recent developments with miniaturized diagnostic NMR (DMR) technologies, we have developed a magnetic nanoparticle based approach to directly measure both target expression and Drug Binding in freshly harvested human cancer cells. The method involves covalent conjugation of a small molecule Drug to a magnetic nanoparticle that is then used as a read-out for target expression and Drug Binding affinity. Using poly(ADP-ribose) polymerase (PARP) inhibition as a model system, we developed an approach to distinguish differential expression of PARP across various cell lines with excellent correlation of DMR measurements to gold standards such as flow cytometry (r 2 = 0.97) and western blotting (r 2 = 0.92). We also sought to mimic Drug pharmacodynamics ex-vivo through competitive target-Drug Binding and could quantify the relative Binding affinities of several PARP inhibitors (e.g. Olaparib, Velaparib, AG-014699, and 3-aminobenzamide) in whole cells. Finally, we demonstrate the potential to perform such measurements directly in clinical samples. Further applications of the assay could result in a Drug development platform to probe Drug Binding in cells, the identification of resistant cancer cells, and the ability to determine whether a Drug has reached its target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1968. doi:1538-7445.AM2012-1968
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nanoparticle mediated measurement of target Drug Binding in cancer cells
ACS Nano, 2011Co-Authors: Adeeti V Ullal, Thomas Reiner, Katherine S Yang, Rostic Gorbatov, Changwook Min, David Issadore, Hakho Lee, Ralph WeisslederAbstract:Responses to molecularly targeted therapies can be highly variable and depend on mutations, fluctuations in target protein levels in individual cells, and Drug delivery. The ability to rapidly quantitate Drug response in cells harvested from patients in a point-of-care setting would have far reaching implications. Capitalizing on recent developments with miniaturized NMR technologies, we have developed a magnetic nanoparticle-based approach to directly measure both target expression and Drug Binding in scant human cells. The method involves covalent conjugation of the small-molecule Drug to a magnetic nanoparticle that is then used as a read-out for target expression and Drug-Binding affinity. Using poly(ADP-ribose) polymerase (PARP) inhibition as a model system, we developed an approach to distinguish differential expression of PARP in scant cells with excellent correlation to gold standards, the ability to mimic Drug pharmacodynamics ex vivo through competitive target–Drug Binding, and the potential to ...
Masaki Otagiri - One of the best experts on this subject based on the ideXlab platform.
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A Site-Directed Mutagenesis Study of Drug-Binding Selectivity in Genetic Variants of Human α1-Acid Glycoprotein
Journal of Pharmaceutical Sciences, 2009Co-Authors: Koji Nishi, Yuka Murakami, Naoko Fukunaga, Daisuke Kadowaki, Ayaka Suenaga, Teruo Akuta, Megumi Ueno, Hiroshi Watanabe, Toru Maruyama, Masaki OtagiriAbstract:Human α1-acid glycoprotein (AGP), a major carrier of many basic Drugs in circulation, consists of at least two genetic variants, namely A and F1*S variant. Interestingly, the variants of AGP have different Drug-Binding properties. The purpose of this study was to identify the amino acid residues that are responsible for the selectivity of Drug Binding to genetic variants of AGP using site-directed mutagenesis. First, we screened amino acid residues in the region proximal to position 100 that are involved in Binding of warfarin and dipyridamole, which are F1*S-specific ligands, and of propafenone, which is an A-specific ligand, using ultrafiltration. In the F1*S variant, His97, His100, and Trp122 were involved in either warfarin- or dipyridamole-Binding, while Glu92, His100, and Trp122 participated in the Binding of propafenone in the A variant. Exchange of the residue at position 92 between AGP variants reversed the relative strength of propafenone Binding to the two variants, but had a markedly different effect on Binding of warfarin and dipyridamole. These findings indicate that the amino acid residue at position 92 plays a significant role in Drug-Binding selectivity in AGP variants, especially for Drugs that preferentially bind to the A variant. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4316–4326, 2009
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structural basis of the Drug Binding specificity of human serum albumin
Journal of Molecular Biology, 2005Co-Authors: J Ghuman, Masaki Otagiri, P A Zunszain, I Petitpas, A A Bhattacharya, Stephen CurryAbstract:Human serum albumin (HSA) is an abundant plasma protein that binds a remarkably wide range of Drugs, thereby restricting their free, active concentrations. The problem of overcoming the Binding affinity of lead compounds for HSA represents a major challenge in Drug development. Crystallographic analysis of 17 different complexes of HSA with a wide variety of Drugs and small-molecule toxins reveals the precise architecture of the two primary Drug-Binding sites on the protein, identifying residues that are key determinants of Binding specificity and illuminating the capacity of both pockets for flexible accommodation. Numerous secondary Binding sites for Drugs distributed across the protein have also been identified. The Binding of fatty acids, the primary physiological ligand for the protein, is shown to alter the polarity and increase the volume of Drug site 1. These results clarify the interpretation of accumulated Drug Binding data and provide a valuable template for design efforts to modulate the interaction with HSA.
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Species Differences of Serum Albumins: I. Drug Binding Sites
Pharmaceutical Research, 1997Co-Authors: Takamitsu Kosa, Toru Maruyama, Masaki OtagiriAbstract:Purpose. The purpose of this study was the classification and identification of Drug Binding sites on albumins from several species in order to understand species differences of both Drug Binding properties and Drug interaction on protein Binding. Methods. Binding properties and types of Drug-Drug interaction on the different albumins were examined using typical site I Binding Drugs, warfarin (WF) and phenylbutazone (PBZ), and site II Binding Drugs, ibuprofen (IP) and diazepam (DZ) on human albumin. Equilibrium dialysis was carried out for two Drugs and the free concentrations of Drugs were then treated using the methods of Kragh-Hansen (Mol. Pharmacol. 34. 160−171, (1988)). Results. Binding affinities of site I Drugs to bovine, rabbit and rat albumins were reasonably similar to human albumin. However, interestingly, those to dog albumin were considerably smaller than human albumin. On the other hand, Binding parameters of DZ to bovine, rabbit and rat albumins were apparently different from those of human albumin. These differences are best explained by microenvironmental changes in the Binding sites resulting from change of size and/or hydrophobicity of the Binding pocket, rather than a variation in amino acid residues. Conclusions. We will propose herein that mammalian serum albumins used in this study contain specific Drug Binding sites: Rabbit and rat albumins contain a Drug Binding site, corresponding to site I on human albumin, and dog albumin contains a specific Drug Binding site corresponding to site II on the human albumin molecule.
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Characterization of Drug Binding Sites on α1-Acid Glycoprotein
Chemical & Pharmaceutical Bulletin, 1990Co-Authors: Toru Maruyama, Masaki Otagiri, Akira TakadateAbstract:The classification of Drug Binding sites on α1-acid glycoprotein (AGP) was studied by displacement experiments using fluorescent probes. Basic Drugs not only displaced basic probes strongly but also acidic probes as well. Acidic probes, on the other hand, were displaced by some acidic Drugs such as phenylbutazone and sulfadimethoxine which had no effect on most of the basic probes. This contradiction suggests that the basic Drugs do not completely share a Binding site with the acidic Drugs. The polarity of the basic Drug Binding site was higher than that of the acidic Drug Binding site. The negative charges were probably located in or near the former, different from the latter. The basic Drug Binding site was more sensitive to the conformational change of AGP. It seems that there are particular Drug bindign sites on the AGP molecule for acidic and basic Drugs. However, all the displacement data do not fully support the possibility of two independent Drug Binding sites. Therefore, it is rather reasonable to consider that these sites are not completely separated but are significantly overlapped and influenced by each other. Accordingly, AGP seems to have one wide and flexible Drug Binding area.
