P-glycoprotein structure and evolutionary homologies (original) (raw)
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Molecular analysis of the multidrug transporter, P-glycoprotein
Cytotechnology, 1998
Inherent or acquired resistance of tumor cells to cytotoxic drugs represents a major limitation to the successful chemotherapeutic treatment of cancer. During the past three decades dramatic progress has been made in the understanding of the molecular basis of this phenomenon. Analyses of drug-selected tumor cells which exhibit simultaneous resistance to structurally unrelated anti-cancer drugs have led to the discovery of the human MDR1 gene product, P-glycoprotein, as one of the mechanisms responsible for multidrug resistance. Overexpression of this 170 kDa N-glycosylated plasma membrane protein in mammalian cells has been associated with ATP-dependent reduced drug accumulation, suggesting that P-glycoprotein may act as an energy-dependent drug efflux pump. P-glycoprotein consists of two highly homologous halves each of which contains a transmembrane domain and an ATP binding fold. This overall architecture is characteristic for members of the ATP-binding cassette or ABC superfami...
The P-glycoprotein multidrug transporter
General Pharmacology: The Vascular System, 1996
1. P-glycoprotein (P-gp) is a transmembrane protein involved in ATP-dependent efflux of various structurally unrelated anticancer drugs. Its overexpression in cancer cells decreases intracellular drug concentrations and, thus, confers a multidrug resistance phenotype.
P-Glycoprotein Efflux Transporters and Its Resistance Its Inhibitors and Therapeutic Aspects
Creatinine - A Comprehensive Update [Working Title], 2020
P-glycoprotein (P-gp) is an active member of the ATP Binding Cassette (ABC) protein subfamily which effluxes a wide range of therapeutic drugs out of the cells commonly known as multidrug resistance. But its protective action towards the normal cells and efflux of the toxic and foreign substances is remarkable. Hence the efflux of the P-gp is a crucial step to overcome for the success of the therapy and in the drug discovery process. Modification of the action of the P-gp through various inducers, inhibitors or the genetic polymorphism is the commonly used methods. When it comes to the inhibitor part the natural inhibitors use is more safe and economical as compared to the synthetic ones. Here we review at the mechanism of action and the pharmacokinetic profile of P-gp, how the P-gp engaged in the Multidrug resistance, the strategy to overcome from its action by using natural inhibitors and formulation perspectives.
Proceedings of the National Academy of Sciences, 1992
Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.
Biochemistry, 1998
P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer drugs. Biochemical and genetic studies with Pgp have identified the putative transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved among Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specificity, seven amino acid residues were identified within TM 12 that were not strictly conserved among the MDR1 and MDR2 family of proteins from different mammalian species. We replaced all seven of these amino acid residues with alanine, one at a time and in combinations, and used a vaccinia virus based transient expression system to analyze function. None of the single replacements caused any alteration in transport function. However, when residues L975, V981, and F983 were replaced collectively, drug transport, drug-stimulated ATP hydrolysis, and photoaffinity labeling with the drug analogue, [ 125 I]iodoarylazidoprazosin (IAAP), were abrogated, with little effect on [R-32 P]-8-azido-ATP labeling and basal ATPase activity. Pairwise alanine substitutuions showed variable effects on function. Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine substitutions of all four nonconserved residues M986, V988, Q990, and V991 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extrusion. These results suggest that nonconserved residues in the putative aminoproximal half of TM 12 of Pgp play a more direct role in determining specificity of drug transport function than those in the putative carboxy-terminal half of TM 12. † Peter Hafkemeyer is the recipient of a grant from the Deutsche Forschungsgemeinschaft (DFG), Germany.
P-glycoprotein and multidrug resistance
Current Opinion in Genetics & Development, 1996
Comwell MM, Gottesman MM, Pastan I: Increased vinblastine binding to membrane vesicles from multidrug resistant KB cells. I Biol Chem 1966, 262:7921-7928. Bruggemann EP, Currier SJ, Gottesman MM, Pastan I: Characterization of the azidopine and vinblastine binding site of P-glycoprotein. I Biol Chem 1992, 267:21020-21026. Ambudkar SV: Purification and reconstitution of functional human P-glycoprotein. J Bioenerg Biomembr 1995, 27:23-29. Roepe PD, Wei LY, Cruz J, Carlson D: Lower electrical membrane potential and altered pHi homeostasis in multidrugresistant (MDR) cells: further characterization of a series of MDR cell lines expressing different levels of P-glycoprotein. Biochemistry 1993, 32:11042-l 1056. Weaver JL, Szabo G, Pine PS, Gottesman MM, Goldenberg S, Aszalos A: The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multi-drug transporter. Int I Cancer 1993, 54:456-461. Shapiro AB, Ling V: Reconstitution of drug transport by purified P-glycoprotein. J B/o/ Chem 1995, 270:16167-l 6175. Hrycyna CA, Zhang S, Ramachandra M, Ni B, Pastan I, Gottesman MM: Functional and molecular characterization of the human multidrug transporter. In Multidrug Resistance in Cancer Cells: Cellular, Biochemical, Molecular and Biological Aspects. Edited by Gupta S, Tsuruo T. New York: John Wiley and Sons; 1996:29-37. Ramachandra M, Ambudkar SV, Pastan I, Gottesman MM, Hrycyna CA: Functional characterization of a glycine 165 to valine substitution in human P-glycoprotein using a vaccinia based transient expression system. MO/ Biol Cell 1996, in press.
Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein
Proceedings of the National Academy of Sciences, 2020
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 t...
Journal of Biological Chemistry, 2016
Edited by Norma Allewell P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-andclose motion alters the surface topology of P-gp within the drugbinding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.
5 Towards P-Glycoprotein StructureActivity Relationships
ABC ATP-binding cassette (transport protein) MDR Multidrug resistance Pgp P-Glycoprotein (MDR1) (Q)SAR (Quantitative) structure activity relationship TM Transmembrane (domain) H-bonding Hydrogen bonding Symbols A Strong H-bond acceptor (or strong electron donor) a Weak H-bond acceptor (or weak electron donor) ∆G aw Free Energy of partitioning into the air-water interface ∆G lw Free Energy of drug partitioning into the lipid membrane ∆G t Free Energy of drug binding to the transporter Pgp from within the lipid phase ∆G app Apparent Free Energy of binding to Pgp from the aqueous phase
PLoS ONE, 2013
P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell-and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [ 125 I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.