Understanding polyspecificity within the substrate‐binding cavity of the human multidrug resistance P‐glycoprotein

Martinez, Lorena; Arnaud, Ophélie; Hénin, Émilie; Tao, Houchao; Chaptal, Vincent; Doshi, Rupak; Andrieu, Thibault; Dussurgey, Sébastien; Tod, Michel; Pietro, Attilio Di; Zhang, Qinghai; Chang, Geoffrey; Falson, Pierre

doi:10.1111/febs.12613

Cited by 63 publications

(38 citation statements)

References 32 publications

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“…The candidate residues identified using this approach were in broad agreement with those identified from docking and MD simulation based studies [20,34,35]. Another investigation using a large number of P-gp isoforms adopted a different approach using arginine enhancer mutations to identify residues within the translocation pathway [36].…”

Section: Discussionsupporting

confidence: 62%

Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

Mittra

Pavy

Subramanian

et al. 2017

Biochemical Pharmacology

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The multidrug resistance P-glycoprotein (P-gp) is characterised by the ability to bind and/or transport an astonishing array of drugs. This poly-specificity is imparted by at least four pharmacologically distinct binding sites within the transmembrane domain. Whether or not these sites are spatially distinct has remained unclear. Biochemical and structural investigations have implicated a central cavity as the likely location for the binding sites. In the present investigation, a number of contact residues that are involved in drug binding were identified through biochemical assays using purified, reconstituted P-gp. Drugs were selected to represent each of the four pharmacologically distinct sites. Contact residues important in rhodamine123 binding were identified in the central cavity of P-gp. However, contact residues for the binding of vinblastine, paclitaxel and nicardipine were located at the lipid-protein interface rather than the central cavity. A key residue (F978) within the central cavity is believed to be involved in coupling drug binding to nucleotide hydrolysis. Data observed in this investigation suggest the presence of spatially distinct drug binding sites connecting through to a single translocation pore in the central cavity.

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Section: Discussionsupporting

confidence: 62%

Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

Mittra

Pavy

Subramanian

et al. 2017

Biochemical Pharmacology

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“…The mouse embryonic fibroblasts, of either wild-type (NIH3T3) or overexpressing Pgp (NIH3T3 ABCB1 ) [39], were maintained under the same conditions. The cell culture media were drug supplemented with either 0.75 mg/mL G418 (HEK293 ABCG2 ), 200 μg/mL hygromycin B (HEK293 pcDNA5 and HEK293 ABCC1 ) or 60 ng/mL colchicin (NIH3T3 ABCB1 ).…”

Section: Methodsmentioning

confidence: 99%

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

et al. 2015

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In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.

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“…Figure 10D) although not precisely at the minima indicated in the PMFs. Studies by Lugo et al 37 and Martinez et al 38 have shown that the TM pore of P-gp is large enough to accommodate multiple substrates/molecules and thus it is plausible for both Hoechst 33342 and Rhodamine 123 to occupy the TM pore.…”

Section: The Effect Of Starting Structure On Substrate Bindingmentioning

confidence: 99%

“…In recent years, a number of computational approaches have been used in an attempt to characterize potential substrate binding sites in P-gp. These include molecular docking and pharmacophore mapping studies [36][37][38][39][40] . In two independent studies, Ferreira et al 36 and Tarcsay and Keserű 37 examined the interaction of various P-gp substrates such as Rhodamine 123, vinblastine, verapamil and Hoechst 33342 with P-gp using molecular docking studies.…”

Section: Introductionmentioning

confidence: 99%

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Understanding multidrug resistance: molecular dynamics studies of ligand recognition by P-glycoprotein

Subramanian¹

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Resistance to various chemotherapeutic agents can arise via a multitude of mechanisms, but one of the common mechanisms of multidrug resistance (MDR) results from the action of ATP-dependent multidrug efflux pumps. P-glycoprotein (P-gp) is the most extensively studied multidrug efflux pump. It is overexpressed in a variety of cancers and associated with the development of MDR. Despite extensive research over almost 40 years, the molecular mechanism by which P-gp transports drugs and other endogenous molecules has not been conclusively resolved.Over the years it has been proposed that different molecules have different binding sites in P-gp, but no specific binding sites, or any targeted inhibitors that prevent the transport of specific substrates, have been identified. In order to understand the molecular basis of substrate binding, molecular dynamics (MD) simulations have been performed to estimate the potential of mean force (PMF) for the process of substrate binding to P-gp. As a proof of concept, this approach was used to identify the binding locations of morphine and nicardipine within the transmembrane (TM) pore. It has been found that morphine and nicardipine bind at different but overlapping sites within the TM pore. The results indicate that their permeation pathways through the TM pore are not well separated. This set of PMF calculations was extended to include the canonical competitive and non-competitive P-gp substrates and inhibitors, Hoechst 33342, Rhodamine 123, paclitaxel, tariquidar and verapamil. The obtained PMF profiles show that all these molecules have an energy minimum within the TM pore. The interactions with a specific set of residues can be identified for each molecule in its minimum energy location. However, none of the molecules have a distinct, well-defined binding site. Instead, the binding locations overlap with several residues interacting with multiple P-gp substrates. This suggests that the binding locations for both competitive and non-competitive substrates are not well separated and cannot be considered as unique.Considering the lipophilic and/or amphipathic nature of many P-gp substrates, it has been suggested that P-gp effluxes drugs directly from the membrane. In addition, it has been observed that the presence of cholesterol increases the drug-stimulated ATPase activity of P-gp. However, whether this is due to the direct effect of cholesterol on the activity of P-gp, its effect on the local concentration of substrate in the membrane, or on the rate of entry of the drug into the cell, is currently unresolved. To understand better, the role of cholesterol in drug-membrane interactions, unrestrained and umbrella-sampling ii simulations examining the spontaneous binding and partitioning of four P-gp substrates into a POPC bilayer in the presence or absence of 10% cholesterol has been investigated.It was found that the presence of cholesterol lowers the free energy associated with entering the middle of the bilayer in a substrate-specific manner, suggesting that P-gp s...

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Understanding polyspecificity within the substrate‐binding cavity of the human multidrug resistance P‐glycoprotein

Cited by 63 publications

References 32 publications

Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

Understanding multidrug resistance: molecular dynamics studies of ligand recognition by P-glycoprotein

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