Human Multidrug Transporter P-glycoprotein Research Articles

Membrane-assisted tariquidar access and binding mechanisms of human ATP-binding cassette transporter P-glycoprotein.

The human multidrug transporter P-glycoprotein (P-gp) is physiologically essential and of key relevance to biomedicine. Recent structural studies have shed light on the mode of inhibition of the third-generation inhibitors for human P-gp, but the molecular mechanism by which these inhibitors enter the transmembrane sites remains poorly understood. In this study, we utilized all-atom molecular dynamics (MD) simulations to characterize human P-gp dynamics under a potent inhibitor, tariquidar, bound condition, as well as the atomic-level binding pathways in an explicit membrane/water environment. Extensive unbiased simulations show that human P-gp remains relatively stable in tariquidar-free and bound states, while exhibiting a high dynamic binding mode at either the drug-binding pocket or the regulatory site. Free energy estimations by partial nudged elastic band (PNEB) simulations and Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method identify two energetically favorable binding pathways originating from the cytoplasmic gate with an extended tariquidar conformation. Interestingly, free tariquidar in the lipid membrane predominantly adopts extended conformations similar to those observed at the regulatory site. These results suggest that membrane lipids may preconfigure tariquidar into an active ligand conformation for efficient binding to the regulatory site. However, due to its conformational plasticity, tariquidar ultimately moves toward the drug-binding pocket in both pathways, explaining how it acts as a substrate at low concentrations. Our molecular findings propose a membrane-assisted mechanism for the access and binding of the third-generation inhibitors to the binding sites of human P-gp, and offer deeper insights into the molecule design of more potent inhibitors against P-gp-mediated drug resistance.

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

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.

Probing the Pharmacological Binding Sites of P-Glycoprotein Using Umbrella Sampling Simulations.

The human multidrug transporter P-glycoprotein (P-gp) transports over 200 chemically diverse substrates, influencing their bioavailability and tissue distribution. Pharmacological studies have identified both competitive and noncompetitive P-gp substrates, but neither the precise location of the substrate binding sites, nor the basis of competitive and noncompetitive interactions has been fully characterized. Here, potential of mean force (PMF) calculations are used to identify the transport-competent minimum free energy binding locations of five compounds, Hoechst 33342, Rhodamine 123, paclitaxel, tariquidar, and verapamil to P-gp. Unrestrained molecular dynamics simulations were also performed to confirm the substrates were stable in the energy wells determined using the PMF calculations. All compounds had energy minima within the P-gp transmembrane (TM) pore. For Hoechst 33342 and Rhodamine 123, a second minimum outside the TM pore was also identified. Based on this and previous studies of nicardipine and morphine [ Subramanian et al. J. Chem. Inf. Model. 2015 , 55 , 1202 ], a general scheme that accounts for the observed noncompetitive and competitive substrate interactions with P-gp is proposed.

The human P-glycoprotein transporter enhances the type I interferon response to Listeria monocytogenes infection.

Human multidrug efflux transporters are known for their ability to extrude antibiotics and toxic compounds out of cells, yet accumulating data indicate they have additional functions in diverse physiological processes not related to drug efflux. Here, we show that the human multidrug transporter P-glycoprotein (P-gp) (also named MDR1 and ABCB1) is transcriptionally induced in the monocytic cell line THP-1 upon infection with the human intracellular bacterial pathogen Listeria monocytogenes. Notably, we found that P-gp is important for full activation of the type I interferon response elicited against L. monocytogenes bacteria. Both inhibition of P-gp function by verapamil and inhibition of its transcription using mRNA silencing led to a reduction in the magnitude of the type I response in infected cells. This function of P-gp was specific to type I interferon cytokines elicited against cytosolic replicating bacteria and was not observed in response to cyclic di-AMP (c-di-AMP), a molecule that was shown to be secreted by L. monocytogenes during infection and to trigger type I interferons. Moreover, P-gp was not involved in activation of other proinflammatory cytokines, such as those triggered by vacuolar-restricted L. monocytogenes or lipopolysaccharide (LPS). Taken together, these findings demonstrate a role for P-gp in proper development of an innate immune response against intracellular pathogens, highlighting the complexity in employing therapeutic strategies that involve inhibition of multidrug resistance (MDR) efflux pumps.

Evidence for Modulatory Sites at the Lipid–Protein Interface of the Human Multidrug Transporter P-Glycoprotein

The human multidrug transporter P-glycoprotein (Pgp or ABCB1) sets up pharmacological barriers to many clinically important drugs, a therapeutic remedy for which has yet to be formulated. For the rational design of mechanism-based inhibitors (or modulators), it is necessary to map the potential sites for modulator interaction and understand their modes of communication with the other functional domains of Pgp. In this study, combining directed mutagenesis with homology modeling, we provide evidence of two modulator-specific sites at the lipid protein interface of Pgp. Targeting 21 variant positions in the COOH-terminal transmembrane (TM) regions, we find residues M948 (in TM11) and F983, M986, V988, and Q990 (all four in TM12) critically involved in substrate-site modulation by a thioxanthene-based allosteric modulator cis-(Z)-flupentixol. Interestingly, for ATP-site modulation by the same modulator, only two (M948 and Q990) of those four residues appear indispensable, together with two additional residues, T837 and I864 in TM9 and TM10, respectively, suggesting independent modes of communication linking the allosteric site with the substrate binding and ATPase domains. None of the seven residues identified prove to be critical for modulation of the substrate or ATP sites by Pgp modulators that are transported by the pump, such as cyclosporin A or verapamil, indicating their specificity for cis-(Z)-flupentixol. On the other hand, ATP-site modulation by verapamil proves to be highly sensitive to replacement at positions F716 (in TM7) and I765 (in TM8), and to a more moderate extent at I764 and L772 (both in TM8). Homology modeling based on the known crystal structures of the bacterial multidrug transporter SAV1866 and the mouse Pgp homologue maps the identified residues primarily at the lipid-protein interface of Pgp, in two spatially distinct modulator-specific clusters. The two modulatory sites demonstrate negative synergism in influencing ATP hydrolysis, consolidating their spatial distinctness. Because Pgp is known to recruit drug molecules directly from the lipid bilayer, identification of modulatory sites at the lipid-protein interface and at the same time outside the conventional central drug binding cavity is mechanistically revealing.

Bivalent Probes of the Human Multidrug Transporter P-Glycoprotein

A small library of bivalent agents was designed to probe the substrate binding sites of the human multidrug transporter P-glycoprotein (P-gp). The bivalent agents were composed of two copies of the P-gp substrate emetine, linked by tethers of varied composition. An optimum distance between the emetine molecules of approximately 10 A was found to be necessary for blocking transport of the known fluorescent substrate rhodamine 123. Additionally, it was determined that hydrophobic tethers were optimal for bridging the bivalent compounds; hydrophilic or cationic moieties within the tether had a detrimental effect on inhibition of transport. In addition to acting as probes of P-gp's drug binding sites, these agents were also potent inhibitors of P-gp. One agent, EmeC5, had IC50 values of 2.9 microM for inhibiting transport of rhodamine 123 and approximately 5 nM for inhibiting the binding of a known P-gp substrate, [125I]iodoarylazidoprazosin. Although EmeC5 is an inhibitor of P-gp and was shown to interact directly with P-gp in one or more of the substrate binding sites, our data suggest that it is either not a P-gp transport substrate itself or a poor one. Most significantly, EmeC5 was shown to reverse the MDR phenotype of MCF-7/DX1 cells when co-administered with a cytotoxic agent, such as doxorubicin.

Allosteric Modulation of Human P-glycoprotein: INHIBITION OF TRANSPORT BY PREVENTING SUBSTRATE TRANSLOCATION AND DISSOCIATION

The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z)-flupentixol, a thioxanthene derivative, occurs through an allosteric mechanism. Unlike competitive inhibitors, such as cyclosporin A and verapamil, cis-(Z)-flupentixol does not interfere with substrate ([(125)I]iodoarylazidoprazosin) recognition by Pgp, instead it prevents substrate translocation and dissociation, resulting in a stable but reversible Pgp-substrate complex. cis-(Z)-Flupentixol-induced complex formation requires involvement of the Pgp substrate site, because agents that either physically compete (cyclosporin A) for or indirectly occlude (vanadate) the substrate-binding site prevent formation of the complex. Allosteric modulation by cis-(Z)-flupentixol involves a conformational change in Pgp detectable by monoclonal antibody UIC2 binding to a conformation-sensitive external epitope of Pgp. The conformational change observed is distinct from that induced by Pgp substrates or competitive inhibitors. A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Taken together, our results describe the molecular mechanism by which the Pgp modulator cis-(Z)-flupentixol allosterically inhibits drug transport.

Multidrug transporters and antibiotic resistance in Lactococcus lactis

The Gram-positive bacterium Lactococcus lactis produces two distinct multidrug transporters, designated LmrA and LmrP, that both confer resistance to a wide variety of cationic lipophilic cytotoxic compounds as well as to many clinically relevant antibiotics. While LmrP is a proton/drug antiporter that belongs to the major facilitator superfamily of secondary transporters, LmrA is an ATP-dependent primary transporter that belongs to the ATP-binding cassette superfamily of transport proteins. Both LmrA and LmrP function as “hydrophobic vacuum cleaners” by excreting lipophilic cationic compounds from the inner leaflet of the membrane directly into the external water phase. LmrA is both functionally and structurally homologous to the human multidrug transporter P-glycoprotein. LmrA is a half ABC transporter that is functional as a homodimer, consistent with the general four-domain organization of ABC transporters, and is proposed to mediate drug transport by an alternating two-site transport mechanism.

Transport Activity and Surface Expression of the Na+-Ca2+ Exchanger NCX1 Are Inhibited by the Immunosuppressive Agent Cyclosporin A and by the Nonimmunosuppressive Agent PSC833

Cyclosporin A (CsA) treatment of HEK 293 cells expressing the rat heart RHE-1 (NCX1.1, EMBL accession number ) or the rat brain RBE-2 (NCX1.5, GenBank(TM) accession number ) Na(+)-Ca(2+) exchanger inhibited their transport activity in a concentration-dependent manner. The inhibition was detectable at 2 microm CsA, and exposure of the cells to 20 microm CsA resulted in a decrease of the Na(+)-dependent Ca(2+) uptake to about 20% relative to that of untreated cells. Determination of the surface expression of the exchanger protein revealed a parallel concentration-dependent reduction in the amount of the immunoreactive protein. No reduction was detected in the amount of total immunoreactive exchanger protein in CsA-treated cells relative to untreated ones. Among the different drugs tested, only PSC833, an analog of cyclosporin D, mimicked the effects of CsA. Exposure of the transfected cells to the chemically related cyclosporin D and macrolide drugs (FK506 or rapamycin) had no effect on the transport activity or the surface expression of the Na(+)-Ca(2+) exchanger. Co-expression of the human multidrug transporter P-glycoprotein (of which both drugs are modulators) with the cloned Na(+)-Ca(2+) exchanger revealed that transport activity and surface expression of each transporter in the co-transfected system were similar to those of each transporter alone in both the presence and absence of CsA or PSC833. CsA and PSC833 inhibited the surface expression of the NCX1 protein but did not alter the surface expression of P-glycoprotein. Unlike some P-glycoprotein endoplasmic reticulum-retained mutants (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 709-712), CsA did not rescue RBE-2/F913-->Stop, an endoplasmic reticulum-retained function-competent mutant of the Na(+)-Ca(2+) exchanger (Kasir, J., Ren, X., Furman, I., and Rahamimoff, H. (1999) J. Biol. Chem. 274, 24873-24880) and did not induce its kinesis to the surface membrane, further demonstrating molecular differences between P-glycoprotein and NCX1 mutants for interaction with CsA.

A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol.

Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.