ATP Binding Enables Substrate Release from Multidrug Resistance Protein 1.

The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellular substrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.

Structural Basis of Substrate Recognition by the Multidrug Resistance Protein MRP1.

The multidrug resistance protein MRP1 is an ATP-binding cassette (ABC) transporter that confers resistance to many anticancer drugs and plays a role in the disposition and efficacy of several opiates, antidepressants, statins, and antibiotics. In addition, MRP1 regulates redox homeostasis, inflammation, and hormone secretion. Using electron cryomicroscopy, we determined the molecular structures of bovine MRP1 in two conformations: an apo form at 3.5 Å without any added substrate and a complex form at 3.3 Å with one of its physiological substrates, leukotriene C4. These structures show that by forming a single bipartite binding site, MRP1 can recognize a spectrum of substrates with different chemical structures. We also observed large conformational changes induced by leukotriene C4, explaining how substrate binding primes the transporter for ATP hydrolysis. Structural comparison of MRP1 and P-glycoprotein advances our understanding of the common and unique properties of these two important molecules in multidrug resistance to chemotherapy.

The structure of Plasmodium falciparum multidrug resistance protein 1 reveals an N-terminal regulatory domain.

Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), an adenosine triphosphate (ATP)-binding cassette (ABC) transporter on the digestive vacuole (DV) membrane of the parasite, is associated with the resistance to antimalarial drugs. To understand the mechanisms of PfMDR1, we determined the cryo-electron microscopy structures of this transporter in different states. The transporter in the apo state shows an inward-facing conformation with a large cavity opening to the cytoplasm. Upon ATP binding and dimerization of the nucleotide-binding domains (NBDs), PfMDR1 displays an outward-facing conformation with a cavity toward the DV lumen. Drug resistance-associated mutations were investigated in both structures for their effects, and Y184F was identified as an allosteric activity-enhancing mutation. The amphiphilic substrate-binding site of PfMDR1 was revealed by the complex structure with the antimalarial drug mefloquine and confirmed by mutagenesis studies. Remarkably, a helical structure was found to hinder NBD dimerization and inhibit PfMDR1 activity. The location of this regulatory domain in the N terminus is different from the well-studied R domain in the internal linker region of other ABC transporter family members. The lack of the phosphorylation site of this domain also suggests a different regulation mechanism.

A macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation.

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC4). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

Coupling the role of lipids to the conformational dynamics of the ABC transporter P-glycoprotein.

The ATP-binding cassette transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug-resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, "straight" and "kinked," respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of two human P-gp inward-open conformers, straight and kinked, employing all-atom molecular dynamics (MD) simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter into the cavity of the straight P-gp conformer solved in detergent. However, access to the cavity of the kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp conformers and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, the findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-gp, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane helix (TM10) of the straight conformer underwent a spontaneous transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between the two P-gp conformers.

Reduced P-glycoprotein recognition of a radioiodine-labeled phosphonium cation by stilbenylation for mitochondrial membrane potential based-imaging.

The accumulation of radiolabeled phosphonium cations in cells is dependent on the mitochondrial membrane potential (MMP). However, the efflux of these cations from tumor cells via P-glycoprotein (P-gp) limits their clinical application as MMP-based imaging tracers. In the present study, we designed (E)-diethyl-4-[125I]iodobenzyl-4-stilbenylphosphonium ([125I]IDESP), which contains a stilbenyl substituent, as a P-gp inhibitor to reduce P-gp recognition, and evaluated its biological properties in comparison with 4-[125I]iodobenzyl dipropylphenylphosphonium ([125I]IDPP). The in vitro cellular uptake ratio of [125I]IDESP in P-gp expressing K562/Vin cells to the parent (P-gp negative) K562 cells was significantly higher than that of [125I]IDPP. The efflux rate of [125I]IDESP was not significantly different between K562 and K562/Vin, while [125I]IDPP was rapidly effluxed from K562/Vin compared with K562, and the efflux of [125I]IDPP from K562/Vin was inhibited by the P-gp inhibitor, cyclosporine A. The cellular uptake of [125I]IDESP was well correlated with the MMP levels. These results suggested that [125I]IDESP was accumulated in cells depending on the MMP levels, without being effluxed via P-gp, while [125I]IDPP was rapidly effluxed from the cells via P-gp. Despite having suitable in vitro properties for MMP-based imaging, [125I]IDESP showed rapid blood clearance and lower tumor accumulation than [125I]IDPP. Improvement in the normal tissue distribution of [125I]IDESP is required to develop an agent for use in in vivo MMP-based tumor imaging.

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

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.

Auxin-transporting ABC transporters are defined by a conserved D/E-P motif regulated by a prolylisomerase.

The plant hormone auxin must be transported throughout plants in a cell-to-cell manner to affect its various physiological functions. ABCB transporters are critical for this polar auxin distribution, but the regulatory mechanisms controlling their function is not fully understood. The auxin transport activity of ABCB1 was suggested to be regulated by a physical interaction with FKBP42/Twisted Dwarf1 (TWD1), a peptidylprolyl cis-trans isomerase (PPIase), but all attempts to demonstrate such a PPIase activity by TWD1 have failed so far. By using a structure-based approach, we identified several surface-exposed proline residues in the nucleotide binding domain and linker of Arabidopsis ABCB1, mutations of which do not alter ABCB1 protein stability or location but do affect its transport activity. P1008 is part of a conserved signature D/E-P motif that seems to be specific for auxin-transporting ABCBs, which we now refer to as ATAs. Mutation of the acidic residue also abolishes auxin transport activity by ABCB1. All higher plant ABCBs for which auxin transport has been conclusively proven carry this conserved motif, underlining its predictive potential. Introduction of this D/E-P motif into malate importer, ABCB14, increases both its malate and its background auxin transport activity, suggesting that this motif has an impact on transport capacity. The D/E-P1008 motif is also important for ABCB1-TWD1 interactions and activation of ABCB1-mediated auxin transport by TWD1. In summary, our data imply a new function for TWD1 acting as a putative activator of ABCB-mediated auxin transport by cis-trans isomerization of peptidyl-prolyl bonds.

The influence of calculated physicochemical properties of compounds on their ADMET profiles.

We analyzed the influence of calculated physicochemical properties of more than 20,000 compounds on their P-gp and BCRP mediated efflux, microsomal stability, hERG inhibition, and plasma protein binding. Our goal was to provide guidance for designing compounds with desired pharmacokinetic profiles. Our analysis showed that compounds with ClogP less than 3 and molecular weight less than 400 will have high microsomal stability and low plasma protein binding. Compounds with logD less than 2.2 and/or basic pKa larger than 5.3 are likely to be BCRP substrates and compounds with basic pKa less than 5.2 and/or acidic pKa less than 13.4 are less likely to inhibit hERG. Based on these results, compounds with MW < 400, ClogP < 3, basic pKa < 5.2 and acidic pKa < 13.4 are likely to have good bioavailability and low hERG inhibition.

Surface plasmon resonance biosensor combined with lentiviral particle stabilization strategy for rapid and specific screening of P-Glycoprotein ligands.

A novel surface plasmon resonance-based P-gp ligand screening system (SPR-PLSS) combined with lentiviral particle (LVP) stabilization strategy was constructed to screen out potential P-gp inhibitors from natural products. Firstly, we constructed LVPs with high and low expression levels of P-gp. The LVPs can ensure the natural conformation of P-gp based on the principle that LVPs germinated from packaging cells will contain cell membrane fragments and P-gp they carry. Then the LVPs with high P-gp expression for active channel and LVPs with low P-gp expression for reference channel were immobilized on CM5 chip respectively. The affinity detection was thus carried out with the signal reduction on the two channels. The P-gp inhibitors, Valspodar (Val) and cyclosporin (CsA), as positive compounds, were detected to characterize the chip's activity, and the KD of Val and CsA were 14.09 µM and 16.41 µM, respectively. Forty compounds from natural product library were screened using the SPR CM5 chip, and magnolol (Mag), honokiol (Hon), and resveratrol (Res) were screened out as potential P-gp ligands, showing a significant response signal. This work presented a novel P-gp ligand screening system based on LVP-immobilized biosensor to rapidly screen out P-gp ligands from natural product library. Compared with traditional cell experiments which the screening time may take up to several days, our method only takes several hours. Furthermore, this study has also provided solid evidences to support that some complicated membrane proteins would apply to the lentivirus-based SPR screening system.

Simultaneous binding mechanism of multiple substrates for multidrug resistance transporter P-glycoprotein.

P-glycoprotein (P-gp), a member of ATP-binding cassette (ABC) transporters, is a multidrug resistance pump. Its promiscuous nature is the main cause of multidrug resistance in cancer cells. P-gp can bind multiple drug molecules simultaneously; however, the binding mechanism is still not clear. Furthermore, the upper limit of the number of substrates that can be accommodated by the binding pocket is not fully understood. In this work, we explore the dynamic process of P-gp binding to multiple substrates by using molecular dynamics (MD) simulations. Our results show that P-gp possesses the ability for simultaneous binding, and that the number of substrates has an upper limit. The accommodating ability of P-gp relates to the size of the binding drugs, and conforms to induced fit theory. In the binding process, the residues 339PHE, 982MET and 986GLN are essential. The pocket of P-gp presents strong flexibility and adaptability features according to the mutation results in this work. Drug molecules tend to gather in the pocket during binding, and interactions between these molecules are beneficial to simultaneous binding. These findings provide new insights into the mechanism of the promiscuous nature of P-gp, and may give us a guideline for inhibiting the process of multidrug resistance.

Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1.

The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.

Cross-linking, DEER-spectroscopy and molecular dynamics confirm the inward facing state of P-glycoprotein in a lipid membrane.

The drug efflux pump P-glycoprotein (P-gp) displays a complex transport mechanism involving multiple drug binding sites and two centres for nucleotide hydrolysis. Elucidating the molecular mechanism of transport remains elusive and the availability of P-gp structures in distinct natural and ligand trapped conformations will accelerate our understanding. The present investigation sought to provide biochemical data to validate specific features of these structures; with particular focus on the transmembrane domain that provides the transport conduit. Hence our focus was on transmembrane helices six and twelve (TM6/TM12), which are believed to participate in drug binding, as they line the central transport conduit and provide a direct link to the catalytic centres. A series of P-gp mutants were generated with a single cysteine in both TM6 and TM12 to facilitate measurement of inter-helical distances using cross-linking and DEER strategies. Experimental results were compared to published structures per se and those refined by MD simulations. This analysis revealed that the refined inward-facing murine structure (4M1M) of P-gp provides a good representation of the proximity, topography and relative motions of TM6 and TM12 in reconstituted human P-gp.

Chrysosporazines F-M: P-Glycoprotein Inhibitory Phenylpropanoid Piperazines from an Australian Marine Fish Derived Fungus, Chrysosporium sp. CMB-F294.

Chemical analysis of the fungus Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugil mullet yielded eight new alkaloids, belonging to a rare class of phenylpropanoid piperazines. Chrysosporazines F-M (1-8) occur as an equilibrium mixture of acetamide rotamers and feature unprecedented carbocyclic and heterocyclic scaffolds. Structures inclusive of absolute configuration were assigned by detailed spectroscopic analysis, supported by biosynthetic considerations. Structure-activity relationship studies determined that selected chrysosporazines were promising noncytotoxic inhibitors of the multidrug resistance efflux pump P-glycoprotein (P-gp), capable of reversing doxorubicin resistance in P-gp-overexpressing human colon carcinoma cells (SW620 Ad300). Chrysosporazine F (1) was particularly noteworthy, with a 2.5 µM cotreatment inducing a doxorubicin gain in sensitivity (GS 14) > 2-fold that of the positive control verapamil (GS 6.1).

Homology Modeling of the Human P-glycoprotein (ABCB1) and Insights into Ligand Binding through Molecular Docking Studies.

The ABCB1 transporter also known as P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette super-family of transporters; it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping the compounds out of cells. P-gp contributes to a decrease of toxicity and possesses broad substrate specificity. It is involved in the failure of numerous anticancer and antiviral chemotherapies due to the multidrug resistance (MDR) phenomenon, where it removes the chemotherapeutics out of the targeted cells. Understanding the details of the ligand-P-gp interaction is therefore crucial for the development of drugs that might overcome the MRD phenomenon and for obtaining a more effective prediction of the toxicity of certain compounds. In this work, an in silico modeling was performed using homology modeling and molecular docking methods with the aim of better understanding the ligand-P-gp interactions. Based on different mouse P-gp structural templates from the PDB repository, a 3D model of the human P-gp (hP-gp) was constructed by means of protein homology modeling. The homology model was then used to perform molecular docking calculations on a set of thirteen compounds, including some well-known compounds that interact with P-gp as substrates, inhibitors, or both. The sum of ranking differences (SRD) was employed for the comparison of the different scoring functions used in the docking calculations. A consensus-ranking scheme was employed for the selection of the top-ranked pose for each docked ligand. The docking results showed that a high number of π interactions, mainly π-sigma, π-alkyl, and π-π type of interactions, together with the simultaneous presence of hydrogen bond interactions contribute to the stability of the ligand-protein complex in the binding site. It was also observed that some interacting residues in hP-gp are the same when compared to those observed in a co-crystallized ligand (PBDE-100) with mouse P-gp (PDB ID: 4XWK). Our in silico approach is consistent with available experimental results regarding P-gp efflux transport assay; therefore it could be useful in the prediction of the role of new compounds in systemic toxicity.

TMPE Derived from Saffron Natural Monoterpene as Cytotoxic and Multidrug Resistance Reversing Agent in Colon Cancer Cells.

Terpenes constitute one of the largest groups of natural products. They exhibit a wide range of biological activities including antioxidant, anticancer, and drug resistance modulating properties. Saffron extract and its terpene constituents have been demonstrated to be cytotoxic against various types of cancer cells, including breast, liver, lung, pancreatic, and colorectal cancer. In the present work, we have studied anticancer properties of TMPE, a newly synthesized monoterpene derivative of ß-cyclocitral-the main volatile produced by the stigmas of unripe crocuses. TMPE presented selective cytotoxic activity to doxorubicin-resistant colon cancer cells and was identified to be an effective MDR modulator in doxorubicin-resistant cancer cells. Synergy between this derivative and doxorubicin was observed. Most probably, TMPE inhibited transport activity of ABCB1 protein without affecting its expression level. Analysis of TMPE physicochemical parameters suggested it was not likely to be transported by ABCB1. Molecular modeling showed TMPE being more reactive molecule than the parental compound-ß-cyclocitral. Analysis of electrostatic potential maps of both compounds prompted us to hypothesize that reduced reactivity as well as susceptibility to electrophilic attack were related to the lower general toxicity of ß-cyclocitral. All of the above pointed to TMPE as an interesting candidate molecule for MDR reversal in cancer cells.

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

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.

Dual P-Glycoprotein and CA XII Inhibitors: A New Strategy to Reverse the P-gp Mediated Multidrug Resistance (MDR) in Cancer Cells.

A new series of N,N-bis(alkanol)amine aryl diesters was synthesized and studied as dual P-glycoprotein (P-gp) and carbonic anhydrase XII inhibitors (CA XII). These hybrids should be able to synergistically overcome P-gp mediated multidrug resistance (MDR) in cancer cells. It was reported that the efflux activity of P-gp could be modulated by CA XII, as the pH reduction caused by CA XII inhibition produces a significant decrease in P-gp ATPase activity. The new compounds reported here feature both P-gp and CA XII binding moieties. These hybrids contain a N,N-bis(alkanol)amine diester scaffold found in P-glycoprotein ligands and a coumarin or benzene sulfonamide moiety to target CA XII. Many compounds displayed a dual activity against P-gp and CA XII being active in the Rhd 123 uptake test on K562/DOX cells and in the hCA XII inhibition test. On LoVo/DOX cells, that overexpress both P-gp and CA XII, some coumarin derivatives showed a high MDR reversal effect in Rhd 123 uptake and doxorubicin cytotoxicity enhancement tests. In particular, compounds 7 and 8 showed higher activity than verapamil and were more potent on LoVo/DOX than on K562/DOX cells overexpressing only P-gp. They can be considered as valuable candidates for selective P-gp/CA XII inhibition in MDR cancer cells.

Overcoming Multidrug Resistance: Flavonoid and Terpenoid Nitrogen-Containing Derivatives as ABC Transporter Modulators.

Multidrug resistance (MDR) in cancer is one of the main limitations for chemotherapy success. Numerous mechanisms are behind the MDR phenomenon wherein the overexpression of the ATP-binding cassette (ABC) transporter proteins P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 1 (MRP1) is highlighted as a prime factor. Natural product-derived compounds are being addressed as promising ABC transporter modulators to tackle MDR. Flavonoids and terpenoids have been extensively explored in this field as mono or dual modulators of these efflux pumps. Nitrogen-bearing moieties on these scaffolds were proved to influence the modulation of ABC transporters efflux function. This review highlights the potential of semisynthetic nitrogen-containing flavonoid and terpenoid derivatives as candidates for the design of effective MDR reversers. A brief introduction concerning the major role of efflux pumps in multidrug resistance, the potential of natural product-derived compounds in MDR reversal, namely natural flavonoid and terpenoids, and the effect of the introduction of nitrogen-containing groups are provided. The main modifications that have been performed during last few years to generate flavonoid and terpenoid derivatives, bearing nitrogen moieties, such as aliphatic, aromatic and heterocycle amine, amide, and related functional groups, as well as their P-gp, MRP1 and BCRP inhibitory activities are reviewed and discussed.

Caffeic Acid Attenuates Multi-Drug Resistance in Cancer Cells by Inhibiting Efflux Function of Human P-glycoprotein.

: Multidrug resistance (MDR) is a complicated ever-changing problem in cancer treatment, and P-glycoprotein (P-gp), a drug efflux pump, is regarded as the major cause. In the way of developing P-gp inhibitors, natural products such as phenolic acids have gotten a lot of attention recently. The aim of the present study was to investigate the modulating effects and mechanisms of caffeic acid on human P-gp, as well as the attenuating ability on cancer MDR. Calcein-AM, rhodamine123, and doxorubicin were used to analyze the interaction between caffeic acid and P-gp, and the ATPase activity of P-gp was evaluated as well. Resistance reversing effects were revealed by SRB and cell cycle assay. The results indicated that caffeic acid uncompetitively inhibited rhodamine123 efflux and competitively inhibited doxorubicin efflux. In terms of P-gp ATPase activity, caffeic acid exhibited stimulation in both basal and verapamil-stimulated activity. The combination of chemo drugs and caffeic acid resulted in decreased IC50 in ABCB1/Flp-InTM-293 and KB/VIN, indicating that the resistance was reversed. Results of molecular docking suggested that caffeic acid bound to P-gp through GLU74 and TRY117 residues. The present study demonstrated that caffeic acid is a promising candidate for P-gp inhibition and cancer MDR attenuation.