ATP-binding Cassette Multidrug Transporters Research Articles

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the wild-type protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by Electron Paramagnetic Resonance spectroscopy supported the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like motion, with a transport mechanism distinct from the related lipid A exporter MsbA.

Waste or die: The price to pay to stay alive.

Microorganisms need to constantly exchange with their habitat to capture nutrients and expel toxic compounds. The ATP-binding cassette (ABC) transporters, a family of membrane proteins especially abundant in microorganisms, are at the core of these processes. Due to their extraordinary ability to expel structurally unrelated compounds, some transporters play a protective role in different organisms. Yet, the downside of these multidrug transporters is their entanglement in the resistance to therapeutic treatments. Intriguingly, some multidrug ABC transporters show a high level of ATPase activity, even in the absence of transported substrates. Although this basal ATPase activity might seem a waste, we surmise that this inherent capacity allows multidrug transporters to promptly translocate any bound drug before it penetrates into the cell.

Residues forming the gating regions of asymmetric multidrug transporter Pdr5 also play roles in conformational switching and protein folding

ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA.

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg2+:vanadate, while the second site is more ‘open’ allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31P–31P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.

Abstract 405: A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance by macropinocytosis-mediated internalization and redox changes

Abstract Drug resistance are responsible for most relapses and up to 90% of cancer related death. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism. In tumor microenvironment, intratumoral extracellular ATP (eATP) levels are 1,000 times or more higher than those in corresponding normal tissues. Our previous studies have shown that eATP can be taken up by cancer cell via macropinocytosis, leading to substantial increase in intracellular ATP levels. We found that this high intracellular ATP levels contribute to drug resistance in several ways: first, it directly fueled and enhanced drug-pumping activity of major ATP-binding cassette (ABC) multidrug transporters, which are located in plasma membrane and can pump out a wide range of substrates including anticancer drugs, leading to reduced intracellular drug retention. Secondly, ATP treatment upregulated gene expression of the major ABC transporters, and this gene regulatory effect of ATP was comparable to TGF-beta, which is a well-established epithelial-mesenchymal transition (EMT) inducer, and EMT is known to induce ABC transporter expression and drug resistance. Moreover, eATP treatment led to an increase in intracellular glutathione (GSH) levels, probably by enhancing GSH synthesis. Increased GSH levels have been described to significantly contribute to drug resistance. We also found eATP altered cellular redox state, which potentially stimulates EMT. Furthermore, we performed RNA-sequencing and identified several genes that may serve as downstream factors of eATP in promoting EMT and drug resistance. We are currently investigating the roles of these genes by knocking down gene expression or knocking out these genes. Citation Format: Haiyun Zhang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: extracellular ATP-induced resistance by macropinocytosis-mediated internalization and redox changes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 405.

P-glycoprotein Mediates Resistance to the Anaplastic Lymphoma Kinase Inhiitor Ensartinib in Cancer Cells.

Simple SummaryP-glycoprotein (P-gp; ABCB1) is the most well-characterized ATP-binding cassette (ABC) multidrug efflux transporter known to actively transport a wide variety of cytotoxic and molecularly targeted drugs out of cancer cells, causing multidrug resistance (MDR) and poor prognosis in cancer patients. In this study, we investigate whether P-gp overexpression can contribute to reduced susceptibility of cancer cells to the anaplastic lymphoma kinase (ALK) inhibitor ensartinib. We discovered that P-gp-overexpressing cancer cells and cells with ectopic expression of P-gp are significantly less sensitive to ensartinib than the respective parental cells. By blocking the drug transport activity of P-gp, the intracellular accumulation and cytotoxic activity of ensartinib were fully restored in P-gp-overexpressing cancer cells. Moreover, in silico molecular docking analysis of ensartinib with the inward-open structure of P-gp provided additional information on the potential binding orientation of ensartinib in the substrate-binding pocket of P-gp.Ensartinib (X-396) is a promising second-generation small-molecule inhibitor of anaplastic lymphoma kinase (ALK) that was developed for the treatment of ALK-positive non-small-cell lung cancer. Preclinical and clinical trial results for ensartinib showed superior efficacy and a favorable safety profile compared to the first-generation ALK inhibitors that have been approved by the U.S. Food and Drug Administration. Although the potential mechanisms of acquired resistance to ensartinib have not been reported, the inevitable emergence of resistance to ensartinib may limit its therapeutic application in cancer. In this work, we investigated the interaction of ensartinib with P-glycoprotein (P-gp) and ABCG2, two ATP-binding cassette (ABC) multidrug efflux transporters that are commonly associated with the development of multidrug resistance in cancer cells. Our results revealed that P-gp overexpression, but not expression of ABCG2, was associated with reduced cancer cell susceptibility to ensartinib. P-gp directly decreased the intracellular accumulation of ensartinib, and consequently reduced apoptosis and cytotoxicity induced by this drug. The cytotoxicity of ensartinib could be significantly reversed by treatment with the P-gp inhibitor tariquidar. In conclusion, we report that ensartinib is a substrate of P-gp, and provide evidence that this transporter plays a role in the development of ensartinib resistance. Further investigation is needed.

Atypical cyclin P regulates cancer cell stemness through activation of the WNT pathway

PurposeCancer stem cells represent a cancer cell subpopulation that has been found to be associated with metastasis and chemoresistance. Therefore, it is vital to identify mechanisms regulating cancer stemness. Previously, we have shown that the atypical cyclin P (CCNP), also known as CNTD2, is upregulated in lung and colorectal cancers and is associated with a worse clinical prognosis. Given that other cyclins have been implicated in pluripotency regulation, we hypothesized that CCNP may also play a role in cancer stemness.MethodsCell line-derived spheroids, ex vivo intestinal organoid cultures and induced-pluripotent stem cells (iPSCs) were used to investigate the role of CCNP in stemness. The effects of CCNP on cancer cell stemness and the expression of pluripotency markers and ATP-binding cassette (ABC) transporters were evaluated using Western blotting and RT-qPCR assays. Cell viability was assessed using a MTT assay. The effects of CCNP on WNT targets were monitored by RNA-seq analysis. Data from publicly available web-based resources were also analyzed.ResultsWe found that CCNP increases spheroid formation in breast, lung and colorectal cancers, and upregulates the expression of stemness (CD44, CD133) and pluripotency (SOX2, OCT4, NANOG) markers. In addition, we found that CCNP promotes resistance to anticancer drugs and induces the expression of multidrug resistance ABC transporters. Our RNA-seq data indicate that CCNP activates the WNT pathway, and that inhibition of this pathway abrogates the increase in spheroid formation promoted by CCNP. Finally, we found that CCNP knockout decreases OCT4 expression in iPSCs, further supporting the notion that CCNP is involved in stemness regulation.ConclusionOur results reveal CCNP as a novel player in stemness and as a potential therapeutic target in cancer.

Abstract 1108: A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters

Abstract Cancer is the second leading cause of death in the US. Multidrug resistance (MDR) and the resulting ineffectiveness of the drug treatment are responsible for most relapses of cancer and cancer-related death. One major mechanism of MDR is the efflux of multiple drugs mediated by ATP-binding Cassette (ABC) multidrug transporters. ABC transporters pump drugs with different structures and mechanisms of action out of cancer cells using ATP-provided energy. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism1. Intratumoral extracellular ATP levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues2. Our previous studies have shown that extracellular ATP is internalized by cancer cell via macropinocytosis, and promotes cancer drug resistance by fueling ABC transporters and increase its efflux activities3. However, whether extracellular ATP can indeed enhance efflux of drugs, and the underlying mechanism besides providing energy remain unclear. Our current study results showed extracellular ATP decreased intracellular accumulation and thereby enhanced efflux of 3H-labeled chemotherapeutic drug and targeted drug in human cancer cells. The results of quantitative reverse transcription PCR, western blot and immunofluorescence microscopy demonstrated that extracellular ATP can also significantly upregulate ABC transporters at mRNA and protein levels, and change the intracellular localization of the latter. These results suggest that extracellular ATP has profound regulatory effects on ABC transporters and thus promotes drug resistance, linking the ATP-rich tumor microenvironment with cancer drug resistance. The completion of the study will expand our understanding of the roles of extracellular ATP in cancer, and reveal new therapeutic targets for reducing drug resistance and enhance anticancer efficacy of chemotherapeutics and targeted drugs. 1. Pavlova N, Thompson C. Cell Metab. (2016) 23:27-47 2. Falzoni S, et al., Interface Focus. (2013) 3: 20120101 3. Wang X, et al., Oncotarget. (2017) 8(50):87860-87877 Citation Format: Haiyun Zhang, Xuan Wang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1108.

Insights into the modulatory effect of magnesium on efflux mechanisms of Candida albicans reveal inhibition of ATP binding cassette multidrug transporters and dysfunctional mitochondria.

Candida infections pose a serious hazard to public healthfollowed bywidespread and prolonged deployment of antifungal drugs has which hasled multidrug resistance (MDR) progress in prevalent human fungal pathogen, Candida albicans. Despite the fact that MDR is multifactorial phenomenon govern by several mechanisms in C. albicans, overexpression of drug efflux transporters by far remains the leading cause of MDR govern by ATP Binding Cassette (ABC) or major facilitator superfamily (MFS) transporters. Hence searching for strategies to target efflux pumps transporter still signifies a promising approach. In this study we analyzed the effect of magnesium (Mg) deprivation, on efflux pump action of C. albicans. We explored that Mg deprivation specially inhibits efflux of transporters (CaCdr1p and CaCdr2p) belonging to ABC superfamily as revealed by rhodamine 6G and Nile red accumulation. Furthermore, Mg deprivation causes mislocalization of CaCdr1p and CaCdr2p and reduced transcripts of CDR1 and CDR2 with no effect on CaMdr1p. Additionally, Mg deprivation causes depletion of ergosterol content in azole sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and F2/F5 of C. albicans. Lastly, we observed that Mg deprivation impairs mitochondrial potential which could be the causal reason for abrogated efflux activity. With growing appreciation of manipulating metal homeostasis to combat MDR, inhibition of efflux activity under Mg deprivation warrants further studies to be utilized as an effective antifungal strategy.

Abstract 3008: A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters

Abstract Cancer is the second leading cause of death in the US. Multidrug resistance (MDR) and the resulting ineffectiveness of the drug treatment are responsible for most relapses of cancer and cancer-related death. One major mechanism of MDR is the efflux of multiple drugs mediated by ATP-binding Cassette (ABC) multidrug transporters. ABC transporters pump drugs with different structures and mechanisms of action out of cancer cells using ATP-provided energy. And ABC activities is also intimately linked to epithelial mesenchymal transition and metastasis. The opportunistic uptake of extracellular molecules has been named as a key emerging hallmark of cancer metabolism. Intratumoral extracellular ATP levels of various cancer types are 103 to 104 times higher than those in their corresponding normal tissues. Our previous studies have shown that extracellular ATP is internalized by cancer cell via macropinocytosis, and promotes cancer drug resistance by directly fueling ABC transporters and increase its drug efflux activities. However, whether extracellular ATP can induce change in the expression of ABC transporters and the underlying mechanisms remain unclear. Our results from quantitative reverse transcription PCR and flow cytometry show that extracellular ATP significantly alters mRNA levels and cell surface expression of ABC transporters. These results suggest that extracellular ATP has profound regulatory effects on ABC transporters and thus promotes drug resistance, linking the ATP-rich tumor microenvironment with cancer drug resistance. On-going studies include identification of mechanisms for the extracellular ATP-induced drug resistance via ABC transporters. The completion of the study will expand our understanding of the roles of extracellular ATP in cancer, and reveal new therapeutic targets for reducing drug resistance and enhancing anticancer efficacy of chemotherapeutics and targeted drugs. Citation Format: Haiyun Zhang, Xuan Wang, Xiaozhuo Chen. A new mechanism of drug resistance in cancer: Extracellular ATP-induced resistance through ATP internalization and upregulation of ATP-binding cassette multidrug transporters [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3008.

Octyl gallate reduces ABC multidrug transporter CaCdr1p expression and leads to its mislocalisation in azole-resistant clinical isolates of Candida albicans

ObjectivesFungal pathogens pose a serious threat to public health. Widespread and prolonged use of antifungal drugs has led to the development of multidrug resistance in the human fungal pathogen Candida albicans. Among several mechanisms leading to drug resistance in C. albicans, overexpression of drug efflux transporters remains by far the leading cause of multidrug resistance, facilitated by overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters. Hence, targeting efflux pumps still represents a promising approach to combat multidrug resistance. In this study, the effect of octyl gallate (OG), a natural food additive, on drug efflux pump activity of C. albicans was analysed. MethodsDrug efflux pump activity was determined by rhodamine 6G (R6G) efflux and Nile red accumulation assay in a Candida drug resistance protein 1 (CaCdr1p)-overexpressing strain. Gene expression and protein expression and localisation were studied by RT-PCR, Western blot and confocal microscopy. Ergosterol content was measured by the alcoholic KOH method. ResultsOG specifically inhibits the activity of CaCdr1p, belonging to the ABC superfamily. The underlying mechanism was confirmed as competitive mode of inhibition by OG as revealed by Lineweaver–Burk plot. Furthermore, OG leads to reduced expression of CDR1 and CaCdr1p and mislocalisation of CaCdr1p. Additionally, OG sensitises azole-susceptible and -resistant clinical matched-pair isolates Gu4 & Gu5 and leads to impeded R6G efflux and depleted ergosterol content. ConclusionThe ability of OG as a potent inhibitor of CaCdr1p that chemosensitises drug-resistant C. albicans warrants further studies to be exploited as an effective antifungal agent.

Energy coupling in ABC exporters

Multidrug transporters are important and interesting molecular machines that extrude a wide variety of cytotoxic drugs from target cells. This review summarizes novel insights in the energetics and mechanisms of bacterial ATP-binding cassette multidrug transporters as well as recent advances connecting multidrug transport to ion and lipid translocation processes in other membrane proteins.

Multidrug ABC transporters in bacteria

Multidrug efflux transporters are a plague in the antibiotic resistance mechanisms as they confer the capacity of bacteria to evade most of current therapies. Although these transporters were initially discovered as proton-motive driven pumps, another class of multidrug efflux transporters has emerged in the mid-90s that are powered by ATP hydrolysis. This new class of transporters belongs to one of the largest families of proteins, the ATP-Binding Cassette (ABC) transporters, which are involved in the influx or efflux of a huge variety of molecules. Tremendous progresses have been made in the recent years regarding the functioning mechanism of multidrug efflux ABC transporters, in particular with the accumulation of 3D structures, but many questions remain unsolved. In this review, we will give an overview of our current knowledge on the structure and function of multidrug ABC transporters with an emphasis on bacterial pumps.

Flexible-to-rigid transition is central for substrate transport in the ABC transporter BmrA from Bacillus subtilis

ATP-binding-cassette (ABC) transporters are molecular pumps that translocate molecules across the cell membrane by switching between inward-facing and outward-facing states. To obtain a detailed understanding of their mechanism remains a challenge to structural biology, as these proteins are notoriously difficult to study at the molecular level in their active, membrane-inserted form. Here we use solid-state NMR to investigate the multidrug ABC transporter BmrA reconstituted in lipids. We identify the chemical-shift differences between the inward-facing, and outward-facing state induced by ATP:Mg2+:Vi addition. Analysis of an X-loop mutant, for which we show that ATPase and transport activities are uncoupled, reveals an incomplete transition to the outward-facing state upon ATP:Mg2+:Vi addition, notably lacking the decrease in dynamics of a defined set of residues observed in wild-type BmrA. This suggests that this stiffening is required for an efficient transmission of the conformational changes to allow proper transport of substrate by the pump.

Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment.

Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild-type cells were not known. Using RNA-Seq and promoter-lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil.

P-Glycoprotein Activity Is Non-Monotonically Modulated by Transmembrane Voltage

P-glycoprotein (Pgp) is a broadly polyspecific exporter of hydrophobic and amphiphilic compounds that plays an important role in the blood-brain barrier and in multidrug resistance in cancer. Several research groups have recently made significant strides toward elucidating the mechanism of Pgp's catalytic cycle, but the biophysical properties of the cellular microenvironment can have strong effects on the function of Pgp and other ATP-binding cassette (ABC) transporters in as-yet poorly defined ways. Here we report that Pgp, MRP1, and BCRP, three of the most clinically relevant multidrug resistance-linked ABC transporters, are strongly and non-monotonically regulated by the transmembrane electrical potential difference. We confirmed this result with several assays, which used fluorescence and electrophysiological methods to measure the efflux of transporter substrates from two human cell lines and giant unilamellar vesicles and controlled the voltage using electrolyte gradients or the patch clamp technique. At hyperpolarized (highly inside-negative) potentials, each transporter displayed a pronounced reduction in efflux activity compared to the activity at the resting potential; Pgp and MRP1 also showed reduced activity at inside-positive potentials. The transport data is well-described by a model in which the transmembrane potential biases the conformational distribution of the proteins in a manner similar to the mechanism of action of voltage-gated ion channels. In this model, which is compatible with the “alternating access” framing of Pgp function, each catalytic cycle contains two transitions with an opposite dependence on the transmembrane voltage due to the movement of an equivalent of 3-4 elementary charges back and forth across the membrane. This finding, which points to an additional axis for modulating transporter activity, underscores the importance of electric field effects on the function of all membrane proteins, particularly those that undergo large conformational changes in the transmembrane region.

A lavender ABC transporter confers resistance to monoterpene toxicity in yeast.

Functional expression of a multidrug resistance-type ABC transporter from Lavandulaangustifolia improved yeast resistance to geraniol, a monoterpene constituent of lavender essential oil. Plant ATP-binding cassette (ABC) transporters are a large family of membrane proteins involved in active and selective transport of structurally diverse compounds. In this study, we functionally evaluated LaABCB1, a multidrug resistance (MDR)-type ABC transporter strongly expressed in the secretory cells of lavender glandular trichomes, where monoterpene essential oil constituents are synthesized and secreted. We used LaABCB1 to complement a yeast knockout mutant in which 16 ABC transporters were deleted. Expression of LaABCB1 enhanced tolerance of yeast mutants to geraniol, a key constituent of essential oils in lavenders and numerous other plants. Our findings suggest a role for the MDR-type ABC transporters in the toxicity tolerance of at least certain essential oil constituents in lavender oil glands.

An Outward-Facing Aromatic Amino Acid Is Crucial for Signaling between the Membrane-Spanning and Nucleotide-Binding Domains of Multidrug Resistance Protein 1 (MRP1; ABCC1).

The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe583 in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe583 is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe583 side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.

Isolation of Cancer Stem Cells by Side Population Method.

The Hoechst side population (SP) method is a flow cytometry technique used to obtain stem cells based on the dye efflux properties of the ATP-binding cassette (ABC) transporters. The SP cells are characterized by their capability to efflux the fluorescent DNA-binding dye Hoechst 33342 through their ABC transporters and are enriched in stem cells, which are endowed with a self-renewal capacity and multilineage differentiation potential and express the stemness genes including ABC multidrug transporters. The protocols outlined in this book chapter describe the isolation method of the SP cells from human lung carcinoma cell lines by using Hoechst 33342. In addition, we refer to the propagation method of SP cells by successive rounds of fluorescence-activated cell sorting analysis for SP cells. These approaches will be helpful for the establishment of novel in vitro and in vivo models using cancer stem cells, which may play a key role during carcinogenesis and/or tumor progression.

Abstract 3242: Synthetic analogs of curcumin as modulators of multidrug resistance-linked ABC transporters

Abstract Cancer patients often develop resistance to anticancer drugs. ATP-binding cassette (ABC) transporter-mediated drug efflux is one of the mechanisms responsible for development of resistance to anticancer drugs. The present study investigates the use of curcumin, a natural product and a dietary constituent of turmeric, which inhibits the function of three ABC drug transporters including ABCB1, ABCC1 and ABCG2. However, limited bioavailability of curcumin prevents its use for modulation of the function of these transporters in the clinic. We focused on curcumin analogs that were synthesized at Tohoku University and have been shown to exhibit higher bioavailability than natural curcumin to test whether the synthetic analogs also inhibit the function of ABC drug transporters. To investigate this, KB-V1 and K562/BCRP cell lines overexpressing ABCB1 and ABCG2 were used for the experiments. For these studies, we screened nineteen synthetic analogs of curcumin for their effect on the transport function of ABCB1 and ABCG2 by flow cytometry. Four curcumin analogs, GO-Y030, GO-Y078, GO-Y168 and GO-Y172 inhibited efflux of mitoxantrone mediated by ABCG2 and sensitized ABCG2-expressing K562 leukemic cells to the anticancer drug, SN-38 in cell toxicity assays. Some of the curcumin analogs (GO-Y030, GO-Y078, GO-Y172) stimulated ATPase activity of ABCG2 at nanomolar concentrations (EC50 = 480 ± 0.06, 790 ± 0.10, 930 ± 0.12 nM). In addition, GO-Y030, GO-Y078, GO-Y168, GO-Y172 analogs also inhibited photolabeling of ABCG2 in MCF-7-FL plasma membranes with iodoarylazidoprazosin, which is transported by this transporter. These data demonstrate that similar to curcumin, synthetic curcumin analogs also interact at the drug-binding pocket of ABCG2. In aggregate, these results suggest that non-toxic synthetic curcumin analogs with increased bioavailability may be useful to reverse ABCG2-mediated resistance to anticancer agents. Citation Format: Megumi Obara, Shinobu Ohnuma, Eduardo E. Chufan, Masaharu Ishida, Katsuyoshi Kudoh, Norihiko Sugisawa, Hideo Ohtsuka, Takeshi Naitoh, Hiroyuki Shibata, Yoshiharu Iwabuchi, Suresh V. Ambudkar, Michiaki Unno. Synthetic analogs of curcumin as modulators of multidrug resistance-linked ABC transporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3242. doi:10.1158/1538-7445.AM2017-3242