Fungal ATP-binding cassette (ABC) transporters in drug resistance & detoxification

Pleiotropic drug resistance (PDR) is a well-described phenomenon occurring in fungi. PDR shares several similarities with processes in bacteria and higher eukaryotes. In mammalian cells, multidrug resistance (MDR) develops from an initial single drug resistance, eventually leading to a broad cross-resistance to many structurally and functionally unrelated compounds. Notably, a number of membrane-embedded energy-consuming ATP-binding cassette (ABC) transporters have been implicated in the development of PDR/MDR phenotypes. The yeast Saccharomyces cerevisiae genome harbors some 30 genes encoding ABC proteins, several of which mediate PDR. Therefore, yeast served as an important model organism to study the functions of evolutionary conserved ABC genes, including those mediating clinical antifungal resistance in fungal pathogens. Moreover, yeast cells lacking endogenous ABC pumps are hypersensitive to many antifungal drugs, making them suitable for functional studies and cloning of ABC transporters from fungal pathogens such as Candida albicans. This review discusses drug resistance phenomena mediated by ABC transporters in the model system S. cerevisiae and certain fungal pathogens.     

介导肿瘤多药耐药的ATP结合盒转运体的研究进展

多药耐药（MDR）是阻碍肿瘤化疗成功的一大障碍,其机制之一就是耐药的肿瘤细胞高表达三磷酸腺苷（ATP）结合盒（ABC）转运体。依据此机制提出克服肿瘤细胞耐药的策略即开发外排转运体抑制剂,以期逆转MDR。最近的研究发现肿瘤干细胞也可能是通过表达外排转运体天然耐药,这就提供了一个新的抗癌药物作用靶点。对介导肿瘤细胞多药耐药的ABC转运体及其抑制剂的开发作一综述。     

Inhibitors of multidrug resistance to antitumor agents (MDR)

Multidrug resistance is one of the main obstacles in the chemotherapy of cancer. Its inhibition by combination of chemosensitizers with antitumor compounds is a very active field of research, since safe and potent reversal agents would be beneficial for clinical use. Most modulators act by binding to membrane transport proteins (specially P-gp and MRP) and inhibiting their drug-effluxing activity, or by indirect mechanisms related to phosphorylation of the transport proteins or expression of the mdr1 and mrp1 genes. The main body of the review focuses on the study of the known MDR modulators, which are classified according to their chemical structures. General structure-activity studies of this therapeutic group are hampered by the very heterogeneous chemical structure of the compounds, although some conclusions have been drawn from the study of homogeneous series of molecules.     

Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines

Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the copolymer concentration and hydrophobicity was found. No inhibitory effect against these ABC pumps was observed with the hydrophilic T1107. These findings further evidence the potential usefulness of these Trojan horses as both drug nanocarriers and ABC inhibitors in hepatic MDR tumors and infections that involve the activity of these efflux transporters.     

Metabolism of ATP-binding cassette drug transporter inhibitors: complicating factor for multidrug resistance

Membrane transport proteins belonging to the ATP-binding cassette (ABC) family of transport proteins play a central role in the defence of organisms against toxic compounds, including anticancer drugs. However, for compounds that are designed to display a toxic effect, this defence system diminishes their effectiveness. This is typically the case in the development of cellular resistance to anticancer drugs. Inhibitors of these transporters are thus potentially useful tools to reverse this transporter-mediated cellular resistance to anticancer drugs and, eventually, to enhance the effectiveness of the treatment of patients with drug-resistant cancer. This review highlights the various types of inhibitors of several multidrug resistance-related ABC proteins, and demonstrates that the metabolism of inhibitors, as illustrated by recent data obtained for various natural compound inhibitors, may have considerable implications for their effect on drug transport and their potential for treatment of drug resistance.     

介导肿瘤多药耐药的ATP结合盒转运体的研究进展

多药耐药（MDR）是阻碍肿瘤化疗成功的一大障碍,其机制之一就是耐药的肿瘤细胞高表达三磷酸腺苷（ATP）结合盒（ABC）转运体。依据此机制提出克服肿瘤细胞耐药的策略即开发外排转运体抑制剂,以期逆转MDR。最近的研究发现肿瘤干细胞也可能是通过表达外排转运体天然耐药,这就提供了一个新的抗癌药物作用靶点。对介导肿瘤细胞多药耐药的ABC转运体及其抑制剂的开发作一综述。     

ATP-binding cassette transporters in human heart failure

Adenosine triphosphate-binding cassette (ABC) transporters are involved in energy-dependent transport of substrates across biological membranes. We hypothesized that their expression is altered during human heart failure, suggesting a pathophysiologic basis. Messenger ribonucleic acid quantification of all known ABC transporters revealed multiple alterations in ABC transporter expression in failing human hearts (New York Heart Association classification III–IV) compared to nonfailing controls. These include a loss of ABCC7 chloride channels and an increased expression of the K_ATP channel regulatory subunits ABCC8. Moreover, ABCG2, an efflux pump for xenobiotics/drugs, was expressed at much higher levels in failing hearts compared to nonfailing control hearts. ABCG2 was found in cardiac capillary endothelial cells and cardiomyocytes. Experiments in cells stably transfected with human ABCG2 revealed that the peroxisome proliferator-activated receptor-γ agonist rosiglitazone was transported by ABCG2 but also inhibited the export of the prototypical ABCG2 substrate pheophorbide A (IC₅₀ 25 μM). These results suggest that altered ABC transporter expression in failing hearts might contribute to impaired channel conductance or might affect the cardiac disposition of drugs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. B.P. and T.F.S. contributed equally.     

ATP-binding cassette efflux transporters in human placenta

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity.     

ATP-binding cassette transporters in inflammatory brain disease

Owing to therapeutic progress, the role of ABC-transporters in infectious and autoimmune inflammatory CNS-diseases has recently gained considerable attention. In HIV-encephalitis and HIV-associated neurocognitive disorders, ABC-transporters are discussed to contribute to limited CNS-penetration and -retention of antiviral agents. In multiple sclerosis and its animal model experimental autoimmune encephalomyelitis, ABC-transporters may be involved in pathogenesis and treatment response alike. A prospective pharmacogenetic study is currently underway to examine the predictive role of genetic variations in ABC-transporters for treatment response and adverse events to mitoxantrone, a therapeutic agent used in aggressive MS. These approaches may aid in individualized treatment with this cytostatic anthracenedione, addressing its narrow therapeutic index with potentially fatal side effects. Finally, understanding regulation and function of ABC-transporters under inflammatory conditions may also optimize ABC-transporter-related treatment strategies in other neurological diseases (e.g. neurodegenerative, and neurovascular) where neuroinflammatory mechanisms have gained considerable attention as important contributors to pathogenesis.     

Implications of ATP-binding cassette transporters for brain pharmacotherapies

By preventing pharmacological compounds from achieving therapeutic levels in tissue, ATP-binding cassette (ABC) transporters complicate new drug discoveries. This has profound implications for pharmacotherapies, which go far beyond the need to deliver higher drug dosages. Comparative studies have recently shown that the expression and functionality of efflux proteins vary strongly both between species and strains, and in response to pathophysiological stimuli. This shatters hopes that it might become possible to predict drug biodistribution across species barriers. From this perspective, there is a need for more precise empirical biodistribution experiments to be performed in preparation for clinical trials. In such studies, the accumulation and elimination of drugs should be tested in various species under conditions resembling, as closely as possible, those in which a drug is clinically planned to be used. This approach should markedly enhance the overall success of new drugs and foster progress in neurological therapies.     

ATP结合盒转运体介导的肝癌多药耐药研究进展

ATP结合盒转运体(ABC转运体)介导的多药耐药(MDR)是肝癌化疗失败的主要原因,研究确定天然的及获得性MDR的产生机制,有利于提出更有效的治疗措施。针对ATP结合盒转运体的肿瘤MDR逆转策略,对于肝癌化疗效果的提高具有重要意义。此外,ATP结合盒转运体可能参与了肿瘤生物学的一些重要过程,发挥药物外排泵以外的作用,为肿瘤的治疗提供了新的研究方向。     

Zafirlukast antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance

ATP-binding cassette (ABC) transporters are present in the majority of human tumors and are involved in multidrug resistance (MDR). Therefore, compounds that inhibit the function of ABC transporters may improve the efficacy of anticancer agents. Previous research has shown that zafirlukast is a reversal drug for multidrug resistance protein (MRP) 1-mediated MDR. In the present study, we assessed whether zafirlukast could be a reversal agent for other ABC transporter-mediated MDR. Using the MTT assay, we found that zafirlukast enhanced the cytotoxicity of several anticancer drugs that are substrates of breast cancer resistance proteins (BCRP/ABCG2), including mitoxantrone and SN-38. Furthermore, zafirlukast could partially reverse P-glycoprotein-mediated (P-gp/ABCB1) and MRP7 (ABCC10)-mediated MDR at nontoxic doses. Studies on [(3)H]-mitoxantrone accumulation and efflux have shown that zafirlukast increases the intracellular accumulation of [(3)H]-mitoxantrone by directly inhibiting ABCG2-mediated drug efflux. Western blot analysis indicated that zafirlukast did not alter the expression of ABCG2. In addition, a docking model predicted the binding conformation of zafirlukast within the transmembrane region of homology-modeled human ABCG2. Our findings suggest a possible strategy to potentially enhance the activity of anticancer drugs using a clinically approved drug with known side effects and drug-drug interactions.     

Structure of multidrug-resistance proteins of the ATP-binding cassette (ABC) superfamily

Multidrug resistance of tumors, characterized by resistance against a variety of chemically unrelated anticancer agents, can be caused by overexpression of ATP-binding cassette (ABC) proteins, such as P-glycoprotein and MRP1. These multidrug-resistance proteins are plasma-membrane proteins that actively extrude chemotherapeutic agents from the cell interior, decreasing drug accumulation and thus, allowing the cells to survive in the presence of toxic levels of anticancer agents. ABC proteins contain multispanning transmembrane domains and nucleotide-binding domains (NBDs). The NBDs are responsible for the ATP binding/hydrolysis that drives drug transport, and their structure is conserved independently of the degree of primary-sequence homology. The transmembrane domains contain the drug-binding sites that are likely located in a flexible internal chamber that is sufficiently large to accommodate different drugs. It has been recently proposed that dimerization of the NBDs induced by ATP binding is a key step for the coupling of ATP hydrolysis to substrate transport. The power stroke for substrate transport can be the formation or the dissociation of the dimers. Since the NBDs and TMDs are tightly associated, association/dissociation of the NBDs may control the "gate" of the translocation pathway, formed by intracellular loops. In the case of P-glycoprotein it seems that the power stroke for transport is ATP binding (and therefore NBD dimerization), and not hydrolysis, because the major conformational and functional changes seem to occur at this step.     

The functions and structure of ABC transporters: implications for the design of new inhibitors of Pgp and MRP1 to control multidrug resistance (MDR)

Multidrug resistance (MDR) is a kind of acquired resistance of microorganisms and cancer cells to chemotherapic drugs that are characterized by different chemical structure and different mechanism of action. Classic MDR is the consequence of the over-expression of a variety of proteins that extrude the chemotherapic from the cell, lowering its concentration below the effective one. The ABC (ATP Binding Cassette) is a ubiquitous and important family of such transporter proteins. Members of this super family are present in mammals as well as in prokaryotic organisms and use ATP as the energy source to activate the extrusion process. P-glycoprotein (Pgp) and Multidrug Resistance Proteins (MRP1 and sister proteins) are the most important and widely studied members of ABC super family. Our knowledge about the structures and functions of transporter proteins has definitely improved in recent years, following the resolution of the structure of bacterial pumps which opened the way to the building of homology models for the more complex Pgp and MRP. It can be anticipated that these results will have a strong impact on the design of more potent and safer MDR reverters. A huge number of small molecules, many of natural origin, are able to reverse multidrug resistance by inhibiting the functions of Pgp, MRP1 and sister proteins and their action has been considered a possible way to reverse MDR. However, while a few compounds have reached clinical trials, none of them has, so far, been cleared for therapeutic use. Two main reasons are at the base of this difficulty: i) MDR is a complex phenomenon that may arise from several different biochemical mechanisms, with the consequence that inhibition of transporter proteins may be insufficient to reverse it; ii) the physiological role of Pgp and sister proteins requires more potent modulators with proper selectivity and pharmacokinetic in order to avoid unwanted side effects. This paper first reviews the most recent discoveries on the structures and functions of the ABC super family, in particular Pgp and MRP. Then, the medicinal chemistry of MDR reverters, in light of these findings, is discussed and the molecules that are presently in development are reviewed.     

Chalcogenopyrylium compounds as modulators of the ATP-binding cassette transporters P-glycoprotein (P-gp/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1)

Twenty-seven chalcogenopyrylium derivatives varying in the heteroatom of the pyrylium core and substituents at the 2-, 4-, and 6-positions were examined for their effect on human MRP1-mediated uptake of tritiated estradiol glucuronide into inside-out membrane vesicles, their affinity for and ability to stimulate the ATPase activity of purified human P-glycoprotein (P-gp)-His(10), and their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant cells. Differences in their effects on MRP1 and P-gp activity were noted, and a second set of thiopyrylium compounds with systematic substituent changes was examined to refine these differences further. Derivatives with tert-butyl substituents in the 2- and 6-positions had the lowest inhibitory activity toward both transporters. Derivatives with thioamide functionality in the 4-position were more active against MRP1 than derivatives with amide functionality. Conversely, derivatives with amide functionality in the 4-position were more active in P-gp than derivatives with thioamide functionality.     

Tyrosine kinase inhibitor resistance in cancer: role of ABC multidrug transporters.

Recent antitumor drug research has seen the development of a large variety of tyrosine kinase inhibitors (TKIs) with increasing specificity and selectivity. These are highly promising agents for specific inhibition of malignant cell growth and metastasis. However, their therapeutic potential also depends on access to their intracellular targets, which may be significantly affected by certain ABC membrane transporters. It has been recently shown that several human multidrug transporter ABC proteins interact with specific TKIs, and the ABCG2 transporter has an especially high affinity for some of these kinase inhibitors. These results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the treatment of cancer patients; moreover, the extrusion of TKIs by multidrug transporters may result in tumor cell TKI resistance. Interaction with multidrug resistance ABC transporters may also significantly modify the pharmacokinetics and toxicity of TKIs in patients.     

Novel insights into targeting ATP-binding cassette transporters for antitumor therapy

ATP-binding cassette (ABC) transporters are a large family of proteins implicated in physiological cellular functions. Selected components of the family play a well-recognized role in extruding conventional cytotoxic antitumor agents and molecularly targeted drugs from cells. Some lines of evidence also suggest links between transporters and tumor cell survival, in part unrelated to efflux. However, the study of the precise mechanisms regulating the function of drug transporters (e.g., posttranslational modifications such as glycosylation) is still in its infancy. A better definition of the molecular events clarifying the regulation of transporter levels including regulation by microRNAs may contribute to provide new molecular tools to target such a family of transporters. The present review focuses on the biological aspects that implicate ABC transporters in resistance of tumor cells, including cancer stem cells. Molecular analysis of well-known preclinical systems as well as of cancer stem cell models supports the notion that ABC transporters represent amenable targets for modulation of the efficacy of antitumor agents endowed with different molecular features. Recent achievements regarding tumor cell biology are expected to provide a rationale for developing novel inhibitors that target ABC transporters implicated in drug resistance.     

New structure model for the ATP-binding cassette multidrug transporter LmrA

Multidrug resistance of pathogenic microorganisms and mammalian tumors can be associated with the overexpression of multidrug transporters. These integral membrane proteins are capable of extruding a wide range of structurally unrelated compounds from the cell. Among the different classes of multidrug transporters are the ATP binding cassette (ABC) transporters, which are dependent on the binding and hydrolysis of ATP. In the past five years, many researchers have built homology models of ABC extrusion systems using the atomic coordinates of crystallized MsbA, a lipopolysaccharide transporter in Gram-negative bacteria. Likewise, we have previously used the Vibrio cholera MsbA structure as a template in the modeling of the multidrug transporter LmrA from Lactococcus lactis. In view of the recently discovered inaccuracies in the MsbA structure, we have remodelled LmrA using the atomic coordinates of the MsbA homologue Sav1866 from Staphylococcus aureus. To compare and test our MsbA-based and Sav1866-based LmrA models we performed cysteine cross-linking at three key positions in LmrA. The pattern of cross-linking at these positions was consistent with the overall topology of transmembrane helices in Sav1866, suggesting that its crystal structure might be physiologically relevant. We recently identified E314 as a residue important in proton conduction by LmrA. The predicted location of this residue at the interface between the two half-transporters in the Sav1866-based homodimer, within the inner leaflet of the phospholipid bilayer, provides a new structural basis for the role of E314 in LmrA-mediated transport.