ATP-binding Cassette Multidrug Transporters Research Articles

Pharmacogenetic Approach to Toxicity in Breast Cancer Patients Treated with Taxanes.

Taxanes are widely used to treat breast cancer patients. Taxanes are metabolized in human liver by the cytochrome CYP3A and are substrate of ATP-binding cassette multidrug transporters ABCB1. Single-nucleotide polymorphisms (SNPs) in genes involved in taxanes' metabolism could affect the inter-individual variability in reported toxicities. In this retrospective study, 152 women, affected by breast cancer and receiving a taxane-based chemotherapy, were enrolled. A peripheral blood sample was taken for genotyping the following polymorphisms: CYP3A4* 1B (A>G), CYP3A5 *3 (G>A) and ABCB1 (C1236T; C3435T). We observed an association between ABCB1 3435 T/T and lower grade of toxicities (p=0.05). No other association were found for CYP 3A4 *1B, 3A5*3 and ABCB1 C1236T. ABCB1 3435 T/T seems to be associated to lower rate of toxicity in patients receiving taxanes. Further prospective and larger studies should be performed to clarify the role of this polymorphism.

EUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells

The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter.

Molecular Mechanisms Involved in Qualitative and Quantitative Resistance to the Dicarboximide Fungicide Iprodione in Sclerotinia homoeocarpa Field Isolates.

The dicarboximide fungicide class is commonly used to control Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrass. Despite frequent occurrences of S. homoeocarpa field resistance to iprodione (dicarboximide active ingredient), the genetic mechanisms of iprodione resistance have not been elucidated. In this study, 15 field isolates (seven suspected dicarboximide resistant, three multidrug resistance (MDR)-like, and five dicarboximide sensitive) were used for sequence comparison of a histidine kinase gene, Shos1, of S. homoeocarpa. The suspected dicarboximide-resistant isolates displayed nonsynonymous polymorphisms in codon 366 (isoleucine to asparagine) in Shos1, while the MDR-like and sensitive isolates did not. Further elucidation of the Shos1 function, using polyethylene glycol-mediated protoplast transformation indicated that S. homoeocarpa mutants (Shos1I366N) from a sensitive isolate gained resistance to dicarboximides but not phenylpyrrole and polyols. The deletion of Shos1 resulted in higher resistance to dicarboximide and phenylpyrrole and higher sensitivity to polyols than Shos1I366N. Levels of dicarboximide sensitivity in the sensitive isolate, Shos1I366N, and Shos1 deletion mutants were negatively correlated to values of iprodione-induced expression of ShHog1, the last kinase in the high-osmolarity glycerol pathway. Increased constitutive and induced expression of the ATP-binding cassette multidrug efflux transporter ShPDR1 was observed in six of seven dicarboximide-resistant isolates. In conclusion, S. homoeocarpa field isolates gained dicarboximide resistance through the polymorphism in Shos1 and the overexpression of ShPDR1.

Clotrimazole Drug Resistance in Candida glabrata Clinical Isolates Correlates with Increased Expression of the Drug:H(+) Antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2.

For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. More recently, a few drug efflux pumps from the Drug:H+ Antiporter (DHA) family have also been shown to play a role in this process, although to date only the Candida albicans Mdr1 transporter has been demonstrated to be relevant in the clinical acquisition of antifungal drug resistance. This work provides evidence to suggest the involvement of the C. glabrata DHA transporters CgAqr1, CgQdr2, CgTpo1_1, and CgTpo3 in the clinical acquisition of clotrimazole drug resistance. A screening for azole drug resistance in 138 C. glabrata clinical isolates, from patients attending two major Hospitals in Portugal, was performed. Based on this screening, 10 clotrimazole susceptible and 10 clotrimazole resistant isolates were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance. As a proof-of-concept experiment, the CgTPO3 gene was deleted in an azole resistant C. glabrata isolate, exhibiting high levels of expression of this gene. The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

Prediction and characterization of P-glycoprotein substrates potentially bound to different sites by emerging chemical pattern and hierarchical cluster analysis

P-glycoprotein (P-gp), an ATP-binding cassette (ABC) multidrug transporter, can actively transport a broad spectrum of chemically diverse substrates out of cells and is heavily involved in multidrug resistance (MDR) in tumors. So far, the multiple specific binding sites remain a major obstacle in developing an efficient prediction method for P-gp substrates. Herein, emerging chemical pattern (ECP) combined by hierarchical cluster analysis was utilized to predict P-gp substrates as well as their potential binding sites. An optimal ECP model using only 3 descriptors was established with prediction accuracies of 0.80, 0.81 and 0.74 for 803 training samples, 120 test samples, and 179 independent validation samples, respectively. Hierarchical cluster analysis (HCA) of the ECPs of P-gp substrates derived 2 distinct ECP groups (ECPGs). Interestingly, HCA of the P-gp substrates based on ECP similarities also showed 2 distinct classes, which happened to be dominated by the 2 ECPGs, respectively. In the light of available experimental proofs and molecular docking results, the 2 distinct ECPGs were proved to be closely related to the binding profiles of R- and H-site substrates, respectively. The present study demonstrates, for the first time, a successful ECP model, which can not only accurately predict P-gp substrates, but also identify their potential substrate-binding sites.

Structural and Pharmacological Studies of an ABC Multidrug Transporter

A drug's effectiveness against a disease depends not only on its interaction with receptors but also its pharmacokinetics (absorption, distribution, metabolism, and extrusion; ADME). ATP binding cassette (ABC) multidrug transporters are important proteins that influence the ADME properties of a drug, especially the ABC transporter subfamily B member 1 (ABCB1). Elucidation of the molecular mechanisms of ABCB1 will contribute to our understanding of the molecular basis of ADME. Human ABCB1 is expressed in many organelles, and exports various substrates from cells using energy generated by its ATP hydrolase (ATPase) activity. The ATPase activity depends on the concentration of the transport substrates, and the characteristic behavior of the substrate-dependent ATPase activity can be related to the molecular mechanism of ABCB1. Recently, we have revealed the molecular mechanisms of a eukaryotic ABCB1 homolog, CmABCB1, based on structural and functional studies. In this review, I discuss the relationship between key structural features and the behavior of transport substrate-dependent ATPase activity of CmABCB1, including its role in determining the molecular basis of ADME.

SALL4 as an Epithelial-Mesenchymal Transition and Drug Resistance Inducer through the Regulation of c-Myc in Endometrial Cancer.

SALL4 plays important roles in the development and progression of many cancers. However, the role and molecular mechanism of SALL4 in endometrial cancer remain elusive. In the present research, we have demonstrated that the expression of SALL4 was upregulated in endometrial cancer and correlated positively with tumor stage, metastases and poor survival of patients. The overexpression of SALL4 promoted the invasiveness in endometrial cancer cells, as indicated by the upregulation of mesenchymal cell marker N-cadherin and downregulation of the epithelial marker E-cadherin, and invasion assays in vitro. Additionally, there was also an increase in drug resistance in these cell models due to the upregulation of ATP-binding cassette multidrug transporter ABCB1 expression. Moreover, we also found that ABCB1 was critical for SALL4-induced drug resistance. In contrast, SALL4 knockdown restored drug sensitivity, reversed EMT, diminished cell metastasis and suppressed the downregulation of E-cadherin and the upregulation of N-cadherin and ABCB1. Furthermore, we showed that SALL4 upregulated c-Myc expression and c-Myc was a direct target for SALL4 by ChIP assay, depletion of c-Myc with siRNA abolished the SALL4-induced downregulation of E-cadherin, upregulation of N-cadherin and ABCB1, suggesting that c-Myc was a downstream target for SALL4 and required for SALL4-induced EMT, invasion and drugs resistance in endometrial cancer cells. These results indicated that SALL4 could induce EMT and resistance to antineoplastic drugs through the regulation of c-Myc. SALL4 and c-Myc may be novel therapeutic targets for endometrial cancer.

The multidrug transporter Pdr5 on the 25th anniversary of its discovery: an important model for the study of asymmetric ABC transporters.

Asymmetric ABC (ATP-binding cassette) transporters make up a significant proportion of this important superfamily of integral membrane proteins. These proteins contain one canonical (catalytic) ATP-binding site and a second atypical site with little enzymatic capability. The baker's yeast (Saccharomyces cerevisiae) Pdr5 multidrug transporter is the founding member of the Pdr subfamily of asymmetric ABC transporters, which exist only in fungi and slime moulds. Because these organisms are of considerable medical and agricultural significance, Pdr5 has been studied extensively, as has its medically important homologue Cdr1 from Candida albicans. Genetic and biochemical analyses of Pdr5 have contributed important observations that are likely to be applicable to mammalian asymmetric ABC multidrug transporter proteins, including the basis of transporter promiscuity, the function of the non-catalytic deviant ATP-binding site, the most complete description of an invivo transmission interface, and the recent discovery that Pdr5 is a molecular diode (one-way gate). In the present review, we discuss the observations made with Pdr5 and compare them with findings from clinically important asymmetric ABC transporters, such as CFTR (cystic fibrosis transmembrane conductance regulator), Cdr1 and Tap1/Tap2.

A novel mathematical model describing adaptive cellular drug metabolism and toxicity in the chemoimmune system.

Cells cope with the threat of xenobiotic stress by activating a complex molecular network that recognizes and eliminates chemically diverse toxic compounds. This “chemoimmune system” consists of cellular Phase I and Phase II metabolic enzymes, Phase 0 and Phase III ATP Binding Cassette (ABC) membrane transporters, and nuclear receptors regulating these components. In order to provide a systems biology characterization of the chemoimmune network, we designed a reaction kinetic model based on differential equations describing Phase 0–III participants and regulatory elements, and characterized cellular fitness to evaluate toxicity. In spite of the simplifications, the model recapitulates changes associated with acquired drug resistance and allows toxicity predictions under variable protein expression and xenobiotic exposure conditions. Our simulations suggest that multidrug ABC transporters at Phase 0 significantly facilitate the defense function of successive network members by lowering intracellular drug concentrations. The model was extended with a novel toxicity framework which opened the possibility of performing in silico cytotoxicity assays. The alterations of the in silico cytotoxicity curves show good agreement with in vitro cell killing experiments. The behavior of the simplified kinetic model suggests that it can serve as a basis for more complex models to efficiently predict xenobiotic and drug metabolism for human medical applications.

Stubborn contaminants: influence of detergents on the purity of the multidrug ABC transporter BmrA.

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.

Inhibition or knockdown of ABC transporters enhances susceptibility of adult and juvenile schistosomes to Praziquantel.

Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease that affects hundreds of millions. Treatment of schistosomiasis depends almost entirely on the drug praziquantel (PZQ). Though essential to treating and controlling schistosomiasis, a major limitation of PZQ is that it is not active against immature mammalian-stage schistosomes. Furthermore, there are reports of field isolates with heritable reductions in PZQ susceptibility, and researchers have selected for PZQ-resistant schistosomes in the laboratory. P-glycoprotein (Pgp; ABCB1) and other ATP binding cassette (ABC) transporters remove a wide variety of toxins and xenobiotics from cells, and have been implicated in multidrug resistance (MDR). Changes in ABC transporter structure or expression levels are also associated with reduced drug susceptibility in parasitic helminths, including schistosomes. Here, we show that the activity of PZQ against schistosome adults and juveniles ex vivo is potentiated by co-administration of either the highly potent Pgp inhibitor tariquidar or combinations of inhibitors targeting multiple ABC multidrug transporters. Adult worms exposed to sublethal PZQ concentrations remain active, but co-administration of ABC transporter inhibitors results in complete loss of motility and disruption of the tegument. Notably, juvenile schistosomes (3–4 weeks post infection), normally refractory to 2 µM PZQ, become paralyzed when transporter inhibitors are added in combination with the PZQ. Experiments using the fluorescent PZQ derivative (R)-PZQ-BODIPY are consistent with the transporter inhibitors increasing effective intraworm concentrations of PZQ. Adult worms in which expression of ABC transporters has been suppressed by RNA interference show increased responsiveness to PZQ and increased retention of (R)-PZQ-BODIPY consistent with an important role for these proteins in setting levels of PZQ susceptibility. These results indicate that parasite ABC multidrug transporters might serve as important targets for enhancing the action of PZQ. They also suggest a potentially novel and readily-available strategy for overcoming reduced PZQ susceptibility of schistosomes.

The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter.

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata.

Acquisition of azole resistance by clinically relevant yeasts in nature may result in a significant, yet undetermined, impact in human health. The main goal of this study was to assess the development of cross-resistance between agricultural and clinical azoles by Candida spp. An in vitro induction assay was performed, for a period of 90 days, with prochloraz (PCZ) - an agricultural antifungal. Afterward, the induced molecular resistance mechanisms were unveiled. MIC value of PCZ increased significantly in all Candida spp. isolates. However, only C.glabrata developed cross-resistance to fluconazole and posaconazole. The increased MIC values were stable. Candida glabrata azole resistance acquisition triggered by PCZ exposure involved the upregulation of the ATP binding cassette multidrug transporter genes and the transcription factor, PDR1. Single mutation previously implicated in azole resistance was found in PDR1 while ERG11 showed several synonymous single nucleotide polymorphisms. These results might explain why C.glabrata is so commonly less susceptible to clinical azoles, suggesting that its exposure to agricultural azole antifungals may be associated to the emergence of cross-resistance. Such studies forward potential explanations for the worldwide increasing clinical prevalence of C.glabrata and the associated worse prognosis of an infection by this species.

Functional analysis of ATP-binding cassette transporter of Streptomyces coelicolor

Multidrug resistance (MDR) - type of transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistance to a range of drugs. ATP - binding cassette (ABC) transporters are characterized as MDR transporters utilizing ATP hydrolysis. The SCO 5113 (GeneID: 1100554) encoding a multidrug resistance ABC transporter in Streptomyces coelicolor was expressed in E. coli BL21 (DE3) plysS, using pET 21 a (+). The expression of SCO 5113 occured under 0.1 M IPTG within 2 to 8 hours, was detected on SDS-PAGE at 65 kDa. The E. coli BL21 (DE3) plysS harboring SCO 5113 was used to test for the antibiotic resistance, using disc diffussion method. The results showed the resistance of E. coli BL21 (DE3) plysS harboring SCO 5113 to five kinds of tested antibiotics such as cephalothin (1mg/ml), kanamycin (1mg/ml), ampicillin (10mg/ml), erythromycin (10mg/ml), and chloramphenicol (1mg/ml). The study reported the function of the SCO 5113 gene of Streptomyces coelicolor A3(2). Keywords: ABC transporter, antibiotic resistance, functional analysis, Streptomyces coelicolor Authors: TU NGUYEN, HUU NGUYEN

In vitro investigations of ABC transporters of the human liver – advantages and surprises

The hepatocyte contains a battery of membrane transporters in the sinusoidal and canalicular membrane that guarantee the regulated import and export of nutrients, messengers and toxic compounds. In the canalicular membrane, a number of important ABC (ATP binding cassette) transporters are located. Three of these primary active transporters, BSEP (bile salt export pump, ABC B11), MDR3 (multidrug resistance protein 3, ABC B4) and ABC G5/G8 catalyze the secretion of bile acids (BSEP), phosphatidylcholine (MDR3) and cholesterol (ABC G5/G8). These three molecules form mixed micelles in bile and thereby reduce the harmful action of unbound bile salts on membranes. BSEP, which was initially named s-Pgp (sister of P-gp) and MDR3 share a high sequence identity with MDR1 (P-glycoprotein, ABC B1), which is the hallmark of the family of multidrug resistance ABC transporter present in every eukaryotic cell. For BSEP the identity is approximately 70% and for MDR3 even 85%, respectively. Nevertheless, MDR3 is specific for phosphatidylcholine lipids and BSEP is specific for bile acids, while MDR1 is capable of translocating a myriad of structurally unrelated compound across biological membranes. This of course raises the question, which residues impose specificity in the case of BSEP and MDR3 and which residues impose promiscuity in the case of MDR1. Homology models of BSEP and MDR3 were generated based on the crystal structures of Sav1866 and mouse P-gp to obtain initial information about the putative three-dimensional location of certain amino acid residues of BSEP and MDR3. However, one has to stress that these “structures” represent only models that have to be used with proper caution. The homology model of MDR3 was helpful in providing a structural explanation for the non-functionality of the MDR3 mutant H1231Y. The mutant protein was properly located to the plasma membrane, but displayed a severe substrate transport phenotype. Histidine 1231 corresponds to the histidine of the H-loop, a residue that is critical for ATP hydrolysis in many other ABC transporter systems. Thus, exchange of His against Tyr in position 1231 of MDR3 will impair ATP hydrolysis in one of the two ATP binding site drastically reducing transport activity of MDR3 H1231Y [1]. To study the transport cycle and the importance of mutations within these transporters on their function, overexpression systems are required. We decided to use the methylotrophic yeast Pichia pastoris for the overexpression of two of the three transporters, BSEP and MDR3, respectively. Initially we encountered severe problems in cloning the transporters genes in E. coli employing standard approaches. Only after establishing a cloning strategy that is entirely based on Saccharomyces cerevisiae, both genes could be successfully cloned [2]. This allowed us to combine the speed and ease of cloning of this yeast system for the wild type genes and mutations that were identified in patients. Having this tool in hand, we overexpressed BSEP in yeast to study its transport function in plasma membrane vesicles. This allowed us to establish structure-function relations for a new clinically relevant mutation (G374S) that resulted in a phenotype between PFIC-2 and BRIC-2. Although membrane targeting was normal, e.g. targeting to the canalicular membrane was evident, transport activity of the G374S was strongly impaired [3]. The Sav1866-based homology model of BSEP suggested that Gly 374 is lining the translocation pore of BSEP. Thus, an exchange to serine would impose steric restriction within the pore, thereby influencing the substrate spectrum as evident from the bile acid transport assays. Based on the expression levels of BSEP and MDR3, purification protocols for both membrane proteins were successfully established [4]. Thus, next to the transport assay that is based only highly enriched yeast plasma membranes, purified protein is now also available in the detergent-solubilized state. While the transport assay can be used for BSEP to analyze a possible influence of a mutation on the transporter activity of the protein, purified MDR3 can be used to determine the influence of a mutation on ATPase activity and / or stimulation or inhibition of this activity. In summary, the established yeast overexpression system combines the ease and speed of yeast cloning approaches with the possibility to overexpress and purify both proteins in a quantity and homogeneity sufficient for biochemical and biophysical investigations of the function of these two ABC transporters. Furthermore, the speed with which mutants can be obtained will allow an in depth characterization of clinically relevant mutations of both transporters with respect to function and spectrum of transported substrates.

MFS multidrug transporters in pathogenic fungi: do they have real clinical impact?

Infections caused by opportunistic fungal pathogens have reached concerning numbers due to the increase of the immunocrompromised human population and to the development of antifungal resistance. This resistance is often attributed to the action of multidrug efflux pumps, belonging to the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS). Although many studies have focused on the role of ABC multidrug efflux transporters, little is still known on the part played by the Drug:H+ Antiporter (DHA) family of the MFS in this context. This review summarizes current knowledge on the role in antifungal drug resistance, mode of action and phylogenetic relations of DHA transporters, from the model yeast S. cerevisiae to pathogenic yeasts and filamentous fungi. Through the compilation of the predicted DHA transporters in the medically relevant Candida albicans, C. glabrata, C. parapsilosis, C. lusitaniae, C. tropicalis, C. guilliermondii, Cryptococcus neoformans, and Aspergillus fumigatus species, the fact that only 5% of the DHA transporters from these organisms have been characterized so far is evidenced. The role of these transporters in antifungal drug resistance and in pathogen-host interaction is described and their clinical relevance discussed. Given the knowledge gathered for these few DHA transporters, the need to carry out a systematic characterization of the DHA multidrug efflux pumps in fungal pathogens, with emphasis on their clinical relevance, is highlighted.

Phytol derivatives as drug resistance reversal agents.

Phytol was chemically transformed into fifteen semi-synthetic derivatives, which were evaluated for their antibacterial and drug resistance reversal potential in combination with nalidixic acid against E. coli strains CA8000 and DH5α. The pivaloyl (4), 3,4,5-trimethoxybenzoyl (9), 2,3-dichlorobenzoyl (10), cinnamoyl (11), and aldehyde (14) derivatives of phytol ((2E,7R,11R)-3,7,11,15-tetramethyl-2-hexadecen-1-ol) were evaluated by using another antibiotic, tetracycline, against the MDREC-KG4 clinical isolate of E. coli. Derivative 4 decreased the maximal inhibitory concentration (MIC) of the antibiotics by 16-fold, while derivatives 9, 10, 11, and 14 reduced MIC values of the antibiotics up to eightfold against the E. coli strains. Derivatives 4, 9, 10, 11, and 14 inhibited the ATP-dependent efflux pump; this was also supported by their in silico binding affinity and down-regulation of the efflux pump gene yojI, which encodes the multidrug ATP-binding cassette transporter protein. This study supports the possible use of phytol derivatives in the development of cost-effective antibacterial combinations.

3D Cryo-Electron Reconstruction of BmrA, a Bacterial Multidrug ABC Transporter in an Inward-Facing Conformation and in a Lipidic Environment

ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3nm and 2.5nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane.

真核生物由来ABC多剤排出トランスポーターの構造と分子メカニズム

ATP-binding cassette (ABC) multidrug transporters are membrane proteins which transport various structurally unrelated substrates using the energy of ATP hydrolysis. The precise transport mechanisms of a human ABC multidrug transporter, P-glycoprotein (hP-gp), are not fully understood based on the crystal structure, because of the difficulty of crystallization of hP-gp. Recently, we have determined the crystal structures of a hP-gp homolog, CmABCB1 from Cyanidioschyzon merolae, at 2.6 Å, and its complex with a novel allosteric inhibitor at 2.4 Å. Here, we present how these high resolution structure determinations were achieved, and explain the detailed architecture of the transmembrane domains of CmABCB1.

Combined QSAR and molecule docking studies on predicting P-glycoprotein inhibitors

P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter. The over expression of P-gp leads to the development of multidrug resistance (MDR), which is a major obstacle to effective treatment of cancer. Thus, designing effective P-gp inhibitors has an extremely important role in the overcoming MDR. In this paper, both ligand-based quantitative structure-activity relationship (QSAR) and receptor-based molecular docking are used to predict P-gp inhibitors. The results show that each method achieves good prediction performance. According to the results of tenfold cross-validation, an optimal linear SVM model with only three descriptors is established on 857 training samples, of which the overall accuracy (Acc), sensitivity, specificity, and Matthews correlation coefficient are 0.840, 0.873, 0.813, and 0.683, respectively. The SVM model is further validated by 418 test samples with the overall Acc of 0.868. Based on a homology model of human P-gp established, Surflex-dock is also performed to give binding free energy-based evaluations with the overall accuracies of 0.823 for the test set. Furthermore, a consensus evaluation is also performed by using these two methods. Both QSAR and molecular docking studies indicate that molecular volume, hydrophobicity and aromaticity are three dominant factors influencing the inhibitory activities.