A mass spectrometry based functional assay for the quantitative assessment of ABC transporter activity

ATP‐Binding Cassette (ABC) transporters are highly expressed in pharmacological barriers limiting the access of drugs to their targets. Since characterization of a compound as a transporter substrate or inhibitor bears significant consequences in drug development, there is a great need for reliable tools that enable the rapid analysis of the transport susceptibility of drugs. Here we describe a simple but very efficient high‐performance liquid chromatography/mass spectrometry (HPLC/MS) assay for measuring the ABC transporter‐dependent vesicular transport of compounds. In addition, we provide evidence that the requirement for sample preparation can be minimized using desorption electrospray ionization (DESI)‐MS, paving the way for a direct, high‐throughput investigation of drug‐transporter interactions. Copyright © 2009 John Wiley & Sons, Ltd.     

Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin

ATP-binding cassette (ABC) transporters have been implicated in a multitude of biological pathways. In plants, some ABC transporters are involved in the polar transport of the plant hormone auxin and the gravitropic response. We previously identified Gravacin as a potent inhibitor of gravitropism in Arabidopsis thaliana. We demonstrate that P-glycoprotein19 (PGP19) is a target for Gravacin and participates in its inhibition of gravitropism. Gravacin inhibited the auxin transport activity of PGP19 and PGP19-PIN complexes. Furthermore, we identified E1174 as an important residue for PGP19 activity and its ability to form active transport complexes with PIN1. Gravacin is an auxin transport inhibitor that inhibits PGPs, particularly PGP19, which can be used to further dissect the role of PGP19 without the inhibition of other auxin transporters, namely PIN proteins.     

Probing of multidrug ABC membrane transporters of single living cells using single plasmonic nanoparticle optical probes

Currently, molecular mechanisms of multidrug ABC (ATP-binding cassette) membrane transporters remain elusive. In this study, we synthesized and characterized purified spherically shaped silver nanoparticles (Ag NPs) (11.8 ± 2.6 nm in diameter), which were stable (non-aggregation) in PBS buffer and inside single living cells. We used the size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs to determine their sizes and to probe the size-dependent transport kinetics of the ABC (BmrA, BmrA-EGFP) transporters in single living cells (Bacillus subtilis) in real time at nanometer resolution using dark-field optical microscopy and spectroscopy (DFOMS). The results show that the smaller NPs stayed longer inside the cells than larger NPs, suggesting size-dependent efflux kinetics of the membrane transporter. Notably, accumulation and efflux kinetics of intracellular NPs for single living cells depended upon the cellular expression level of BmrA, NP concentrations, and a pump inhibitor (25 μM, orthovanadate), suggesting that NPs are substrates of BmrA transporters and that passive diffusion driven by concentration gradients is the primary mechanism by which the NPs enter the cells. The accumulation and efflux kinetics of intracellular NPs for given cells are similar to those observed using a substrate (Hoechst dye) of BmrA, demonstrating that NPs are suitable probes for study of multidrug membrane transporters of single living cells in real-time. Unlike fluorescent probes, single Ag NPs exibit size-dependent LSPR spectra and superior photostability, enabling them to probe the size-dependent efflux kinetics of membrane transporters of single living cells in real-time for better understanding of multidrug resistance.     

Intrinsic and Chemotherapeutic Stressors Modulate ABCC-Like Transport in Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent for Chagas disease, which affects 6–7 million people worldwide. The biological diversity of the parasite reflects on inefficiency of benznidazole, which is a first choice chemotherapy, on chronic patients. ABC transporters that extrude xenobiotics, metabolites, and mediators are overexpressed in resistant cells and contribute to chemotherapy failure. An ABCC-like transport was identified in the Y strain and extrudes thiol-conjugated compounds. As thiols represent a line of defense towards reactive species, we aimed to verify whether ABCC-like transport could participate in the regulation of responses to stressor stimuli. In order to achieve this, ABCC-like activity was measured by flow cytometry using fluorescent substrates. The present study reveals the participation of glutathione and ceramides on ABCC-like transport, which are both implicated in stress. Hemin modulated the ABCC-like efflux which suggests that this protein might be involved in cellular detoxification. Additionally, all strains evaluated exhibited ABCC-like activity, while no ABCB1-like activity was detected. Results suggest that ABCC-like efflux is not associated with natural resistance to benznidazole, since sensitive strains showed higher activity than the resistant ones. Although benznidazole is not a direct substrate, ABCC-like efflux increased after prolonged drug exposure and this indicates that the ABCC-like efflux mediated protection against cell stress depends on the glutathione biosynthesis pathway.     

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.     

Molecular phylogenetic study and expression analysis of ATP-binding cassette transporter gene family in Oryza sativa in response to salt stress

ATP-binding cassette (ABC) transporter is a large gene superfamily that utilizes the energy released from ATP hydrolysis for transporting myriad of substrates across the biological membranes. Although many investigations have been done on the structural and functional analysis of the ABC transporters in Oryza sativa, much less is known about molecular phylogenetic and global expression pattern of the complete ABC family in rice. In this study, we have carried out a comprehensive phylogenetic analysis constructing neighbor-joining and maximum-likelihood trees based on various statistical methods of different ABC protein subfamily of five plant lineages including Chlamydomonas reinhardtii (green algae), Physcomitrella patens (moss), Selaginella moellendorffii (lycophyte), Arabidopsis thaliana (dicot) and O. sativa (monocot) to explore the origin and evolutionary patterns of these ABC genes. We have identified several conserved motifs in nucleotide binding domain (NBD) of ABC proteins among all plant lineages during evolution. Amongst the different ABC protein subfamilies, ‘ABCE’ has not yet been identified in lower plant genomes (algae, moss and lycophytes). The result indicated that gene duplication and diversification process acted upon these genes as a major operative force creating new groups and subgroups and functional divergence during evolution. We have demonstrated that rice ABCI subfamily consists of only half size transporters that represented highly dynamic members showing maximum sequence variations among the other rice ABC subfamilies. The evolutionary and the expression analysis contribute to a deep insight into the evolution and diversity of rice ABC proteins and their roles in response to salt stress that facilitate our further understanding on rice ABC transporters.     

Endogenous Biomarkers for SLC Transporter-Mediated Drug-Drug Interaction Evaluation

Membrane transporters play an important role in the absorption, distribution, metabolism, and excretion of xenobiotic substrates, as well as endogenous compounds. The evaluation of transporter-mediated drug-drug interactions (DDIs) is an important consideration during the drug development process and can guide the safe use of polypharmacy regimens in clinical practice. In recent years, several endogenous substrates of drug transporters have been identified as potential biomarkers for predicting changes in drug transport function and the potential for DDIs associated with drug candidates in early phases of drug development. These biomarker-driven investigations have been applied in both preclinical and clinical studies and proposed as a predictive strategy that can be supplanted in order to conduct prospective DDIs trials. Here we provide an overview of this rapidly emerging field, with particular emphasis on endogenous biomarkers recently proposed for clinically relevant uptake transporters.     

Determination of fexofenadine in Hank's balanced salt solution by high‐performance liquid chromatography with ultraviolet detection: application to Caco‐2 cell permeability studies

The drug‐transporting proteins can affect the pharmacokinetics and pharmacodymanics of many drugs, resulting in an erratic and unpredictable pharmacological response. The Caco‐2 monolayer is routinely applied to investigate the carrier‐mediated transport of drugs. Therefore, the selection of a marker compound able to characterize the activity of such transporters is crucial. Fexofenadine (FEX), a P‐gp/OATP substrate, can be considered a suitable probe. However, in order to use be used as a marker compound, it is mandatory to develop an analytical method able to quantify this drug during the in vitro permeability assay. An HPLC method with ultraviolet detection was developed; the mobile phase consisted of phosphate buffer (pH 3.2) containing 10 m m of sodium octanosulphonate and acetonitrile (60:40) and the flow rate was set at 1.2 mL/min. Fexofenadine was eluted at 40°C, the retention time was about 4.6 min. The LOD and LOQ values were 1.9 and 6.2 ng/mL, respectively. Verapamil and ketoconazole, the most common P‐gp inhibitors, were eluted as distinct peaks of that corresponding to fexofenadine The method was successfully applied to quantify the amount of FEX transported across the Caco‐2 monolayer and could be an additional tool for those investigating the role of membrane transporters on drug absorption. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolic stability of the nucleoside transport system of Novikoff rat hepatoma cells

Rates of transport of uridine and thymidine, estimated with a rapid sampling technique, did not change with culture age. Inhibition of cellular RNA and protein synthesis for periods up to 6 h, did not lead to a loss of nucleoside transport activity. Mild treatment of cell suspensions with trypsin or neuraminidase had no effect on the kinetics of thymidine transport. Thus we conclude, contrary to previous reports, that nucleoside transporters are metabolically stable and that the decreases in nucleoside uptake rates observed with decreased protein synthesis reflect loss of nucleoside kinase activities. These kinases (which have narrow substrate specificity) rather than the membrane-associated, transport apparatus (which has broad substrate specificity) are the most likely sites for regulation of nucleoside uptake.     

Classifying substrate specificities of membrane transporters from Arabidopsis thaliana

Membrane transporters catalyze the active transport of molecules across biological barriers such as lipid bilayer membranes. Currently, the experimental annotation of which proteins transport which substrates is far from complete and will likely remain so for much longer. Therefore, it is highly desirable to develop computational methods that may aid in the substrate annotation of putative membrane transport proteins. Here, we measured the similarity of membrane transporters from Arabidopsis thaliana by their amino acid composition, higher sequence order information, amino acid characteristics, or sequence conservation. We considered the substrate classes amino acids, oligopeptides, phosphates, and hexoses. Substrate classification based on the amino acid frequency yielded an accuracy of 75% or higher. Integrating additional information improved the prediction performance to 90% and higher.     

Mapping Conformational Heterogeneity of Mitochondrial Nucleotide Transporter in Uninhibited States

One of the less well understood aspects of membrane transporters is the dynamic coupling between conformational change and substrate transport. NMR approaches are used herein to investigate conformational heterogeneity of the GTP/GDP carrier (GGC) from yeast mitochondria. NMR residual dipolar coupling (RDC) analysis of GGC in a DNA‐origami nanotube liquid crystal shows that several structured segments have different generalized degrees of order (GDO), thus indicating the presence of conformational heterogeneity. Complete GDO mapping reveals asymmetry between domains of the transporter and even within certain transmembrane helices. Nucleotide binding partially reduces local structural heterogeneity, and the substrate binds to multiple sites along the transport cavity. These observations suggest that mitochondrial carriers in the uninhibited states are intrinsically plastic and structural plasticity is asymmetrically distributed among the three homologous domains.     

5-Aminolaevulinic acid methyl ester transport on amino acid carriers in a human colon adenocarcinoma cell line

The transport mechanisms of 5-aminolevulinic acid methyl ester (5-ALA-ME) have been studied in a human adenocarcinoma cell line (WiDr) by means of 14[C]-labeled 5-ALA-ME. The transport was found to be partly Na+ dependent, while the extracellular Cl- concentration did not affect the uptake. The transport of 5-ALA-ME into WiDr cells was dependent on the incubation temperature and was found to be completely blocked by the inhibitors of energy metabolism, 2-deoxyglucose and sodium azide. WiDr cells were treated with 10 mM of 14 different amino acids and the substrate specificity of the 5-ALA-ME transporter(s) was analyzed by treating the cells with 23 microM or 1 mM 14[C]-labeled 5-ALA-ME. The transport of 5-ALA-ME was found to be inhibited to the highest extent, i.e. about 60%, by the nonpolar amino acids L-alanine, L-methionine, L-tryptophan and glycine. The uptake of 5-ALA-ME followed an exponential decay with increasing concentration of glycine, reaching a maximum inhibition of uptake of 5-ALA-ME of 55%. Sarcosine, a specific inhibitor of system Gly, did not significantly inhibit 5-ALA-ME transport. In contrast to transport of 5-ALA, 5-ALA-ME does not seem to be taken up by system BETA transporters. In conclusion, the cellular uptake of 5-ALA-ME into WiDr cells seems to be due to active transport mechanisms, involving transporters of nonpolar amino acids.     

2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.     

Quantification of the transporter substrate fexofenadine in cell lysates by liquid chromatography/tandem mass spectrometry

Drug–drug interactions at transporters present a significant and under‐investigated clinical problem. Investigations of specific transporter functions and screening for potential drug‐drug interactions, both in vitro and especially in vivo, will require validated experimental probes. Fexofenadine, an approved, well‐tolerated drug, is a promising probe for studies of membrane transporter function. Although fexofenadine pharmacokinetics are known to be controlled by transporters, the contributions of individual transporters have not been defined. We have developed a rapid, specific, and sensitive analytical method for quantitation of fexofenadine to support this work. This liquid chromatography/tandem mass spectrometry (LC/MS/MS) method quantifies fexofenadine in cell lysates from in vitro studies using cetirizine as the internal standard. Cell lysates were prepared for analysis by acetonitrile precipitation. Analytes were then separated by gradient reversed‐phase chromatography and analyzed by tandem mass spectrometry using the m/z 502.17/466.2 transition for fexofenadine and m/z 389.02/201.1 for cetirizine. The method exhibited a linear dynamic range of 1–500 ng/mL for fexofenadine in cell lysates. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 5%. Intra‐ and inter‐day precision and accuracy were within the limits presented in the FDA guidelines for bioanalysis. We also will validate this method to support not only the quantification of fexofenadine, but also other probe drugs for drug–drug interaction studies. This method for quantification will facilitate the use of fexofenadine as a probe drug for characterization of transporter activity. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of membrane transport mechanisms of endogenous substrates using chromatographic techniques

Membrane transporters are expressed in various bodily tissues and play essential roles in the homeostasis of endogenous substances and the absortion, distribution and/or excretion of xenobiotics. For transporter assays, radioisotope‐labeled compounds have been mainly used. However, commercially available radioisotope‐labeled compounds are limited in number and relatively expensive. Chromatographic analyses such as high‐performance liquid chromatography with ultraviolet absorptiometry and liquid chromatography with tandem mass spectrometry have also been applied for transport assays. To elucidate the transport properties of endogenous substrates, although there is no difficulty in performing assays using radioisotope‐labeled probes, the endogenous background and the metabolism of the compound after its translocation across cell membranes must be considered when the intact compound is assayed. In this review, the current state of knowledge about the transport of endogenous substrates via membrane transporters as determined by chromatographic techniques is summarized. Chromatographic techniques have contributed to our understanding of the transport of endogenous substances including amino acids, catecholamines, bile acids, prostanoids and uremic toxins via membrane transporters.     

Structural modeling of human organic cation transporters

Human organic cation transporters (hOCTs) belong to solute carriers (SLC) 22 family of membrane proteins that play a central role in transportation of chemotherapeutic drugs for several clinical and pathological conditions, including cancer and diabetes. These transporters mediate drug transport; however, the precise mechanism of drug-binding and transport by them is not fully uncovered yet, partly due to unavailability of any crystal structure record. In this work, we performed a multi-phasic approach to compute the 3D structural models of seven human organic cation transporters (hOCTs) starting from primary protein sequence. Our structure modeling approach included 1) I-TASSER based comparative sequence alignment, threading and ab-initio protein modeling; 2) models comparison with PSIPRED secondary structure prediction; 3) loop modeling for incongruent secondary structure in Chimera 1.10.1; 4) high resolution structure simulation, refinement, energy minimization using ModRefiner, and 5) validation of the structure models using PROCHECK at SAVEs. From structural point, the computed 3D structures of hOCTs consist of a typical major facilitator superfamily (MFS) fold of twelve α-transmembrane helix domains arranged in a manner rendering hOCTs a barrel shaped structure with a large cleft that opens in cytoplasm. The modeled 3D structure of all hOCTs closely resemble to human SLC2A3 (GLUT3) transporter (PDB ID: 5c65) and displayed an outward-open confirmation and putative cyclic C1 protein symmetry. In addition, hOCTs has a large (>100 amino acids) unique extracellular loop between TMH1 and TMH2 having potential glycosylation sites (Asn-Xaa-Ser/Thr) and cysteine residues, both features indicative of putative role in drug binding and uptake. There is an intracellular three/four-helix loop between TMH6 and TMH7 containing putative phosphorylation sites for precise regulation of hOCTs function as drug transporters. There are nine loops of 4 to 11 amino acids length that protrude from membrane, both intracellularly and extracellularly, and connect adjacent TMHs. The 2D structure prediction showed N_in-C_in topology of all hOCTs. In the unavailability of the crystal structures of hOCTs, the 3D structural models computed in-silico and presented herein can be used for studying the mechanism of drug binding and transport by hOCTs.     

Role of the D-loops in allosteric control of ATP hydrolysis in an ABC transporter

ABC transporters couple ATP hydrolysis to movement of substrates across cell membranes. They comprise two transmembrane domains and two cytosolic nucleotide-binding domains forming two active sites that hydrolyze ATP cooperatively. The mechanism of ATP hydrolysis is controversial and the structural dynamic basis of its allosteric control unknown. Here we report molecular dynamics simulations of the ATP/apo and ATP/ADP states of the bacterial ABC exporter Sav1866, in which the cytoplasmic region of the protein was simulated in explicit water for 150 ns. In the simulation of the ATP/apo state, we observed, for the first time, conformers of the active site with the canonical geometry for an in-line nucleophilic attack on the ATP γ-phosphate. The conserved glutamate immediately downstream of the Walker B motif is the catalytic base, forming a dyad with the H-loop histidine, whereas the Q-loop glutamine has an organizing role. Each D-loop provides a coordinating residue of the attacking water, and comparison with the simulation of the ATP/ADP state suggests that via their flexibility, the D-loops modulate formation of the hydrolysis-competent state. A global switch involving a coupling helix delineates the signal transmission route by which allosteric control of ATP hydrolysis in ABC transporters is mediated.