Specific inhibition of the ABCG2 transporter could improve the efficacy of photodynamic therapy

Photodynamic therapy is based on the selective accumulation of a photosensitizer in tumors, followed by destruction of the target tissue by a light source. Protoporphyrin IX, a well-known photosensitizer, was recently reported as an endogenous substrate for the multidrug transporter ABCG2. We investigated the role of ABCG2 protein in the porphyrin extrusion ability of keratinocytes, with regard to the impact of the specific inhibition of ABCG2 by a non-toxic fumitremorgin C analog, Ko-134, on photodynamic therapy efficacy. We studied the level of porphyrin accumulation in response to delta-aminolevulinic acid pretreatment in proliferating and highly differentiated HaCaT keratinocytes. An in vitro model of photodynamic therapy on HaCaT cells was established with a therapeutically approved narrow-bandwidth red-light source. The porphyrin extrusion ability of HaCaT cells proved to correlate with their ABCG2 expression which was higher in proliferating cells than in differentiated cells. Moreover, the specific inhibition of ABCG2 by Ko-134 enhanced the sensitivity of keratinocytes to photodynamic therapy in vitro. These results suggest that ABCG2 may serve as a target molecule via which to improve the photodynamic therapy of skin lesions: its inhibition by the non-toxic Ko-134 is a promising therapeutic modality.     

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.     

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.     

Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy

Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

Crucial role of HMGA1 in the self-renewal and drug resistance of ovarian cancer stem cells

Cancer stem cells are a subpopulation of cancer cells characterized by self-renewal ability, tumorigenesis and drug resistance. The aim of this study was to investigate the role of HMGA1, a chromatin remodeling factor abundantly expressed in many different cancers, in the regulation of cancer stem cells in ovarian cancer. Spheroid-forming cancer stem cells were isolated from A2780, SKOV3 and PA1 ovarian cancer cells by three-dimensional spheroid culture. Elevated expression of HMGA1 was observed in spheroid cells along with increased expression of stemness-related genes, such as SOX2, KLF4, ALDH, ABCB1 and ABCG2. Furthermore, spheroid A2780 cells, compared with adherent cells, showed higher resistance to chemotherapeutic agents such as paclitaxel and doxorubicin. HMGA1 knockdown in spheroid cells reduced the proliferative advantage and spheroid-forming efficiency of the cells and the expression of stemness-related genes. HMGA1 overexpression in adherent A2780 cells increased cancer stem cell properties, including proliferation, spheroid-forming efficiency and the expression of stemness-related genes. In addition, HMGA1 regulated ABCG2 promoter activity through HMGA1-binding sites. Knockdown of HMGA1 in spheroid cells reduced resistance to chemotherapeutic agents, whereas the overexpression of HMGA1 in adherent ovarian cancer cells increased resistance to chemotherapeutic agents in vitro. Furthermore, HMGA1-overexpressing A2780 cells showed a significant survival advantage after chemotherapeutic agent treatment in a xenograft tumorigenicity assay. Together, our results provide novel insights regarding the critical role of HMGA1 in the regulation of the cancer stem cell characteristics of ovarian cancer cells, thus suggesting that HMGA1 may be an important target in the development of therapeutics for ovarian cancer patients.     

Structure-Based Discovery of ABCG2 Inhibitors: A Homology Protein-Based Pharmacophore Modeling and Molecular Docking Approach

ABCG2 is an ABC membrane protein reverse transport pump, which removes toxic substances such as medicines out of cells. As a result, drug bioavailability is an unexpected change and negatively influences the ADMET (absorption, distribution, metabolism, excretion, and toxicity), leading to multi-drug resistance (MDR). Currently, in spite of promising studies, screening for ABCG2 inhibitors showed modest results. The aim of this study was to search for small molecules that could inhibit the ABCG2 pump. We first used the WISS MODEL automatic server to build up ABCG2 homology protein from 655 amino acids. Pharmacophore models, which were con-structed based on strong ABCG2 inhibitors (IC₅₀ < 1 μM), consist of two hydrophobic (Hyd) groups, two hydrogen bonding acceptors (Acc2), and an aromatic or conjugated ring (Aro|PiR). Using molecular docking method, 714 substances from the DrugBank and 837 substances from the TCM with potential to inhibit the ABCG2 were obtained. These chemicals maybe favor synthesized or extracted and bioactivity testing.     

Profiling and comprehensive expression analysis of ABC transporter solute-binding proteins of Bacillus subtilis membrane based on a proteomic approach

We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).     

ABC triblock polymethacrylates: Group transfer polymerization synthesis of the ABC,ACB, and BAC topological isomers and solution characterization

ABC triblock copolymers of methyl methacrylate (MMA), (dimethylamino)-ethyl methacrylate (DMAEMA), and tetrahydropyranyl methacrylate (THPMA) consisting of 12 units of each type of monomer were synthesized by group transfer polymerization (GTP). These were the three topological isomers with differentblock sequences: DMAEMA₁₂-THPMA₁₂-MMA₁₂, DMAEMA₁₂-MMA₁₂-THPMA₁₂, and THPMA₁₂-DMAEMA₁₂-MMA₁₂. The molecular weights and molecular weight distributions of the copolymers were determined by gel permeation chromatography (GPC) in tetrahydrofuran, and their number-average degrees of polymerization and copolymer compositions were calculated by proton nuclear magnetic resonance spectroscopy (¹H-NMR). These molecular weights and degrees of polymerization corresponded closely to the values expected from the monomer/initiator ratios. The polydispersities were low as expected for GTP, and ranged from 1.09 to 1.25. The three triblocks were chemically modified by converting the THPMA units to methacrylic acid (MAA) units either by thermolysis or acid hydrolysis. The resulting ABC triblock poly-ampholytes were characterized by ¹H-NMR spectroscopy and hydrogen ion titration. Aqueous GPC studies in 1.0M NaCl at pH 8.5 showed that the triblock copolymers form micelles whose size depends on their block sequence. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 617–631, 1998     

Binding of serotonin to the human serotonin transporter. Molecular modeling and experimental validation

Molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-HT) to the human serotonin transporter (hSERT). Homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from Aquifex aeolicus, as the template and three slightly different sequence alignments. Induced fit docking of 5-HT into hSERT homology models resulted in two different binding modes. Both show a salt bridge between Asp98 and the charged primary amine of 5-HT, and both have the 5-HT C6 position of the indole ring pointing toward Ala173. The difference between the two orientations of 5-HT is an enantiofacial discrimination of the indole ring, resulting in the 5-hydroxyl group of 5-HT being vicinal to either Ser438/Thr439 or Ala169/Ile172/Ala173. To assess the binding experimentally, binding affinities for 5-HT and 17 analogues toward wild type and 13 single point mutants of hSERT were measured using an approach termed paired mutant-ligand analogue complementation (PaMLAC). The proposed ligand-protein interaction was systematically examined by disrupting it through site-directed mutagenesis and re-establishing another interaction via a ligand analogue matching the mutated residue, thereby minimizing the risk of identifying indirect effects. The interactions between Asp98 and the primary amine of 5-HT and the interaction between the C6-position of 5-HT and hSERT position 173 was confirmed using PaMLAC. The measured binding affinities of various mutants and 5-HT analogues allowed for a distinction between the two proposed binding modes of 5-HT and biochemically support the model for 5-HT binding in hSERT where the 5-hydroxyl group is in close proximity to Thr439.     

Comparing the morphology and phase diagram of H-shaped ABC block copolymers and linear ABC block copolymers

By using a combinatorial screening method based on the self-consistent field theory (SCFT) for polymers, we have investigated the morphology of H-shaped ABC block copolymers (A2BC2) and compared them with those of the linear ABC block copolymers. By changing the ratios of the volume fractions of two A arms and two C arms, one can obtain block copolymers with different architectures ranging from linear block copolymer to H-shaped block copolymer. By systematically varying the volume fractions of block A, B, and C, the triangle phase diagrams of the H-shaped ABC block copolymer with equal interactions among the three species are constructed. In this study, we find four different morphologies (lamellar phase (LAM), hexagonal lattice phase (HEX), core-shell hexagonal lattice phase (CSH), and two interpenetrating tetragonal lattice (TET2)). Furthermore, the order-order transitions driven by architectural change are discussed.     

Proteomic and redox‐proteomic study on the role of glutathione reductase in human lung cancer cells

Glutathione reductase (GR), a cytosolic protein, plays a vital role in maintaining a correct redox status in cells. However, comprehensive investigations of GR‐modulated cellular responses, including protein level alteration and redox regulation, have yet to be performed. In this study, we cultured a human lung adenocarcinoma line transfected with empty pLKO.1 vector as a control, CL1‐0shControl, and its GR‐knockdown derivative, CL1‐0shΔGR, to evaluate differential protein level alteration and redox regulation of these two cell lines. We identified 34 spots that exhibited marked changes in intensities, and 13 proteins showing significant changes in thiol reactivity, in response to GR depletion. Several proteins involved in redox regulation, calcium signaling, cytoskeleton regulation, and protein folding showed significant changes in expression, whereas proteins involved in redox regulation, protein folding, and glycolysis displayed changes in thiol reactivity. Interestingly, GR knockdown induces peroxiredoxin‐1 overexpression in the air‐exposed tissue and high oxygen consuming tissue such as cornea and liver, but not in the low oxygen consuming tissues such as breast and uterine. In summary, we used a comprehensive lung adenocarcinoma based proteomic approach for identifying GR‐modulated protein expression alteration and redox modification. Based on our research, this is the first comprehensive proteomic and redox‐proteomic analysis used to investigate the role of GR in a mammalian cell model.     

Simultaneous binding of anti-tuberculosis and anti-thrombosis drugs to a human transporter protein: a FRET study

Although rifampicin (Rf) is one of the most effective antibiotics against infection caused by Mycobacterium tuberculosis, interaction of the drug with universal carrier protein in human blood plasma is not fully understood. Reduction of medicinal efficacy of other drugs, including anti-thrombosis drug warfarin (Wf), to the patients on Rf therapy also needs molecular understanding. In the present work we have studied interaction of Rf with one of the model carrier protein (human serum albumin). By using circular dichroism (CD) spectroscopy we have characterized the change in the secondary structure of the protein. The consequence of the simultaneous binding of the two drugs, Rf and Wf, on the structure of the protein has also been explored. Picosecond resolved F?rster resonance energy transfer (FRET) from Wf to Rf explores possible binding sites of the anti-tuberculosis drug on the protein. In this report, we have discussed the potential problem of using the single tryptophan of the protein (Trp 214) as energy donor in FRET experiment for the characterization of the binding site of the drug Rf on the protein.     

Resolvin E2: identification and anti-inflammatory actions: pivotal role of human 5-lipoxygenase in resolvin E series biosynthesis

The family of resolvins consists of omega-3 fatty acid-derived mediators, including E series resolvins generated from eicosapentaenoic acid (EPA), and carry potent anti-inflammatory properties. Here, we report the isolation, identification, and bioactions of resolvin E2 (RvE2), which is 5S,18-dihydroxy-eicosapentaenoic acid. RvE2 stopped zymosan-induced polymorphonuclear (PMN) leukocyte infiltration and displayed potent anti-inflammatory properties in murine peritonitis. We also demonstrate that human recombinant 5-lipoxygenase generates RvE2 from a common precursor of E series resolvins, namely, 18-hydroxyeicosapentaenoate (18-HEPE). Furthermore, the initial 5-hydroperoxide intermediate was also converted to a 5(6)-epoxide intermediate in RvE1 formation. These results demonstrate that RvE2, together with RvE1, may contribute to the beneficial actions of omega-3 fatty acids in human diseases. Moreover, they indicate that the 5-lipoxygenase in human leukocytes is a pivotal enzyme that can produce both pro- and anti-inflammatory chemical mediators.