Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis.

We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 degrees C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein.     

Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus

Purified trehalose synthase from Thermus caldophilus GK24 produced 18–86% trehalose from 10 mM–1 M maltose. The enzyme also catalyzed the conversion of ,-trehalose into maltose but did not act on other disaccharides. The yield of trehalose from maltose by this enzyme increased 30% more at 40°C than at 80°C and was independent of the substrate concentration. The maximum yield of ,-trehalose from 10 mM maltose reached 86% at 40°C. In addition, ,-trehalose was also formed from maltose or ,-trehalose at 3.5% yield at 80°C. © Rapid Science Ltd. 1998     

A survey of detergents for the purification of stable,active human cystic fibrosis transmembrane conductance regulator (CFTR)

Structural knowledge of the cystic fibrosis transmembrane conductance regulator (CFTR) requires developing methods to purify and stabilize this aggregation-prone membrane protein above 1 mg/ml. Starting with green fluorescent protein- and epitope-tagged human CFTR produced in mammalian cells known to properly fold and process CFTR, we devised a rapid tandem affinity purification scheme to minimize CFTR exposure to detergent in order to preserve its ATPase function. We compared a panel of detergents, including widely used detergents (maltosides, neopentyl glycols (MNG), C₁₂E₈, lysolipids, Chaps) and innovative detergents (branched alkylmaltosides, facial amphiphiles) for CFTR purification, function, monodispersity and stability. ATPase activity after reconstitution into proteoliposomes was 2–3 times higher when CFTR was purified using facial amphiphiles. ATPase activity was also demonstrated in purified CFTR samples without detergent removal using a novel lipid supplementation assay. By electron microscopy, negatively stained CFTR samples were monodisperse at low concentration, and size exclusion chromatography showed a predominance of monomer even after CFTR concentration above 1 mg/ml. Rates of CFTR aggregation quantified in an electrophoretic mobility shift assay showed that detergents which best preserved reconstituted ATPase activity also supported the greatest stability, with CFTR monomer half-lives of 6–9 days in MNG or Chaps, and 12–17 days in facial amphiphile. Cryoelectron microscopy of concentrated CFTR in MNG or facial amphiphile confirmed mostly monomeric protein, producing low resolution reconstructions in conformity with similar proteins. These protocols can be used to generate samples of pure, functional, stable CFTR at concentrations amenable to biophysical characterization.     

Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies.     

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.     

Human P-glycoprotein is active when the two halves are clamped together in the closed conformation

The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the two homologous halves of P-gp is composed of a transmembrane domain (TMD) with six TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures show drug pumps in the open and closed conformations, where the drug-binding pocket and NBDs are open or closed at the cytoplasmic side, respectively. Although it has been postulated that drug substrates enter the drug-binding pocket in the open conformation, it is unknown if they can enter in the closed conformation. To determine this, we introduced cysteines into regions of TM3 (residues 175-178) and TM9 (residues 820-822) that extend into the cytoplasm and are 4 Å and 20 Å apart in the closed and open conformations, respectively. The 12 double cysteine mutants were then cross-linked with a short cross-linker, M1M (4 Å) at 0 °C to reduce thermal motion in the protein. Only mutant L175C/N820C was cross-linked. Cross-linking was not increased in the presence of ATP or drug substrates. Cross-linking increased its basal ATPase activity about 3-fold. Activity could be increased further by drug substrates such as verapamil and rhodamine B. These results suggest that P-gp in the membrane is in the closed conformation that has a high affinity for drug substrates.     

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (K_d = 0.18 μm) and an ATPase activity with a K_m of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.     

Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1

Nucleotide Binding Domains (NBDs) are responsible for the ATPase activity of the multidrug resistance protein 1 (MRP1). A series of NBD1-linker-NBD2 chimeric fusion proteins were constructed, expressed and purified, and their ATPase activities were analyzed. We report here that a GST linked NBD1_642-890-GST-NBD2_1286-1531 was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (K_m = 2.17 mM, V_max = 12.36 nmol/mg/min), which was comparable to the purified and reconstituted MRP1. In contrast, neither a mixture of NBD1 and GST-NBD2 nor the NBD1-GST-NBD1 fusion protein showed detectable ATPase activity. Additionally, the E1455Q mutant was found to be nonfunctional. Measurements by both MIANS labeling and circular dichroism spectroscopy revealed significant conformational differences in the NBD1-GST-NBD2 chimeric fusion protein compared to the mixture of NBD1 and GST-NBD2. The results suggest a direct interaction mediated by GST between the two NBDs of MRP1 leading to conformational changes which would enhance its ATPase activity.     

Improved biovar test for Ralstonia solanacearum

Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test uses bromothymol blue as a pH indicator in 15 ml culture tubes containing 3 to 5 ml of test media, and takes weeks to complete at 24 or 28 °C. We improved the biovar test by using phenol red as a pH indicator that changes color at a higher pH when a carbohydrate is utilized. We also conducted the test at 32 °C in 0.2 ml of 8-tube strips that reduced the medium needed by at least 20 fold. Using the improved test, biovars of R. solanacearum strains can be determined in 4 days when a panel of seven carbohydrates is used including glucose, trehalose, mannitol, sorbitol, dulcitol, maltose and cellobiose. To differentiate biovars 1, 2, 3 and 4, the test can be further simplified and completed in 3 days using a panel of four carbohydrates containing glucose, trehalose, maltose and dulcitol, significantly saving money, space and time.     

Multiple Effects of Viscosity,Water Activity and Glass Transition Temperature on Peroxidase Activity in Binary and Ternary Carbohydrate Solutions

The individual and combined effects of water activity (a_w), bulk viscosity and glass transition temperature (Tg’) on the activity of horseradish peroxidase (HRP) in buffered sugars (glucose, trehalose and maltose) and maltodextrin solutions were investigated. Viscosity was the most important factor in the inhibition of HRP activity; however, when Tg’ was changed by the using solutes with different molecular weight, it became a key factor in the modulation of enzyme activity. Viscosity being equal, the sugar addition to maltodextrin solution lowered a_w and lowered Tg’ causing an increase of the enzymatic activity. Nevertheless, an inhibition of the HRP activity occurred when a_w values of 0.87 were reached due to the addition of glucose, which, among the tested sugars, showed the lowest molecular weight. Among disaccharides, maltose was more effective than trehalose in impairing the enzyme activity both in binary and ternary systems, and this is due to a non competitive biochemical inhibition exerted by this sugar on HRP. When compared to glucose, maltose and trehalose were more effective in reducing HRP activity only in the low viscosity range whilst in the high viscosity range (1–4 10^?6 m² s^?1) glucose, despite its lower Tg’ value, was slightly more efficient than disaccharides due to its a_w lowering effect.     

Cloning, expression and functional characterization of a novel trehalose synthase from marine Pseudomonas sp. P8005

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose. A novel treS gene with a length of 3,369 bp, encoding a protein of 1,122 amino acid residues with a predicted molecular mass of 126 kDa, was cloned from a marine Pseudomonas sp. P8005 (CCTCC: M2010298) and expressed in Escherichia coli. The amino acid sequence identities between this novel TreS and other reported TreS is relatively low. The purified recombinant TreS showed an optimum pH and temperature of 7.2 and 37 °C, respectively. The enzyme displayed a high conversion rate (70 %) of maltose to trehalose during equilibrium and had a higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, suggesting its application in the production of trehalose. In addition to maltose and trehalose, this enzyme can also act on sucrose, although this activity is relatively low. Mutagenesis studies demonstrated that enzymatic activity was reduced dramatically by individually substitution with alanine for D78, Y81, H121, D219, E261, H331 or D332, which implied that these residues might be important in P8005-TreS. Experiments using isotope-labeled substrates showed that [²H₂]trehalose combined with unlabeled trehalose was converted to [²H₂]maltose and maltose, but without any production of [²H]maltose or [²H]trehalose and with no incorporation of exogenous [²H₇]glucose into the disaccharides during the conversion catalyzed by this enzyme. This finding indicated the involvement of an intramolecular mechanism in P8005-TreS catalyzing the reversible interconversion of maltose and trehalose.     

Isopetasin and S-isopetasin as novel P-glycoprotein inhibitors against multidrug-resistant cancer cells

BackgroundA major problem of cancer treatment is the development of multidrug resistance (MDR) to chemotherapy. MDR is caused by different mechanisms such as the expression of the ABC-transporters P-glycoprotein (P-gp, MDR1, ABCB1) and breast cancer resistance protein (BCRP, ABCG2). These transporters efflux xenobiotic toxins, including chemotherapeutics, and they were found to be overexpressed in different cancer types.PurposeIdentification of novel molecules that overcome MDR by targeting ABC-transporters.MethodsResazurin reduction assay was used for cytotoxicity test. AutoDock 4.2. was used for molecular docking. The function of P-gp and BCRP was tested using a doxorubicin uptake assay and an ATPase assay. ROS generation was detected using flow cytometry for the measurement of H₂DCFH-DA fluorescence. Annexin/PI staining was applied for the detection of apoptosis. Bioinformatic analyses were performed using LigandScout 3.12. software and DataWarrior software.ResultsIn our search for new molecules that selectively act against resistant phenotypes, we identified isopetasin and S-isopetasin, which are bioactive natural products from Petasites formosanus. They exerted collateral sensitivity towards leukemia cells with high P-gp expression in CEM/ADR5000 cells, compared to sensitive wild-type CCRF-CEM leukemia cells. Also, they revealed considerable activity towards breast cancer cells overexpressing breast cancer resistance protein, MDA-MB-231-BCRP clone 23. This motivated us to investigate whether the function of P-gp was inhibited. In-silico results showed the compounds bound with high affinity and interacted with key amino acid residues in P-gp . Then, we found that the two compounds increased doxorubicin accumulation in P-gp overexpressing CEM/ADR5000 by three-fold compared to cells without inhibitor. P-gp-mediated drug efflux was ATP-dependent. Isopetasin and S-isopetasin increased the ATPase activity of human P-gp in a comparable fashion as verapamil used as control P-gp inhibitor. As isopetasin and S-isopetasin exerted dual roles, first as cytotoxic compounds and then as P-gp inhibitors, we suggested that their P-gp inhibition is part of a larger complex of mechanisms to induce cell death in cancer patients. P-gp dysfunction induces mitochondrial stress to generate ATP. Upon continuing stress by P-gp inhibition, the mitochondria generate reactive oxygen species (ROS). Initially established for verapamil, this theory was validated in the present study for isopetasin and S-isopetasin, as treatment with the two candidates increased ROS levels in CEM/ADR5000 cells followed by apoptosis.ConclusionOur study highlights the importance of isopetasin and S-isopetasin as novel ROS-generating and apoptosis-inducing P-gp inhibitors.     

Enzymatic Synthesis of Trehalose from Maltose

Synthesis of trehalose from maltose by a coupled enzyme system with trehalose Phosphorylase and maltose Phosphorylase has been studied. Trehalose Phosphorylase was partially purified from Euglena gracilis and maltose Phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5~7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.

Immobilized maltose and trehalose Phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.     

The ATPase Activity of the P-glycoprotein Drug Pump Is Highly Activated When the N-terminal and Central Regions of the Nucleotide-binding Domains Are Linked Closely Together

The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 Å) or long (22 Å) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.     

Overexpression and characterization of a thermostable trehalose synthase from Meiothermus ruber

A thermostable trehalose synthase (TreS) gene from Meiothermus ruber CBS-01 was cloned and overexpressed in Escherichia coli. The purified recombinant TreS could utilize maltose to produce trehalose, and showed an optimum pH and temperature of 6.5 and 50°C, respectively. Kinetic analysis showed that the enzyme had a twofold higher catalytic efficiency (k _cat/K _m) for maltose than for trehalose, indicating maltose as the preferred substrate. The TreS also had a weak hydrolytic property with glucose as the byproduct, and glucose was a strong competitive inhibitor of the enzyme. The maximum production of trehalose by the enzyme reached 65% at 20°C. The most importantly the enzyme could maintain very high activity (above 90%) at pH 4.0–8.0 and 60°C 5 h. These results provided that the stable TreS was suitable for the industrial production of trehalose from maltose in a one-step reaction.     

The detergent-soluble maltose transporter is activated by maltose binding protein and verapamil

The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V_max of the ATPase 2.7-fold but had no effect on the K_m of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN₃, as well as a mercurial, p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.     

Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation

One of the recent approaches to enhance desiccation tolerance in red blood cells (RBCs) is by loading trehalose. This process has been shown to increase the recovery of lyophilized RBCs; conversely, it results in cellular damage including hemoglobin oxidation and loss of membrane integrity. The purpose of this study was to further investigate the extent of oxidative injury during the loading of trehalose into RBCs.RBCs were incubated in the absence (control) or presence of trehalose (0.8 mol/l) at 4 °C or 37 °C for different time scales. Oxidative damage was monitored by flow cytometry using dichlorofluorescin for reactive oxygen species formation, Annexin V-FITC for phosphatidylserine translocation and fluorescein-DHPE for lipid peroxidation. Percent methemoglobin, percent hemolysis and thiobarbituric acid reactive substances were measured by spectrophotometry. The extent of oxidative damage during trehalose loading is affected by the incubation temperature, incubation time and the presence of trehalose. Incubation at 4 °C was relatively innocuous; however, oxidative injury was evident at 37 °C in both RBC groups. The addition of trehalose is correlated with high osmotic pressure, which had minor effects during incubation at 4 °C, but seemed to have exacerbated the severity of cellular injury at 37 °C, as measured by higher levels of hemolysis, methemoglobin and lipid peroxidation.The process of trehalose-loading is problematic due to its requirement for prolonged incubations at 37 °C. These conditions are correlated with oxidative injury, even in the absence of trehalose. While trehalose is believed to be crucial for stabilizing biomembranes, the consequences of its introduction into the cells require further investigation.     

A novel flavonoid from Fissistigma cupreonitens, 5‑hydroxy‑7,8‑dimethoxyflavanone,competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance

BackgroundP-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems.Purpose and study designThe aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well.MethodsCalcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses.Results5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively.ConclusionThe present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.     

CJX1, an amlodipine derivative, interacts with ATPase of human P-glycoprotein

Our aim has been to elucidate the possible mechanism of CJX1, an amlodipine derivative, in the modulation of P-gp function by determining its effect on P-gp ATPase activity. Basal P-gp ATPase activity was increased by CJX1 with half-maximal activity concentration (Km) of 8.6 ± 1.4 μM. Kinetic analysis indicated a non-competitive inhibition of Verapamil (Ver)-stimulated P-gp ATPase activity by CJX1 and competitive inhibition of CJX1-stimulated P-gp ATPase activity by tetrandrine (Tet). The effect of CsA on CJX1-stimulated and Ver-stimulated P-gp ATPase activity was non-competitive and competitive inhibition, respectively. These findings implying that CJX1 and Tet can bind P-gp either on overlapping sites or distinct but interacting sites, while CJX1 and Ver as well as CsA can bind P-gp on separated sites in K562/DOX cells. Furthermore, the combined effect of CJX1 and Ver has been evaluated isobolographically in numerous fixed-ratio combinations of 1:1, 1:2, 1:4, 1:8, 1:10 in K562/DOX cells. The results show that mixtures of both drugs at these fixed-ratios exerted synergistic interactions, indicating that when the two reverses that bind P-gp on separated sites are combined, each can contribute to the overall interaction with P-gp, leading to the greater effect than that by either agent alone.