ABSTRACT
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast Pichia pastoris, a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated the Pichia ABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter from P. pastoris in the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function.
Subject(s)
ATP-Binding Cassette Transporters , Sterols , Humans , ATP-Binding Cassette Transporters/metabolism , Adenylyl Imidodiphosphate/metabolism , Sterols/metabolism , Phylogeny , Adenosine Triphosphate/metabolism , Saccharomyces cerevisiae/metabolism , Verapamil/pharmacology , Verapamil/metabolism , Triazoles/metabolism , Rhodamines/metabolismABSTRACT
We examined whether the α1L-adrenoceptor (AR), which shows low affinity (pA2 < 9) for prazosin (an α1-AR antagonist) and high affinity (pA2 ≈ 10) for tamsulosin/silodosin (α1A-AR antagonists), is involved in phenylephrine-induced contractions in the guinea pig (GP) thoracic aorta (TA). Intracellular signaling induced by α1L-AR activation was also examined by focusing on Ca2+ influx pathways. Tension changes of endothelium-denuded TAs were isometrically recorded and mRNA encoding α-ARs/Ca2+ channels and their related molecules were measured using RT-quantitative PCR. Phenylephrine-induced contractions were competitively inhibited by prazosin/tamsulosin, and their pA2 value were calculated to be 8.53/9.74, respectively. These contractions were also inhibited by silodosin concentration-dependently. However, the inhibition was not competitive fashion with the apparent pA2 value being 9.48. In contrast, phenylephrine-induced contractions were not substantially suppressed by L-765314 (an α1B-AR antagonist), BMY 7378 (an α1D-AR antagonist), yohimbine, and idazoxan (α2-AR antagonists). Phenylephrine-induced contractions were markedly inhibited by YM-254890 (a Gq protein inhibitor) or removal of extracellular Ca2+, and partially inhibited by verapamil (a voltage-dependent Ca2+ channel (VDCC) inhibitor). The residual contractions in the presence of verapamil were slightly inhibited by LOE 908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) and strongly inhibited by SKF-96365 (a store-operated Ca2+ channel (SOCC) and ROCC inhibitor). Among the mRNA encoding α-ARs/SOCC-related molecules, α1A-AR (Adra1a)/Orai3, Orai1, and Stim2 were abundant in this tissue. In conclusion, phenylephrine-induced contractions in the GP TA can be triggered by stimulation of Gq protein-coupled α1L-AR, followed by activation of SOCCs and VDCCs.
Subject(s)
Adrenergic alpha-Antagonists , Aorta, Thoracic , Guinea Pigs , Animals , Phenylephrine/pharmacology , Adrenergic alpha-Antagonists/metabolism , Adrenergic alpha-Antagonists/pharmacology , Tamsulosin/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Prazosin/pharmacology , Verapamil/pharmacology , Verapamil/metabolism , RNA, Messenger/metabolism , Muscle ContractionABSTRACT
Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRtPCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 µg/mL, 1-128 µg/mL, 2-32 µg/mL, 4-16 µg/mL and 15.62-250 µg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Tuberculosis , Humans , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Verapamil/pharmacology , Verapamil/metabolism , DNA, Complementary/metabolism , DNA, Complementary/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Tuberculosis/microbiology , Isoniazid/pharmacology , Rifampin/pharmacology , Microbial Sensitivity Tests , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: (R,R), (S,S), (R,S), or (S,R). The (R,R)/(S,S) and (R,S)/(S,R) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N-protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.
Subject(s)
Protons , Verapamil/analysis , Ion Mobility Spectrometry , Verapamil/metabolismABSTRACT
Radioresistance significantly decreases the efficacy of radiotherapy, which can ultimately lead to tumor recurrence and metastasis. As a novel type of nano-radiosensitizer, silver nanoparticles (AgNPs) have shown promising radiosensitizing properties in the radiotherapy of glioma, but their ability to efficiently enter and accumulate in tumor cells needs to be improved. In the current study, AS1411 and verapamil (VRP) conjugated bovine serum albumin (BSA) coated AgNPs (AgNPs@BSA-AS-VRP) were synthesized and characterized. Dark-field imaging and inductively coupled plasma mass spectrometry were applied to investigate the accumulation of AgNPs@BSA-AS and AgNPs@BSA-AS-VRP mixed in different ratios in U251 glioma cells. To assess the influences of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP on the P-glycoprotein (P-gp) efflux activity, rhodamine 123 accumulation assay was carried out. Colony formation assay and tumor-bearing nude mice model were employed to examine the radiosensitizing potential of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP. Thioredoxin Reductase (TrxR) Assay Kit was used to detect the TrxR activity in cells treated with different functionally modified AgNPs. Characterization results revealed that AgNPs@BSA-AS-VRP were successfully constructed. When AgNPs@BSA-AS and AgNPs@BSA-AS-VRP were mixed in a ratio of 19:1, the amount of intracellular nanoparticles increased greatly through AS1411-mediated active targeting and inhibition of P-gp activity. In vitro and in vivo experiments clearly showed that the radiosensitization efficacy of 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP was much stronger than that of AgNPs@BSA and AgNPs@BSA-AS. It was also found that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP significantly inhibited intracellular TrxR activity. These results indicate that 19:1 mixed AgNPs@BSA-AS and AgNPs@BSA-AS-VRP can effectively accumulate in tumor cells and have great potential as high-efficiency nano-radiosensitizers in the radiotherapy of glioma.
Subject(s)
Aptamers, Nucleotide/metabolism , Brain Neoplasms/radiotherapy , Glioma/radiotherapy , Metal Nanoparticles/chemistry , Oligodeoxyribonucleotides/metabolism , Radiation Tolerance , Radiation-Sensitizing Agents/pharmacology , Silver/chemistry , Verapamil/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/pharmacology , Brain Neoplasms/pathology , Cell Line, Tumor , Glioma/pathology , Humans , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/pharmacology , Verapamil/chemistry , Verapamil/pharmacologyABSTRACT
1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism.
Subject(s)
Drugs, Chinese Herbal/metabolism , Animals , Biological Transport , Dogs , Flavanones/metabolism , Glucosides/metabolism , Glycyrrhizic Acid/metabolism , Humans , Madin Darby Canine Kidney Cells , Models, Biological , Monoterpenes/metabolism , Oleanolic Acid/analogs & derivatives , Oleanolic Acid/metabolism , Permeability , Saponins/metabolism , Verapamil/metabolismABSTRACT
Multidrug resistance (MDR) caused by overexpressed permeability-glycoprotein (P-gp) in cancer cells is the main barrier for the cure of cancers. P-gp can pump many chemotherapeutic drugs, which is a viable target to overcome P-gp-mediated MDR by efficient inhibitors of P-gp. However, limited understanding of the efflux mechanism by human P-gp hinders the development of efficient inhibitors. Herein, the transport of a P-gp inhibitor, verapamil, by human P-gp has been investigated using targeted molecular dynamics simulations and energetics analysis based on our previous research on the transport of a drug (doxorubicin). The energetics analysis identifies that the driving forces for the transport of verapamil are electrostatic repulsions contributed by the positively charged residues in the initial stage and then hydrophobic interactions contributed by the important residues in the later stage. This scenario is generally consistent with that in the transport of doxorubicin. However, the positively charged residues and the important residues for the transport of verapamil are incompletely consistent with the relative residues for the transport of doxorubicin. Moreover, the binding free energy contributions of the positively charged residues for the transport of verapamil are generally higher than them for the transport of doxorubicin, while the important residues constitute significantly different binding free energy compositions in the transports of the two substrates. Consequently, the pathway for the transport of verapamil is identified, which shares only two residues (F336 and M986) with the pathway of doxorubicin. This may imply the weak competitiveness of verapamil with doxorubicin in the substrate efflux. Taken together, this work provided new insights into the efflux mechanisms by human P-gp and would be beneficial in the design of potent P-gp inhibitors.
Subject(s)
Verapamil/metabolism , ATP Binding Cassette Transporter, Subfamily B/chemistry , ATP Binding Cassette Transporter, Subfamily B/metabolism , Amino Acids/chemistry , Biological Transport , Doxorubicin/chemistry , Doxorubicin/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Dynamics Simulation , Protein Binding , Static Electricity , Thermodynamics , Verapamil/chemistryABSTRACT
OBJECTIVE: Overexpression of the drug transporter P-glycoprotein (P-gp) is thought to be involved in drug-resistance in epilepsy by extrusion of antiepileptic drugs (AEDs). We used positron emission tomography (PET) and the P-gp substrate radiotracer (R)-[11 C]verapamil (VPM) together with the third-generation P-gp inhibitor tariquidar (TQD) to evaluate P-gp function in individuals with drug-resistant epileptogenic developmental lesions. METHODS: Twelve healthy controls (7 male, median age 45, range 35-55 years), and two patients with epileptogenic developmental lesions (2 male, aged 24 and 62 years) underwent VPM-PET scans before and 60 minutes after a 30-minute infusion of 2 and 3 mg/kg TQD. The influx rate constant, VPM-K1 , was estimated from the first 10 minutes of dynamic data using a single-tissue compartment model with a VPM plasma input function. Statistical parametric mapping (SPM) analysis was used to compare individual patients with the healthy controls. RESULTS: At baseline, SPM voxel-based analysis revealed significantly lower uptake of VPM corresponding to the area of the epileptogenic developmental lesion compared to 12 healthy controls (P < .048). This was accentuated following P-gp inhibition with TQD. After TQD, the uptake of VPM was significantly lower in the area of the epileptogenic developmental lesion compared to controls (P < .002). SIGNIFICANCE: This study provides further evidence of P-gp overactivity in patients with drug-resistant epilepsy, irrespective of the type of lesion. Identifying P-gp overactivity as an underlying contributor to drug-resistance in individual patients will enable novel treatment strategies aimed at overcoming or reversing P-gp overactivity.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Carbon Radioisotopes/metabolism , Drug Resistant Epilepsy/diagnostic imaging , Drug Resistant Epilepsy/metabolism , Positron-Emission Tomography/methods , Verapamil/metabolism , Adult , Female , Humans , Male , Middle Aged , Vasodilator Agents/metabolism , Young AdultABSTRACT
High-resolution mass spectrometry (HRMS) is an important technology for studying biotransformations of drugs in biological systems. In order to process complex HRMS data, bioinformatics, including data-mining techniques for identifying drug metabolites from liquid chromatography/high-resolution mass spectrometry (LC/HRMS) or multistage mass spectrometry (MSn ) datasets as well as elucidating the detected metabolites' structure by spectral interpretation software, are important tools. Data-mining technologies have widely been used in drug metabolite identification, including mass defect filters, product ion filters, neutral-loss filters, control sample comparisons and extracted ion chromatographic analysis. However, the metabolites identified by current different technologies are not the same, indicating the importance of technique integration for efficient and complete identification of metabolic products. In this study, a universal, high-throughput workflow for identifying and verifying metabolites by applying the drug metabolite identification software UNIFI is reported, to study the biotransformation of verapamil in rats. A total of 71 verapamil metabolites were found in rat plasma, urine and faeces, including two metabolites that have not been reported in the literature. Phase I metabolites of verapamil were identified as N-demethylation, O-demethylation, N-dealkylation and oxidation and dehydrogenation metabolites; phase II metabolites were mainly glucuronidation and sulfate conjugates, indicating that UNIFI software could be effective and valuable in identifying drug metabolites.
Subject(s)
Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Verapamil , Animals , Biotransformation , High-Throughput Screening Assays , Male , Models, Molecular , Rats , Rats, Wistar , Software , Verapamil/analysis , Verapamil/chemistry , Verapamil/metabolismABSTRACT
BACKGROUND: Multidrug resistance (MDR) is a pressing obstacle in clinical chemotherapy for breast cancer. Based on the fact that the drug efflux is an important factor in MDR, we designed a codelivery system to guide the drug efflux inhibitor verapamil (VRP) and the chemotherapeutic agent novantrone (NVT) synergistically into breast cancer cells to reverse MDR. RESULTS: This co-delivery system consists of following components: the active targeting peptide RGD, an inorganic calcium phosphate (CaP) shell and an organic inner core. VRP and NVT were loaded into CaP shell and phosphatidylserine polyethylene glycol (PS-PEG) core of nanoparticles (NPs) separately to obtain NVT- and VRP-loaded NPs (NV@CaP-RGD). These codelivered NPs allowed VRP to prevent the efflux of NVT from breast cancer cells by competitively combining with drug efflux pumps. Additionally, NV@CaP-RGD was effectively internalized into breast cancer cells by precise delivery through the effects of the active targeting peptides RGD and EPR. The pH-triggered profile of CaP was also able to assist the NPs to successfully escape from lysosomes, leading to a greatly increased effective intracellular drug concentration. CONCLUSION: The concurrent administration of VRP and NVT by organic/inorganic NPs is a promising therapeutic approach to reverse MDR in breast cancer.
Subject(s)
Antineoplastic Agents/chemistry , Breast Neoplasms/drug therapy , Mitoxantrone/chemistry , Nanocapsules/chemistry , Verapamil/chemistry , Animals , Calcium Phosphates/chemistry , Cell Line, Tumor , Cell Membrane Permeability , Cell Survival , Drug Compounding/methods , Drug Liberation , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Therapy, Combination/methods , Female , Humans , Mice, Inbred BALB C , Mice, Nude , Mitoxantrone/pharmacology , Molecular Targeted Therapy , Oligopeptides/chemistry , Oligopeptides/metabolism , Phosphatidylserines/chemistry , Polyethylene Glycols/chemistry , Verapamil/metabolismABSTRACT
Contributions of cytochrome P450 (CYP450) isoforms to drug metabolism are often predicted using relative activity factor (RAF) method, assuming RAF values were independent of probe. We aimed to report probe-dependent characteristic of RAF values using CYP3A4 or CYP2C9 probes. Metabolism of four CYP3A4 probes (testosterone, midazolam, verapamil and atorvastatin) and three CYP2C9 probes (tolbutamide, diclofenac and S-warfarin) in human liver microsomes (HLM) and cDNA-expressed recombinant CYP450 (Rec-CYP450) systems were characterized and RAFCL value was estimated as ratio of probe intrinsic clearance in HLM to that in Rec-CYP450. CYP450i contributions to metabolic reaction of a probe were predicted using other probes and compared with data from specific inhibitions. Contributions of CYP3A4 and CYP2C9 to metabolism of deoxypodophyllotoxin and nateglinide were also predicted. RAF values were dependent on probes, leading to probe-dependently predicted contributions. Predicted contributions of CYP3A4 to formations of 6ß-hydroxytestosterone, 1'-hydroxymidazolam, norverapamil, ortho-hydroxyatorvastatin and para-hydroxyatorvastatin using other probes were 47.46-219.46%, 21.62-98.87%, 186.49-462.44%, 21.87-101.15% and 53.62-247.97%, respectively. Predicted contributions of CYP3A4 and CYP2C9 to nateglinide metabolism were 8.18-37.84% and 36.08-94.04%, separately. In conclusion, CYP450i contribution to drug metabolism in HLM estimated using RAF approach were probe-dependent. Therefore, contribution of each isoform must be confirmed by multiple probes.
Subject(s)
Cytochrome P-450 Enzyme System/physiology , Microsomes, Liver/metabolism , Atorvastatin/metabolism , Cytochrome P-450 Enzyme System/metabolism , Diclofenac/metabolism , Humans , Kinetics , Midazolam/metabolism , Protein Isoforms/metabolism , Protein Isoforms/physiology , Testosterone/metabolism , Tolbutamide/metabolism , Verapamil/metabolism , Warfarin/metabolismABSTRACT
P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD-NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Adenosine Triphosphate/metabolism , Calcium Channel Blockers/metabolism , Lipid Bilayers/chemistry , Models, Molecular , Verapamil/metabolism , ATP Binding Cassette Transporter, Subfamily B/chemistry , ATP Binding Cassette Transporter, Subfamily B/genetics , Adenosine Triphosphate/chemistry , Amino Acid Substitution , Animals , Binding Sites , Biological Transport, Active , Bioluminescence Resonance Energy Transfer Techniques , Calcium Channel Blockers/chemistry , Cysteine/chemistry , Europium/chemistry , Hydrolysis , Mice , Mutation , Nanostructures/chemistry , Protein Conformation , Protein Interaction Domains and Motifs , Protein Refolding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Terbium/chemistry , Verapamil/chemistryABSTRACT
Cytochromes P450 (CYPs) oxidize alkylated amines commonly found in drugs and other biologically active molecules, cleaving them into an amine and an aldehyde. Metabolic studies usually neglect to report or investigate aldehydes, even though they can be toxic. It is assumed that they are efficiently detoxified into carboxylic acids and alcohols. Nevertheless, some aldehydes are reactive and escape detoxification pathways to cause adverse events by forming DNA and protein adducts. Herein, we modeled N-dealkylations that produce both amine and aldehyde metabolites and then predicted the reactivity of the aldehyde. This model used a deep learning approach previously developed by our group to predict other types of drug metabolism. In this study, we trained the model to predict N-dealkylation by human liver microsomes (HLM), finding that including isozyme-specific metabolism data alongside HLM data significantly improved results. The final HLM model accurately predicted the site of N-dealkylation within metabolized substrates (97% top-two and 94% area under the ROC curve). Next, we combined the metabolism, metabolite structure prediction, and previously published reactivity models into a bioactivation model. This combined model predicted the structure of the most likely reactive metabolite of a small validation set of drug-like molecules known to be bioactivated by N-dealkylation. Applying this model to approved and withdrawn medicines, we found that aldehyde metabolites produced from N-dealkylation may explain the hepatotoxicity of several drugs: indinavir, piperacillin, verapamil, and ziprasidone. Our results suggest that N-dealkylation may be an under-appreciated bioactivation pathway, especially in clinical contexts where aldehyde detoxification pathways are inhibited. Moreover, this is the first report of a bioactivation model constructed by combining a metabolism and reactivity model. These results raise hope that more comprehensive models of bioactivation are possible. The model developed in this study is available at http://swami.wustl.edu/xenosite/ .
Subject(s)
Indinavir/metabolism , Liver/metabolism , Microsomes, Liver/metabolism , Piperacillin/metabolism , Piperazines/metabolism , Thiazoles/metabolism , Verapamil/metabolism , Aldehydes/chemistry , Aldehydes/metabolism , Amines/chemistry , Amines/metabolism , Dealkylation , Humans , Indinavir/pharmacology , Liver/drug effects , Microsomes, Liver/chemistry , Microsomes, Liver/drug effects , Models, Molecular , Molecular Structure , Piperacillin/pharmacology , Piperazines/pharmacology , Thiazoles/pharmacology , Verapamil/pharmacologyABSTRACT
PURPOSE: The organic cation transporters (OCTs) and multidrug and toxin extrusions (MATEs) together are regarded as an organic cation transport system critical to the disposition and response of many organic cationic drugs. Patient response to the analgesic morphine, a characterized substrate for human OCT1, is highly variable. This study was aimed to examine whether there is any organic cation transporter-mediated drug and drug interaction (DDI) between morphine and commonly co-administrated drugs. METHODS: The uptake of morphine and its inhibition by six drugs which are commonly co-administered with morphine in the clinic were assessed in human embryonic kidney 293 (HEK293) cells stably expressing OCT1, OCT2 and MATE1. The in vivo interaction between morphine and the select irinotecan was determined by comparing the disposition of morphine in the absence versus presence of irinotecan treatment in mice. RESULTS: The uptake of morphine in the stable HEK293 cells expressing human OCT1 and OCT2 was significantly increased by 3.56 and 3.04 fold, respectively, than that in the control cells, with no significant uptake increase in the cells expressing human MATE1. All of the six drugs examined, including amitriptyline, fluoxetine, imipramine, irinotecan, ondansetron, and verapamil, were inhibitors of OCT1/2-mediated morphine uptake. The select irinotecan significantly increased the plasma concentrations and decreased hepatic and renal accumulation of morphine in mice. CONCLUSIONS: Morphine is a substrate of OCT1 and OCT2. Clinician should be aware that the disposition of and thus the response to morphine may be altered by co-administration of an OCT1/2 inhibitor, such as irinotecan.
Subject(s)
Irinotecan/metabolism , Morphine/metabolism , Narcotics/metabolism , Organic Cation Transporter 1/antagonists & inhibitors , Organic Cation Transporter 2/antagonists & inhibitors , Amitriptyline/metabolism , Amitriptyline/pharmacology , Animals , Drug Interactions , Fluoxetine/metabolism , Fluoxetine/pharmacology , HEK293 Cells , Humans , Imipramine/metabolism , Imipramine/pharmacology , Irinotecan/pharmacology , Mice, Inbred C57BL , Ondansetron/metabolism , Ondansetron/pharmacology , Organic Cation Transport Proteins/metabolism , Organic Cation Transporter 1/metabolism , Organic Cation Transporter 2/metabolism , Tissue Distribution , Verapamil/metabolism , Verapamil/pharmacologyABSTRACT
Drug metabolism in the intestine is considered to substantially contribute to the overall first-pass metabolism, which has been neglected for a long time. It is highly desirable to develop a reliable model to evaluate drug metabolism in the intestine in vitro. In this work, we made the first attempt to develop a biomimetic human gut-on-a-chip for modeling drug metabolism in intestine. In this chip, constant flow, together with porous nitrocellulose membrane and collagen I, mimics an in vivo-like intestinal microenvironment. The Caco-2 cells grown in the chip formed a compact intestinal epithelial layer with continuous expression of the tight junction protein, ZO-1. Furthermore, higher gene expression of villin, sucrase-isomaltase, and alkaline phosphatase demonstrated that cells in the biomimetic human gut-on-a-chip device were more mature with near-physiological functions compared to the control on planar substrate. In particular, cellular metabolic activity was assessed on different substrates, indicating higher metabolic efficiency of ifosfamide and verapamil in the biomimetic human gut-on-a-chip model. Taken together, our results suggested that this biomimetic human gut-on-a-chip promoted the differentiation of intestinal cells with enhanced functionality by creating a biomimetic 3D microenvironment in vitro. It might offer a bioactive, low-cost, and flexible in vitro platform for studies on intestinal metabolism as well as preclinical drug development.
Subject(s)
Intestinal Mucosa/metabolism , Lab-On-A-Chip Devices , Pharmaceutical Preparations/metabolism , Biomimetics , Caco-2 Cells , Gene Expression , Humans , Ifosfamide/metabolism , Verapamil/metabolismABSTRACT
The ecotoxicological consequences of residues from pharmaceutical drugs on aquatic biota have necessitated the development of sensitive and reliable techniques to assess the impact of these xenobiotics on aquatic organisms. This study investigated the alteration in DNA structure, molecular responses and the activities of Na+ -K+ -ATPase and antioxidant enzymes in the gill of Nile tilapia, Oreochromis niloticus, exposed to long-term effects at the concentrations (0.14, 0.28 and 0.57mgL-1) of verapamil in static renewal system for 15, 30, 45 and 60 days. Evaluation of DNA structure, using single cell gel electrophoresis, revealed certain degree of DNA damages in the gill in a time and concentration-dependent relationship. Transcription of mRNA of superoxide dismutase (sod), catalase (cat) and heat shock protein (hsp70) genes in the gill of the fish showed the genes were up-regulated. Na+-K+-ATPase activity was inhibited in a concentration and time dependent manner. The indices of oxidative stress biomarkers (lipid peroxidation and carbonyl protein) as well as superoxide dismutase, glutathione peroxidase, glutathione-S-transferase were elevated in the treated fish in comparison to the control. Further, the level of reduced glutathione and catalase activity were inhibited at 0.28mgL-1 after day 30. Long-term exposure to sub-lethal concentration of verapamil can cause DNA damages, molecular effects and oxidative stress in O. niloticus. The biomarkers analysed can be used as early warning signals in environmental biomonitoring and assessment of drug contamination in aquatic ecosystem.
Subject(s)
Cichlids/metabolism , DNA Damage , Gills/drug effects , Sodium-Potassium-Exchanging ATPase/metabolism , Verapamil/toxicity , Water Pollutants, Chemical/toxicity , Animals , Antioxidants/metabolism , Biomarkers/metabolism , Cichlids/genetics , Dose-Response Relationship, Drug , Environmental Monitoring/methods , Gills/metabolism , Oxidative Stress/drug effects , Verapamil/metabolism , Water Pollutants, Chemical/metabolismABSTRACT
P-glycoprotein affects the transport of numerous drugs including chemotherapeutic drugs vincristine sulfate (VCR) and docetaxel (DTX), and is one of the main causes for multidrug resistance. Our previous studies have shown that oxypeucedanin (OPD) can enhance the intestinal transit of puerarin and VCR. However, the underlying mechanism is unclear. This study investigated the potential mechanism by which OPD improves P-gp-mediated drug transport. Molecular docking was performed to predict the binding force between OPD and P-gp and the contribution of OPD on P-gp activity. We observed the effect of OPD on the transport of VCR in MDCK-MDR1 cell monolayer and also measured the plasma pharmacokinetic parameters of DTX in the presence and absence of OPD by LC-MS/MS. Moreover, we further investigated the reversal mechanism of OPD on P-gp-mediated drug transport by determining the intracellular accumulation of Rhodamine-123 (Rh123) and P-gp ATPase activity as well as protein expression and mRNA level of P-gp. Our molecular docking results revealed that the binding force between OPD and P-gp was much lower than that between P-gp and verapamil (a P-gp substrate). The transport study in vitro indicated that OPD increased the flux of VCR across MDCK-MDR1 cell monolayer. The in vivo pharmacokinetic parameters data showed OPD increased the absorption of DTX. OPD activated P-gp ATPase activity and enhanced intracellular accumulation of Rh123 in MDCK-MDR1 cells. Western blotting and qRT-PCR outcomes indicated that OPD suppressed P-gp protein expression as well as downregulated P-gp mRNA level. Thus, OPD reverse P-gp-mediated drug transport via inhibition of P-gp activity and P-gp protein expression as well as downregulation of P-gp mRNA level. Our results suggest that OPD could reverse P-gp-mediated drug resistance in tumor cells.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Drug Resistance, Multiple/drug effects , Furocoumarins/pharmacology , RNA, Messenger/antagonists & inhibitors , Taxoids/metabolism , Vincristine/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Biological Transport/drug effects , Docetaxel , Dogs , Drug Resistance, Multiple/genetics , Fluorescent Dyes/metabolism , Fluorescent Dyes/pharmacology , Furocoumarins/chemistry , Furocoumarins/metabolism , Gene Expression/drug effects , Kinetics , Madin Darby Canine Kidney Cells , Molecular Docking Simulation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rhodamine 123/metabolism , Rhodamine 123/pharmacology , Taxoids/pharmacology , Verapamil/metabolism , Verapamil/pharmacology , Vincristine/pharmacologyABSTRACT
The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking. Different drugs likely occupy partially overlapping but distinct sites and are therefore variably coupled to nucleotide binding and hydrolysis. Many fluorescent drug analogues are used in cell-based transport models; however, their specific interactions with P-gp have not been studied, and this limits interpretation of transport assays in terms of molecular models. Here we monitor binding of the fluorescent probe substrates BODIPY-verapamil, BODIPY-vinblastine, and Flutax-2 at low occupancy to murine P-gp in lipid nanodiscs via fluorescence correlation spectroscopy, in variable nucleotide-bound states. Changes in affinity for the different nucleotide-dependent conformations are probe-dependent. For BODIPY-verapamil and BODIPY-vinblastine, there are 2-10-fold increases in KD in the nucleotide-bound or vanadate-trapped state, compared to that in the nucleotide-free state. In contrast, the affinity of Flutax-2 is unaffected by nucleotide or vanadate trapping. In further contrast to BODIPY-verapamil and BODIPY-vinblastine, Flutax-2 does not cause stimulation of ATP hydrolysis despite the fact that it is transported in vesicle-based transport assays. Whereas the established substrates verapamil, paclitaxel, and vinblastine displace BODIPY-verapamil or BODIPY-vinblastine from their high-affinity sites, the transport substrate Flutax-2 is not displaced by any of these substrates. The results demonstrate a unique binding site for Flutax-2 that allows for transport without stimulation of ATP hydrolysis.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Lipid Bilayers/chemistry , Models, Molecular , ATP Binding Cassette Transporter, Subfamily B/chemistry , ATP Binding Cassette Transporter, Subfamily B/genetics , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Binding, Competitive , Biological Transport , Boron Compounds/metabolism , Dimyristoylphosphatidylcholine/chemistry , Fluorescent Dyes/metabolism , Humans , Hydrolysis , Kinetics , Ligands , Mice , Nanostructures/chemistry , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Taxoids/metabolism , Verapamil/analogs & derivatives , Verapamil/metabolism , Vinblastine/analogs & derivatives , Vinblastine/metabolismABSTRACT
Resistance to the anticancer antibiotics, doxorubicin and daunorubicin, in the producer organism Streptomyces peucetius is conferred by an ABC transporter made of two proteins, DrrA and DrrB, which together form a dedicated exporter for these two antibiotics. Surprisingly, however, the DrrAB system exhibits broad substrate specificity overlapping with well-studied multidrug resistance transporters, including P-glycoprotein. Therefore, it provides an excellent model for studying the molecular basis of multispecificity in a prototype efflux system with the potential to unravel the origin and evolution of multidrug resistance. It has been suggested that multispecificity in multidrug exporters may be generally determined by the number and location of aromatic residues. Strategically placed negatively charged residues may also be critical for binding of cationic lipophilic drugs. We selected 13 aromatic and four negatively charged residues on the basis of their location in and/or near the predicted drug-binding pocket of DrrB for analysis. Indeed, mutations of most tested residues drastically inhibited doxorubicin efflux. Interestingly, several mutants lost resistance to doxorubicin and verapamil simultaneously but retained resistance to Hoechst 33342 and/or ethidium bromide, suggesting the presence of overlapping as well as independent drug-binding sites in a common drug-binding pocket of DrrB. This study provides the first comprehensive analysis of residues involved in drug binding in a bacterial multidrug resistance protein of the ABC superfamily, and it shows strong similarity in the molecular mechanism of polyspecific drug recognition between DrrAB and Pgp. Altogether, we conclude that aromatic residue-based multidrug specificity is conserved across domains and over long evolutionary periods. The significance of these findings is discussed.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , ATP-Binding Cassette Transporters/chemistry , Bacterial Proteins/chemistry , Daunorubicin/chemistry , Doxorubicin/chemistry , Verapamil/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Benzimidazoles/chemistry , Benzimidazoles/metabolism , Daunorubicin/metabolism , Doxorubicin/metabolism , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Ethidium/chemistry , Ethidium/metabolism , Evolution, Molecular , Gene Expression , Models, Molecular , Multidrug Resistance-Associated Proteins , Mutation , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Streptomyces/genetics , Streptomyces/metabolism , Structural Homology, Protein , Substrate Specificity , Verapamil/metabolismABSTRACT
BACKGROUND/AIMS: The human-voltage gated Kv1.3 channel (hKv1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hKv1.3 channels. We characterized the effect of verapamil on currents through hKv1.3 channels paying special attention to the on-rate (kon) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated. METHODS: Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hKv1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate kon of verapamil. RESULTS: The on-rate of verapamil to block current through hKv1.3_T419C mutant channels is similar to that obtained for hKv1.3_wt channels whereas the on-rate of verapamil to block currents through hKv1.3_L417C and hKv1.3_L418C mutant channels was â¼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hKv1.3_L346C and the double mutant hKv1.3_L346C_L418C channel was â¼ 2 times slower compared to that obtained in the wt channel. The hKv1.3_I420C mutant channel reduced the on-rate of verapamil to block currents â¼ 6 fold. CONCLUSIONS: We conclude that position 420 in hKv1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hKv1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hKv1.3_L346C and hKv1.3_L346C_L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current.