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1.
Toxicol Sci ; 131(2): 502-11, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23104431

ABSTRACT

Digitalis-like compounds (DLCs), or cardiac glycosides, are produced and sequestered by certain plants and animals as a protective mechanism against herbivores or predators. Currently, the DLCs digoxin and digitoxin are used in the treatment of cardiac congestion and some types of cardiac arrhythmia, despite a very narrow therapeutic index. P-glycoprotein (P-gp; ABCB1) is the only known ATP-dependent efflux transporter that handles digoxin as a substrate. Ten alanine mutants of human P-gp drug-binding amino acids-Leu(65), Ile(306), Phe(336), Ile(340), Phe(343), Phe(728), Phe(942), Thr(945), Leu(975), and Val(982)-were generated and expressed in HEK293 cells with a mammalian baculovirus system. The uptake of [(3)H]-N-methyl-quinidine (NMQ), the P-gp substrate in vesicular transport assays, was determined. The mutations I306A, F343A, F728A, T945A, and L975A abolished NMQ transport activity of P-gp. For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol) were determined. The affinities of digoxin, proscillaridin A, peruvoside, and cymarin for mutants F336A and I340A were decreased two- to fourfold compared with wild type, whereas that of digitoxin and strophanthidol did not change. In addition, the presence of a hydroxyl group at position 12ß seems to reduce the apparent affinity when the side chain of Phe(336) and Phe(942) is absent. Our results showed that a δ-lactone ring and a sugar moiety at 3ß of the steroid body are favorable for DLC binding to P-gp. Moreover, DLC inhibition is increased by hydroxyl groups at positions 5ß and 19, whereas inhibition is decreased by those at positions 1ß, 11α, 12ß, and 16ß. The understanding of the P-gp-DLC interaction improves our insight into DLCs toxicity and might enhance the replacement of digoxin with other DLCs that have less adverse drug effects.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cardiac Glycosides/metabolism , Digitalis/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Blotting, Western , HEK293 Cells , Humans , Kinetics , Mutagenesis, Site-Directed
2.
Biochem Pharmacol ; 84(3): 366-73, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22542979

ABSTRACT

Multidrug resistance-associated protein 4 (MRP4) is a membrane transporter that mediates the cellular efflux of a wide range of anionic drugs and endogenous molecules. MRP4 transport can influence the pharmacokinetics of drugs and their metabolites, therefore more knowledge about the molecular determinants important for its transport function would be of relevance. Here, we substituted amino acids Phe(368), Trp(995), and Arg(998) with conservative or non-conservative residues, and determined the effect on transport of the model substrates estradiol 17-ß-d-glucuronide (E(2)17ßG), cyclic guanosine monophosphate (cGMP), methotrexate (MTX), and folic acid into membrane vesicles isolated from baculovirus transduced HEK293 cells overexpressing the mutant MRP4 proteins. This revealed that all Arg(998) mutations appeared to be deleterious, whereas the effect of a Phe(368) or Trp(995) replacement was dependent on the amino acid introduced and the substrate studied. Substitution of Phe(368) with Trp (F368W) induced a gain-of-function of E(2)17ßG transport and a loss-of-function of MTX transport, which could not be attributed to an altered substrate binding. Moreover, we did not observe any modification in ATP or ADP handling for F368W. These results, in combination with docking of substrates in a homology model of MRP4 in the inward- and outward-facing conformation, suggest that Phe(368) and Trp(995) do not play an important role in the initial binding of substrates. They, however, might interact with the substrates during rearrangement of helixes for substrate translocation, funneling the substrates to the exit site in the outward-facing conformation.


Subject(s)
Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/physiology , Phenylalanine/physiology , Amino Acid Sequence , Amino Acid Substitution/genetics , Down-Regulation/genetics , HEK293 Cells , Humans , Molecular Sequence Data , Multidrug Resistance-Associated Proteins/genetics , Phenylalanine/chemistry , Phenylalanine/genetics , Protein Structure, Secondary/genetics , Protein Transport/genetics , Substrate Specificity/genetics
3.
Drug Metab Dispos ; 40(6): 1076-9, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22415933

ABSTRACT

Coumarin (1,2-benzopyrone) is a natural compound that has been used as a fragrance in the food and perfume industry and could have therapeutic usefulness in the treatment of lymphedema and different types of cancer. Several previous pharmacokinetic studies of coumarin have been performed in humans, which revealed extensive first-pass metabolism of the compound. 7-Hydroxycoumarin (7-HC) and its glucuronide (7-HC-G) are the main metabolites formed in humans, and via this route, 80 to 90% of the absorbed coumarin is excreted into urine, mainly as 7-HC-G. Active transport processes play a role in the urinary excretion of 7-HC-G; however, until now, the transporters involved remained to be elucidated. In this study, we investigated whether the efflux transporters multidrug resistance-associated proteins (MRP)1-4, breast cancer resistance protein, or P-glycoprotein play a role in 7-HC and 7-HC-G transport. For this purpose, we measured uptake of the metabolites into membrane vesicles overexpressing these transporters. Our results showed that 7-HC is not transported by any of the efflux transporters tested, whereas 7-HC-G was a substrate of MRP3 and MRP4. These results are in line with the pharmacokinetic profile of coumarin and suggest that MRP3 and MRP4 are the main transporters involved in the excretion of the coumarin metabolite 7-HC-G from liver and kidney.


Subject(s)
Coumarins/metabolism , Multidrug Resistance-Associated Proteins/physiology , Umbelliferones/metabolism , Biological Transport, Active/physiology , HEK293 Cells , Humans
4.
Mol Pharm ; 9(5): 1351-60, 2012 May 07.
Article in English | MEDLINE | ID: mdl-22428727

ABSTRACT

Although the CB1 receptor antagonist/inverse agonist rimonabant has positive effects on weight loss and cardiometabolic risk factors, neuropsychiatric side effects have prompted researchers to develop peripherally acting derivatives. Here, we investigated for a series of 3,4-diarylpyrazoline CB1 receptor antagonists if transport by the brain efflux transporter P-gp could be used as a selection criterion in the development of such drugs. All 3,4-diarylpyrazolines and rimonabant inhibited P-gp transport activity in membrane vesicles isolated from HEK293 cells overexpressing the transporter, but only the 1,1-dioxo-thiomorpholino analogue 23 exhibited a reduced accumulation (-38 ± 2%) in these cells, which could be completely reversed by the P-gp/BCRP inhibitor elacridar. In addition, 23 appeared to be a BCRP substrate, whereas rimonabant was not. In rats, the in vivo brain/plasma concentration ratio of 23 was significantly lower than for rimonabant (0.4 ± 0.1 vs 6.2 ± 1.6, p < 0.001). Coadministration of elacridar resulted in an 11-fold increase of the brain/plasma ratio for 23 (p < 0.01) and only 1.4-fold for rimonabant (p < 0.05), confirming the involvement of P-gp and possibly BCRP in limiting the brain entrance of 23 in vivo. In conclusion, these data support the conception that efflux via transporters such as P-gp and BCRP can limit the brain penetration of CB1 receptor antagonists, and that this property could be used in the development of peripheral antagonists.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cannabinoid Receptor Antagonists/pharmacology , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/metabolism , Acridines/pharmacology , Animals , Biological Transport/drug effects , Blood-Brain Barrier , Blotting, Western , Cannabinoid Receptor Antagonists/metabolism , Cell Line , Humans , Kinetics , Male , Neoplasm Proteins/metabolism , Piperidines/pharmacology , Pyrazoles/pharmacology , Quinidine/pharmacology , Rats , Rats, Wistar , Rimonabant , Tandem Mass Spectrometry , Tetrahydroisoquinolines/pharmacology
5.
Drug Metab Dispos ; 39(7): 1294-302, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21511945

ABSTRACT

Cannabinoid type 1 (CB1) receptor antagonists have been developed for the treatment of obesity, but a major disadvantage is that they cause unwanted psychiatric effects. Selective targeting of peripheral CB1 receptors might be an option to circumvent these side effects. Multidrug resistance-associated proteins (MRPs) can influence the pharmacokinetics of drugs and thereby affect their disposition in the body. In this study, we investigated the interaction of the prototypic CB1 receptor antagonist rimonabant and a series of 3,4-diarylpyrazoline CB1 receptor antagonists with MRP1, MRP2, MRP3, and MRP4 in vitro. Their effect on ATP-dependent transport of estradiol 17-ß-D-glucuronide (E(2)17ßG) was measured in inside-out membrane vesicles isolated from transporter-overexpressing human embryonic kidney 293 cells. Rimonabant inhibited MRP1 transport activity more potently than MRP4 (K(i) of 1.4 and 4 µM, respectively), whereas the 3,4-diarylpyrazolines were stronger inhibitors of MRP4- than MRP1-mediated transport. A number of CB1 receptor antagonists, including rimonabant, stimulated MRP2 and MRP3 transport activity at low substrate concentrations but inhibited E(2)17ßG transport at high substrate concentrations. The interaction of 3,4-diarylpyrazolines and rimonabant with MRP1-4 indicates their potential for drug-drug interactions. Preliminary in vivo data suggested that for some 3,4-diarylpyrazolines the relatively lower brain efficacy may be related to their inhibitory potency against MRP4 activity. Furthermore, this study shows that the modulatory effects of the 3,4-diarylpyrazolines were influenced by their chemical properties and that small variations in structure can determine the affinity of these compounds for efflux transporters and thereby affect their pharmacokinetic behavior.


Subject(s)
Multidrug Resistance-Associated Proteins/metabolism , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Blotting, Western , Cell Line , Chromatography, Liquid , Humans , Protein Transport , Tandem Mass Spectrometry
6.
Am J Physiol Renal Physiol ; 296(1): F204-11, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18842822

ABSTRACT

A recently described novel controllable method to regulate protein expression is based on a mutated FK506-binding protein-12 (mtFKBP) that is unstable and rapidly degraded in mammalian cells. This instability can be conferred to other proteins directly fused to mtFKBP. Binding of a synthetic cell-permeant ligand (Shield-1) to mtFKBP reverses the instability, allowing conditional expression of mtFKBP-fused proteins. We adapted this strategy to study multimeric plasma membrane proteins using the ion channel TRPV5 as model protein. mtFKBP-TRPV5 forms functional ion channels and its expression can be controlled in a time- and dose-dependent fashion using Shield-1. Moreover, in the presence of Shield-1, mtFKBP-TRPV5 formed heteromultimeric channels with untagged TRPV5, which were codegraded upon washout of Shield-1, providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.


Subject(s)
Gene Expression Regulation/physiology , Kidney/metabolism , Protein Engineering/methods , Protein Multimerization , Small Molecule Libraries/metabolism , TRPV Cation Channels/metabolism , Tacrolimus Binding Protein 1A/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Cell Line , Cell Membrane/metabolism , Humans , Kidney/cytology , Kidney/embryology , Ligands , Protein Structure, Tertiary , TRPV Cation Channels/genetics
7.
Melanoma Res ; 17(6): 400-9, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17992124

ABSTRACT

Oxidative phosphorylation in the mitochondria is an important energy-producing process for eukaryotic cells, but this process can also result in producing potentially cell-damaging side products. Oxygen is the final proton acceptor in this cascade of electron/proton transfer and results in harmless water. The electron transfer, however, is not completely efficient and results in the production of reactive oxygen species (ROS). Low amounts of these ROS are important for cellular-signalling pathways. Excessive ROS, however, can induce cell damage that can culminate in cell death. Therefore, the cell has developed an antioxidant network to scavenge excessively produced ROS. In general, the balance between the production and scavenging of ROS leads to homeostasis. Disturbance of this equilibrium can alter normal cellular processes; it often occurs in tumour cells. In this review, the role of ROS in cutaneous melanoma development and progression is described. Cutaneous melanoma arises from epidermal melanocytes in skin, which is a relatively hypoxic tissue. ROS are generated as a result of increased metabolism of transformed cells, immune reaction against the developing tumour, ultraviolet radiation, melanin production and an altered antioxidant system. Knowledge of the role of ROS in melanoma development and the mechanisms that alleviate oxidative stress can aid in the development of better antimelanoma therapies.


Subject(s)
Antioxidants/metabolism , Melanins/biosynthesis , Melanoma/drug therapy , Melanoma/metabolism , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Skin Neoplasms/metabolism , Animals , Electron Transport , Humans , Melanoma/immunology , Melanoma/secondary , Oxidation-Reduction , Oxidative Phosphorylation , Oxidative Stress , Signal Transduction , Skin Neoplasms/drug therapy , Skin Neoplasms/immunology
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