Synthesis and Characterization of a BODIPY Conjugate of the BCR-ABL Kinase Inhibitor Tasigna (Nilotinib): Evidence for Transport of Tasigna and Its Fluorescent Derivative by ABC Drug Transporters (original) (raw)
Related papers
Biochemical and Biophysical Research Communications, 2008
ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.
PLoS ONE, 2013
P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell-and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [ 125 I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.
Molecular Cancer Therapeutics, 2012
Ponatinib is a novel tyrosine kinase inhibitor with potent activity against BCR-ABL with mutations including T315I, and also against fms-like tyrosine kinase 3 (FLT3). We tested interactions between ponatinib at pharmacologically relevant concentrations of 50 to 200 nM and the multidrug resistance-associated ATPbinding cassette (ABC) proteins ABCB1, ABCC1 and ABCG2. Ponatinib enhanced uptake of substrates of ABCG2 and ABCB1, but not ABCC1, in cells overexpressing these proteins, with a greater effect on ABCG2 than on ABCB1.
Residues contributing to drug transport by ABCG2 are localised to multiple drug-binding pockets
The Biochemical journal, 2018
Multidrug binding and transport by the ATP-binding cassette transporter ABCG2 is a factor in the clinical resistance to chemotherapy in leukaemia, and a contributory factor to the pharmacokinetic profiles of many other prescribed drugs. Despite its importance, the structural basis of multidrug transport, i.e. the ability to transport multiple distinct chemicals, has remained elusive. Previous research has shown that at least two residues positioned towards the cytoplasmic end of transmembrane helix 3 (TM3) of the transporter play a role in drug transport. We hypothesised that other residues, either in the longitudinal span of TM3, or a perpendicular slice through the intracellular end of other TM helices would also contribute to drug binding and transport by ABCG2. Single-point mutant isoforms of ABCG2 were made at ∼30 positions and were analysed for effects on protein expression, localisation (western blotting, confocal microscopy) and function (flow cytometry) in a mammalian stabl...
ABCG2/BCRP: variants, transporter interaction profile of substrates and inhibitors
Expert Opinion on Drug Metabolism & Toxicology, 2019
Introduction: ABCG2 has a broad substrate specificity and is one of the most important efflux proteins modulating pharmacokinetics of drugs, nutrients and toxicokinetics of toxicants. ABCG2 is an important player in transporter-mediated drug-drug interactions (tDDI). Areas covered: The aims of the review are i) to cover transporter interaction profile of substrates and inhibitors that can be utilized to test interaction of drug candidates with ABCG2, ii) to highlight main characteristics of in vitro testing and iii) to describe the structural basis of the broad substrate specificity of the protein. Preclinical data utilizing Abcg2/Bcrp1 knockouts and clinical studies showing effect of ABCG2 c.421C>A polymorphism on pharmacokinetics of drugs have provided evidence for a broad array of drug substrates and support drug-ABCG2 interaction testing. A consensus on using rosuvastatin and sulfasalazine as intestinal substrates for clinical studies is in the formation. Other substrates relevant to the therapeutic area can be considered. Monolayer efflux assays and vesicular transport assays have been extensively utilized in vitro. Expert opinion: Clinical substrates display complex pharmacokinetics due to broad interaction profiles with multiple transporters and metabolic enzymes. Substrate-dependent inhibition has been observed for several inhibitors. Harmonization of in vitro and in vivo testing makes sense. However, rosuvastatin and sulfasalazine are not efficiently transported in either MDCKII or LLC-PK1-based monolayers. Caco-2 monolayer assays and vesicular transport assays are potential alternatives.
¹⁸FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2014
2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer c...
Leukemia, 2014
Letter to the Editor Nilotinib, imatinib (structures shown in Supplementary Figure S1) and other tyrosine kinase inhibitors (TKIs) have been shown to be transported by the ABC drug transporters Pglycoprotein (P-gp) and ABCG2 (1, 2). This is clinically important, as the transporters not only hamper the bioavailability of these TKIs but may also cause the emergence of drug resistance in patients. We have previously shown that imatinib and nilotinib interact at the substrate-binding pocket of ABC transporters, but do not interact at the ATP sites of these transporters (3). Identification of the key structural features of nilotinib and similar TKIs is essential for understanding their interaction with P-gp. Towards this goal, molecular docking, mutational mapping and quantitative structure-activity relationships were used to identify nilotinib's binding site on P-gp. Nilotinib was docked in a human P-gp homology model that was developed based on the mouse P-gp crystal structure (4) using the XP-Glide docking method to understand the orientation and the complementarity of pharmacophore features of nilotinib with respect to the residues in the drug-binding pocket of P-gp (Figure 1a). Comparison of binding energy data for the docked poses of nilotinib at sites 1-4 (5) suggested site-1 (QZ59-RRR site) (4, 6) as the most favorable site (binding energy score of −9.52 kcal/mol). The binding pocket is Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
High-Affinity Interaction of Tyrosine Kinase Inhibitors with the ABCG2 Multidrug Transporter
Molecular Pharmacology, 2004
Tyrosine kinase inhibitors (TKIs) are promising new agents for specific inhibition of malignant cell growth and metastasis formation. Because most of the TKIs have to reach an intracellular target, specific membrane transporters may significantly modulate their effectiveness. In addition, the hydrophobic TKIs may interact with so-called multidrug transporters and thus alter the cellular distribution of unrelated pharmacological agents. In the present work, we show that certain TKIs, already in the clinical phase of drug development, directly interact with the ABCG2 multidrug transporter protein with a high affinity. We found that in several in vitro assay systems, STI-571 (Gleevec; imatinib mesylate), ZD1839 (Iressa; gefitinib), and N- [4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (EKI-785) interacted
Transmembrane Inhibitors of P-Glycoprotein, an ABC Transporter
Journal of Medicinal Chemistry, 2005
Drug resistance mediated by ABC transporters such as P-glycoprotein (P-gp) continues to be a major impediment to effective cancer chemotherapy. We have developed a panel of highly specific peptide inhibitors of P-gp based on the structure of the transmembrane domains of the transporter. These peptides are thought to exert their inhibitory action by disrupting the proper assembly of P-gp. A novel 96-well-plate assay based on the efflux of fluorescent P-gp substrate DiOC 2 (3-ethyl-2-[3-(3-ethyl-2(3H)-benzoxazolylidene)-1-propenyl]benzoxazolium iodide) was developed and used for structure-functional characterization of transporter inhibitors. The studies strongly suggest that potent and selective inhibitors of ABC transporters can now be developed solely on the basis of the primary structures of the target proteins. The inhibition of P-gp with transmembrane peptides was shown to be chirality-independent. A 25-residue long retroinverso D-analogue of transmembrane domain 5 inhibited the efflux of the fluorescent P-gp substrate with an IC 50 of 500 nM. Transmembrane peptides effectively sensitized resistant cancer cells to doxorubicin in vitro without demonstrating any cell toxicity of their own. The newly synthesized P-gp antagonists appear to be promising nontoxic drug resistance inhibitors that merit further development.