ABCG2 (Breast Cancer Resistance Protein/Mitoxantrone Resistance-Associated Protein) ATPase Assay: A Useful Tool to Detect Drug-Transporter Interactions

Glavinas, Hristos; Kis, Emese; Ákos, Pál; Kovács, Rita; Jani, Márton; Vági, Erika; Molnár, Éva; Bansághi, Száva; Kele, Zoltán; Janáky, Tamás; Báthori, György; Richter, Oliver von; Koomen, Gerrit‐Jan; Krajcsi, Péter

doi:10.1124/dmd.106.014605

Cited by 57 publications

(40 citation statements)

References 21 publications

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“…Of the drugs tested, Ko143 was the most effective inhibitor of ABCG2 function. It had previously been shown to inhibit ABCG2 at nanomolar concentrations by blocking ATPase activity of the transporter (35). We derived an ED 50 for maximal inhibition of ABCG2 at the BBB of 6.3 ± 1.8 mg/kg.…”

Section: Discussionmentioning

confidence: 99%

Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

Bakhsheshian

Wei

Chang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter. P rovision of nutrients and maintenance of chemical homeostasis in the brain is performed by the endothelial cells of brain capillaries within a neurovascular unit termed the bloodbrain barrier (BBB) (1). In contrast to endothelial cells of capillaries elsewhere in the body, those in the brain are joined by tight junctions forming a physiologic barrier. Drug delivery to the brain depends on physicochemical characteristics such as lipophilicity, molecular weight, and ionic state. For many compounds, brain entry is lower than other tissues/organs because of the presence of ATP-binding cassette (ABC) efflux transporters at the apical surface of endothelial cells at the BBB (2, 3). These transporters maintain chemical homeostasis in the brain, and prevent toxins from interfering with neural processes by regulating the compounds that can enter the brain.ABC transporters contribute to the clinical challenge of drug delivery to the brain, and it has been estimated that only 2% of drug discovery compounds can cross the BBB to reach therapeutic targets (4). ABCG2 (also known as breast cancer resistance protein) and ABCB1 (also called P-glycoprotein) are the two most highly expressed efflux transporters at the BBB (5). Altered expression of ABC transporters at the BBB has been associated with a range of pathophysiological conditions (2, 6). ABC efflux transporters at the BBB also play a major role in limiting effective concentrations of chemotherapeutic agents to treat primary and metastatic tumors in the brain (7). ABCG2 has been shown to work in tandem with ABCB1 at the BBB (8, 9). However, its individual contribution is not understood.Molecular imaging allows the measurement of the individual contribution and function of transporters in vivo (10). Efflux of a substrate by transporters at the BBB is reflected by...

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Section: Discussionmentioning

confidence: 99%

Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

Bakhsheshian

Wei

Chang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…To verify further the interaction of the analogs with ABCG2, an ATPase assay was used. This is another useful assay for studying the interaction of transport substrates with ABCG2, since ATP hydrolysis is coupled with the transport function of this transporter (Glavinas et al, 2007;Gallus et al, 2014). The analogs were found to stimulate ATP hydrolysis in a dose-dependent manner.…”

Section: Discussionmentioning

confidence: 99%

Synthetic Analogs of Curcumin Modulate the Function of Multidrug Resistance–Linked ATP-Binding Cassette Transporter ABCG2

et al. 2017

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Multidrug resistance (MDR) caused by the overexpression of ATPbinding cassette (ABC) transporters in cancer cells is a major obstacle in cancer chemotherapy. Previous studies have shown that curcumin, a natural product and a dietary constituent of turmeric, inhibits the function of MDR-related ABC transporters, including ABCB1, ABCC1, and especially ABCG2. However, the limited bioavailability of curcumin prevents its use for modulation of the function of these transporters in the clinical setting. In this study, we investigated the effects of 24 synthetic curcumin analogs with increased bioavailability on the transport function of ABCG2. The screening of the 24 synthetic analogs by means of flow cytometry revealed that four of the curcumin analogs (GO-Y030, GO-Y078, GO-Y168, and GO-Y172) significantly inhibited the efflux of the ABCG2 substrates, mitoxantrone and pheophorbide A, from ABCG2-overexpressing K562/breast cancer resistance protein (BCRP) cells. Biochemical analyses showed that GO-Y030, GO-Y078, and GO-Y172 stimulated the ATPase activity of ABCG2 at nanomolar concentrations and inhibited the photolabeling of ABCG2 with iodoarylazidoprazosin, suggesting that these analogs interact with the substrate-binding sites of ABCG2. In addition, when used in cytotoxicity assays, GO-Y030 and GO-Y078 were found to improve the sensitivity of the anticancer drug, SN-38, in K562/BCRP cells. Taken together, these results suggest that nontoxic synthetic curcumin analogs with increased bioavailability, especially GO-Y030 and GO-Y078, inhibit the function of ABCG2 by directly interacting at the substrate-binding site. These synthetic curcumin analogs could therefore be developed as potent modulators to overcome ABCG2-mediated MDR in cancer cells.

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“…It was reported that sulfasalazine activates ABCG2 ATPase 10) in membrane based assays. In this work the detailed kinetic characterization of sulfasalazine-ABCG2 interaction is described using direct vesicular transport and ATPase assays at pH 7.0 and pH 5.5.…”

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confidence: 99%

Kinetic Characterization of Sulfasalazine Transport by Human ATP-Binding Cassette G2

Jani

Szabó

Kis

et al. 2009

Biological & Pharmaceutical Bulletin

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Sulfasalazine is an anti-inflammatory agent used for the treatment of chronic inflammatory bowel diseases and rheumatoid arthritis. It displays low passive permeability and low oral bioavailability. ATP-binding cassette G2 (ABCG2) (breast cancer resistance protein (BCRP), mitoxantrone resistance protein (MXR)), an ATP driven efflux pump present at many important pharmacological barriers, most notably in the apical membrane of enterocytes, brain microcapillary endothelial cells, placenta, as well as in the canalicular membrane of hepatocytes 1) is known to influence the pharmacokinetic properties of many low and high permeability drugs. 2)Cells overexpressing the transporter show drug resistance to multiple anti-proliferative drugs used in cancer treatment. 3)Earlier studies indicated that sulfasalazine is a substrate of ABCG2. Its high efflux ratio on Caco-2 monolayer was unresponsive to verapamil, an ATP-binding cassette B1 (ABCB1) (P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)) inhibitor, 4) but was decreased by fumitremorgin C, an ABCG2 inhibitor. 5) Resistance to sulfasalazine was linked to ABCG2 overexpression in T-lymphocytes. 6) In abcg2 knock out mice compared to control animals the area under curve (area under blood concentration vs. time curve) (AUC ) and the C max level of sulfasalazine was over 100 fold and over 70 fold elevated respectively. 7) Clinical studies revealed that in vitro sulfasalazine transport 8) and in vivo measured plasma concentrations 9) correlate to single nucleotide polymorphisms in the ABCG2 gene. The studies suggest that sulfasalazine is an ideal probe substrate for ABCG2.Yet, hitherto the kinetic parameters of the interaction were still undetermined. Due to its low passive permeability, sulfasalazine requires a presently unknown uptake transporter. Therefore, its intracellular concentration is also largely unknown. Thus, kinetic characterization of the ABCG2-sulfasalazine interaction in cell based experiments is difficult. In contrast, membrane preparations contain inside out vesicles with the ABCG2 binding sites facing the external buffer. This enables precise control of the sulfasalazine concentration the transporter is exposed to.It was reported that sulfasalazine activates ABCG2 ATPase 10) in membrane based assays. In this work the detailed kinetic characterization of sulfasalazine-ABCG2 interaction is described using direct vesicular transport and ATPase assays at pH 7.0 and pH 5.5. MATERIALS AND METHODSAll chemicals were purchased from Sigma (Sigma-Aldrich Kft, Budapest, Hungary). Membrane preparations from mammalian cells selectively overexpressing ABCG2 were obtained from Solvo (Solvo Biotechnology, Budaors, Hungary).ATPase activity was measured as described earlier. 11)Membrane vesicles (SB-BCRP-M-ATPase; 20 mg/well) were incubated in 10 mM MgCl 2 , 40 mM MOPS-Tris (pH 7.0), 50 mM KCl, 5 mM dithiothreitol, 0.1 mM EGTA, 4 mM sodium azide, 1 mM ouabain, 5 mM ATP, and in the presence of various concentrations of test drugs with or without 1.2 mM sodium ort...

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ABCG2 (Breast Cancer Resistance Protein/Mitoxantrone Resistance-Associated Protein) ATPase Assay: A Useful Tool to Detect Drug-Transporter Interactions

Cited by 57 publications

References 21 publications

Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

Synthetic Analogs of Curcumin Modulate the Function of Multidrug Resistance–Linked ATP-Binding Cassette Transporter ABCG2

Kinetic Characterization of Sulfasalazine Transport by Human ATP-Binding Cassette G2

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