ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

Iram, Surtaj H.; Gruber, Simon; Raguimova, Olga N.; Thomas, David D.; Robia, Seth L.

doi:10.1124/mol.114.096792

Cited by 18 publications

(27 citation statements)

References 61 publications

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“…The high-resolution FRET approach, coupled to functional assays, is applicable to a wide range of protein targets, including the ryanodine receptor 29 , myosin 18,30 , phospholamban 10 , multiple-drug resistance receptor 31 , and the tumor necrosis receptor 32 . The ability to quickly and reliably assess structural perturbations from biosensors in relation to physiologically-relevant functional changes holds high promise for the development of allosteric effectors and potentially valuable lead compounds.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

et al. 2017

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A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

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Section: Discussionmentioning

confidence: 99%

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

et al. 2017

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“…Effects of Test Compounds on MRP1 Activity in Cell-Based Assays. We previously engineered a two-color MRP1 protein that reports intramolecular FRET changes as an index of structural changes in the nucleotide binding domains of MRP1 (Iram et al, 2015). Specifically, a structural change that brings the nucleotide binding domains closer together increases FRET efficiency from a basal to higher level, reflecting…”

Section: Resultsmentioning

confidence: 99%

“…Our results show that collateral sensitivity of MRP1 over-expressing cell lines toward calcitriol and calcipotriol can be eliminated by MRP1 inhibitor MK571. We initiated the present work as a follow up of our previous study, which used a genetically-engineered two-color MRP1 for high-throughput identification of potential MRP1 substrates and modulators (Iram et al, 2015). The two-color MRP1 reports ligand-induced structural changes through changes in intramolecular FRET efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…The genetically encoded two-color MRP1 recombinant protein can measure intramolecular fluorescence resonance energy transfer (FRET) efficiency as an index of protein conformational changes upon ligand binding in live cells. Using this approach, a compound library was screened and eight compounds were identified that directly interact, bind and induce a conformational change in the structure of MRP1 (Iram et al, 2015). In the present work, we evaluated the effects of the hit compounds on MRP1 transport activity using different substrates of MRP1 in various cell-and membrane-based assays.…”

Section: Introductionmentioning

confidence: 99%

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Calcitriol and Calcipotriol Modulate Transport Activity of ABC Transporters and Exhibit Selective Cytotoxicity in MRP1-overexpressing Cells

et al. 2018

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Efflux transporters P-glycoprotein (P-gp/ABCB1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) can affect the efficacy and toxicity of a wide variety of drugs and are implicated in multidrug resistance (MDR). Eight test compounds, recently identified from an intramolecular FRET-based high throughput screening, were characterized for their interaction with MRP1. We report that the active metabolite of vitamin D, calcitriol, and its analog calcipotriol are selectively cytotoxic to MRP1-overexpressing cells, besides inhibiting transport function of P-gp, MRP1, and BCRP. Calcitriol and calcipotriol consistently displayed a potent inhibitory activity on MRP1-mediated doxorubicin and calcein efflux in MRP1-overexpressing H69AR and HEK293/MRP1 cells. Vesicular transport studies confirmed a strong inhibitory effect of calcitriol and calcipotriol on MRP1-mediated uptake of tritium-labeled estradiol glucuronide and leukotriene C In cytotoxicity assays, MRP1-overexpressing cells exhibited hypersensitivity toward calcitriol and calcipotriol. Such collateral sensitivity, however, was not observed in HEK293/P-gp and HEK293/BCRP cells, although the vitamin D analogs inhibited calcein efflux in P-gp-overexpressing cells, and mitoxantrone efflux in BCRP-overexpressing cells. The selective cytotoxicity of calcitriol and calpotriol toward MRP1 over-expressing cells can be eliminated with MRP1 inhibitor MK571. Our data indicate a potential role of calcitriol and its analogs in targeting malignancies in which MRP1 expression is prominent and contributes to MDR.

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“…On the other hand, various types of chemical libraries are currently available, and they have been used to explore potent MRP inhibitors. 35,36) Thus, further screening of high-affinity MRP3 inhibitors using larger chemical libraries may lead to the discovery of better candidates for agents with highly neuroblastoma-selective cytotoxicity. a) Obtained in the present study.…”

Section: Discussionmentioning

confidence: 99%

Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

Nakamichi

Ishimoto

Yamauchi

et al. 2016

Biological & Pharmaceutical Bulletin

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The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.

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ATP–Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening

Cited by 18 publications

References 61 publications

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

Calcitriol and Calcipotriol Modulate Transport Activity of ABC Transporters and Exhibit Selective Cytotoxicity in MRP1-overexpressing Cells

Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

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