Multiplexing Terbium- and Europium-Based TR-FRET Readouts to Increase Kinase Assay Capacity

Horton, Robert A.; Vogel, Kurt W.

doi:10.1177/1087057110368993

Cited by 12 publications

(10 citation statements)

References 11 publications

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“…Traditional approaches rely on the use of specific antibodies using detection techniques such as time-resolved luminescence resonance energy transfer (TR-LRET). [2][3][4] Universal assays for enzyme activity have been developed using nucleotide detection methods for a variety of enzymes depending on the product being formed such as AMP for ligases, ADP for kinases, and UDP for glycosyltransferases. 5,6 However, these non-direct approaches are limited to nucleotide-dependent enzymes and secondary products are detected.…”

mentioning

confidence: 99%

Toward universal protein post-translational modification detection in high throughput format

et al. 2018

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Post-translational modification (PTM) of proteins plays essential regulatory roles in a variety of pathological conditions. Reliable and practical assays are required to accelerate the discovery of inhibitors and activators for PTM related diseases. Today, methodologies are based on specific or group-specific PTM recognition of e.g. phosphate for kinase activity without extending to other type of PTMs. Here we have established a universal time-resolved luminescence assay on a peptide-break platform for the direct detection of wide variety of PTMs. The developed assay is based on the leucine zipper concept wherein a europium-chelate labeled detection peptide and a non-labeled peptide substrate form a highly luminescent dimer. As an active PTM enzyme at sub or low nanomolar concentration modifies the substrate peptide, the luminescent signal of the detached detection peptide is quenched in the presence of soluble quenchers. The functionality of this universal assay technique has been demonstrated for the monitoring of phosphorylation, dephosphorylation, deacetylation, and citrullination with high applicability also to other PTMs in a high throughput format.

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confidence: 99%

Toward universal protein post-translational modification detection in high throughput format

et al. 2018

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“…A similar strategy for assessing selectivity for two kinases simultaneously has been explored using TR FRET. 42…”

Section: Discussionmentioning

confidence: 99%

“…A similar strategy for assessing selectivity for two kinases simultaneously has been explored using TR FRET. 42 A final way that multiplexed FP FRET assays could increase assay power is by allowing multiple nodes in a complex process to be examined in a single assay step, making it possible to directly screen for functions that would otherwise require multiple follow-up experiments after screening. For example, using DAGR, CKAR, and DKAR, screens could be conducted to look for direct activators or inhibitors of PKD.…”

Section: Discussionmentioning

confidence: 99%

Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening

et al. 2016

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Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

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“…Stock solutions of compounds were prepared in DMSO, with a final DMSO concentration of 0.1%. HEK293 cells were cultured in 6-well plates for immunoblotting and used at passages P [3][4][5][6][7][8][9][10][11][12][13][14][15] .…”

Section: Methodsmentioning

confidence: 99%

“…9) Various kinase detection formats have been developed for high throughput screening (HTS) to identify RSK inhibitors. 10,11) Fluorescence-based biochemical assays such as fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) are widely used to screen compound libraries for drug discovery because these methods are homogenous, sensitive, and convenient. 12,13) Despite their popularity in HTS, all single assays using tagged-fluorescein are vulnerable to interference from the intrinsic fluorescence of compounds or from scattering light.…”

mentioning

confidence: 99%

A Dual Readout Assay Based on Fluorescence Polarization and Time-Resolved Fluorescence Resonance Energy Transfer to Screen for RSK1 Inhibitors

Jeong

Lee

et al. 2016

Biological & Pharmaceutical Bulletin

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A dual readout assay based on fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) exhibits many advantages over single assay technology in terms of screening quality and efficiency. In this study, we developed a dual readout assay combining FP and TR-FRET to identify ribosomal S6 kinase 1 (RSK1) inhibitors. This dual readout assay can monitor both FP and TR-FRET signals from a single RSK1 kinase reaction by using the immobilized metal affinity for phosphochemical (IMAP)-based assay. The Z' value and signal to background (S/B) ratio were 0.85 and 4.0 using FP, and 0.79 and 10.6 using TR-FRET, which led to performance of a pilot library screening against the drug repositioning set consisting of 2320 compounds with a reasonable reproducibility. From this screening, we identified 16 compounds showing greater than 50% inhibition against RSK1 for both FP and TR-FRET; 6 compounds with greater than 50% inhibition only for FP; and 4 compounds with greater than 50% inhibition only for TR-FRET. In a cell-based functional assay to validate the hit compounds, 10 compounds identified only in a single assay had little effect on the RSK-mediated phosphorylation of liver kinase B1, whereas 5 compounds showing greater than 80% inhibition for both FP and TR-FRET reduced the phosphorylation of liver kinase B1. These results demonstrate that the dual readout assay can be used to identify hit compounds by subsequently monitoring both FP and TR-FRET signals from one RSK1 reaction.

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Multiplexing Terbium- and Europium-Based TR-FRET Readouts to Increase Kinase Assay Capacity

Cited by 12 publications

References 11 publications

Toward universal protein post-translational modification detection in high throughput format

Toward universal protein post-translational modification detection in high throughput format

Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening

A Dual Readout Assay Based on Fluorescence Polarization and Time-Resolved Fluorescence Resonance Energy Transfer to Screen for RSK1 Inhibitors

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