Homogeneous single-label tyrosine kinase activity assay for high throughput screening

Tong-Ochoa, Natalia; Kopra, Kari; Syrjänpää, Markku; Legrand, Nicolas; Härmä, Harri

doi:10.1016/j.aca.2015.09.032

Cited by 5 publications

(5 citation statements)

References 36 publications

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“…To confirm that EGF and TGF increase PS-ASO activity through a mechanism involving EGFR, we co-treated cells with EGF and a specific EGFR tyrosine kinase inhibitor, PD174265 ( 32 ), to block EGFR internalization and signaling. PS-ASO activities were compared among cells that were pre-treated with EGF alone or EGF with PD174265 or cells without any treatment before they were incubated with either Drosha - or Malat1 -specific PS-ASOs.…”

Section: Resultsmentioning

confidence: 99%

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

Wang

Allen

Vickers

et al. 2018

Nucleic Acids Research

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Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.

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Section: Resultsmentioning

confidence: 99%

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

Wang

Allen

Vickers

et al. 2018

Nucleic Acids Research

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“…10,11 The universal peptide-break detection platform for PTMs was constructed on our quenching resonance energy transfer (QRET) detection technology. [12][13][14][15][16] The QRET technique distinguishes between the leucine zipper complex and the dissociated posttranslationally modified peptides in the presence of a soluble quencher molecule (Fig. 1b).…”

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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“…Using the QRET detection technology (signal-off) in peptide-break concept, high TRF signal is monitored upon peptide complex formation. Enzymatic addition of PTM disrupts the binding between peptides and disposes the Eu 3+ -label to soluble quencher to produce low TRF-signal. , The previously introduced QRET detection technology has proved its functionality in different HTS-compatible assay formats. − In the current study, the introduced signal-on method, called modulated time-resolved fluorescence resonance energy transfer (mTR-FRET), was constructed by modifying the QRET detection to provide a selectable positive signal change upon choice, also with dissociated peptides and not only with peptide complex. In the mTR-FRET approach, high TRF-signal is obtained, as the peptide carrying a PTM is incapable to form a peptide complex with the Eu 3+ -chelate conjugated reporter peptide (EuLZ) detection peptide.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously developed a signaling technique, named as quenching resonance energy transfer (QRET) technology, which is so far mainly performed in the signal-off format. , The single-label QRET technique is based on energy transfer between lanthanide (Eu 3+ or Tb 3+ ) chelate conjugated small-molecular-weight molecule and soluble quencher molecule. − Upon binding, the Ln 3+ -conjugated molecule is protected from the soluble quencher, and the binding is monitored for high time-resolved fluorescence (TRF) signal. Previously, we have utilized QRET for multiple targets and also to monitor enzymatic PTM reactions. ,− Now, we have edited the QRET principle to enable signal-on and signal-off sensing by simply introducing different soluble Eu 3+ -signal-modulating molecule but without any further changes in the assay conditions or Eu 3+ -reporter peptide. The method was named as modulated time-resolved fluorescence resonance energy transfer (mTR-FRET), whose principle is now demonstrated using peptide-break platform for enzymatic phosphorylation/dephosphorylation monitoring.…”

Section: Introductionmentioning

confidence: 99%

Peptic Fluorescent “Signal-On” and “Signal-Off” Sensors Utilized for the Detection Protein Post-Translational Modifications

et al. 2019

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Protein post-translational modifications (PTMs) are typically enzymecatalyzed events generating functional diversification of proteome; thus, multiple PTM enzymes have been validated as potential drug targets. We have previously introduced energy-transfer-based signal-modulation method called quenching resonance energy transfer (QRET), and utilize it to monitor PTM addition or removal using the developed peptide-break technology. Now we have reinvented the QRET technology, and as a model, we introduced the tunable fluorescent "signal-on" and "signal-off" detection scheme in the peptide-break PTM detection. Taking the advantage of timeresolved fluorescence-based single-label detection technology, we were able to select the signal direction upon PTM addition or removal by simply introducing different soluble Eu 3+ -signal-modulating molecule. This enables the selection of positive signal change upon measurable event, without any additional labeling steps, changes in assay condition or Eu 3+ -reporter. The concept functionality was demonstrated with four Eu 3+ -signal modulators in a high-throughput compatible kinase and phosphatase assays using signal-on and signal-off readout at 615 nm or time-resolved Forster resonance energy transfer at 665 nm. Our data suggest that the introduced signal modulation methodology provides a transitional fluorescence-based single-label detection concept not limited only to PTM detection.

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Homogeneous single-label tyrosine kinase activity assay for high throughput screening

Cited by 5 publications

References 36 publications

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

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

Peptic Fluorescent “Signal-On” and “Signal-Off” Sensors Utilized for the Detection Protein Post-Translational Modifications

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