Rapid kinase activity detection and kinetic analysis using a convenient EGFR FRET-based detection probe.
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| Title: | Rapid kinase activity detection and kinetic analysis using a convenient EGFR FRET-based detection probe. |
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| Authors: | Jaengwang K; Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand., Pommayon S; Department of Biotechnology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, Bangkok 10800, Thailand., Sonklin C; Department of Industrial Chemistry, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, Bangkok 10800, Thailand; Food and Agro-Industrial Research Center, King Mongkut's University of Technology North Bangkok, Bangkok 10800, Thailand., Choowongkomon K; Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; Center for Advanced Studies in Nanotechnology for Chemical, Food and Agricultural Industries, KU Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand., Waraho-Zhmayev D; Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand., Tabtimmai L; Department of Biotechnology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, Bangkok 10800, Thailand; Food and Agro-Industrial Research Center, King Mongkut's University of Technology North Bangkok, Bangkok 10800, Thailand. Electronic address: Lueacha.t@sci.kmutnb.ac.th. |
| Source: | Biochimica et biophysica acta. General subjects [Biochim Biophys Acta Gen Subj] 2025 Dec; Vol. 1869 (12), pp. 130854. Date of Electronic Publication: 2025 Sep 08. |
| Publication Type: | Journal Article |
| Language: | English |
| Journal Info: | Publisher: Elsevier Country of Publication: Netherlands NLM ID: 101731726 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1872-8006 (Electronic) Linking ISSN: 03044165 NLM ISO Abbreviation: Biochim Biophys Acta Gen Subj Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Amsterdam : Elsevier |
| MeSH Terms: | Fluorescence Resonance Energy Transfer*/methods , ErbB Receptors*/metabolism , ErbB Receptors*/genetics , Biosensing Techniques*/methods, Kinetics ; Humans ; Protein Kinase Inhibitors/pharmacology ; Fluorescent Dyes/chemistry |
| Abstract: | Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest. In modern drug discovery, there is a pressing need for rapid, cost-effective, and accessible methods to evaluate the biological activities of newly synthesized compounds. Traditional kinase assay platforms are often labor-intensive, time-consuming, and require specialized equipment or expertise. To address these limitations, we developed and validated a convenient in vitro kinase assay based on a recombinant biosensor, Picchu-B, constructed using a bacterial expression system. Picchu-B, derived from the live-cell EGFR FRET biosensor Picchu, was successfully expressed as a highly soluble, fluorescent protein. It served as a direct Probe for EGFR kinase, enabling real-time FRET-based detection of kinase activity. Optimization of Picchu-B concentration revealed a linear correlation between FRET signal intensity and EGFR-TK levels. The biosensor demonstrated high selectivity for EGFR and its clinically relevant mutants (e.g., T790M/L858R), with minimal cross-reactivity to unrelated kinases such as JAK-2. Enzyme kinetic studies confirmed nucleotide specificity of EGFR-TK in the presence of Picchu-B. This study highlights Picchu-B as a practical and scalable tool for EGFR-targeted drug screening, offering significant advantages in speed, and simplicity over conventional approaches. (Copyright © 2025 Elsevier B.V. All rights reserved.) |
| Contributed Indexing: | Keywords: EGFR; FRET; Kinase; Phosphorylation; cancer |
| Substance Nomenclature: | EC 2.7.10.1 (ErbB Receptors) EC 2.7.10.1 (EGFR protein, human) 0 (Protein Kinase Inhibitors) 0 (Fluorescent Dyes) |
| Entry Date(s): | Date Created: 20250910 Date Completed: 20251106 Latest Revision: 20251106 |
| Update Code: | 20251107 |
| DOI: | 10.1016/j.bbagen.2025.130854 |
| PMID: | 40930331 |
| Database: | MEDLINE |
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Nájsť tento článok vo Web of Science | Abstract: | Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest.<br />In modern drug discovery, there is a pressing need for rapid, cost-effective, and accessible methods to evaluate the biological activities of newly synthesized compounds. Traditional kinase assay platforms are often labor-intensive, time-consuming, and require specialized equipment or expertise. To address these limitations, we developed and validated a convenient in vitro kinase assay based on a recombinant biosensor, Picchu-B, constructed using a bacterial expression system. Picchu-B, derived from the live-cell EGFR FRET biosensor Picchu, was successfully expressed as a highly soluble, fluorescent protein. It served as a direct Probe for EGFR kinase, enabling real-time FRET-based detection of kinase activity. Optimization of Picchu-B concentration revealed a linear correlation between FRET signal intensity and EGFR-TK levels. The biosensor demonstrated high selectivity for EGFR and its clinically relevant mutants (e.g., T790M/L858R), with minimal cross-reactivity to unrelated kinases such as JAK-2. Enzyme kinetic studies confirmed nucleotide specificity of EGFR-TK in the presence of Picchu-B. This study highlights Picchu-B as a practical and scalable tool for EGFR-targeted drug screening, offering significant advantages in speed, and simplicity over conventional approaches.<br /> (Copyright © 2025 Elsevier B.V. All rights reserved.) |
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| ISSN: | 1872-8006 |
| DOI: | 10.1016/j.bbagen.2025.130854 |