Rapid kinase activity detection and kinetic analysis using a convenient EGFR FRET-based detection probe

• Published in: Biochimica et biophysica acta. General subjects Vol. 1869; no. 12; p. 130854
• Main Authors: Jaengwang, Kittitat, Pommayon, Suleeporn, Sonklin, Chanikan, Choowongkomon, Kiattawee, Waraho-Zhmayev, Dujdaun, Tabtimmai, Lueacha
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2025
• Subjects: cancer; EGFR; FRET; Kinase; Phosphorylation; Phosphorylation; FRET; cancer; Kinase; EGFR
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2025.130854

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. [Display omitted] •FRET-based probes (TRE-Picchu-X and Picchu-B) enable real-time EGFR activity monitoring.•Bacterial expression produced selective, rapid and non-invasive EGFR detection probes.•Kinetic parameters of EGFR-TK were characterized using recombinant Picchu-B probe