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

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 laborintensive, 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, con­ structed 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 selec­ tivity for EGFR and its clinically relevant mutants (e.g., T790M/L858R), with minimal cross-reactivity to un­ related 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.