2.3. Measurement of cell-free EGFR kinase activity

Cell-free EGFR kinase activity was performed by LANCE® Ultra time-resolved fluorescence resonance energy transfer technology. Briefly, different EGFR-TKI solutions (including aumolertinib, afatinib, and osimertinib) were prepared by serial dilution and mixed with each enzyme (WT or mutant EGFR), the U-Light-PolyGT/ATP mixture (substrate and ATP in a kinase buffer) into 384-well plate. Kinase reactions were incubated at room temperature for one hour and then quenched by EDTA. The specific LANCE® Eu-W1024 anti-phosphotyrosine (PT66) was added to the reaction mixture in the LANCE® detection buffer, followed by incubation for 1 h. The LANCE® signal was recorded on the microplate reader (BioTek) at the emission wavelength of 665 nm using the time-resolved fluorescence program. The half-maximal inhibitory concentration (IC₅₀) value was determined by GraphPad Prism 9.3 software.