PhosphoSens EGFR Protein Kinase Assays, Substrates & Recombinant Enzymes

PhosphoSens generates continuous fluorescent progress curves by monitoring EGFR substrate phosphorylation in real time, allowing direct observation of the time-dependent loss of enzyme activity characteristic of covalent inhibitors. By fitting progress curves at multiple inhibitor concentrations, kinact and KI parameters are extracted from a single experiment without the ambiguity introduced by fixed-time endpoint methods. This makes PhosphoSens uniquely suited to rank-ordering covalent warhead efficiency during medicinal chemistry campaigns.

Yes, PhosphoSens peptide substrates and assay formats are compatible with wild-type EGFR and common oncogenic or resistance mutants including L858R, T790M/L858R double mutant, and exon 19 deletion constructs. Running parallel assays with mutant and wild-type enzyme on the same continuous-read platform enables direct selectivity profiling with no changes to the assay protocol. This is critical for understanding whether next-generation inhibitors overcome resistance while maintaining activity against activating mutations.

ADP-Glo and HTRF assays capture activity at a single timepoint, which can introduce significant errors if the reaction is not in a linear phase or if reagent coupling reactions interfere with inhibitors. PhosphoSens monitors the full reaction progress curve in real time, ensuring IC50 values are derived from true initial-rate kinetics and revealing mechanistic information such as slow-binding or covalent inhibition that endpoint assays completely miss. Additionally, PhosphoSens uses physiological ATP concentrations, improving the translatability of inhibitor potency data to cellular and in vivo settings.