EGFR affinity and selectivity for the phosphorylation codes of pseudo triad tyrosine (YYY) motif and its extensions in lung cancer-related substrate-inhibitor alignment: an integrated molecular simulation-QSAR modelling–in vitro assay approach

Human epidermal growth factor receptor (EGFR) has been approved as a well-established druggable target of lung carcinoma. The binding peptide segments of both substrate and inhibitory proteins contain a phosphorylatable tandem YY motif but interact with EGFR kinase domain in different manners. Here, the two tandem substrate Y0Y+1 and inhibitor Y+1Y+2 motifs were aligned to define a new pseudo triad tyrosine Y0Y+1Y+2 (PtriY) motif, in which the Y0, Y+1 and Y+2 residues bind to catalytic, priming and priming pockets on EGFR kinase domain surface, respectively. Here, we examined the effects of different PtriY phosphorylation codes on EGFR binding and created a systematic single-point substitution–binding energy change profile of its N- and C-terminal extensions, which was then used to develop and validate quantitative structure–activity relationship (QSAR) models. The best model was utilized to guide rational peptidic inhibitor design, from which more than 40 promising hits were selected to perform affinity and/or kinase assays. The QSAR-designed PH2[Y0pY+1pY+2] peptide (ENGHY0pY+1pY+2AL) was identified to have the strongest binding potency (Kd = 0.26 ± 0.07 μM) and the highest inhibitory activity (IC50 = 5.8 ± 0.9 nM), which consists of an amphiphilic N-terminal extension, a double-phosphorylated PtriYmotif and hydrophobic C-terminal extension.