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 YY and inhibitor YY motifs were aligned to define a new pseudo triad tyrosine YYY (PtriY) motif, in which the Y, Y and Y 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[YpYpY] peptide (ENGHYpYpYAL) was identified to have the strongest binding potency ( = 0.26 ± 0.07 μM) and the highest inhibitory activity (IC = 5.8 ± 0.9 nM), which consists of an amphiphilic N-terminal extension, a double-phosphorylated PtriYmotif and hydrophobic C-terminal extension.