Computational Design, Combinatorial Screening and Experimental Analysis of Lung Cancer EGFRVIII-binding Peptides

Human epidermal growth factor receptor mutation variant III (EGFR[Formula: see text] is a cancer-specific cell surface oncogenic marker and has been observed to be widely involved in the formation, progression and metastasis of lung cancer and some other tumors. Previously, a massive quantity of EGFR[Formula: see text]-binding peptides were enriched via random phage display (RPD) targeted against the protein. In this study, we reported rational discovery of 12-mer peptides with high affinity to EGFR[Formula: see text] and strong selectivity for EGFR[Formula: see text] over wild-type EGFR (EGFR[Formula: see text]. A combinatorial peptide library was designed to target EGFR[Formula: see text] based on over ten thousands of known EGFR[Formula: see text] binders enriched from RPD analysis, and a virtual high-throughput screening protocol was then systematically performed against the library to derive those potential candidates, which were further examined rigorously at structural and energetic levels to identify few promising hits. Anisotropy binding assays were carried out to substantiate the computational findings. Consequently, eight 12-mer peptides were designed as effective binders that can target the EGFR[Formula: see text] extracellular subdomain 3 (SD3). In particular, two potent peptides (T1: FLHRYEIVTSYF and T3: FLQKYEWNTSYW) were found to have a high affinity to EGFR[Formula: see text] and a good selectivity for EGFR[Formula: see text] over EGFR WT . Structural analysis revealed that the peptide-binding site can be divided into hydrophobic, polar and mixed regions, which correspond to the nonpolar [Formula: see text]-terminal section, polar/charged middle section and hybrid C-terminal section of the peptide. The peptide selectivity originated from the middle section, which can form a different hydrogen bond network between the two proteins upon the mutating perturbation, whereas the N- and C-terminal sections are primarily responsible for the peptide stability but not specificity.