Epidermal growth factor receptor tyrosine kinase domain (EGFR-TK) has been one of the prominent targets for therapeutics of several human cancers, in particular non-small cell lung cancer. Although several small chemical compounds targeting EGFR-TK have been approved by FDA for treatment of such a cancer, the discovery of a new class of EGFR-TK inhibitors, for example, small peptides, is still desired. In this study, using molecular docking-based virtual screening, we selected five small peptides with high docking scores from eight thousand peptides as candidate compounds against EGFR-TK. Among five, the tripeptide WFF had the most potency to suppress the survival of non-small cell lung cancer cells but had the least toxicity to human liver cancer cells. Our in vitro kinase assays showed that WFF exhibited much lower inhibitory activity against purified EGFR-TK than the drug erlotinib (i.e., IC 50 values of ≈ 0.62 μM vs ≈ 7.57 nM, respectively). The relative free binding energies estimated from molecular dynamic simulations were consistent with the in vitro experiments in which the WFF bound had a lower affinity than erlotinib bound to EGFR-TK (i.e., ΔG bind values of −20.3 kJ/mol vs ≈ −126.8 kJ/mol, respectively). In addition, the simulation analyses demonstrated the difference in EGFR binding preference between the drug and tripeptide in which erlotinib was stably bound in the ATP-binding pocket for 4-anilinoquinazoline class of inhibitors, while WFF moved out of that pocket to interact with polar amino acid residues on the αC-helix, activation loop,

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Aberrations of the epidermal growth factor receptor (EGFR), for example, mutations and overexpression, play pivotal roles in various cellular functions, such as proliferation, migration, and cell differentiation. Approved small molecule‐based inhibitors, including gefitinib and erlotinib, are used clinically to target the tyrosine kinase domain of EGFR (TK‐EGFR). However, the severity of the side effects, off‐target effects, and drug resistance is a concern. Cyclic peptides are a well‐known peptide format with high stability and are promising molecules for drug development. Herein, the Ph.D.™‐C7C phage display library was used to screen cyclic peptides against TK‐EGFR. Biopanning, both with and without propagation methods, was performed to assess the highest capacity peptides using the enzymatic activity of TK‐EGFR. Interestingly, NP1, a peptide selected during biopanning without propagation demonstrated an inhibitory effect against TK‐EGFR at IC50 within the nanomolar range; this effect was better than that of P1 obtained using biopanning with propagation. Moreover, NP1 elicited EGFR with an affinity binding (KD) value of 18.40 ± 5.50 µM by surface plasmon resonance (SPR). Introducing cell‐penetrating peptides or Arginine‐9 (Arg9) at the N‐terminus of NP1 thus improves cell‐penetrability and can lead to the inhibition of EGFR‐driven cancer cell lines; however, it exhibits no hepatotoxicity. Furthermore, NP1 caused a decrease in phosphorylated EGFR after activation within cells. A docking model shows that NP1 interacted primarily with TK‐EGFR via hydrogen bonding. Together, this suggests that NP1 is a novel EGFR peptide inhibitor candidate with specificity and selectivity toward TK‐EGFR, and may be applied to targeted therapy.

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