Protein Chemical Synthesis Combined with Mirror‐Image Phage Display Yields d‐Peptide EGF Ligands that Block the EGF–EGFR Interaction

Abstract

The epidermal growth factor (EGF) pathway, being overactive in a number of cancers, is a good target for clinical therapy. Although several drugs targeting the EGF receptor (EGFR) are on the market, tumours acquire resistance very rapidly. As an alternative, small molecules and peptides targeting EGF have been developed, although with moderate success. Herein, we report the use of mirror‐image phage display technology to discover protease‐resistant peptides with the capacity to inhibit the EGF–EGFR interaction. After the chemical synthesis of the enantiomeric protein d‐EGF, two phage‐display peptide libraries were used to select binding sequences. The d versions of these peptides bound to natural EGF, as confirmed by surface acoustic waves (SAWs). High‐field NMR spectroscopy showed that the best EGF binder, d‐PI_4, interacts preferentially with an EGF region that partially overlaps with the receptor binding interface. Importantly, we also show that d‐PI_4 efficiently disrupts the EGF–EGFR interaction. This methodology represents a straightforward approach to find new protease‐resistant peptides with potential applications in cancer therapy.

D-Peptide and D-Protein technology: Recent advances, challenges, and opportunities

“…[68] Consequently, enantiomeric segments of both the epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF-A) were synthesized for MIPD. [57,69] A 12-residue linear D-peptide ligand for EGF, D-PI_4, was identified with both binding affinity and half-maximal inhibitory concentration (IC50) in micromolar range. [69] In the case of VEGF-A, the mini-protein GB1 was used as a template scaffold to create a 56 residue D-mini-protein RFX001.D that has a binding affinity as low as 85 nM.…”

Selection of a d-Enantiomeric Peptide Specifically Binding to PHF6 for Inhibiting Tau Aggregation in Transgenic Mice

“…36 Mirror image phage display mainly takes advantage of chiral amino acids and, consequently, of the peptides or proteins that they form. 37 Compared to Lenantiomeric peptides, D-enantiomeric peptides are known to be less or even not at all immunogenic and protease sensitive as well as more resistant to degradation in vivo. 38−40 Therefore, D-enantiomeric peptides screened from mirror image phage display may be well-suited to diagnosis and treatment in living animals and humans.…”

“…Currently, it has been widely used in disease diagnosis, study of protein interactions, development of new vaccines, peptide drug development, and clinical diagnosis and treatment. − In our laboratory, we have screened a specific Zn­(II)-binding peptide to improve the cognitive dysfunction of APP/PS1 transgenic mice by inhibiting Zn 2+ -mediated amyloid protein aggregation and neurotoxicity . Mirror image phage display mainly takes advantage of chiral amino acids and, consequently, of the peptides or proteins that they form . Compared to l -enantiomeric peptides, d -enantiomeric peptides are known to be less or even not at all immunogenic and protease sensitive as well as more resistant to degradation in vivo . − Therefore, d -enantiomeric peptides screened from mirror image phage display may be well-suited to diagnosis and treatment in living animals and humans.…”

“…Compared to l -enantiomeric peptides, d -enantiomeric peptides are known to be less or even not at all immunogenic and protease sensitive as well as more resistant to degradation in vivo . − Therefore, d -enantiomeric peptides screened from mirror image phage display may be well-suited to diagnosis and treatment in living animals and humans. With the rapid development of chemical synthesis and peptide ligation technology, far more d -enantiomeric peptides have been obtained through the use of mirror image phage display. ,,− In AD, representative applications of mirror image phage display have been focused on the identification of d -enantiomeric peptide ligands of Aβ. − However, increasing evidence indicates that some neurodegenerative diseases display Tau deposits independently of Aβ (e.g., PiD, PSP, AD) . These findings establish the role of Tau itself as a causative agent.…”

Evaluating the therapeutic efficacy of nano-drugs targeting epidermal growth factor receptor

“…5 EGFR is widely distributed on the membrane of mammalian epithelial cells, fibroblasts and glial cells, and is also overexpressed in many malignancies, providing the basis for selective ligand targeting of cancer cells. 6–8 As the ligand of EGFR, EGF could specifically bind to EGFR, and plays an important role in biological activity, cell proliferation and differentiation. 9,10 Meanwhile, studies have shown that a small molecule peptide GE11 (YHWYGYTPQNVI) can specifically bind to EGFR without cell growth side-effects.…”