A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

Abstract

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides.

Design Strategy to Create Antibody Mimetics Harbouring Immobilised Complementarity Determining Region Peptides for Practical Use

“…Strategy for creating FLAPs. Our previous study revealed that it is crucial for target-binding peptides to be structurally constrained in appropriate scaffolds to ensure that these peptides have high binding affinity 15 . Based on these findings, we aimed to develop a strategy to computationally design small antibody mimetics, named FLAPs, by selecting adequate small protein scaffolds and extracting target-binding sequences from mAb drugs ( Fig.…”

“…In this report, we aimed to develop a clinically applicable small antibody mimetic termed fluctuation-regulated affinity proteins (FLAPs). The approach for developing FLAPs is based on our recent study that showed the binding activity of target-binding peptides increases by two logs when their structure is immobilised by grafting to a particular site of a fluorescent protein scaffold 15 . Our fast and easy method for developing FLAPs includes computational methods for selecting short peptides from the CDR that help with binding to the target and for determining the graft acceptor (GA) sites, which are crucial for proper immobilisation of the grafted short peptide structure, in small protein scaffolds.…”

Strategic design to create HER2-targeting proteins with target-binding peptides immobilized on a fibronectin type III domain scaffold

“…Our previous research demonstrated that short peptides graed in a particular region of protein scaffold were structurally immobilized and showed increased resistance to proteases. 17 This nding prompted us to develop clinically applicable HER2targeting small proteins with a structurally immobilized HBP. Fig.…”

“…15,16 Recently, we found that the affinity and proteolysis-resistance of peptides was greatly improved aer their structure was immobilized by graing into a particular site of a protein scaffold. 17 Based on this nding, we reported a computational design strategy for developing antibody mimetics named FLAP, which is uctuation-regulated affinity protein (FLAP) having structurally immobilized mAb-derived peptide. 18 In this study, we focused on the development of a HER2targeting small protein with a structurally immobilized HBP, HBP-FLAP.…”

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning

“…The lack of cetuximab binding observed for Md2 and Md3, which did respond in the fluorescence polarization assay, suggests that these peptides were not cyclized efficiently by the yeast secretory machinery. Yeast display has been used extensively on different types of proteins (25,(32)(33)(34)(35)(36) as well as cysteine knot cyclic peptides (37), but those molecules possess a well defined tertiary structure that will favor correct disulfide pairing during oxidative folding in the endoplasmic reticulum. Simple disulfide-constrained cyclic peptides lack such a stabi- lizing scaffold, and therefore they may form incorrect disulfide bridges with other proteins.…”