Title

Design, Synthesis, and In Vitro Binding Studies of Novel Peptides for HER2 Targeting Based on Knob-Socket Model

Poster Number

3

Lead Author Affiliation

School of Pharmacy

Introduction

Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) protein is found in about 30% of breast cancers. HER2 has been considered as a target for receptor-mediated drug delivery system and antibodies for cancer treatment. Pertuzumab is an antibody that inhibits cell proliferation and differentiation by binding to the extracellular domain of HER2 and has been successfully used in the treatment of breast cancer. Compared to antibodies, peptides that selectively recognize tumor cells have advantages in solubility, stability, immunogenicity and cost.

Purpose

To design, synthesize and characterize binding specificity and affinity of novel peptides that bind to HER2 based on Knob-Socket model.

Method

Based on Knob-Socket analysis of interface from crystalline structure of Pertuzumab-HER2 complex, simple patterns of single residue knobs packing into 3 residue sockets that contribute to Pertuzumab-HER2 complex were identified. Linking the knob residues from Pertuzumab that pack into sockets on HER2 surface served as the basis to design over twenty potential peptides bind to HER2. From simulations in MOE software, five peptides showing low binding energies and significant number of interaction sites were identified and synthesized for binding specificity and affinity studies. A control peptide was created with scrambled sequence from one of the five peptides. In vitro binding and uptake of peptides were studied using HER2 over-expressed MDA-MB-361 and ZR-75-1 cells and non-HER2 expressing HEK293 cells as control. Cells were incubated with the five peptides and control peptide at concentration of 10μM at 37°C for 15 minutes. The fluorescence intensities were determined using flow cytometry. The binding affinities of peptides towards HER2 and control protein BSA were determined using surface plasmon resonance (SPR) spectroscopy.

Results

Flow cytometry studies showed mean fluorescence intensity (MFI) of MDA-MB-361 and ZR-75-1 cells after treating with these five peptides were 4~60 fold higher than control peptide, while no significant difference of MFI for HEK293 cells was observed between five peptides and control peptide. SPR results showed that the KD for the peptides designed using Knob- Socket model towards HER2 were in the range of 55.4 nM to 525.5 nM. No significant binding was observed for scrambled sequence and BSA. Significant improvement of HER2 binding was achieved by using Knob-Socket model when comparing with two peptides previously designed based on residues from complementary determining regions (CDRs) of Pertuzumab and their KD towards HER2 were 323.0 nM and 410.0 nM.

Significance

Current methods to develop antibody alternatives all suffer from the drawbacks of tedious, labor intensive, and time consuming processes, uncertainty of outcome and high costs of production. This study created novel strategy to design mimics for antibodies without involving massive experimental screening trials.

Location

DeRosa University Center, Stockton campus, University of the Pacific

Format

Poster Presentation

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Design, Synthesis, and In Vitro Binding Studies of Novel Peptides for HER2 Targeting Based on Knob-Socket Model

DeRosa University Center, Stockton campus, University of the Pacific

Overexpression of Human Epidermal Growth Factor Receptor 2 (HER2) protein is found in about 30% of breast cancers. HER2 has been considered as a target for receptor-mediated drug delivery system and antibodies for cancer treatment. Pertuzumab is an antibody that inhibits cell proliferation and differentiation by binding to the extracellular domain of HER2 and has been successfully used in the treatment of breast cancer. Compared to antibodies, peptides that selectively recognize tumor cells have advantages in solubility, stability, immunogenicity and cost.