Rational Design of EGFR Antibody Mimics Based on Knob-Socket Model
S. Sachdeva, D. Su, H. Joo, J. Tsai, B. Jasti, X. Li
University of the Pacific Purpose To design and characterize peptides to mimic antibody binding to Epidermal Growth Factor Receptor (EGFR) based on Knob-Socket model. Methods Antibody mimic molecules were designed by mapping the Cetuximab-EGFR epitope surface and identifying the sockets. Each socket is formed by three amino acid residues on the EGFR surface and binds with the amino acid knob residues from the antibodys complementary binding region loops. Antibody mimics were designed by selecting the knob residues with high probability to pack into sockets on the EGFR surface. Twenty five molecules (Pep1-Pep25) were designed and analyzed using Molecular Operating Environment (MOE) software to determine the binding energy, total number of interactions and preserved interactions between EGFR and designed molecules. A pair of peptides (Pep6 and Pep24) with reverse sequence was designed to test the sequence requirement for binding and a scrambled peptide (Pep25) was created as a control. The specific binding of antibody mimics was evaluated by observing uptake of the FITC labeled antibody mimics in A431, MDA-MB468 cells (overexpressed-EGFR) and HEK293 (control) using confocal microscope and flow cytometry. Equilibrium dissociation constant (KD) was determined by using surface plasmon resonance for antibody mimics binding to EGFR and bovine serum albumin. Cell based ELISA was performed to analyze the percentage phosphorylation inhibition by antibody mimic molecules. Results Pep6, Pep11, Pep22, and Pep24 showed low calculated binding energy and high number of total and preserved interactions. Pep11 containing high probability interaction knobs F and Y for sockets VSS and ISI, respectively, exhibited the lowest KD of 252nM. Pep6 containing lower probability reversed sequence knobs (Y and F) showed a higher KD. Pep 6 and Pep24 packed into sockets in similar fashion, showed comparable KD values. Confocal microscopy images and mean fluorescence intensities in flow cytometry analysis displayed significantly higher cellular uptake of antibody mimics in EGFR overexpressed cells and negligible uptake in control cells. Percentage inhibition of phosphorylation for antibody mimics was found to be in the range of 4.400.60 to 8.130.12 as compared with 37.900.07 inhibition by Cetuximab. Conclusion Antibody mimics against EGFR have been successfully designed using a rational approach based on the Knob-Socket model. These antibody mimics can specifically bind to EGFR and display partial EGFR phosphorylation inhibition.
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