Title

DESIGN OF PEPTIDE LIGANDS FOR EGFR TARGETING

Lead Author Affiliation

Department of Pharmaceutics and Medicinal Chemistry

Second Author Affiliation

Department of Pharmaceutics and Medicinal Chemistry

Third Author Affiliation

Department of Pharmaceutics and Medicinal Chemistry

Purpose

To design peptide ligands derived from the heavy chain of Cetuximab for EGFR targeting and determine the key design parameters.

Method

Peptides were designed based on the interactions between Cetuximab-EGFR complex. Two peptides, PEP1 with sequence THR-TYR-TYR-ASP-TYR-GLU-PHE (100-106 residues-Cetuximab) from CDR-H3 region and PEP2 with sequence ILE-TRP-SER-GLY-GLY-ASN-THR-ASP-TYR (51-59 residues-Cetuximab) from CDR-H2 region were designed. Docking studies were performed to identify binding interactions in simulated environment. Specific interactions between peptides and EGFR for number of H-bonds, number of binding sites and docking scores were identified based on these studies. Peptides were synthesized by solid phase peptide synthesis using Fmoc-chemistry on Wang resin and coupling of amino acids was performed with HATU (2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HOBT (N-Hydroxybenzotriazole) and DIPEA (N,N-Diisopropylethylamine). Fluorescein isothiocyanate (FITC) was conjugated to N-terminus of the peptides. Peptides were lyophilized and purity was analyzed by reverse-phase HPLC. Targeting capability of peptides was evaluated for cellular uptake by using confocal microscope and flow cytometer using A431, MDA-MB468 and A549 cells with overexpressed EGFR and HEK293 cells which does not overexpress EGFR as control.

Results

Molecular interactions showed that two hydrogen bonds were involved in the binding of both PEP1 and PEP2 to EGFR domain III. Salt-bridge formation by ASP of PEP1 and PEP2 was also observed.PEP1 interacts with four amino acids in EGFR whereas PEP2 with only two amino acids of EGFR. Docking scores for thirty confirmations were in the range of -8.47 to -6.45 and -10.27 to -4.93 for PEP1 and PEP2, respectively. Confocal microscopy images display significantlyhigher cellular uptake of both peptides in EGFR overexpressed cells and negligible uptake in control cells. Higher uptake of PEP1 was observed in EGFR overexpressed cells when compared to PEP2. Percentage of fluorescent positive cells analyzed by flow cytometer were 89.51±3.96, 80.45±1.56, 69.75±9.16, 37.43±1.73 for PEP1 and were 76.86±6.31, 65.86±3.42, 68.29±2.70, 22.06±2.89 for PEP2 in A431, MDA-MB468, A549 and HEK293, respectively.

Significance

Peptides that are capable of specific binding/uptake to EGFR can be designed based on the interactions between CDR-H2, CDR-H3 regions of the antibody and EGFR. Number of binding residues to the peptides plays an important role in binding/uptake of the peptide. In vitro cellular uptake studies demonstrated that the designed peptides can be used to target EGFR overexpressed cancers.

Location

DeRosa University Center, Stockton campus, University of the Pacific

Format

Poster Presentation

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Mar 25th, 10:00 AM Mar 25th, 3:00 PM

DESIGN OF PEPTIDE LIGANDS FOR EGFR TARGETING

DeRosa University Center, Stockton campus, University of the Pacific