Phage Display Technology in Identification of Novel Peptide Ligands for hEGFR with the Aim of Developing Anticancer Agents and Investigation of peptide_ Based GI Drug Delivery Vehicles

Abstract

Phage display technology, a biology-based combinatorial approach, is a multi-purpose and versatile tool which has may applications in drug discovery and development. This technique could provide a rapid molecular biology means for discovery of novel peptides and proteins from genetically engineered variants which may bind to various targets with high specificity and affinity or act as specific vehicles for drug delivery particularly through the intestinal barrier. Studies presented in this thesis cover two aspects of phage display application in pharmaceutical science. In the first approach (Chapter 2), we have investigated the possibility of identification of novel peptides capable of targeting epidermal growth factor receptor. These receptors are overexpressed in the surface of human epithelial cancer cells, and therefore is a suitable target for drug discovery in cancer therapy. To this end, A-431 cells expressing EGFR were used as the matrix in a cell based subtractive biopanning approach using a 7-mer peptide displaying phage library. Two novel peptide ligands were identified and tested for their affinities and functional effects on EGFR. The identified peptides were able to inhibit the EGF-induced phosphorylation of EGFR in a concentration dependent manner. The results of affinity binding experiments showed that the natural ligand, i.e., EGF, was able to competitively inhibit the binding of peptide bearing phages to EGFR expressing A-431 cells. In this investigation molecular modeling studies were also used to calculate the binding free energies for peptide-receptor complexes and to propose the modes of interactions. The results of molecular modeling studies support the experimental results obtained in the current work as well as the previously reported mutagenesis data. Moreover, a three dimensional model was proposed for a previously discovered EGFR specific 12-mer peptide (named GE11) using ab initio approach and its mode of interaction with the receptor was predicted via molecular modeling methods. The binding similarities for the identified peptides and GE11 were discussed in line with the experimentally observed interactions in the previously reported crystal structure of EGF-EGFR complex. In Chapter 3 we used phage display technology to examine the possibility of identifying peptides capable of transmucosal transport of phage particles in GI tract, which then can be used as a tool for facilitating the GI delivery of pharmaceuticals. We hypothesized that the introduction of a library of peptide displaying phages into the intestine may lead to the identification of sequences that could be responsible for the transmucosal transport of phage particles bearing these sequences. A biopanning protocol was performed by applying a 7-mer random amino acid phage library to mice by gavage and then assessing their absorption via phage recovery from the spleen and blood. Following isolation of 77 different phages, the sequences of the displayed peptides were identified. Statistical treatment of the obtained sequences did not support the notion that the GI translocation depends on the presence of any particular peptide sequence fused on the pIII coat proteins of the M13 phages. There are, however, some residue types under represented which could be due to specific GI selection mechanisms and/or their effects on the amplification rate for phages bearing those residues.In summary, we have used phage display technology in drug discovery oriented projects and the results can be used for the development of new pharmaceuticals.