Novel Screen for Suppressors of Breast Tumor Cell Growth Using an Oriented Random Peptide Library Method to Identify Inhibitors of the ErbB2 Tyrosine Kinase

摘要

The aberrant activation of the ErbB2 receptor tyrosine kinase activity appears to play an active role in the progression of some mammary tumors to malignancy. It is therefore of value to identity compounds that efficiently and specifically inhibit ErbB2 kinase activation. Using cyclic peptide library technology we have developed a method for screening up to 17 billion related compounds in a single experiment for the ability to bind with high affinity to the ErbB2 receptor extracellular domain. Libraries were designed based on the Loop3 structure of the EGF-like growth factors because preliminary observations indicated that a modified form of this subdomain could act as an antagonist of ligand-stimulated ErbB2 activity. Cyclic peptide libraries were synthesized containing ten to 15 residues where up to eight positions were each 19-fold degenerate in the natural ammo acids (excluding cysteine), and at least five positions were fixed with non-degenerate orienting amino acids. Libraries were then incubated at 100-fold molar excess with purified ErbB2 extracellular domain, so that those peptides that bind to ErbB2 with highest affinity were selected from the mixture. Receptor-bound peptides were then isolated and sequenced by Edman degradation to determine the optimal receptor binding motif provided by that library. While specific binding to the receptor extracellular domain was observed, these did not yield any significant high affinity binding motifs.