Identification of peptide substrates and development of pseudosubstrate-based peptide inhibitors for p60(C-SRC) protein tyrosine kinase

Protein tyrosine kinases (PTKs) mediate important signaling events associated with cellular growth, differentiation, and mitogenesis. The p60c-src protein is the first described cellular protein tyrosine kinase. Human p60c-src PTK has been implicated in the development of colon and breast cancer, and leukemia. However, the exact physiological role of p60c-src PTK or its physiological target proteins are not well known, and the mechanism by which the p60c-src PTK activity is regulated is not completely understood. Peptide substrates can be used to determine the substrate specificity and kinetic parameters, and therefore to provide important information for understanding of the physiological role and mechanism of action of this enzyme. Peptide substrates can also be used to develop pseudosubstrate-based peptide inhibitors for p60c-src PTK. Combinatorial peptide library methods have proven to be very powerful in identifying ligands for receptors and in discovering peptide substrates for protein kinases. In this dissertation, a "one-bead one-compound" combinatorial peptide library method was applied to identify peptide substrates for p60c-src PTK, the structure-activity relationship of the identified peptide substrates was studied, and the pseudosubstrate-based peptide inhibitors for p60c-src PTK were developed. Using the "one-bead one-compound" combinatorial peptide library method, a novel peptide, YIYGSFK, was identified as an efficient substrate for p60c-src PTK. The structure-activity relationship study was performed on over 70 analogs of YIYGSFK. It was determined that -Ile-Tyr- were the two critical residues required for activity. Based on this dipeptide motif a secondary library was synthesized (XIYXXXX, wherein X = all 19 eukaryotic amino acids except cysteine, I = isoleucine, Y = tyrosine) and screened with p60c-src PTK. One of the identified peptides, GIYWHHY, was found to be more efficient for p60c-src PTK than the parental compound, YIYGSFK. Several potent psedosubstrate based inhibitors were developed using GIYWHHY as a template. Some of the more potent inhibitors have branched structure indicating the enzyme active site can accommodate more than a linear motif. These data demonstrate that the "one-bead one-compound" combinatorial library method is a powerful tool to discover novel peptide substrates, and to develop pseudosubstrate-based peptide inhibitors for PTKs