IDENTIFICATION OF EFFICIENT PENTAPEPTIDE SUBSTRATES FOR THE TYROSINE KINASE PP60(C-SRC)

The development of inhibitors of protein tyrosine kinases (PTKs) is a promising approach to obtaining new therapeutic agents for a variety o f diseases, particularly cancer. However, the discovery of peptide-bas ed inhibitors has been hindered by the lack of small peptide substrate sequences (i.e. five residues or less) with which a variety of inhibi tor designs could be readily evaluated by replacing the Tyr with natur al and unnatural amino acids. These prototypical small peptide inhibit ors could then form the basis for designing analogous conformationally constrained, peptide-mimetic or non-peptide inhibitors with improved therapeutic potential. In this study we have identified the best known small peptide substrate for the PTK pp60(c-src) which is the parent o f the src family of nonreceptor PTKs. This pentapeptide substrate, Ac- Ile-Tyr-Gly-Glu-Phe-NH2, has a K-m of 368 mu M and V-max of 1.02 mu mo l/min/mg when tested utilizing the assay methodology of Budde et al. ( Anal. Biochem. 1992, 200, 347-351) after a series of modifications wer e made to more closely simulate the conditions inside a typical mammal ian cell. This substrate was designed from information obtained by Son gyang et al. (Nature 1995, 373, 536-539) with a 2.5 billion member com binatorial library of peptide substrates for pp60(c-src). A second pen tapeptide substrate, Ac-Glu-Asp-Ala-Ile-Tyr-NH2, with a weaker binding affinity (K-m = 880 mu M) but improved V-max (1.86 mu mol/min/mg), wa s also identified. This peptide was designed from the pp60(c-src) auto phosphorylation sequence and information obtained by Songyang et al. ( Ibid.) and Till et al. (J. Biol. Chem. 1994, 269, 7423-7428) with comb inatorial libraries of peptide substrates. These new substrates provid e sufficient binding affinities and rates of phosphorylation to be uti lized for evaluating the relative effectiveness of various reversible and mechanism-based irreversible inhibitor designs for pp60(c-src) whi le appended to easily prepared small peptides.