Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Abstract

Using a combinatorial peptide library method, we identified Y1YGSFK as an efficient and specific peptide substrate for pp60 c-src protein tyrosine kinase (PTK) [Lain et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60 c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2'-Nal)GSFK, selectively inhibited the kinase activity of pp60 °sr~, with a K i of 24 BM. This peptide inhibitor exhibited selectivity for pp60 ..... as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.

Discovery of a Novel Series of Potent and Selective Substrate-Based Inhibitors of p60c-src Protein Tyrosine Kinase: Conformational and Topographical Constraints in Peptide Design

“…To preserve the hydrophobic nature at position 2, shown to be important for activity, we introduced the chimeric amino acid penicillamine (Pen) at that position, which provided as well the SH group necessary to form the disulfide bond with Cys 7 . Recently, Lam et al have reported that replacement of Tyr 3 with (2‘)Nal in substrate 1 resulted in a moderately potent and specific inhibitor of p60 c - src PTK with an IC 50 value of 66 μM. YI-(2‘)Nal-GSFK was shown to be a competitive inhibitor for p60 c - src PTK, when 1 was used as a substrate, and showed no activity toward cAMP-dependent protein kinase (a representative of serine/threonine kinases).…”

Development and characterization of potent and specific peptide inhibitors of p60c‐src protein tyrosine kinase using pseudosubstrate‐based inhibitor design approach

“…An important strategy for the development of substrate‐based inhibitors for PTKs is to replace the l ‐tyrosine residue of the substrate with tyrosine mimetics such as l ‐phenylalanine (47), l ‐phosphonomethylphenylalanine (48), l ‐1‐naphthylalanine (1′Nal), l ‐2‐naphthylalanine (2′Nal) (29), and (3,5‐diiodo)Tyr [(3,5‐di I)Tyr] (28). In the last few years, we have successfully applied this strategy to develop p60 c‐src PTK inhibitors based on substrate motifs (28,29).…”

Development of inhibitors for protein tyrosine kinases

“…The smallest and most active peptide inhibitor is a nonapeptide, GRTGRRNAI, with a Ki value of 0.036 mM. We (Wu et al, 1996;Lou et al, 1997;Alfaro-Lopez et al, 1998) and others (Fry et al, 1994;Niu and Lawrence 1997a, b;Walsh and Glass 1991;Petrakis and Nagabhushan 1987;Burke et al, 1993;Yuan et al, 1990) have applied the same strategy to Figure 1 Chemical structures of natural products and their derivatives with kinases inhibitory activity develop pseudosubstrate-based peptide inhibitors for PTK. The following Tyr analogues have been used to replace Tyr in these studies: p-¯uorophenylalanine, pchlorophenylalanine, 1-naphthylalanine, 2-naphthylalanine, phenylalanine, D-tyrosine, methyl-tyrosine, 1,6-dichloro-tyrosine, tetra¯uorotyrosine, 4-phosphonophenylalanine, phosphomethyl-phenylalanine, tetrauorotyrosine,…”