Discovery of a Novel Series of Potent and Selective Substrate-Based Inhibitors of p60^c-src Protein Tyrosine Kinase:  Conformational and Topographical Constraints in Peptide Design

Abstract

On the basis of the efficient substrate for p60c-src protein tyrosine kinase (PTK) YIYGSFK-NH2 (1) (Km = 55 microM) obtained by combinatorial methods, we have designed and synthesized a series of conformationally and topographically constrained substrate-based peptide inhibitors of this enzyme, which showed IC50 values in the low-micromolar range (1-3 microM). A "rotamer scan" was performed by introducing the four stereoisomers of beta-Me(2')Nal in the postulated interaction site of the peptide inhibitor 23(IC50 = 1.6 microM). This substitution led to selective and potent inhibitors of p60c-src PTK; however, no substantial difference in potency was observed among them. This and the results of the "stereochemical scan" performed at residues 2 and 7 of 3 (peptides 19-21), which form the disulfide bond, may suggest that the enzyme active site does not have rigid topographic requirements and thus is able to achieve important conformational changes to bind the ligand as long as the pharmacophore pattern in the inhibitor is conserved. Two new potent iodo-containing nonphosphorylatable tyrosine analogues were also incorporated into our lead inhibitory sequence 23, producing the most potent inhibitors for p60c-src PTK identified thus far in our studies. Compounds 29 and 30 exhibit IC50 values of 0.13 and 0.54 microM, respectively. Peptide 29 is 420-fold more potent than the parent peptide 1. Selectivity studies of peptides 23-30 toward p60c-src, Lyn, and Lck PTK showed in general high Lyn/Src and moderate Lck/Src selectivity ratios. We found that the chi1 space constraints of the specialized amino acids, introduced at position 3 of the peptide lead 23, were not as important as the configuration of the Calpha of that residue to recognize the subtle chemical environment surrounding the active site of Src and Lck PTK, as reflected on the obtained Lck/Src selectivity ratios.

Regulation of Adipocyte Differentiation by Distinct Subcellular Pools of Protein Kinase B (PKB/Akt)

“…1, b and c). This is consistent with the ability of pseudosubstrate inhibitors to inhibit other kinases with high specificity, including JNK (39), p60 c-Src protein-tyrosine kinase (40), and PKC (41).…”

Synthesis and structure–activity relationship studies of peptidomimetic PKB/Akt inhibitors: The significance of backbone interactions

“…[2][3][4][5][6] Peptidic substrate-based inhibitors have a much greater potential to become selective inhibitors, due to comprehensive specific interactions with the protein kinase binding site. [7][8][9] Recently, a library of peptides derived from a PKB/Akt substrate, the protein Glycogen Synthase Kinase 3 (GSK-3), was developed and the interactions of the peptides with PKB/ Akt was studied. The peptide Arg-Pro-Arg-Nva-Tyr-Dap-Hol, (PTR6154), derived from the GSK-3 substrate peptide Arg-ProArg-Thr-Ser-Ser-Phe, was found to be a selective, potent PKB/Akt inhibitor.…”

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,…”