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

On the basis of the efficient substrate for p60(c-src) protein tyrosin e kinase (PTK) YIYGSFK-(NH2 (1) (K-m = 55 mu M) obtained by combinator ial methods, we have designed and synthesized a series of conformation ally and topographically constrained substrate-based peptide inhibitor s of this enzyme, which showed IC50 values in the low-micromolar range (1-3 mu M). 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 mu M) This substitution led to se lective and potent inhibitors of p60(c-src) PTK; however, no substanti al difference in potency was observed among them. This and the results ;of the ''stereochemical scan'' performed at residues 2 and 7 of 3 (pe ptides 19-21), which farm the disulfide bond, may suggest that the enz yme 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. T wo new potent iodo-containing nonphosphorylatable tyrosine analogues w ere also incorporated into our lead inhibitory sequence 23, producing the most potent inhibitors for p60(c-src) PTK identified thus far in o ur studies. Compounds 29 and 30 exhibit IC50 values of 0.13 and 0.54 m u M, respectively. Peptide 29 is 420-fold more potent than the parent peptide 1. Selectivity studies of peptides 23-30 toward p60(c-src), Ly n, and Lck PTK showed in general high Lyn/Src and moderate Lck/Src sel ectivity ratios.We found that the chi(1) space constraints of the spec ialized amino acids, introduced at position 3 of the peptide lead 23, were not as important as the configuration of the C-alpha, of that res idue to recognize the subtle chemical environment surrounding the acti ve site of Src and Lck PTK, as reflected on the obtained Lck/ Src sele ctivity ratios.