Cyclic peptides incorporating 4-carboxyphenylalanine and phosphotyrosine are potent inhibitors of pp60(c)-(src)

The protein tyrosine kinase, pp60(c-src), is involved in cellular signaling and is activated during mitosis and in various tumors. We have been employ ing cyclic decapeptides to identify the determinants for substrate binding and phosphorylation to develop inhibitors competitive with protein substrat es of Src. A structure-activity study [McMurray, J. S., Budde, R. J. A., Ke , S., Obeyesekere, O. U., Wang, W., Ramdas, L., and Lewis, C. A. (1998) Arc h. Biochem. Biophys. 355, 124] revealed that, at the position 3 residues C- terminal to the phosphorylated tyrosine (Y + 3), both glutamic acid and phe nylalanine gave identical K-i, K-m, and V-max values. We hypothesized that the area of Src that binds the Y + 3 residue contains either a positively c harged lysine or an arginine, capable of ionic interactions with glutamic a cid or cation-pi interactions with phenylalanine. To test this hypothesis, a series of phenylalanine analogues were substituted at position 7 (the Y 3 residue) in cyclo(Asp(1)-Asn(2)-Glu(3)-Tyr(4)-Ala(5)-Phe(6)-Phe(7)- Gln( 8)-D-Phe(9)-Pro(10)). Of these, 4-carboxyphenylalanine (4-Cpa) and phosphot yrosine resulted in high affinity peptides exhibiting K-i values of 0.85 an d 1.1 mu M, respectively, 180- and 130-fold increases in potency over the p arent cyclic peptide (K-i = 150 mu M). These peptides were noncompetitive w ith respect to ATP and competitive against the phosphate-accepting substrat e, polyGlu(4)Tyr. The truncated cyclic peptide, cyclo(Phe-4-Cpa-Gln-D-Phe-P ro-Asp-Aca) (Aca = epsilon-aminocaproic acid), which did not contain tyrosi ne, was also a competitive inhibitor with a K-i value of 24 mu M. We conclu de that these cyclic peptides bind to a positively charged area that is nea r the phosphate transfer region of the active site of Src but does not nece ssarily include the tyrosine-binding pocket. Furthermore, the 4-Cpa-contain ing cyclic decapeptide shows remarkable selectivity in the inhibition of Sr c versus the src family members Yes and Lck, as well as other protein tyros ine kinases, Ser/Thr kinases, and other ATP-utilizing enzymes.