N-myristoylation of a peptide substrate for Src converts it into an apparent slow-binding bisubstrate-type inhibitor

The conversion of a peptide substrate to a potent inhibitor by chemical mod ification is a promising approach in the development of inhibitors for prot ein tyrosine kinases. N-acylation of the synthetic peptide substrate NH2-Gl u-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH2 (EFLYGVFD) resulted in synergistic inhi bition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was d ependent upon the peptide sequence and the length of the acyl chain. The mi nimum length of the fatty acyl chain to synergistically inhibit Src was a l auryl (C11H23CO) group. N-myristoylated EFLYGVFD (myr-EFLYGVFD) inhibited t he phosphorylation of poly E4Y by Src with an apparent K-i of 3 mu m, where as EFLYGVFD and myristic acid inhibited with K-i values of 260 and 35 mu m, respectively. The nonacylated EFLYGVFD was a substrate for Src with K-m an d V-max values of 100 mu m and 400 nmol/min/mg protein, respectively. Howev er, upon myristoylation, the peptide was no longer a substrate for Src. Bot h the acylated and non-acylated peptides were competitive inhibitors agains t the substrate poly E4Y. The nonacylated free peptide showed mixed inhibit ion against ATP while the myristoylated peptide was competitive against ATP . Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a r eversible slow-binding inhibitor with two binding sites on Src. The myristo ylated 8-mer peptide was reduced in size to a myristoylated 3-mer without l osing the affinity or characteristics of a bisubstrate-type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow-binding multisubstrate analogue has improved the potency of inhibition by the pept ide.