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 modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N-acylation of the synthetic peptide substrate NH₂-Glu-Phe-Leu-Tyr-Gly-Val-Phe-Asp-CONH₂ (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C₁₁H₂₃CO) group. N-myristoylated EFLYGVFD (myr- EFLYGVFD) inhibited the phosphorylation of poly E₄Y by Src with an apparent K(i) of 3 μm, whereas EFLYGVFD and myristic acid inhibited with K(i) values of 260 and 35 μm, respectively. The nonacylated EFLYGVFD was a substrate for Src with K(m) and V(max) values of 100 μm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non-acylated peptides were competitive inhibitors against the substrate poly E₄Y. The non-acylated free peptide showed mixed inhibition 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 reversible slow-binding inhibitor with two binding sites on Src. The myristoylated 8-mer peptide was reduced in size to a myristoylated 3-mer without losing 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 peptide.