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 Ki of 3 microm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 microm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 microm 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.
Ramdas
L
L
Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Obeyesekere
N U
NU
Sun
G
G
McMurray
J S
JS
Budde
R J
RJ
D000215:Acylation; D000081247:CSK Tyrosine-Protein Kinase; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D008074:Lipoproteins / Q000138:chemical synthesis / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D018909:src Homology Domains; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D008074:Lipoproteins; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases