Cyclic Peptides as Protein Kinase Inhibitors: Structure-Activity Relationship and Molecular Modeling

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR]₅, a peptide containing alternative arginine (R) and tryptophan (W) residues as a non-competitive c-Src tyrosine kinase inhibitor. A number of larger cyclic peptides containing alternative hydrophobic and positively charged residues [WR] (x = 6-9) and hybrid cyclic-linear peptides, [R₆K]W₆ and [R₅K]W₇, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR]₉ was found to be the most potent tyrosine kinase inhibitor. [WR]₉ showed higher inhibitory activity (IC₅₀ = 0.21 μM) than [WR]₅, [WR]₆, [WR]₇, and [WR]₈ with IC₅₀ values of 0.81, 0.57, 0.35, and 0.33 μM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-³³P]ATP. Consistent with the result above, [WR]₉ inhibited other protein kinases such as Abl kinase activity with an IC₅₀ value of 0.35 μM, showing 2.2-fold higher inhibition than [WR]₅ (IC₅₀ = 0.79 μM). [WR]₉ also inhibited PKCa kinase activity with an IC₅₀ value of 2.86 μM, approximately threefold higher inhibition than [WR]₅ (IC₅₀ = 8.52 μM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR]₉ exhibited IC₅₀ values of <0.25 μM against Akt1, Alk, and Btk. These data suggest that [WR]₉ is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [R₆K]W₆ and [R₅K]W₇ against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR]_(5-9). The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR]₅ or [WR]₉ affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR]₉.