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. 2012 Feb 23;55(4):1526-37.
doi: 10.1021/jm201265f. Epub 2012 Feb 10.

Testing the Promiscuity of Commercial Kinase Inhibitors Against the AGC Kinase Group Using a Split-Luciferase Screen

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Free PMC article

Testing the Promiscuity of Commercial Kinase Inhibitors Against the AGC Kinase Group Using a Split-Luciferase Screen

Benjamin W Jester et al. J Med Chem. .
Free PMC article

Abstract

Using a newly developed competitive binding assay dependent upon the reassembly of a split reporter protein, we have tested the promiscuity of a panel of reported kinase inhibitors against the AGC group. Many non-AGC targeted kinase inhibitors target multiple members of the AGC group. In general, structurally similar inhibitors consistently exhibited activity toward the same target as well as toward closely related kinases. The inhibition data was analyzed to test the predictive value of either using identity scores derived from residues within 6 Å of the active site or identity scores derived from the entire kinase domain. The results suggest that the active site identity in certain cases may be a stronger predictor of inhibitor promiscuity. The overall results provide general guidelines for establishing inhibitor selectivity as well as for the future design of inhibitors that either target or avoid AGC kinases.

Figures

Figure 1
Figure 1
A dendrogram of the 27 protein kinases screened in this study. Six families are highlighted.
Figure 2
Figure 2
Scheme of the competitive binding assay using split-luciferase reassembly. (A) A protein kinase and the Fos peptide were fused to complementary halves of firefly luciferase, Cfluc and Nfluc respectively. The addition of a Jun-staurosporine conjugate leads to luciferase activation. Small molecules capable of binding in the active site of the kinase prevent complex formation and result in a loss of luminescence. (B) Structures of staurosporine (1) and its modified analog attached to Jun (2).
Figure 3
Figure 3
Selectivity profiles for the screened compounds 3–14, with the results mapped to the AGC dendrogram. Each pink circle is proportional in size to the measured % inhibition for each kinase, with only % inhibition >25% being shown.
Figure 4
Figure 4
Selectivity profiles for the screened compounds 15–26, with the results mapped to the AGC dendrogram. Each pink circle is proportional in size to the measured % inhibition for each kinase, with only % inhibition >25% being shown.
Figure 5
Figure 5
Selectivity profiles for the screened compounds 27–38, with the results mapped to the AGC dendrogram. Each pink circle is proportional in size to the measured % inhibition for each kinase, with only % inhibition >25% being shown.
Figure 6
Figure 6
Structures of compounds 39–74, which were found to demonstrate <25% inhibition for every kinase tested.
Figure 7
Figure 7
Comparison of inhibition frequencies between kinase domain or active site identity groups. (A) Average F values for incrementally binned identity groups were plotted for either the full kinase domain or active site residues. (B) The crystal structures and pseudosequence of residues within 6 Å of an ATP analog (green) aligned for PKA, AKT2, and AURKA. Homology maps generated using a percent identity cutoff that results in the formation of nine groups for the (C) kinase domain and (D) active site residues.

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