A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases

Abstract

Protein kinases play a pivotal role in cell signaling, and dysregulation of many kinases has been linked to disease development. A large number of kinase inhibitors are therefore currently under investigation in clinical trials, and so far seven inhibitors have been approved as anti-cancer drugs. In addition, kinase inhibitors are widely used as specific probes to study cell signaling, but systematic studies describing selectivity of these reagents across a panel of diverse kinases are largely lacking. Here we evaluated the specificity of 156 validated kinase inhibitors, including inhibitors used in clinical trials, against 60 human Ser/Thr kinases using a thermal stability shift assay. Our analysis revealed many unexpected cross-reactivities for inhibitors thought to be specific for certain targets. We also found that certain combinations of active-site residues in the ATP-binding site correlated with the detected ligand promiscuity and that some kinases are highly sensitive to inhibition using diverse chemotypes, suggesting them as preferred intervention points. Our results uncovered also inhibitor cross-reactivities that may lead to alternate clinical applications. For example, LY333'531, a PKCbeta inhibitor currently in phase III clinical trials, efficiently inhibited PIM1 kinase in our screen, a suggested target for treatment of leukemia. We determined the binding mode of this inhibitor by x-ray crystallography and in addition showed that LY333'531 induced cell death and significantly suppressed growth of leukemic cells from acute myeloid leukemia patients.

Fig. 1.

Phylogenetic tree of the human protein kinase family (1). Screened targets are indicated by a yellow dot, and structure-determined catalytic domains by the Structural Genomics Consortium or other laboratories are indicated by a red dot and a blue dot with a yellow sphere, respectively. The kinase dendrogram is adapted from ref. and has been reproduced with permission from Science and Cell Signaling.

Fig. 2.

Inhibitor array of the screened kinases. Yellow fields indicate a T_m shift >4°C and red field of >8°C, respectively. White fields represent data that have not been determined. A complete table with T_m values is available in SI Table 3. Some targets and inhibitors discussed in the text have been annotated in the figure.

Fig. 3.

Alignment and active site. (A) Pseudosequence alignment and phylogenetic tree of the ATP-binding site residues for proteins studied including promiscuity score. (B) A 2D projection of a typical protein kinase ATP-binding site viewed down the axis from the N-terminal lobe through to the C-terminal lobe. The backbone is shown as a black line, which is thickest in the foreground. The residues found to form the ATP-binding site are marked and numbered, and arrows point along the consensus direction of the side chains. “bb” indicates that the residue interacts only via backbone atoms. (C) A cut through the plane of the adenosine ring of ATP of a typical ATP-binding site with ATP included. Upper is viewed from the N-terminal lobe to the C-terminal lobe, and Lower is from the C-terminal lobe up to the N-terminal lobe.

Fig. 4.

Binding of LY333′531 to PIM1 and effects on primary tumor cells. (A) Active site of PIM1 in complex with LY333′531 solved at 1.9-Å resolution. Hydrogen bonds are indicated by dotted lines, and interacting residues are labeled and shown in ball-and-stick representation. (B) Phosphorylation status of BAD (Ser-112) upon treatment of LY333′531, detected by Western blot analysis. α-Actin levels and total BAD levels are shown as a control. (C) Effects of LY333′531 on the survival of primary tumor cells from five AML patients (Left). A concentration of 0 μM (DMSO), 1 μM, 5 μM, and 10 μM was used, and the increasing concentration of inhibitor has been indicated by black, dark gray, light gray, and white bars. Effect of LY333′531 on clonogenic growth of cells from patient 1 is shown in Right.