High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Abstract

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.

Figure 1. Chemical scaffolds explored in this study

The hydrogen bond contacts to the kinase hinge region residues are marked as dashed bonds

Figure 2. Kinases targeted by the furan and pyridone thiazolidinone-type compounds

(A) Percent of kinases targeted by this scaffold (64 compounds) with an Ambit score of <1% of DMSO control are highlighted in red circles. The size of the circle represents the percentage a particular kinase is targeted. DRAK1 is the most highly targeted kinase with 28% followed by HIPK1 and PIM2 at 27%. Only targeted kinases of 10% and higher are indicated. (B&C) The primary ambit data for all the furan-type compounds (B) and pyridone-type compounds (C) were clustered using MultiExperiment Viewer. Only kinases with at least 3 compound hits and with a score of <5% of DMSO control were considered for this clustering analysis. Kinome Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

Figure 3. Cellular data of the PIM1 inhibitor A47

(A) Ba/F3 wild-type (WT) cells and stably expressing hPIM1 and/or hPIM2 cells were exposed to increasing amounts of A47 and cell viability was determined 24 h and 48 h later (expressed as a percentage normalized to viability of cells treated with 0.1% DMSO only (n=3). Data is shown for cell viability after 24h. (B) MV4;11 cells were incubated with increasing concentrations of A47 for indicated times, harvested, and protein extracts separated by SDS-PAGE. The effect of the compound on PIM endogenous target was followed by Western blotting with indicated phosphospecific antibodies. Membranes were stripped and re-probed with non-phosphospecific and anti-actin antibodies to check for equal loading. (C) View of co-crystal structure between PIM1 kinase and furan thiazolidinedione A54. The inhibitor binds in a typical type I fashion and its trifluoromethylaniline portion is directed towards the solvent exposed region.

Figure 4. Kinases targeted by the pyrimido diazepines and pyrimido benzodiazepines

(A) Percent of kinases targeted by the pyrimido diazepines and pyrimido benzodiazepines with an Ambit score of <1% of DMSO control are highlighted in red circles. The size of the circle represents the percentage a particular kinase is targeted. ERK5 is the most highly targeted kinase with 23% followed by PLK3 and DCAMKL1 at 15%. Only targeted kinases of 8% and higher are indicated. (B) The primary ambit data was clustered using MultiExperiment Viewer. Only kinases with at least 3 compound hits and with a score of <5% of DMSO control were considered for this clustering analysis. Kinome Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

Figure 5. Cellular data of the ERK5 inhibitor B46 and ACK1 inhibitor B19

(A) HeLa cells were serum starved overnight followed by treatment with different concentrations of B46 for 1 hr. Cells were then stimulated with EGF for 17 min and BMK1 activation was detected by mobility retardation. (B) ACK1 or TNK1 cDNA in pcDNA3 vector was transfected into HEK293 cells for 36 hrs. DMSO (control), B19 or B20 (N-Me amide version) at indicated concentration was added into the culture medium for 12 hrs. The cells were lysed and the cell lysates were subjected to SDS-PAGE. Tyrosine phosphorylated ACK was detected with immunoblotting with anti-phosphotyrosine antibody (4G10). (C) A549 cells were seeded in a 6-well culture plate at a density of 2000/well. EGF (50 ng/ml), AG1478 (1 μM), B19 (10 μM), or DMSO (control) was added into culture medium and refreshed every three days. The cells were cultured for 7 days and the number of cells was counted with Bright-Line Hemacytomer (Hausser Scientific) under microscope.

Figure 6. Cellular data of the dual MPS1/Polo-like kinases inhibitor B13 and pan-Aurora kinase inhibitor B54

(A) Schematic of the treatment regimen used to assess the effect of B13 treatment on spindle checkpoint-induced mitotic arrest in U2OS cells. (B) Immunoblot of cyclin B from U2OS cells treated with B13. U2OS cells were arrested in mitosis by combination treatment of thymidine and nocodazole prior to treatment with nocodazole or co-administration with B13 ± MG132 for 2 hrs. (C) Immunoblot of mitotic markers from U2OS cells treated with B54. U2OS cells were arrested in mitosis by combination treatment of thymidine and taxol prior to treatment with taxol or co-administration with B54 (or VX-680) ± MG132 for 2 hrs.