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Setting up a kinase profiler with IMAP

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RESULTS OF SCREENING WITH 3 INHIBITORS
The histogram in Figure 19 shows what a powerful tool kinase profiling can be. In one assay, inhibition data for 12 different kinases was generated. An assay concentration of 20 nM staurosporine was selected, as this inhibitor is generally known to inhibit Ser/Thr kinases in the low nanomolar range.1

Several Ser/Thr kinases in the profiling assay, including AMPK, Aurora B, IRAK4 and PAK4, were inhibited by staurosporine when tested with at least the low dose of ATP. A representative Aurora B assay is shown in Figure 20, Panel A. Notably, the Ser/Thr kinases CK1 (Figure 20, Panel C) and PKCx (Figure 20, Panel D) were relatively resistant to staurosporine inhibition, which correlates well with published reports.(1,2) Staurosporine clearly reduced the activity of CDK5/p25, as shown in Figure 20, Panel B, consistent with the published in vitro staurosporine IC50 for CDK5/p25 of 4 nM.3

The IMAP profiling assay outlined in Appendix A was next performed with the compound SB203580, which is known to be a relatively specific inhibitor of p38. In the profiling assay shown in Figure 21, SB203580 had little or no effect on several of the kinases, including AMPK, which correlates well with Davies results.4 Conversely, casein kinases are strongly inhibited by this compound,6 and the profili
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