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Assay to evaluate factors that contribute to the correlation between enzyme and cellular potency for ATP competitive inhibitors of p38 kinase

A major challenge in kinase drug discovery is accurately relating a compound’s inhibitory activity in vitro to its potency within the cell. A number of factors can contribute to the relationship including those that influence the drug’s intrinsic affinity for the kinase, such as choice of substrate, and those that influence the concentration of drug required to achieve the affinity in a cellular environment, such as serum protein binding and competition with ATP for ATP competitive inhibitors. In this study we evaluated the effect of different substrates as well as human serum protein (tested up to 40%) on the apparent affinity in the p38α enzyme assay and compared the results to those found in human whole blood and THP1 cellular assays measuring a reduction in IL1‐β and TNFα production, respectively, following stimulation with LPS. The enzyme assays were conducted with active p38α, ATP at Km and saturating concentrations, with MBP or MK2 as substrate, with and without human serum protein under initial velocity conditions. We observed that the differences in inhibition potency between the enzyme and cellular assays were, in general, accounted for by choice of substrate and serum protein. A shift in potency between the enzyme and cellular assays due to competition of the inhibitors with ATP was not observed. The KI value for the ATP competitive p38 inhibitors in the enzyme assays was similar to the IC50 value for inhibition of IL1‐β in human whole blood.