Development of a p38 kinase binding assay for high throughput screening

p38 is a member of the mitogen-activated protein kinase (MAPK) family of se rine/threonine kinases, which is activated by cellular stressors and has be en shown to be a critical enzyme in the synthesis and action of proinflamma tory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 b eta (IL-1 beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to t he ATP pocket of the enzyme. These compounds inhibit the p38 kinase activit y, block TNF-alpha and IL-1 beta secretion both in vivo and in vitro and ar e found to be effective in animal models of arthritis, bone resorption, and endotoxin shock. We postulated that other classes of compounds capable of competing the binding of pyridinyl imidazole with p38 enzyme could have eff icacy in the treatment of inflammatory diseases. Therefore, a simple and ro bust assay was developed to measure the ability of small molecules to inhib it the binding of tritium-labeled pyridinyl imidazole, SB202190, to recombi nant p38 kinase, For assay development, the human p38 gene was cloned in ba culovirus and then expressed in insect cells. Tritiated SB202190 was synthe sized and used as the p38 ligand for a competitive filter binding assay. Th is assay has been used successfully to screen both synthetic and combinator ial chemical libraries for other classes of p38 kinase inhibitors.