1. Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher, T. F.; Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.; Heys, J. R.; Landvatter, S. W.; Stricker, J. E.; McLaughlin, M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J. R.; Adams, J. L.; Yound, P. R. Nature 1994, 372, 739. Han, J.; Lee, J.-D.; Bibbs, L.; Ulevitch, R. J. Science 1994, 265, 808.
2. Gallagher, T. F.; Fier-Thompson, S. M.; Garigipati, R. S.; Sorenson, M. E.; Smietana, J. M.; Lee, D.; Bender, P. E.; Lee, J. C.; Laydon, J. T.; Chabot-Fletcher, M. C.; Breton, J. J.; Adams, J. L. Bioorg. Med. Chem. Lett. 1995, 5, 1171. Boehm, J. C.; Smietana, J. M.; Sorenson, M. E.; Gallagher, T. F.; Sheldrake, P. L.; Bradbeer, J.; Badger, A. M.; Laydon, J. T.; Lee, J. C.; Hillegrass, L. M.; Griswold, D. E.; Breton, J. J.; Chabot-Fletcher, M. C.; Adams, J. L. J. Med. Chem. 1996, 39, 3929.
3. The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase
4. Purified and His-tagged p38 α2 (expressed in E. coli) was activated in vitro by MMK-6 to a high specific activity. Using a microtiter format, all reactions were conducted in 100 μL volumes with reagents diluted to yield 0.05 μg/well of activated p38 α2 and 10 μg/well of myelin basic protein in assay buffer (25 mM HEPES 7.4, 20 mM MgCl2, 150 mM NaCl). Test compounds (5 μL of a 10% DMSO solution in water) were prepared and diluted into the assay to cover a final concentration range from 5 nM to 2.5 μM. The kinase assay was initiated by addition of 25 μL of an ATP cocktail to give a final concentration of 10 μM cold ATP and 0.2 μCi [γ-33P] ATP per well (200–400 dpm/pmol of ATP). The plate was incubated at 32°C for 35 min and the reaction quenched with 7 μL of a 1 N aq HCl solution. The samples were harvested onto a P30 Filtermat (Wallac, Inc.) using a TomTec 1295 Harvester (Wallac, Inc.), and counted in a LKB 1205 Betaplate Liquid Scintillation Counter (Wallac, Inc.). Negative controls included substrate plus ATP alone. SmithKline Beecham's SB203580 (1) was used as a standard (IC50=20 nM). At least two independent IC50 determinations were performed on each compound, and the mean value is reported. In all cases, standard deviations were less than 50% of the mean IC50 value.