1. Biphenyl amide p38 kinase inhibitors 1: Discovery and binding mode
2. Fluorescence polarisation assays used GST-tagged p38α (activated using MKK6 and re-purified) or GST-tagged truncated JNK3 (residues 39–402) and an ATP-competitive rhodamine-green labelled fluoroligand (2-(6-amino-3-imino-3H-xanthen-9-yl)-5-{[({4-[4-(4-Cl-3-hydroxyphenyl)-5-(4-pyridinyl)-1H-imidazol-2yl]phenyl}methyl)amino]carbonyl}benzoic acid). These components were dissolved in a buffer of final composition 62.5mM Hepes, pH 7.5, 1.25mM CHAPS, 1mM DTT, 12.5mM MgCl2, with final concentrations of 12nM of p38α or 50nM JNK3 and 5nM fluoroligand. Thirty microliters of this mixture were added to wells containing 1μl of test compound (0.28nM–16.6μM) and incubated for 30–60min at room temperature. Fluorescence anisotropy was read in a Molecular Devices Acquest (excitation 485nm/emission 535nm).
3. The Cheng–Prusoff equation (Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099–3108), Ki=IC50*(1+([S]/Km)), was used to calculate Ki from determined IC50 for activity assays. To calculate Ki from determined IC50 for fluorescence polarisation assays a modification of the Cheng–Prusoff equation was used (Cheng, H. C. Pharmacol. Res. 2005, 50, 21–40). Ki=IC50*(1+((n[E])/Kd)) where n is the fraction of enzyme competent to bind fluoroligand. Ki values for 381 compounds in the fluorescence polarisation assay correlate with those from an activity assay (details to be published) with r2=0.9.