Molecular basis for p38 protein kinase inhibitor specificity

p38 is a member of the mitogen-activated protein (MAP) kinase family and is a critical enzyme in the proinflammatory cytokine pathway. Other MAP kinas e group members that share both structural and functional homology to p38 i nclude the c-Jun NH2-terminal kinases (JNKs or SAPKs) and the extracellular -regulated protein kinases (ERKs). In this study, we determined the molecul ar basis for p38 alpha inhibitor specificity exhibited by five compounds in the diarylimidazole, triarylimidazole, and triarylpyrrole classes of prote in kinase inhibitors. These compounds are significantly more potent inhibit ors of p38 compared to the JNKs and ERKs. Three active site ATP-binding dom ain residues in p38; T106, M109, and A157, selected based on primary sequen ce alignment, molecular modeling, and X-ray crystal structure data, were mu tated to assess their role in inhibitor binding and enzymatic catalysis. Al l mutants, with the exception of T106M, had kinase activity within 3-fold o f wild-type p38. Mutation of T106 to glutamine. the residue present at the corresponding position in ERK-2, or methionine, the corresponding residue i n p38 gamma, p38 delta, and the JNKs, rendered all five inhibitors ineffect ive. The diarylimidazoles had approximately a 6-fold decrease in potency to ward M109A p38. For the mutant A157V, all diarylimidazoles and triarylimida zoles tested were 5-10-fold more potent compared with wild-type p38. In con trast, two triarylpyrroles were 15-40-fold less potent versus A157V p38. Th ese results showed that the molecular basis for the specificity of the p38 inhibitors was attributed largely to threonine 106 in p38 and that methioni ne 109 contributes to increased binding affinity for imidazole based inhibi tors.