Molecular Basis for p38 Protein Kinase Inhibitor Specificity

Abstract

p38 is a member of the mitogen-activated protein (MAP) kinase family and is a critical enzyme in the proinflammatory cytokine pathway. Other MAP kinase group members that share both structural and functional homology to p38 include the c-Jun NH2-terminal kinases (JNKs or SAPKs) and the extracellular-regulated protein kinases (ERKs). In this study, we determined the molecular basis for p38alpha inhibitor specificity exhibited by five compounds in the diarylimidazole, triarylimidazole, and triarylpyrrole classes of protein kinase inhibitors. These compounds are significantly more potent inhibitors of p38 compared to the JNKs and ERKs. Three active site ATP-binding domain residues in p38, T106, M109, and A157, selected based on primary sequence alignment, molecular modeling, and X-ray crystal structure data, were mutated to assess their role in inhibitor binding and enzymatic catalysis. All mutants, with the exception of T106M, had kinase activity within 3-fold of wild-type p38. Mutation of T106 to glutamine, the residue present at the corresponding position in ERK-2, or methionine, the corresponding residue in p38gamma, p38delta, and the JNKs, rendered all five inhibitors ineffective. The diarylimidazoles had approximately a 6-fold decrease in potency toward M109A p38. For the mutant A157V, all diarylimidazoles and triarylimidazoles tested were 5-10-fold more potent compared with wild-type p38. In contrast, two triarylpyrroles were 15-40-fold less potent versus A157V p38. These results showed that the molecular basis for the specificity of the p38 inhibitors was attributed largely to threonine 106 in p38 and that methionine 109 contributes to increased binding affinity for imidazole based inhibitors.

Purification and Molecular Characterization of cGMP-dependent Protein Kinase from Apicomplexan Parasites

“…1). Compound 1 is structurally similar to known protein kinase inhibitors (29,30,39,40) and we show here that this compound does inhibit a kinase in the parasite, namely PKG. Like other protein kinase inhibitors, Compound 1 is competitive with respect to ATP.…”

“…Compound 1-binding Protein Is a cGMP-dependent Protein Kinase-The structural similarity between Compound 1 and protein kinase inhibitors that have been described in the literature (29,30) prompted us to assay for and detect kinase activity (using the peptide substrate Kemptide) in the ligand binding fractions during the purification protocol. However, protein kinase activity was marginal (data not shown), even in the final fractions from the MonoQ column pictured in Fig.…”

Chemical Genetics Define the Roles of p38α and p38β in Acute and Chronic Inflammation

“…Previous in vitro studies demonstrated that replacement of Thr 106 with Met in p38␣ had no measurable affect on enzymatic activity but decreased the affinity of certain p38 inhibitors by several orders of magnitude (25)(26)(27)(28). We generated in vivo models to study the specific roles of these p38 isoforms by creating genetically engineered mice expressing a Thr 106 to Met substitution in either p38␣ or p38␤.…”

Urokinase Plasminogen Activator/Urokinase-specific Surface Receptor Expression and Matrix Invasion by Breast Cancer Cells Requires Constitutive p38α Mitogen-activated Protein Kinase Activity

“…SB203580, a member of this group, is a highly selective inhibitor of p38 MAPK, which does not alter the activity of other MAPKs including ERKs and JNKs (41)(42)(43). We thus used this inhibitor to investigate the role of p38 MAPK activity in invasion of BT549 breast cancer cells.…”