The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.

The crystal structure of human p38 mitogen-activated protein (MAP) kinase in complex with a potent and highly specific pyridinyl-imidazole inhibitor has been determined at 2.0 A resolution. The structure of the kinase, which is in its unphosphorylated state, is similar to that of the closely-related ERK2. The inhibitor molecule is bound in the ATP pocket. A hydrogen bond is made between the pyridyl nitrogen of the inhibitor and the main chain amido nitrogen of residue 109, analogous to the interaction from the N1 atom of ATP. The crystal structure provides possible explanations for the specificity of this class of inhibitors. Other protein kinase inhibitors may achieve their specificity through a similar mechanism. The structure also reveals a possible second binding site for this inhibitor, with currently unknown function.

We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.

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p38 MAPK and MAPK-activated protein kinase 2 (MK2) are key components of signaling pathways leading to many cellular responses, notably the proinflammatory cytokine production. The physical association of p38␣ isoform and MK2 is believed to be physiologically important for this signaling. We report the 2.7-Å resolution crystal structure of the unphosphorylated complex between p38␣ and MK2. These protein kinases bind ''head-to-head,'' present their respective active sites on approximately the same side of the heterodimer, and form extensive intermolecular interactions. Among these interactions, the MK2 Ile-366 -Ala-390, which includes the bipartite nuclear localization signal, binds to the p38␣-docking region. This binding supports the involvement of noncatalytic regions to the tight binding of the MK2:p38␣ binary assembly. The MK2 residues 345-365, containing the nuclear export signal, block access to the p38␣ active site. Some regulatory phosphorylation regions of both protein kinases engage in multiple interactions with one another in this complex. This structure gives new insights into the regulation of the protein kinases p38␣ and MK2, aids in the better understanding of their known cellular and biochemical studies, and provides a basis for understanding other regulatory protein-protein interactions involving signal transduction proteins.X-ray structure T he MAPK has been directly associated with proinflammatory cytokine production. Inhibition of p38␣ (and its homolog, p38␤) by inhibitors such as SB203580 and BIRB 796, reduces LPS-induced production of proinflammatory cytokines, including TNF␣ and IL-1, that are implicated in the etiology of chronic inflammation (1-5). Recent genetic evidence suggests that p38␣, not p38␤, is the major p38 isoform involved in the LPS-induced immune response (6). Embryonic stem cells derived from mice that lack the p38␣ function exhibit impaired IL-1 signaling (7). Biological therapies directed against inflammatory cytokines, e.g., anti-TNF␣ and anti-IL-1␤, have also proven effective at ameliorating the symptoms of inflammatory diseases such as arthritis and psoriasis (not anti-IL-1␤-mediated) (8-11).MAPK-activated protein kinase-2 (MK2), the primary substrate of p38␣, is also a key participant in modulating proinflammatory cytokine production (12, 13). MK2 knockout mice (MK2 Ϫ/Ϫ ) challenged with LPS produce significantly less TNF␣, IL-6, IFN␥, and IL-1 than LPS-stimulated wild-type mice (13). MK2Ϫ/Ϫ mice are also resistant to collagen-induced arthritis (14). The transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 protein expression and a subsequent, significant reduction in the production of TNF␣, . The catalytic activity of MK2 has further been shown to be necessary for TNF␣ production (16). Taken together, these data suggest that the inhibition of the p38␣ pathway, particularly MK2, is desirable for the potential treatment of chronic inflammation.In resting cells, p38␣ forms a physiological complex with MK2, w...