John C. Lee

p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases

The p38 MAP kinases are a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals. Since their identification about 10 years ago, much has been learned of the activation and regulation of the p38 MAP kinase pathways. Inhibitors of two members of the p38 family have been shown to have anti-inflammatory effects in preclinical disease models, primarily through the inhibition of the expression of inflammatory mediators. Several promising compounds have also progressed to clinical trials. In this review, we provide an overview of the role of p38 MAP kinases in stress-activated pathways and the progress towards clinical development of p38 MAP kinase inhibitors in the treatment of inflammatory diseases.

Restraint of Proinflammatory Cytokine Biosynthesis by Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages

Exposure of macrophages to LPS elicits the production of proinflammatory cytokines, such as TNF-α, through complex signaling mechanisms. Mitogen-activated protein (MAP) kinases play a critical role in this process. In the present study, we have addressed the role of MAP kinase phosphatase-1 (MKP-1) in regulating proinflammatory cytokine production using RAW264.7 macrophages. Analysis of MAP kinase activity revealed a transient activation of c-Jun N-terminal kinase (JNK) and p38 after LPS stimulation. Interestingly, MKP-1 was induced concurrently with the inactivation of JNK and p38, whereas blocking MKP-1 induction by triptolide prevented this inactivation. Ectopic expression of MKP-1 accelerated JNK and p38 inactivation and substantially inhibited the production of TNF-α and IL-6. Induction of MKP-1 by LPS was found to be extracellular signal-regulated kinase dependent and involved enhanced gene expression and increased protein stability. Finally, MKP-1 expression was also induced by glucocorticoids as well as cholera toxin B subunit, an agent capable of preventing autoimmune diseases in animal models. These findings highlight MKP-1 as a critical negative regulator of the macrophage inflammatory response, underscoring its premise as a potential target for developing novel anti-inflammatory drugs.

IL‐1‐ and TNF‐induced bone resorption is mediated by p38 mitogen activated protein kinase

We have previously shown that p38 mitogen‐activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin‐1 (IL‐1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL‐1 and TNF, but not parathyroid hormone, transforming growth factor‐β (TGF‐β), 1,25(OH)2D3, insulin‐like growth factor‐1 (IGF‐1), or IGF‐II. Similar results were obtained using primary bovine chondrocytes and an SV40‐immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL‐1 and TNF‐induced p38 MAPK activity and IL‐6 production (IC50s 0.3–0.5 μM) in osteoblasts and chondrocytes. In addition, IL‐1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL‐1‐ and TNF‐stimulated bone resorption in vitro in a dose‐dependent manner (IC50s 0.3–1 μM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL‐1 and TNF‐stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).

Pyrimidinylimidazole inhibitors of p38: cyclic N-1 imidazole substituents enhance p38 kinase inhibition and oral activity.

Optimization of a series of N-1-cycloalkyl-4-aryl-5-(pyrimidin-4-yl)imidazole inhibitors of p38 kinase is reported. Oral administration of inhibitors possessing a cyclohexan-4-ol or piperidin-4-yl group at N-1 in combination with alkoxy, amino(alkyl), phenoxy and anilino substitution at the 2-position of the pyrimidine was found to potently inhibit LPS-induced TNF in mice and rats. The selectivity of these new inhibitors for p38 kinase versus eight other protein kinases is high and in all cases exceeds that of SB 203580.

N-Phenyl-N-purin-6-yl ureas: The design and synthesis of p38α MAP kinase inhibitors

The design, synthesis and SAR of a series of 2,6,9-trisubstituted purine inhibitors of p38alpha kinase is reported. Synthetic routes were devised to allow for array synthesis in which all three points of diversity could be facilely explored. The binding of this novel series to p38alpha kinase, which was predicted to have several key interactions in common with SB-203580, was confirmed by X-ray crystallography of 19 (p38 IC(50)=82 nM).

Isolation and characterization of a unique C57BL B-tropic virus

Abstract A unique B-tropic virus (CS43) has been isolated from a C57BL mouse. This virus differs from those we have previously characterized and from several isolates we have obtained from normal and leukemic C57BL mice. Unlike other C57BL B-tropic viruses, CS43 does not have a p12 polypeptide homologous to that of the class II xenotropic virus, but does have a p12 homologous to that of AKR MuLV (class I). In common with the other C57BL B-tropic isolates, CS43 has an altered major core protein (p30), which competes only weakly in a class-specific assay that distinguishes ecotropic MuLV p30s from those of xenotropic origin. The data are compatible with a unique recombinational event in the generation of this isolate, which should prove useful in studying the origin of B-tropic viruses and the properties that determine N/B tropism.

The immune response of (C57BL/6 X C3H)F1 mice to the endogenous AKR-MuLV.

Murine leukemia viruses (MuLV) are endogenous, genetically transmitted and associated with spontaneous leukemias in strains such as AKR [14,15]. In AKR mice, virus expression begins shortly after birth and viremia persists until death. In addition to AKR mice, most inbred strains of mice harbor a genetically transmitted AKR-type MuLV but in contrast, virus expression does not result in either viremia or leukemia. Although it was initially postulated that mice would be tolerant to an endogenous virus, recent experiments have demonstrated an immune response in most strains of mice harboring the virus [12]. In this report we characterize the humoral and cellular immune response of one strain of mice to the endogenous AKR-MuLV.

PROGRESS IN ACHIEVING PROOF OF CONCEPT FOR p38 KINASE INHIBITORS

The year 1994 marked the discovery by three independent research groups of a novel mammalian stress-induced kinase (variously named as p38, RK and CSBP), which was subsequently elucidated to be central to a signaling pathway parallel to—but distinct from—the classical growth factor–induced MAP kinase cascade. This discovery, along with the identification of the JNK family of kinases, paved the way for intense research in the molecular and cellular aspects of these important signaling events in health and disease. In the ensuing years, an impressive list of discoveries was made, ranging from the identification of p38 homologues to a detailed understanding of the complex cell-signaling pathways and the biological significance of p38. These findings were facilitated by a pivotal study linking inflammatory cytokine biosynthesis to the p38 pathways using p38 inhibitors as chemical tools. Advances in our understanding of the kinome and other technology platforms have further facilitated the rapid progress in our understanding of p38 MAP kinases as drug targets. This chapter will review important achievements towards the development of p38 kinase inhibitors to treat disease.