Jeffrey Charles Boehm

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.

Structural basis of inhibitor selectivity in MAP kinases.

BACKGROUND The mitogen-activated protein (MAP) kinases are important signaling molecules that participate in diverse cellular events and are potential targets for intervention in inflammation, cancer, and other diseases. The MAP kinase p38 is responsive to environmental stresses and is involved in the production of cytokines during inflammation. In contrast, the activation of the MAP kinase ERK2 (extracellular-signal-regulated kinase 2) leads to cellular differentiation or proliferation. The anti-inflammatory agent pyridinylimidazole and its analogs (SB [SmithKline Beecham] compounds) are highly potent and selective inhibitors of p38, but not of the closely-related ERK2, or other serine/threonine kinases. Although these compounds are known to bind to the ATP-binding site, the origin of the inhibitory specificity toward p38 is not clear. RESULTS We report the structural basis for the exceptional selectivity of these SB compounds for p38 over ERK2, as determined by comparative crystallography. In addition, structural data on the origin of olomoucine (a better inhibitor of ERK2) selectivity are presented. The crystal structures of four SB compounds in complex with p38 and of one SB compound and olomoucine in complex with ERK2 are presented here. The SB inhibitors bind in an extended pocket in the active site and are complementary to the open domain structure of the low-activity form of p38. The relatively closed domain structure of ERK2 is able to accommodate the smaller olomoucine. CONCLUSIONS The unique kinase-inhibitor interactions observed in these complexes originate from amino-acid replacements in the active site and replacements distant from the active site that affect the size of the domain interface. This structural information should facilitate the design of better MAP-kinase inhibitors for the treatment of inflammation and other diseases.

Disease-modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis

OBJECTIVE To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). METHODS Male Lewis rats with AIA were orally treated either prophylactically (days 0-20) or therapeutically (days 10-20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. RESULTS SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. CONCLUSION Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.

Regulation of stress-induced cytokine production by pyridinylimidazoles; inhibition of CSBP kinase

Members of three classes of pyridinylimidazoles bind with varying affinities to CSBP (p38) kinase which is a member of a stress-induced signal transduction pathway. Based upon SAR and protein homology modeling, the pharmacophore and three potential modes of binding to the enzyme are presented. For a subset of pyridinylimidazoles, binding is shown to correlate with inhibition of CSBP kinase activity, whereas no significant inhibition of PKA, PKC alpha and ERK kinase activity is observed.

Pyrimidinylimidazole inhibitors of CSBP/P38 kinase demonstrating decreased inhibition of hepatic cytochrome P450 enzymes

Pyrimidine analogs of the pyrimidinylimidazole class of CSBP/p38 kinase inhibitors were prepared in an effort to reduce the potent inhibition of hepatic cytochrome P450 observed for the pyridinyl compounds. The substitution of pyrimidin-4-yl, 2-methoxypyrimidin-4-yl, or 2-methylaminopyrimidin-4-yl for pyridin-4-yl effectively dissociates CSBP/p38 kinase from P450 inhibition for this series and furthermore achieves an increase in oral activity.

New inhibitors of p38 kinase

In the short time since the 1994 report that the molecular target for the 4-aryl-5-pyridin-4-yl imidazole class of cytokine suppressive anti-inflammatory agents (exemplified by SB-203580) was the human homologue of the stress-induced kinase p38, there has been an explosion of patent literature disclosing related inhibitor analogues. These compounds fall into the general structural class of vicinal aryl/pyridin-4-yl heterocycles. Compounds of this class have been claimed as p38 inhibitors or inhibitors of cytokine biosynthesis. Since cytokines mediate a variety of disease processes, inhibition of cytokine biosynthesis has potential as a therapeutic target. The SAR of binding to p38 for this intensively studied class of compounds is now understandable on the basis of recent x-ray crystallographic and mutagenesis studies. The inhibitors can be sub-classified based upon a variety of structural characteristics. Recently new inhibitor structural classes have been disclosed. Although distinctly different structurally, several of these new compounds appear to bind with the same key interactions as the vicinal aryl/pyridin-4-yl heterocycles.

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).

Phenoxypyrimidine inhibitors of p38α kinase: synthesis and statistical evaluation of the p38 inhibitory potencies of a series of 1-(piperidin-4-yl)-4-(4-fluorophenyl)-5-(2-phenoxypyrimidin-4-yl) imidazoles

As a continuation of our work with 1,4,5 substituted imidazole inhibitors of p38alpha, we report a series of 1-(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-phenoxy-4-pyrimidinyl) imidazoles related to 7. The compounds have IC50s for inhibition of p38alpha ranging from 6.0 to 650nM. Statistical analysis of the p38beta inhibitor potencies shows a correlation of IC50s with the electron donating strength of low molecular weight substituents.

Synthesis of C2-symmetric and pseudosymmetric HIV-1 protease inhibitors from D-arabitol

Facile stereocontrolled syntheses of the potent HIV-a protease inhibitors 1 and 2 are described, employing a unified synthetic route from carbohydrate precursors.