BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo

The compound BIRB796 inhibits the stress-activated protein kinases p38alpha and p38beta and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38gamma. This occurs at higher concentrations of BIRB796 than those that inhibit p38alpha and p38beta and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38gamma. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38alpha and p38beta, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38gamma as well as those of p38alpha and p38beta.     

p38 Regulates Pigmentation via Proteasomal Degradation of Tyrosinase

The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by α-melanocyte-stimulating hormone (α-MSH) and ultraviolet irradiation. Previous studies have shown that α-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and α-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA.     

Synthesis and SAR of p38alpha MAP kinase inhibitors based on heterobicyclic scaffolds

The synthesis and structure-activity relationships (SAR) of p38alpha MAP kinase inhibitors based on heterobicyclic scaffolds are described. This effort led to the identification of compound (21) as a potent inhibitor of p38alpha MAP kinase with good cellular potency toward the inhibition of TNF-alpha production. X-ray co-crystallography of an oxalamide analog (24) bound to unphosphorylated p38alpha is also disclosed.     

p38 MAP kinase inhibitors. Part 5: discovery of an orally bio-available and highly efficacious compound based on the 7-amino-naphthyridone scaffold

A new sub-class of p38 inhibitors represented by 7-amino-naphthyridone have been discovered. Benchmark compound 16 potently inhibited p38 in vitro, was functionally active, and displayed excellent pharmacokinetic profiles in two animal species. Compound 16 reduced inflammation in animal disease models at EC(50) doses as low as 0.2mpk.     

Discovery of a novel class of non-ATP site DFG-out state p38 inhibitors utilizing computationally assisted virtual fragment-based drug design (vFBDD)

Discovery of a new class of DFG-out p38α kinase inhibitors with no hinge interaction is described. A computationally assisted, virtual fragment-based drug design (vFBDD) platform was utilized to identify novel non-aromatic fragments which make productive hydrogen bond interactions with Arg 70 on the αC-helix. Molecules incorporating these fragments were found to be potent inhibitors of p38 kinase. X-ray co-crystal structures confirmed the predicted binding modes. A lead compound was identified as a potent (p38α IC(50)=22 nM) and highly selective (≥ 150-fold against 150 kinase panel) DFG-out p38 kinase inhibitor.     

Synthesis,antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-hydrazone derivatives

In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth inhibition and apoptosis induction characteristics are being strongly screened for their cancer chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to evaluate for their anti-proliferative and apoptosis induction potential against cervical (HeLa), breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293 cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further, mechanistic studies were conducted on compound 14a to understand the biochemical mechanisms and functional interactions with various signaling pathways triggered in HeLa and MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen species. In downstream signaling pathway, activated p53 protein interacted with MAPK pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38 on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125) prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing and were dependent on p53 activity. Based on the results, compound 14a was identified as a promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo experiments on anti-proliferative activity.     

Biphenyl amide p38 kinase inhibitors 2: Optimisation and SAR

The biphenyl amides are a novel series of p38 MAP kinase inhibitors. Structure-activity relationships of the series against p38alpha are discussed with reference to the X-ray crystal structure of an example. The series was optimised rapidly to a compound showing oral activity in an in vivo disease model.     

Dual effects of p38 MAPK on TNF-dependent bronchoconstriction and TNF-independent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome

The administration of endotoxins from Gram-negative bacteria induces manifestations reminding of acute respiratory distress syndrome. p38 MAPKs have been implicated in this pathology. In this study, we show that the specific p38 alpha,beta MAPK inhibitor, compound 37, prevents LPS-induced bronchoconstriction and neutrophil recruitment into the lungs and bronchoalveolar space in a dose-dependent manner in C57BL/6 mice. Furthermore, TNF induction and TNF signals were blocked. In TNF-deficient mice, bronchoconstriction, but not neutrophil sequestration, in the lung was abrogated after LPS administration. Therefore, TNF inhibition does not explain all of the effects of the p38 MAPK inhibitor. The p38 alpha,beta MAPK inhibitor also prevented LPS-induced neutrophilia in TNF-deficient mice. In conclusion, LPS provokes acute bronchoconstriction that is TNF dependent and p38 MAPK mediated, whereas the neutrophil recruitment is independent of TNF but depends on LPS/TLR4-induced signals mediated by p38 MAPK.     

Synthesis and biological evaluation of p38α kinase-targeting dialkynylimidazoles

Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical intermediates, a series of 1,2-dialkynylimidazoles were designed as potential irreversible p38 MAP kinase α-isoform (p38α) inhibitors. The synthesis of these dialkynylimidazoles and their kinase inhibition activity is reported. The 1-ethynyl-substituted dialkynylimidazole 14 is a potent (IC₅₀ = 200 nM) and selective inhibitor of p38α. Moreover, compound 14 covalently modifies p38α as determined by ESI-MS after 12 h incubation at 37 °C. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450 2D6 inhibition by compound 14 demonstrate that dialkynylimidazoles are a new, promising class of p38α inhibitors.     

Novel triazolopyridylbenzamides as potent and selective p38α inhibitors

A new class of p38α inhibitors based on a biaryl-triazolopyridine scaffold was investigated. X-ray crystallographic data of the initial lead compound cocrystallised with p38α was crucial in order to uncover a unique binding mode of the inhibitor to the hinge region via a pair of water molecules. Synthesis and SAR was directed towards the improvement of binding affinity, as well as ADME properties for this new class of p38α inhibitors and ultimately afforded compounds showing good in vivo efficacy.     

Identification of a novel inhibitor of LPS-induced TNF-alpha production with antiproliferative activity in monocyte/macrophages

An isoquinoline derivative, 5-methyl-7,8-dimethoxy-1-phenylpyrazolo[5,4-c]isoquinoline (compound 1), was identified as a novel inhibitor of LPS-induced TNF-alpha production by cell-based screening. Compound 1 suppressed LPS-induced TNF-alpha production in RAW264.7 cells and murine peritoneal macrophages in a dose-dependent manner similar to SB203580, known as a specific inhibitor of p38 MAPK. It also inhibited an LPS-induced increase in serum TNF-alpha in a mouse endotoxic shock model with an ED(50) of approximately 10 mg/kg. Compound 1 had little effect on the incorporation of [3H]-leucine into the cells, while it suppressed LPS-induced TNF-alpha mRNA levels in RAW264.7 cells. The results indicate that suppression of TNF-alpha production was not a result of nonspecific inhibition of de novo translation but was based on the decreased TNF-alpha mRNA levels. The in vitro kinase assay revealed that compound 1 did not strongly inhibit p38 MAPK activity, its potency being much lower than that of SB203580, suggesting that the TNF-alpha-suppressive action of compound 1 cannot be attributed to the inhibition of p38 MAPK. Furthermore, in contrast to SB203580, it significantly inhibited the growth of RAW264.7 and THP-1 cells in a cytostatic manner. Compound 1 is likely to have antiinflammatory and antiproliferative effects by acting on some molecule other than p38 MAPK that contributes to both LPS-induced TNF-alpha production and the cell growth of monocyte/macrophages.     

New derivative of 2-(2,4-dihydroxyphenyl)thieno-1,3-thiazin-4-one (BChTT) elicits antiproliferative effect via p38-mediated cell cycle arrest in cancer cells

2-(2,4-Dihydroxyphenyl)thieno-1,3-thiazin-4-ones are a group of new compounds with potential anticancer activity. This type of derivatives was poorly investigated in the area of synthesis and biological activities. In the present study the antiproliferative action of the most active derivative BChTT was described. The aim of biological evaluation was to investigate the ability of the compound to inhibit cancer cell proliferation and identify mechanism involved in its action on the molecular level. BChTT inhibited the proliferation of lung cancer A549, colon cancer HT-29 and glioma C6 cells in the concentration-dependent manner. It was not toxic to normal cells including skin fibroblasts, hepatocytes and oligodendrocytes in the antiproliferative concentrations. BChTT decreased the DNA synthesis in the treated cancer cells and induced cell cycle arrest in the G0/G1 phase. Moreover, the ability of the compound to activate p38 kinase and decrease cyclin D1 expression was estimated. Participation of p38 kinase in the antiproliferative action of the compound was confirmed by the analysis of BChTT activity in the cells with the p38 silenced gene. The obtained results may suggest the ability of the tested derivative to inhibit cancer cells proliferation by induction of p38-mediated cyclin D1 downregulation.     

Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite.

It is well-known that p38 mitogen-activated protein kinase (p38MAPK) participates in cellular responses to mitogenic stimuli, environmental and genotoxic stresses, and apoptotic agents. Although there are several reports on p38MAPK in relation to cell growth and apoptosis, the exact mechanism of p38MAPK-mediated cell growth regulation remains obscure. Here, we examined possible roles of p38MAPK in the sodium arsenite-induced cell growth inhibition in NIH3T3 cells. Sodium arsenite induced transient cell growth delay with marked activation of p38MAPK. In addition, arsenite induced CDK inhibitor p21(CIP1/WAF1) and enhanced its binding to the CDK2, which resulted in inhibition of CDK2 activity. The levels of cyclin D1 expression and the CDK4 kinase activity were also significantly reduced. pRB was hypophosphorylated by sodium arsenite. SB203580, a specific inhibitor of p38MAPK, blocked arsenite-induced growth inhibition as well as the arsenite-induced p21(CIP1/WAF1) expression. Expression of dominant negative p38MAPK also blocked arsenite-induced p21(CIP1/WAF1) expression. Inhibited-CDK2 activity was also completely reversed by SB203580 or expression of dominant negative p38MAPK, while the decreased-cyclin D1 protein by the compound was not restored. These data demonstrate a possible link between the activation of p38MAPK and induction of p21(CIP1/WAF1), suggesting that the activation of p38MAPK is, at least in part, related to the cell growth inhibition by sodium arsenite.     

Pyrazolo-pyrimidines: a novel heterocyclic scaffold for potent and selective p38 alpha inhibitors

The synthesis and structure-activity relationships (SAR) of p38 alpha MAP kinase inhibitors based on a pyrazolo-pyrimidine scaffold are described. These studies led to the identification of compound 2x as a potent and selective inhibitor of p38 alpha MAP kinase with excellent cellular potency toward the inhibition of TNFalpha production. Compound 2x was highly efficacious in vivo in inhibiting TNFalpha production in an acute murine model of TNFalpha production. X-ray co-crystallography of a pyrazolo-pyrimidine analog 2b bound to unphosphorylated p38 alpha is also disclosed.     

5-amino-pyrazoles as potent and selective p38α inhibitors

The synthesis and structure-activity relationships (SAR) of p38α MAP kinase inhibitors based on a 5-amino-pyrazole scaffold are described. These studies led to the identification of compound 2j as a potent and selective inhibitor of p38α MAP kinase with excellent cellular potency toward the inhibition of TNFα production. Compound 2j was highly efficacious in vivo in inhibiting TNFα production in an acute murine model of TNFα production. X-ray co-crystallography of a 5-amino-pyrazole analog 2f bound to unphosphorylated p38α is also disclosed.     

Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells

The formation of glucose-derived methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. We examined whether MG was capable of inducing apoptosis in Schwann cells (SCs), since recent studies have suggested a potential involvement of apoptotic cell death in the development of diabetic neuropathy. MG induced apoptosis in SCs in a dose-dependent manner, accompanied by a reduction of intracellular glutathione content and activation of the p38 MAPK. Inhibiting the p38 MAPK activation by SB203580 successfully suppressed the MG-induced apoptosis in SCs. Aminoguanidine and N-acetyl-l-cysteine also inhibited the MG-induced p38 MAPK activation and apoptosis along with restoration of the intracellular glutathione content. These results suggest a potential role for MG in SC injury through oxidative stress-mediated p38 MAPK activation under diabetic conditions, and it may serve as a novel insight into therapeutic strategies for diabetic neuropathy.     

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.     

Activation of p90RSK and cAMP response element binding protein in stimulated neutrophils: novel effects of the pyridinyl imidazole SB 203580 on activation of the extracellular signal-regulated kinase cascade.

Neutrophils stimulated with the chemoattractant FMLP or the phorbol ester PMA are known to exhibit activation of a 90-kDa renaturable protein kinase. Activation of this kinase was maximal at approximately 1-3 min after cell stimulation and the time course for activation was similar to that of the extracellular-regulated kinases (ERKs) and p38-mitogen activated protein kinase (p38MAPK). Compounds that block activation of ERK-1/2 (PD 98059) or that inhibit the activity of p38MAPK (SB 203580) blocked activation of this 90-kDa kinase. SB 203580 is a highly selective inhibitor of p38MAPK in vitro and is under intense study as a lead compound for developing novel anti-inflammatory agents. However, we demonstrate that SB 203580 at concentrations >/=10 microM can also inhibit activation of ERK-1/2 in neutrophils. An Ab to the protein kinase p90RSK2 (also referred to as MAPKAP-K1b, or p90rsk) immunoprecipitated the active 90-kDa kinase from lysates of stimulated neutrophils. No activity was observed for this enzyme in immunoprecipitates obtained from unstimulated cells, and the amounts of activity were markedly reduced if the cells were treated with PD 98059 or SB 203580 before stimulation. Neutrophils stimulated with FMLP exhibited phosphorylation of the cAMP response element binding protein (CREB), and this reaction was inhibited by SB 203580 and PD 98059. These data establish that the renaturable 90-kDa protein kinase is p90RSK2 and that CREB may be a substrate for this enzyme in these cells. Novel effects of compound SB 203580 on stimulated neutrophils are also described.