P38α MAP Kinase Research Articles

Quantification of p38αMAP kinase: A prognostic marker in HNSCC with respect to radiation therapy

BackgroundTranslation of early findings from basic research is aimed to benefit cancer therapeutics. We report the p38α level in serum of head and neck squamous cell carcinoma (HNSCC) patients indicating it as a prognostic marker and established its correlation with radiation therapy (RT). MethodsThe case-controlled study was performed on 120 HNSCC patients from whom 81 patients and 45 controls were statistically analyzed. The p38α estimation was done at pre-RT, during-RT and post-RT using a real time Surface Plasmon Resonance (SPR) technology, ELISA and western blot. ResultsHNSCC patients showed threefold increase in p38α level when compared to control (p value<0.0001). The estimated concentration of p38α in a temporal manner, before-RT, during-RT and post-RT was 0.61ng/μl (95%CI: 0.53–0.69), 0.35ng/μl (95%CI: 0.31–0.38) and 0.30ng/μl (95%CI: 0.26–0.33), respectively. Among the 81 cases, 70 patients (86.42%) showed a declined p38α in response to RT as evaluated by SPR and were responding clinically (clinical tumor regression). ConclusionsThis study showed elevated p38α level at cancer diagnosis and a statistically significant decline during-RT and post-RT periods. Hence, it can emerge as a prognostic marker supporting the candidature of p38α as a suitable serum marker in HNSCC.

P38α MAP Kinase Dimers with Swapped Activation Segments and a Novel Catalytic Loop Conformation

Many protein kinase functions, including autophosphorylation in trans, require dimerization, possibly by activation segment exchange. Such dimers have been reported for a few autophosphorylating protein kinases, but not for mitogen-activated protein kinases (MAPKs). Activation of MAPKs proceeds not only via the well-characterized action of dual T/Y specificity MAPK kinases, phosphorylating both residues of the MAPK TxY activation loop motif, but also via a noncanonical activation pathway triggered by phosphorylation at Tyr323 and homodimerization. Here, we report the 2. 7-Å-resolution structure of p38α MAPK from Salmo salar in a novel domain-swapped homodimeric form. The tyrosines of the conserved sequence YxAPE anchor the swapped activation segments in a configuration suitable for autophosphorylation in trans and provide a model for the noncanonical pathway. In the dimer, a structural unit containing Tyr323 is formed at a dimerization contact region that stabilizes the HRD catalytic loop in a unique inactive geometry. This feature is consistent with the requirement of Tyr323 phosphorylation for the initiation of the noncanonical pathway. Despite the interacting surface of more than 2600 Å 2, the dimer is not obligate, as gel filtration shows the dimerization to occur only at relatively high concentrations. The transition from monomer to dimer involves a relatively simple hinged displacement of helix EF and adjacent residues. Thus, dimer formation is likely to be transient, compatible with functional requirements for autophosphorylation, allowing further modulation, for example, by scaffolding mechanisms.

Discovery of pyrrolo[2,1-f][1,2,4]triazine C6-ketones as potent, orally active p38α MAP kinase inhibitors

Pyrrolo[2,1-f][1,2,4]triazine based inhibitors of p38α have been prepared exploring functional group modifications at the C6 position. Incorporation of aryl and heteroaryl ketones at this position led to potent inhibitors with efficacy in in vivo models of acute and chronic inflammation.

ChemInform Abstract: 5‐Amino‐pyrazoles as Potent and Selective p38α Inhibitors.

AbstractExtensive SAR studies on p38α MAP kinase inhibitors based on a 5‐aminopyrazole scaffold result in the identification of compound (I) as one of the most potent p38α inhibitor with single digit nanomolar potency for the inhibition of TNF‐α release in human peripheral blood mononuclear cells.

Alternate Modes of Cognate RNA Recognition by Human PUMILIO Proteins

Human PUMILIO1 (PUM1) and PUMILIO2 (PUM2) are members of the PUMILIO/FBF (PUF) family that regulate specific target mRNAs posttranscriptionally. Recent studies have identified mRNA targets associated with human PUM1 and PUM2. Here, we explore the structural basis of natural target RNA recognition by human PUF proteins through crystal structures of the RNA-binding domains of PUM1 and PUM2 in complex with four cognate RNA sequences, including sequences from p38α and erk2 MAP kinase mRNAs. We observe three distinct modes of RNA binding around the fifth RNA base, two of which are different from the prototypical 1 repeat:1 RNA base binding mode previously identified with model RNA sequences. RNA-binding affinities of PUM1 and PUM2 are not affected dramatically by the different binding modes in vitro. However, these modes of binding create structurally variable recognition surfaces that suggest a mechanism in vivo for recruitment of downstream effector proteins defined by the PUF:RNA complex.

Prioritization of charge over geometry in transition state analogues of a dual specificity protein kinase.

The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.

Conformational effects on potency of thioimidazoles and dihydrothiazolines

We investigated the effect of steric restriction and hindrance on inhibitors directed against the biological activity of p38α MAP kinase. The structural reduction of the early lead compound SKF86002 into three structural classes of optimized SKF86002-like inhibitors produced a strong effect on inhibitory potency. This effect was investigated by conformational analysis and docking experiments.

Stress induced gene expression: a direct role for MAPKAP kinases in transcriptional activation of immediate early genes

Immediate early gene (IEG) expression is coordinated by multiple MAP kinase signaling pathways in a signal specific manner. Stress-activated p38α MAP kinase is implicated in transcriptional regulation of IEGs via MSK-mediated CREB phosphorylation. The protein kinases downstream to p38, MAPKAP kinase (MK) 2 and MK3 have been identified to regulate gene expression at the posttranscriptional levels of mRNA stability and translation. Here, we analyzed stress-induced IEG expression in MK2/3-deficient cells. Ablation of MKs causes a decrease of p38α level and p38-dependent IEG expression. Unexpectedly, restoration of p38α does not rescue the full-range IEG response. Instead, the catalytic activity of MKs is necessary for the major transcriptional activation of IEGs. By transcriptomics, we identified MK2-regulated genes and recognized the serum response element (SRE) as a common promoter element. We show that stress-induced phosphorylation of serum response factor (SRF) at serine residue 103 is significantly reduced and that induction of SRE-dependent reporter activity is impaired and can only be rescued by catalytically active MK2 in MK2/3-deficient cells. Hence, a new function of MKs in transcriptional activation of IEGs via the p38α-MK2/3-SRF-axis is proposed which probably cooperates with MKs’ role in posttranscriptional gene expression in inflammation and stress response.

Effects of Water Placement on Predictions of Binding Affinities for p38α MAP Kinase Inhibitors

Monte Carlo free energy perturbation (MC/FEP) calculations have been applied to compute the relative binding affinities of 17 congeneric pyridazo-pyrimidinone inhibitors of the protein p38α MAP kinase. Overall correlation with experiment was found to be modest when the complexes were hydrated using a traditional procedure with a stored solvent box. Significant improvements in accuracy were obtained when the MC/FEP calculations were repeated using initial solvent distributions optimized by the water placement algorithm JAWS. The results underscore the importance of accurate placement of water molecules in a ligand binding site for the reliable prediction of relative free energies of binding.

Synthesis and biological activity of pyridopyridazin-6-one p38 MAP kinase inhibitors. Part 1

The development and synthesis of potent p38α MAP kinase inhibitors containing a pyridazinone platform is described. Evolution of the p38α selective pyridopyridazin-6-one series from the p38α/β dual inhibitor 2H-quinolizin-2-one series will be discussed in full detail.

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.

Tri- and tetrasubstituted imidazoles as p38α mitogen-activated protein kinase inhibitors

The synthesis of 2,4,5-trisubstituted and 1,2,4,5-tetrasubstituted imidazoles as potent p38α mitogen-activated protein kinase inhibitors is described. The trisubstituted imidazole series was found to be more potent than the tetrasubstituted imidazole series. Many of these compounds show low-nanomolar activities in the isolated p38α MAP kinase inhibition assay. The structure–activity relationships between these two series are different and not comparable.

Discovery of 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a Clinical p38α MAP Kinase Inhibitor for the Treatment of Inflammatory Diseases

The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp(2) character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38α enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38α was confirmed by X-ray crystallographic analysis.

Utilization of a nitrogen–sulfur nonbonding interaction in the design of new 2-aminothiazol-5-yl-pyrimidines as p38α MAP kinase inhibitors

The design, synthesis, and structure–activity relationships (SAR) of a series of 2-aminothiazol-5-yl-pyrimidines as novel p38α MAP kinase inhibitors are described. These efforts led to the identification of 41 as a potent p38α inhibitor that utilizes a unique nitrogen–sulfur intramolecular nonbonding interaction to stabilize the conformation required for binding to the p38α active site. X-ray crystallographic studies that confirm the proposed binding mode of this class of inhibitors in p38α and provide evidence for the proposed intramolecular nitrogen–sulfur interaction are discussed.

Glucagon-Like Peptide-1 Increases Myocardial Glucose Uptake via p38α MAP Kinase–Mediated, Nitric Oxide–Dependent Mechanisms in Conscious Dogs With Dilated Cardiomyopathy

Background— We have shown that glucagon-like peptide-1 (GLP-1[7–36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1–induced myocardial glucose uptake are unknown. Methods and Results— Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7–36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20±7 nmol/min/g; DCM+GLP-1: 61±12 nmol/min/g; P =0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98±2 density units; DCM: 256±58 density units; P =0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1–induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38α MAP kinase activity (DCM+vehicle: 97±22 pmol ATP/mg/min; DCM+GLP-1: 170±36 pmol ATP/mg/min; P =0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151±13 density units; DCM+GLP-1: 306±12 density units; P =0.001), and GLUT-1 translocation (DCM+vehicle: 21±3% membrane bound; DCM+GLP-1: 39±3% membrane bound; P =0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38α MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro- l -arginine. Conclusions— GLP-1 stimulates myocardial glucose uptake through a non–Akt-1–dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Unexpected Reaction of 2-Alkylsulfanylimidazoles to Imidazol-2-ones: Pyridinylimidazol-2-ones as Novel Potent p38α Mitogen-Activated Protein Kinase Inhibitors

While optimizing the synthesis of 2-alkylsulfanyl-5-(2-aminopyridin-4-yl)imidazoles, we identified an unexpected reaction to pyridinylimidazol-2-ones. 2-Alkylsulfanylimidazoles, bearing a 2-hydroxyethyl or a 2,3-dihydroxypropyl moiety at the imidazole C2-S position, were converted by heating into imidazol-2-ones. These imidazol-2-ones were tested for their ability to inhibit p38alpha MAP kinase and LPS-stimulated TNF-alpha release in HWB. Introduction of an amino moiety at the pyridine C2 position led to compounds showing potent enzyme inhibitory activity with double-digit nanomolar IC(50) values (5a: IC(50) = 23 nM).

The discovery and initial optimisation of pyrrole-2-carboxamides as inhibitors of p38α MAP kinase

A novel pyrrole-2-carboxamide series of p38α inhibitors, discovered through the application of virtual screening, is presented. Following evaluation of activity, selectivity and developability properties of commercially available analogues, a synthesis program enabled rapid assessment of the series’ suitability for further lead optimisation studies.

Acute Lymphoid and Gastrointestinal Toxicity Induced by Selective p38α Map Kinase and Map Kinase–Activated Protein Kinase-2 (MK2) Inhibitors in the Dog

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Abstract 132: Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Abstract Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that kill tumor and tumor vasculature cells. Nitric oxide (NO) produced by these cells could be pro-carcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. Our previous studies showed that NO from a chemical donor made COH-BR1 breast tumor cells hyperresistant to apoptotic photokilling induced by photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX localized in mitochondria. Hyperresistance was associated with inhibition of proapoptotic JNK and p38α MAP kinase phosphorylation-activation. The purpose of this study was to determine whether tumor cells upregulate NOS-generated NO as a cytoprotective measure during PDT.COH-BR1 cells sensitized in mitochondria with ALA-derived protoporphyrin IX died apoptotically following irradiation as measured by Hoechst staining. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated &amp;gt;3-fold within 4 h after ALA/light treatment, while other NOS isoforms were unaffected. Exposing cells to the iNOS inhibitor, 1400W during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2-3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by &amp;gt;2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. Using Hoechst and TUNEL staining, we found that short hairpin RNA (shRNA)-induced knockdown of iNOS enhanced the apoptotic kill of ALA/light treated COH-BR1 cells. ALA/light-treated COH-BR1cells exhibited a transient post-irradiation activation of JNK and p38α as measured by Western blot analysis. Consistently, both effects were intensified and prolonged by 1400W. The survival MAP kinase ERK1/2 was deactivated more rapidly when 1400W was present during a PpIX/light challenge. Similar effects on MAP kinase activation/deactivation were observed for iNOS knockdown cells under photostress cinfirming iNOS's protective role. As demonstrated for non-photodynamic stress systems, NO could have interfered with apoptosis by inactivating participating MAP kinases and/or caspases. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy, and suggest NOS-based pharmacologic interventions for preventing this. (Supported by NIH Grant CA70823) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 132.

Synthesis and biological activity of 2 H-quinolizin-2-one based p38α MAP kinase inhibitors

The development and synthesis of potent p38α MAP kinase inhibitors containing a 2 H-quinolizin-2-one platform is described. Evolution of the 2 H-quinolizin-2-one series from an early lead to solving off target activity and pharmacokinetic issues is also discussed.