Synthesis and SAR of aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors

The development of a series of novel aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, JNK2 and CDK2, and the in vitro inhibitory value for a c-Jun cellular assay are discussed.     

The role of c-Jun N-terminal kinase (JNK) in Parkinson's disease

Given the critical role that the c-Jun N-terminal kinase (JNK) pathway plays in regulating many of the cellular processes which are affected in Parkinson's disease (PD), the possible importance of JNK in disease pathogenesis is being increasingly recognized. Here we review recent findings implicating the JNK signaling pathway in animal models of Parkinson's disease and discuss the relationship between this pathway and the prominent pathological processes observed in the disease state. We suggest that regulation of the JNK signaling pathway may be a central facet in potential treatments for the disease.     

c-Jun N-terminal kinase (JNK) signaling: Recent advances and challenges

c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.     

Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors

The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c, whose kinase selectivity is reported.     

The regulation of Bax by c-Jun N-terminal protein kinase (JNK) is a prerequisite to the mitochondrial-induced apoptotic pathway

The signaling mechanism by which JNK affects mitochondria is critical to initiate apoptosis. Here we show that the absence of JNK provides a partial resistance to the toxic effect of the heavy metal cadmium. Both wild type and jnk−/− fibroblasts undergoing death exhibit cytosolic cytochrome c but, unlike wild type cells, the JNK-deficient fibroblasts do not display increased caspase activity and DNA fragmentation. The absence of apoptotic death correlates with a specific defect in activation of Bax. We conclude that JNK-dependent regulation of Bax is essential to mediate the apoptotic release of cytochrome c regardless of Bid and Bim activation.     

Mitochondrial c-Jun N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.     

c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade

Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) (Ser(312) in human IRS1). Here we show that JNK mediated feedback inhibition of the insulin signal in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity required phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin was inhibited by a cell-permeable peptide that disrupted the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 was not required for its activation by insulin, whereas direct binding was required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation was directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1.     

Ligation of IFN-gamma-induced HLA-DR molecules on fibroblasts induces RANTES expression via c-Jun N-terminal kinase (JNK) pathway

The role of human leukocyte antigen (HLA) class II molecules on non-antigen presenting cells has been a matter of controversy. We recently reported that ligation of HLA-DR molecule with anti-HLA-DR antibodies (L243) and/or antigenic peptide/T cell receptor complex resulted in a secretion of several chemokines such as RANTES. In the present study, we aimed to detect putative signal transduction pathway leading to RANTES production from fibroblasts when the DR molecules were ligated with L243. Protein tyrosine kinase inhibitor (GF109203X) suppressed RANTES expression in a dose dependent manner for up to 50% from gingival fibroblasts (GF), while protein kinase C inhibitor (genistein) had no inhibitory effect. Ligation of DR molecules with L243 resulted in tyrosine phosphorylation of 54 kDa cellular protein. Thus, we suspected that either Jun N-terminal kinase-2 (JNK-2) or Src family proteins were involved in HLA-DR-mediated signaling. JNK inhibitor (SP600125), but not Src inhibitor (PP2), suppressed both L243 stimulated RANTES mRNA expression and protein secretion. The maximum inhibition for RANTES production by SP600125 was more than 80%. Additionally, JNK inhibitor nearly completely blocked tumor necrosis factor-alpha (TNF-alpha)-induced RANTES production in GF. Furthermore, ligation of GF HLA-DR with L243 induced selective phosphorylation of JNK-2. We concluded that JNK-2 was one of the HLA-DR-mediated signal transduction pathways.     

Activation of c-Jun N-terminal kinase (JNK) during mitosis in retinal progenitor cells

Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.     

Docking interactions in the c-Jun N-terminal kinase pathway

The c-Jun N-terminal kinase (JNK) signaling pathway is a major mediator of stress responses in cells. Similar to other mitogen-activated protein kinases (MAPKs), JNK activity is controlled by a cascade of protein kinases and by protein phosphatases, including dual-specificity MAPK phosphatases. Components of the JNK pathway associate with scaffold proteins that modulate their activities and cellular localization. The JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAPK kinase 7 (MKK7), and members of the mixed lineage kinase (MLK) family, and regulates JNK activation in neurons. In this study we demonstrate that distinct regions within the N termini of MKK7 and the MLK family member dual leucine zipper kinase (DLK) mediate their binding to JIP-1. We have also identified amino acids in JNK required for: (a) binding to JIP-1 and for JIP-1-mediated JNK activation, (b) docking to MAPK kinase 4 (MKK4) and efficient phosphorylation by MKK4, and (c) docking to its substrate c-Jun and efficient c-Jun phosphorylation. None of the amino acids identified were essential for JNK docking to MKK7 or the dual-specificity phosphatase MAPK phosphatase 7 (MKP7). These findings uncover molecular determinants of JIP-1 scaffold complex assembly and demonstrate that there are overlapping, but also distinct, binding determinants within JNK that mediate interactions with scaffold proteins, activators, phosphatases, and substrates.     

Phosphorylation of microtubule-associated protein tau by isoforms of c-Jun N-terminal kinase (JNK)

Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases commonly referred to as tauopathies. Hyperphosphorylation of tau at most sites appears to precede filament assembly. Many of the hyperphosphorylated sites are serine/threonine-proline sequences. Here we show that c-Jun N-terminal kinases JNK1, JNK2 and JNK3 phosphorylate tau at many serine/threonine-prolines, as assessed by the generation of the epitopes of phosphorylation-dependent anti-tau antibodies. Of the three protein kinases, JNK2 phosphorylated the most sites in tau, followed by JNK3 and JNK1. Phosphorylation by JNK isoforms resulted in a greatly reduced ability of tau to promote microtubule assembly. These findings extend the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.     

Synergistic interaction of MEK kinase 2, c-Jun N-terminal kinase (JNK) kinase 2, and JNK1 results in efficient and specific JNK1 activation

Mitogen-activated protein kinases (MAPKs) are activated through cascades or modules consisting of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Investigating the molecular basis of activation of the c-Jun N-terminal kinase (JNK) subgroup of MAPK by the MAPKKK MEKK2, we found that strong and specific JNK1 activation by MEKK2 was mediated by the MAPKK JNK kinase 2 (JNKK2) rather than by JNKK1 through formation of a tripartite complex consisting of MEKK2, JNKK2, and JNK1. No scaffold protein was required for the MEKK2-JNKK2-JNK1 tripartite-complex formation. Expression of JNK1, JNKK2, and MEKK2 significantly augmented the coprecipitation of, respectively, MEKK2-JNKK2, MEKK2-JNK1, and JNKK2-JNK1, indicating that the interaction of MEKK2, JNKK2, and JNK1 is synergistic. Finally, the JNK1 was activated more efficiently in the MEKK2-JNKK2-JNK1 complex than was the JNK1 excluded from the complex. Thus, formation of a signaling complex through synergistic interaction of a MAPKKK, a MAPKK, and a MAPK molecule like MEKK2-JNKK2-JNK1 is likely to be responsible for the efficient, specific flow of information via MAPK cascades.     

Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) signaling cascade. They are activated through dual phosphorylation of two residues in the activation loop, a threonine and a tyrosine, by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock, as well as by cytokines and growth factors. Only small amounts of phosphorylated, active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in Escherichia coli, which lack the appropriate upstream kinases. We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active, phosphorylated JNKs suitable for a variety of enzymatic, biophysical and structural characterizations. We utilize N-terminally His-tagged MKK4 that is coexpressed in E. coli with a constitutively active form of MEKK1. This phosphorylated, active His-MKK4 is purified by Ni–NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1α1, JNK1α2 and JNK2α2) that had separately been expressed and purified from E. coli in their inactive forms. These in vitro activated JNKs are phosphorylated on both residues (T183, Y185) in their activation loops and are active towards their substrate, ATF2.     

JNK pathway in osteoarthritis: pathological and therapeutic aspects

Context: Osteoarthritis (OA) is a common chronic degenerative joint disease resulting in physical disability and reduced quality of life. Different biochemical signaling pathways are involved in the progression of OA, including the c-Jun NH2-terminal kinase (JNK) signal transduction pathway.

Objective: In this study, we have reviewed the recent updates on the association of JNK pathway with OA.

Methods: In this review, we have explored the databases like PubMed, Google Scholar, Medline, Scopus, etc., and collected the most relevant papers of JNK signaling pathway involved in the pathogenesis and therapeutics of OA

Results: JNK has been shown by scientific studies to be activated (phosphorylated) in OA that can play a key role in the cartilage destruction. Activation of JNK causes the phosphorylation of c-Jun that causes decreased proteoglycan synthesis and enhanced production of matrix metalloproteinase 13 (MMP-13). Overproduction of MMP-13 by chondrocytes plays a central role in cartilage degeneration in OA. Thus, targeting JNK pathway might be a promising therapeutic application for the prevention and treatment of OA. A number of JNK-inhibitors have been used in vitro and in vivo studies; however, not yet been translated into human use.

Conclusions: This review study indicates that JNK pathway plays an important role in development and progression of OA, and targeting the JNK pathway might be a potential approach for the treatment of OA in future.     

Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells

The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells.     

c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, is essential for tumor necrosis factor alpha-induced c-Jun kinase activation and apoptosis

Two ubiquitously expressed isoforms of c-Jun N-terminal protein kinase (JNK), JNK1 and JNK2, have shared functions and different functions. However, the molecular mechanism is unknown. Here we report that JNK1, but not JNK2, is essential for tumor necrosis factor alpha (TNF-alpha)-induced c-Jun kinase activation, c-Jun expression, and apoptosis. Using mouse fibroblasts deficient in either Jnk1 or Jnk2, we found that JNK1 was activated by TNF-alpha, whereas JNK2 activation was negligible. In addition, JNK2 interfered with JNK1 activation via its "futile" phosphorylation by upstream kinases. Consequently, expression and activation of c-Jun, which depends on JNK activity, were impaired in Jnk1 null cells but enhanced in Jnk2 null cells. TNF-alpha-induced apoptosis was also suppressed in Jnk1 null fibroblasts but increased in Jnk2 null cells. Thus, our results provide a molecular mechanism underlying the different biological functions of JNK isoforms.