Syntheses and biological evaluation of 1-heteroaryl-2-aryl-1H-benzimidazole derivatives as c-Jun N-terminal kinase inhibitors with neuroprotective effects

1-Heteroaryl-2-aryl-1H-benzimidazole derivatives were synthesized as inhibitors of c-Jun N-terminal kinases, JNK3. Their activities were evaluated through measurement of K_d using SPR, JNK3 kinase assay, and cell-viability of human neuroblastoma cells. Most tested compounds showed high affinity (10 μM–46 nM) to JNK3. Among them, compound 16f exhibited potent activities (K_d = 46 nM). Especially, 16f was also found to present a potent cell protective effect (IC₅₀ = 1.09 μM) against toxicity induced by anisomycin, showing a possibility as protective therapeutics in neuronal cell apoptosis.     

Ricin A-chain requires c-Jun N-terminal kinase to induce apoptosis in nontransformed epithelial cells

Ricin is a toxin isolated from castor beans that has potential as a weapon of bioterrorism. This glycoprotein consists of an A-chain (RTA) that damages the ribosome and inhibits protein synthesis and a B-chain that plays a role in cellular uptake. Ricin activates the c-Jun N-terminal kinase (JNK) and p38 signaling pathways; however, a role for these pathways in ricin-induced cell death has not been investigated. Our goals were to determine if RTA alone could activate apoptosis and if the JNK and p38 pathways were required for this response. Comparable caspase activation was observed with both ricin and RTA treatment in the immortalized, nontransformed epithelial cell line, MAC-T. Ribosome depurination and inhibition of protein synthesis were induced in 2–4 h with 1 μg/ml RTA and within 4–6 h with 0.1 μg/ml RTA. Apoptosis was not observed until 4 h of treatment with either RTA concentration. RTA activated JNK and p38 in a time- and concentration-dependent manner that preceded increases in apoptosis. Inhibition of the JNK pathway reduced RTA-induced caspase activation and poly(ADP-ribose) polymerase cleavage. In contrast, inhibition of the p38 pathway had little effect on RTA-induced caspase 3/7 activation. These studies are the first to demonstrate a role for the JNK signaling pathway in ricin-induced cell death. In addition, the MAC-T cell line is shown to be a sensitive in vitro model system for future studies using RTA mutants to determine relationships between RTA-induced depurination, ribotoxic stress, and apoptosis in normal epithelial cells.     

Inhibition of CYP2E1 attenuates chronic alcohol intake-induced myocardial contractile dysfunction and apoptosis

Alcohol intake is associated with myocardial contractile dysfunction and apoptosis although the precise mechanism is unclear. This study was designed to examine the effect of the cytochrome P450 enzyme CYP2E1 inhibition on ethanol-induced cardiac dysfunction. Adult male mice were fed a 4% ethanol liquid or pair-fed control diet for 6 weeks. Following 2 weeks of diet feeding, a cohort of mice started to receive the CYP2E1 inhibitor diallyl sulfide (100 mg/kg/d, i.p.) for the remaining feeding duration. Cardiac function was assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate CYP2E1, heme oxygenase-1 (HO-1), iNOS, the intracellular Ca^2 + regulatory proteins sarco(endo)plasmic reticulum Ca^2 +-ATPase, Na⁺Ca^2 + exchanger and phospholamban, pro-apoptotic protein cleaved caspase-3, Bax, c-Jun-NH₂-terminal kinase (JNK) and apoptosis signal-regulating kinase (ASK-1). Ethanol led to elevated levels of CYP2E1, iNOS and phospholamban, decreased levels of HO-1 and Na⁺Ca^2 + exchanger, cardiac contractile and intracellular Ca^2 + defects, cardiac fibrosis, overt O₂^? production, and apoptosis accompanied with increased phosphorylation of JNK and ASK-1, the effects were significantly attenuated or ablated by diallyl sulfide. Inhibitors of JNK and ASK-1 but not HO-1 inducer or iNOS inhibitor obliterated ethanol-induced cardiomyocyte contractile dysfunction, substantiating a role for JNK and ASK-1 signaling in ethanol-induced myocardial injury. Taken together, these findings suggest that ethanol metabolism through CYP2E1 may contribute to the pathogenesis of alcoholic cardiomyopathy including myocardial contractile dysfunction, oxidative stress and apoptosis, possibly through activation of JNK and ASK-1 signaling.     

Baicalein inhibits nuclear factor-κB and apoptosis via c-FLIP and MAPK in d-GalN/LPS induced acute liver failure in murine models

The hepatoprotective effects and molecular mechanisms of baicalein on acute liver failure induced by d-galactosamine (d-GalN)/lipopolysaccharides (LPS) were investigated in vivo. Mice were administered with different doses of baicalein (50, 100 or 150 mg/kg, p.o.) 1 h before injection of d-GalN (700 mg/kg)/LPS (10 μg/kg) and then sacrificed 6 h after treatment with d-GalN/LPS. Pretreatment with baicalein prevented d-GalN/LPS-induced liver damage by preventing associated increases of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and by reducing serum tumor necrosis factor α (TNF-α), nitric oxide (NO) or inducible nitric oxide synthase (iNOS) expressions. The molecular mechanisms involved in baicalein-induced inhibition of d-GalN/LPS-caused apoptosis were associated with the protection of mitochondria, increasing the Bcl-2/Bax ratio, blocking the release of cytochrome c, and suppressing the phosphorylation of IκBα, ERK and JNK. Moreover, baicalein activated c-FLIP_L, XIAP and cIAP2 proteins, potentially blocking the recruitment of NF-κB signaling molecules. The results support the investigation of baicalein as a therapeutic candidate for acute liver apoptosis or injury and indicate that baicalein might inhibit liver apoptosis by mediating one or more of these pathways.     

ONTD induces apoptosis of human hepatoma Bel-7402 cells via a MAPK-dependent mitochondrial pathway and the depletion of intracellular glutathione

3-Oxo-29-noroleana-1,9(11),12-trien-2,20-dicarbonitrile (ONTD) is a novel synthetic derivative of glycyrrhetinic acid (GA), which has the ability to inhibit the proliferation of human hepatocellular carcinoma (HCC) cells. However, the mechanisms by which ONTD exerts its inhibitory effects remain elusive. The present study was conducted to investigate the cytotoxicity of ONTD in Bel-7402 cells and its molecular mechanisms. We found that ONTD depleted intracellular GSH, increased the level of ROS, and consequently induced mitochondrial permeability transition (MPT) leading to the release of apoptosis-inducing factor (AIF) and cytochrome c (Cyt c) to the cytosol. Mitochondrial alteration and subsequent apoptotic cell death in ONTD-treated Bel-7402 cells could be blocked by addition of exogenous antioxidants N-acetylcystein (NAC), GSH and the MTP inhibitor cyclosporin A (CsA). In addition, ONTD activated the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPK) but not extracellular signal-regulated protein kinases (ERK 1/2). When the cells were exposed to SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), the deregulation of the expression of apoptotic proteins was attenuated. Furthermore, 40 mg/kg ONTD significantly reduced tumor weight (?70.62%, p < 0.01) in the H22 tumor-bearing mouse model in vivo. Taken together, these findings provide the first experimental evidence supporting that ONTD could induce apoptosis of Bel-7402 cells via MAPK-mediated mitochondrial pathway and ONTD has the potential to be developed as a therapeutic agent for the treatment of HCC.     

3D-QSAR studies of 1,2-diaryl-1H-benzimidazole derivatives as JNK3 inhibitors with protective effects in neuronal cells

JNKs (c-Jun N-terminal kinases) have the potential to serve as a therapeutic target for various inflammatory, vascular, neurodegenerative, metabolic and oncological diseases. In particular, ATP-competitive JNK3 inhibitors act as neuroprotective agents. Here we introduce 1,2-diaryl-1H-benzimidazole derivatives as selective JNK3 inhibitors from among our in-house compounds and describe our elucidation of their SAR using 3D-QSAR models. A predictive CoMFA model (q² = 0.795, r² = 0.931) and a CoMSIA model (q² = 0.700, r² = 0.937) were used to describe the non-linearly combined affinity of each functional group in the inhibitors.     

Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): Optimization for JNK potency and physicochemical properties

A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC₅₀ of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties.     

Telmisartan attenuates aortic hypertrophy in hypertensive rats by the modulation of ACE2 and profilin-1 expression

Profilin-1 has recently been linked to vascular hypertrophy and remodeling. Here, we assessed the hypothesis that angiotensin (Ang) II type I receptor antagonist telmisartan improves vascular hypertrophy by modulation of expression of profilin-1 and angiotensin-converting enzyme 2 (ACE2). Ten-week-old male spontaneously hypertensive rats (SHR) were received oral administration of telmisartan (5 or 10 mg/kg; daily) or saline for 10 weeks. Compared with Wistar–Kyoto (WKY) rats, there were marked increases in systolic blood pressure and profilin-1 expression and reduced ACE2 and peroxisome proliferator activated receptor-γ (PPARγ) levels in aorta of SHR, associated with elevated extracellular-signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) phosphorylation signaling and aortic hypertrophy characterized with increased media thickness, which were strikingly reversed by telmisartan. In cultured human umbilical artery smooth muscle cells (HUASMCs), Ang II induced a dose-dependent increase in profilin-1 expression, along with decreased ACE2 protein expression and elevated ERK1/2 and JNK phosphorylation. In addition, blockade of ERK1/2 or JNK by either specific inhibitor was able to abolish Ang II-induced ACE2 downregulation and profilin-1 upregulation in HUASMCs. Importantly, treatment with telmisartan (1 or 10 μM) or recombinant human ACE2 (2 mg/ml) largely ameliorated Ang II-induced profilin-1 expression and ERK1/2 and JNK phosphorylation and augmented PPARγ ?expression in the cultured HUASMCs. In conclusion, telmisartan treatment attenuates vascular hypertrophy in SHR by the modulation of ACE2 and profilin-1 expression with a marked reversal of ERK1/2 and JNK phosphorylation signaling pathways.     

Expression of Na+/K+-ATPase α-subunit mRNA during embryonic development of the crayfish Astacus leptodactylus

Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na⁺/K⁺-ATPase α-subunit, and one putative housekeeping gene, β-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na⁺/K⁺-ATPase α-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). β-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na⁺/K⁺-ATPase α-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI = 100 μm), and one in embryos just before hatching (at EI = 420 μm), continuing in the freshly hatched juveniles. The Na⁺/K⁺-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.     

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors

As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC₅₀ = 160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC₅₀ = 47 nM) was a highly specific JNK inhibitor.     

Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-phenylurea derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor treatment

Based on the roles of Raf1 and JNK1 in hepatocarcinoma development, scaffold-based drug design was employed to produce a series of compounds, which subsequently were synthesized and explored as potential dual inhibitors Raf1 and JNK1 kinases for anti-tumor treatment. The compound 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chloro-phenyl)urea (3d) showed 66%, 67% and 13% inhibition rate at 50 μM against Raf1, JNK1 and p38-alpha, respectively, but no effect on ERK1 and ERK2, and inhibited the expression of pERK1/2 markedly and HepG2 cells proliferation with IC₅₀ at 8.3 μM. Furthermore, 3d showed lower toxicity against normal liver cell-lines QSG7701 and HL7702. Molecular docking study further showed that 3d can fit into binding domain of JNK1 and Raf1. Our data suggested the activities of 3d were associated with dual inhibition of JNK1 and Raf1 kinases.     

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P < 0.01) down-regulated, while SLC19A1 was up-regulated (P < 0.01) in FD group. FD cells exhibited significantly (P < 0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P < 0.01) down-regulated and IGF-1 concentration was decreased (P < 0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P < 0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24 h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.     

Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases

The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway.     

Differences in egg deposition of corticosterone and embryonic expression of corticosterone metabolic enzymes between slow and fast growing broiler chickens

Glucocorticoids (GCs) are vital for embryonic development and their bioactivity is regulated by the intracellular metabolism involving 11β-hydroxysteroid dehydrogenases (11β-HSDs) and 20-hydroxysteroid dehydrogenase (20-HSD). Here we sought to reveal the differences in egg deposition of corticosterone and embryonic expression of corticosterone metabolic enzymes between slow and fast growing broiler chickens (Gallus gallus). Eggs of fast-growing breed contained significantly higher (P < 0.05) corticosterone in the yolk and albumen, compared with that of a slow-growing breed. 11β-HSD1 and 11β-HSD2 were expressed in relatively higher abundance in the liver, kidney and intestine, following similar tissue-specific ontogenic patterns. In the liver, expression of both 11β-HSD1 and 11β-HSD2 was upregulated (P < 0.05) towards hatching, yet 20-HSD displayed distinct pattern showing a significant decrease (P < 0.05) on posthatch day 1 (D1). Hepatic mRNA expression of 11β-HSD1 and 11β-HSD2 was significantly higher in fast-growing chicken embryos at all the embryonic stages investigated and so was the hepatic protein content on embryonic day of 14 (E14) for 11β-HSD1 and on E14 and D1 for 11β-HSD2. 20-HSD mRNA was higher in fast-growing chicken embryos only on E14. Our data provide the first evidence that egg deposition of corticosterone, as well as the hepatic expression of glucocorticoid metabolic enzymes, differs between fast-growing and slow-growing chickens, which may account, to some extent, for the breed disparities in embryonic development.     

Poly(ADP-ribose) polymerase-1 inhibition protects the brain against systemic inflammation

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, but its overactivation can induce cell death. Our aim was to investigate the role of PARP-1 in activation of programmed cell death processes in the brain during systemic inflammation.Our data indicated that lipopolysaccharide (1 mg/kg b.w., i.p.)-evoked systemic inflammation enhanced PARP-1 activity in the mouse brain, leading to the lowering of β-NAD⁺ concentration, to translocation of apoptosis inducing factor from mitochondria to the nucleus, and to enhanced lipid peroxidation. Inhibitor of PARP-1, 3-aminobenzamide (30 mg/kg b.w., i.p.), protected the brain against prooxidative and cell death processes, suggesting involvement of PARP-1 in systemic inflammation-related processes in the brain.