Design, synthesis, and biological evaluation of indolin-2-one derivatives as novel cyclin-dependent protein kinase 8 (CDK8) inhibitors.

Cyclin-dependent protein kinase 8 (CDK8) plays important roles in regulating fibrotic growth factors and inflammatory signaling pathways. Long-term chronic inflammation of the lungs can lead to idiopathic pulmonary fibrosis (IPF). Abnormal alveolar epithelial regeneration leads to the release of various fibrotic growth factors and the activation of inflammatory cells. CDK8 regulates profibrotic cytokines broadly implicated in the pathogenesis of fibrosis. Therefore, inhibition of CDK8 is considered a promising strategy for treating IPF. Here, CDK8 inhibitors were designed and optimized using a fragment-based drug design strategy. Testing results revealed that 71% of the synthesized compounds inhibited CDK8 activity better than the original compound E966-0530. Of these compounds, compound 4k exhibited the strongest CDK8 enzyme-inhibiting activity (IC50 =129 nM). Notably, it displayed a 13-fold increase in potency when compared to E966-0530. Experiments on toxicity and inhibition of epithelial-mesenchymal transition (EMT) protein expressions showed that compound 4k can inhibit EMT protein expressions, but with no significant cytotoxicity for alveolar epithelial cells. Compound 4k showed a potent inhibitory effect in cell migration assays. Furthermore, compound 4k significantly inhibited the phosphorylation of p-Smad3 and RNA Pol II, which are critical mediators in the fibrotic response signaling pathway. Compound 4k remarkably reduced TGF-ß1-induced oxidative stress. The above results reveal optimized CDK8 inhibitors with potential use for IPF therapeutic treatment.

Ibuprofen treatment ameliorates memory deficits in rats with collagen-induced arthritis by normalizing aberrant MAPK/NF-κB and glutamatergic pathways.

Many studies have indicated that the risk of cognitive impairment is higher in patients with rheumatoid arthritis (RA). Additionally, patients with RA may have a lower incidence of cognitive impairment with long-term use of ibuprofen. This study was aimed at investigating the impacts of RA on memory function and the mechanisms that ibuprofen may exhibit to improve memory function in rats with collagen-induced arthritis (CIA). Ibuprofen (30 mg/kg) was given twice daily to CIA rats for two weeks starting from Day 18 following the first immunization. Memory function was measured by the Morris water maze (MWM) test and long-term potentiation (LTP). The proinflammatory cytokine levels and downstream signaling pathways, including mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), were examined. Furthermore, the glutamatergic system, including glutamate transporters/receptors and brain extracellular levels of glutamate, was investigated. The results showed that the impaired learning memory in CIA rats, examined by the MWM test and LTP, can be ameliorated by ibuprofen treatment. Along with the improvement in memory deficits, ibuprofen attenuated both neuroinflammation and the associated elevated levels of phosphorylated p38, JNK, and p65 in the hippocampus of CIA rats. In addition, the decreased excitatory amino acid transporter 2 level, the increased extracellular glutamate, and the upregulated hippocampal NMDA receptor 2B of CIA rats were all normalized by ibuprofen treatment. These findings suggest that the effect of ibuprofen on the memory improvement in CIA rats is associated with the normalization of the activated MAPK and NF-κB pathways and the aberrant glutamatergic system.

Discovery of a novel cyclin-dependent kinase 8 inhibitor with an oxindole core for anti-inflammatory treatment.

Chronic inflammation is an underlying cause in a number of diseases. Cyclin-dependent kinase 8 (CDK8) has been implicated as an inflammatory mediator, indicating its potential as an anti-inflammatory target. Herein, we performed structure-based virtual screening (SBVS) to identify novel CDK8 inhibitors. The pharmacological interactions for CDK8 were identified and incorporated into a SBVS protocol. Selected compounds were tested in enzymatic assays, and one compound was confirmed to be a CDK8 inhibitor with a 50% inhibitory concentration (IC50) value of 1684.4 nM. Comparing structural analogs identified a compound, F059-1017, with greater potency (IC50 558.1 nM). When tested in cell lines, the compounds displayed low cytotoxicity. Cellular assays revealed that the identified CDK8 inhibitors can reduce phosphorylation and expression of signaling mediators associated with inflammation. In addition, results of kinase profiling showed that compound F059-1017 is selective towards CDK8. These findings suggest that the new inhibitors have great potential as lead compounds for developing novel anti-inflammatory therapeutics.

Neuroprotective Studies of Evodiamine in an Okadaic Acid-Induced Neurotoxicity.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease, and it manifests as progressive memory loss and cognitive decline. However, there are no effective therapies for AD, which is an urgent problem to solve. Evodiamine, one of the main bioactive ingredients of Evodia rutaecarpa, has been reported to ameliorate blood-brain barrier (BBB) permeability and improve cognitive impairment in ischemia and AD mouse models. However, whether evodiamine alleviates tauopathy remains unclear. This study aimed to examine whether evodiamine ameliorates tau phosphorylation and cognitive deficits in AD models. METHODS: A protein phosphatase 2A inhibitor, okadaic acid (OA), was used to induce tau phosphorylation to mimic AD-like models in neuronal cells. Protein expression and cell apoptosis were detected using Western blotting and flow cytometry, respectively. Spatial memory/cognition was assessed using water maze, passive avoidance tests, and magnetic resonance imaging assay in OA-induced mice models, and brain slices were evaluated further by immunohistochemistry. RESULTS: The results showed that evodiamine significantly reduced the expression of phosphor-tau, and further decreased tau aggregation and neuronal cell death in response to OA treatment. This inhibition was found to be via the inhibition of glycogen synthase kinase 3ß, cyclin-dependent kinase 5, and mitogen-activated protein kinase pathways. In vivo results indicated that evodiamine treatment ameliorated learning and memory impairments in mice, whereas Western blotting and immunohistochemical analysis of the mouse brain also confirmed the neuroprotective effects of evodiamine. CONCLUSIONS: Evodiamine can decrease the neurotoxicity of tau aggregation and exhibit a neuroprotective effect. Our results demonstrate that evodiamine has a therapeutic potential for AD treatment.

Anti-Inflammatory and Tau Phosphorylation-Inhibitory Effects of Eupatin.

Alzheimer's disease (AD), which is among the most prevalent neurodegenerative diseases, manifests as increasing memory loss and cognitive decline. Tau phosphorylation and aggregation are strongly linked to neurodegeneration, as well as associated with chronic neuroinflammatory processes. The anti-inflammation effects of natural products have led to wide recognition of their potential for use in treating and preventing AD. This study investigated whether eupatin, a polymethoxyflavonoid found in Artemisia species, has inhibitory effects on neuroinflammation and tau phosphorylation. We treated mouse macrophages and microglia cells with lipopolysaccharides (LPSs) to activate inflammatory signals, and we treated neuronal cells with a protein phosphatase 2A inhibitor, okadaic acid (OA), or transfection with pRK5-EGFP-Tau P301L plasmid to induce tau phosphorylation. The results indicated that eupatin significantly reduced the LPS-induced protein expression and phosphorylation of p65 and inducible nitric oxide synthase as well as downstream products interleukin 6 and nitrite, respectively. Furthermore, eupatin markedly inhibited the expression of phospho-tau in response to OA treatment and plasmid transfection. We discovered that this inhibition was achieved through the inhibition of glycogen synthase kinase 3ß (GSK3ß), and molecular docking results suggested that eupatin can sufficiently bind to the GSK3ß active site. Our results demonstrate that eupatin has neuroprotective effects, making it suitable for AD treatment.