Structural Insight into the Binding Pattern and Interaction Mechanism of Antagonist MCC950 and Agonist BMS986299 with NLRP3 by Molecular Dynamics Simulation.

OBJECTIVE: The NLRP3 inflammasome mediates a range of inflammatory responses that are associated with an increasing number of pathological mechanisms. Over-activation of NLRP3 can exacerbate many diseases. However, NLRP3 antagonists have significant therapeutic potential. Moreover, NLRP3 plays an important role in limiting the growth and spread of some tumors, and NLRP3 agonists also have clinical value. MCC950 and BMS986299 are an antagonist and agonist of NLRP3, respectively. In light of the important clinical applications of NLRP3, especially for NLRP3 inhibitors, a computational method was used to investigate the interaction modes of MCC950 and BMS986299 with NLRP3 in order to design and develop more potent NLRP3 regulators. METHODS: In this study, the conformational behaviors of NLRP3 bound to the antagonist MCC950 in an inactive state and the agonist BMS986299 in an active state were investigated using 200 ns equilibrium all-atom molecular dynamics (MD) simulations, and then the analyses of the MD trajectories (RMSD, Rg, RMSF, SASA, PCA, and DCCM) were carried out to explore the mechanism of the antagonist and agonist on NLRP3 in the two different states. RESULTS: The RMSD, RMSF, Rg, SASA, and PCA analyses indicated that NLRP3 was more dispersive and less energetically stable in the active state than in the inactive state and that MCC950 significantly reduced the fluctuations of the interactive residues while BMS986299 did not. The antagonist MCC950 interacted with residues mainly in the NBD, HD1, WHD, and HD2 domains of NLRP3, whereas the agonist BMS986299 mainly in the NBD and WHD of NLRP3. Additionally, both compounds did not interact with residues located in the FISNA domain. The conformation of the FISNA domain appeared to change significantly when NLRP3 was translated from an inactive state to an active state. CONCLUSION: The antagonist may interact with residues mainly in the NBD, HD1, WHD, and HD2 domains, and the agonist may interact in the NBD and WHD domains. Our study provided new insights into the development of NLRP3 regulators.

Design, Synthesis, Antitumor Activity Evaluation, and Molecular Dynamics Simulation of Some 2-Aminopyrazine Derivatives.

OBJECTIVE: Cancer poses a great threat to human health, and effective drugs to treat it are always needed. Several compounds containing a 2-aminopyrazine framework have been identified as antitumor agents with SHP2 inhibition activities. This current work aimed to search for more potent novel compounds possessing a 2-aminopyrazine moiety with antitumor activities. METHODS: A series of 12 novel 2-aminopyrazine derivatives was synthesized, and their structures were confirmed by spectroscopic techniques. The inhibitory activities of all the synthesized compounds against MDA-MB-231 and H1975 cancer cell lines were evaluated by an MTT assay. The most potent compound 3e was analyzed by flow cytometry. Subsequently, computational studies were performed to investigate the possible antitumor mechanisms of compound 3e. RESULTS: The results indicated that compound 3e exhibited potent antitumor activities with IC50 values of 11.84±0.83µM against H1975 cells and 5.66±2.39µM against MDA-MB-231 cells, which were more potent than the SHP2 inhibitor GS493 (IC50 = 19.08±1.01 µM against H1975 cells and IC50 = 25.02±1.47 µM against MDA-MB-231 cells). Further analysis by flow cytometry demonstrated that compound 3e induced cell apoptosis in H1975 cells. The results of the molecular docking and MD simulations, including RMSD, RMSF, PCA, DCCM and binding energy and decomposition analyses, revealed that compound 3e probably selectively inhibited SHP2. CONCLUSION: A new compound having a 2-aminopyrazine substructure with potent inhibitory activities against the H1975 and MDA-MB-231 cancer cells was obtained, meriting further investigation as an antitumor drug.

In silico-determined compound from the root of Pueraria lobate alleviates synaptic plasticity injury induced by Alzheimer's disease via the p38MAPK-CREB signaling pathway.

Pueraria lobata is utilized as a food source in China. The aim of this study is to combine virtual screening and molecular dynamics predictive model to screen out the potential synaptic plasticity-maintaining components from the root of P. lobate and to verify it by employing the amyloid ß-injected rats' model. Eighteen compounds were identified by HPLC-MS/MS; puerarin manifested the most potential to form a stable complex with calcium/calmodulin kinase IIα (CaMK IIα), which is the key protein in synaptic plasticity by the in silico study. The further in vivo assay showed that puerarin could elevate the synaptic thickness, density, and length, relieve calcium overload, regulate the expression of CaMK IIα, and other p38MAPK-CREB signaling pathway-related biochemical criteria. The behavioral test also verified the results. Results have confirmed that the root of P. lobate can work anti-AD by maintaining the synaptic plasticity and proved the reliability of using the in silico predictive model to determine active ingredients from the natural product.

Synthesis and Biological Evaluations of Monocarbonyl Curcumin Inspired Pyrazole Analogues as Potential Anti-Colon Cancer Agent.

PURPOSE: The monocarbonyl analogs of curcumin (MCACs) have been widely studied for their promising antitumor activity. Pyrazole is a five-membered aromatic heterocyclic system with various bioactivities incorporated frequently in drugs. However, few of MCACs inspired pyrazole analogues were investigated. To search for more potent cytotoxic agents based on MCACs, a series of new 1,5-diaryl/heteroaryl-1,4-pentadien-3-ones inspired pyrazole moiety was synthesized and evaluated on their anti-colon cancer activities. METHODS: Fifteen new compounds were synthesized and characterized by spectral datum, and then they were tested preliminarily by MTT assay for their cytotoxic activities against a panel of four human cancer cell lines, namely, gastric (SGC-7901), liver (HepG2), lung (A549), and colon (SW620) cancer cells. Compound 7h exhibited excellent selectivity and outstanding anti-proliferation activity against SW620 cells among these 15 compounds. Further, the mechanisms were investigated by transwell migration and invasion assay, clonogenic assay, cell apoptosis analysis, cell cycle analysis, Western blot analysis. RESULTS: The IC50 value of 7h against SW620 cells was 12 nM, being more potent than curcumin (IC50 = 9.36 µM), adriamysin (IC50 = 3.28 µM) and oxaliplatin (IC50 = 13.33 µM). Further assays showed that 7h inhibited SW620 cell migration, invasion and colony formation obviously, which was due to its ability to induce cell cycle arrest in the G2/M and S phases and apoptosis. Western blot assay revealed that 7h decreased the protein expression of ATM gene, which may primarily contribute to its anticancer activity against SW620 cells. CONCLUSION: A new MCACs 7h was synthesized and found to exhibit excellent anti-proliferation activity against SW620 cells. Further studies indicated that 7h exerted its anticancer activity against SW620 cells probably via decreasing the ATM protein expression. The present study suggested that 7h was a promising candidate as an anti-colon cancer drug for future development.

miR-1247-3p mediates apoptosis of cerebral neurons by targeting caspase-2 in stroke.

Brain stroke is one of the leading causes of death worldwide. We explored a potential stroke-related role for a newly found microRNA, miR-1247-3p, and one of its target genes, caspase-2, predicted by TargetScanVert. In the present study, we found that miR-1247-3p was downregulated during ischemia/reperfusion (I/R) and that LV-miR-1247-3p overexpression attenuated brain impairment induced by I/R. Similar results were observed in neuro2a (N2a) cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R). Caspase-2 was upregulated in the I/R and OGD/R model, while Z-VDVAD-FMK - the inhibitor of caspase-2-inhibited apoptosis of N2a cells induced by OGD/R. An miR-1247-3p mimic inhibited caspase-2 expression and attenuated apoptosis of N2a cells induced by OGD/R. Myocardin-related transcription factor-A (MRTF-A) overexpression upregulated miR-1247 and mature miR-1247-3p levels and attenuated apoptosis induced by OGD/R, whereas its anti-apoptotic function could be blocked by a miR-1247-3p inhibitor. Hence, we conclude that miR-1247-3p may protect cells during brain stroke. This study offers insights for the development of effective therapeutics for promoting the survival of cerebral neurons during brain I/R injury.

Synthesis, preliminarily biological evaluation and molecular docking study of new Olaparib analogues as multifunctional PARP-1 and cholinesterase inhibitors.

A series of new Olaparib derivatives was designed and synthesized, and their inhibitory activities against poly (ADP-ribose) polymerases-1 (PARP-1) enzyme and cancer cell line MDA-MB-436 in vitro were evaluated. The results showed that compound 5l exhibited the most potent inhibitory effects on PARP-1 enzyme (16.10 ± 1.25 nM) and MDA-MB-436 cancer cell (11.62 ± 2.15 µM), which was close to that of Olaparib. As a PARP-1 inhibitor had been reported to be viable to neuroprotection, in order to search for new multitarget-directed ligands (MTDLs) for the treatment of Alzheimer's disease (AD), the inhibitory activities of the synthesized compounds against the enzymes AChE (from electric eel) and BChE (from equine serum) were also tested. Compound 5l displayed moderate BChE inhibitory activity (9.16 ± 0.91 µM) which was stronger than neostigmine (12.01 ± 0.45 µM) and exhibited selectivity for BChE over AChE to some degree. Molecular docking studies indicated that 5l could bind simultaneously to the catalytic active of PARP-1, but it could not interact well with huBChE. For pursuit of PARP-1 and BChE dual-targeted inhibitors against AD, small and flexible non-polar groups introduced to the compound seemed to be conducive to improving its inhibitory potency on huBChE, while keeping phthalazine-1-one moiety unchanged which was mainly responsible for PARP-1 inhibitory activity. Our research gave a clue to search for new agents based on AChE and PARP-1 dual-inhibited activities to treat Alzheimer's disease.

The emerging role of microRNA-4487/6845-3p in Alzheimer's disease pathologies is induced by Aß25-35 triggered in SH-SY5Y cell.

BACKGROUND: Accumulation of amyloid ß-peptide (Aß) is implicated in the pathogenesis and development of Alzheimer's disease (AD). Neuron-enriched miRNA was aberrantly regulated and may be associated with the pathogenesis of AD. However, regarding whether miRNA is involved in the accumulation of Aß in AD, the underlying molecule mechanism remains unclear. Therefore, we conduct a systematic identification of the promising role of miRNAs in Aß deposition, and shed light on the molecular mechanism of target miRNAs underlying SH-SY5Y cells treated with Aß-induced cytotoxicity. RESULTS: Statistical analyses of microarray data revealed that 155 significantly upregulated and 50 significantly downregulated miRNAs were found on the basis of log2 | Fold Change | ≥ 0.585 and P < 0.05 filter condition through 2588 kinds of mature miRNA probe examined. PCR results show that the expression change trend of the selected six miRNAs (miR-6845-3p, miR-4487, miR-4534, miR-3622-3p, miR-1233-3p, miR-6760-5p) was consistent with the results of the gene chip. Notably, Aß25-35 downregulated hsa-miR-4487 and upregulated hsa-miR-6845-3p in SH-SY5Y cell lines associated with Aß-mediated pathophysiology. Increase of hsa-miR-4487 could inhibit cells apoptosis, and diminution of hsa-miR-6845-3p could attenuate axon damage mediated by Aß25-35 in SH-SY5Y. CONCLUSIONS: Together, these findings suggest that dysregulation of hsa-miR-4487 and hsa-miR-6845-3p contributed to the pathogenesis of AD associated with Aß25-35 mediated by triggering cell apoptosis and synaptic dysfunction. It might be beneficial to understand the pathogenesis and development of clinical diagnosis and treatment of AD. Further, our well-designed validation studies will test the miRNAs signature as a prognostication tool associated with clinical outcomes in AD.

In silico identification of AChE and PARP-1 dual-targeted inhibitors of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disease of the elderly that seriously affects the quality of life and the life expectancy of those affected. There is, as yet, no effective drug treatment of AD, although several acetylcholinesterase (AChE) inhibitors and a glutamate antagonist can provide relief from its symptoms. Recent studies have indicated that the overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) may promote nerve cell death in the brains of AD patients, implying that PARP-1 inhibition may have therapeutic value for the treatment of AD. Therefore, it is important to investigate novel agents with both AChE- and PARP-1-inhibitory bioactivities. In this study, the structure-based virtual screening of PARP-1 inhibitors was performed to search for potential agents with high affinities for AChE. The dynamic stability of the selected AChE-ligand complexes was investigated by molecular dynamics (MD) simulation. Two compounds, CID57390505 and CID71605390, showed high affinities for and stability in complex with AChE in docking and MD simulations. Thus, our in silico research identified two compounds with AChE and PARP-1 dual-targeted activities, indicating that this technique could aid attempts to develop more potent agents against AD.

Effects of fucoxanthin on autophagy and apoptosis in SGC-7901cells and the mechanism.

Autophagy and apoptosis are involved in the development of a variety of cancers. Fucoxanthin is a natural compound known to have antitumor effects, so we aimed to explore its effects on autophagy and apoptosis in gastric cancer SGC7901 cells. Specifically, we performed methyl thiazolyl tetrazolium assay, transmission electron microscopy, real-time polymerase chain reaction, Western blot analysis, immunofluorescence assay, and cell apoptosis analysis to clarify the role of fucoxanthin in SGC-7901 cells. Our results indicate that fucoxanthin significantly inhibits the viability of SGC-7901 cells, effectively inducing both autophagy and apoptosis by up-regulating the expressions of beclin-1, LC3, and cleaved caspase-3 (CC3), and by down regulating Bcl-2. Fucoxanthin-induced autophagy also seems to occur before, and may promote apoptosis.

Opposite effects of HDAC5 and p300 on MRTF-A-related neuronal apoptosis during ischemia/reperfusion injury in rats.

Our recent study has revealed that the myocardin-related transcription factor-A (MRTF-A) is involved in the apoptosis of cortical neurons induced by ischemia/reperfusion (I/R). Histone deacetylase 5 (HDAC5) and histone acetyltransferase p300 (P300) are two well-known regulators for transcription factors; however, their roles in MRTF-A-related effect on neuronal injuries during I/R are still unclear. In this study, in a model rat cerebral I/R injury via middle cerebral artery occlusion and reperfusion, we found that the expression and activity of HDAC5 was upregulated, whereas p300 and MRTF-A were downregulated both in expression and activity during I/R. Their expression changes and the interaction of the MRTF-A with HDAC5 or p300 were further verified by double immunofluorescence and co-immunoprecipitation. In cultured neuronal apoptosis model induced by H2O2, MRTF-A exhibited an anti-apoptotic effect by enhancing the transcription of Bcl-2 and Mcl-1 via CArG box binding. MRTF-A-induced anti-apoptotic effect was effectively inhibited by HDAC5, but was significantly enhanced by p300. The results suggest that both HDAC5 and p300 are involved in MRTF-A-mediated effect on neuronal apoptosis during ischemia/reperfusion injury, but with opposite effects.

SIRT2 mediated antitumor effects of shikonin on metastatic colorectal cancer.

SIRT2 is involved in the development of a variety of cancers. Shikonin is a natural compound that is known to have antitumor effects. This study aims to assess the effects of shikonin on the development and metastatic progression of colorectal cancer (CRC) through regulation of SIRT2 expression and whether this effect is related to the phosphorylation of extracellular signal-regulated kinases (ERKs). The results demonstrated that SIRT2 is downregulated in CRC biopsy samples (n=31) compared with the adjacent non-cancerous tissues (ANCT, n=26). Furthermore, CRC metastases were positive for SIRT2 despite a lack of expression in the primary tumor. In addition, data from an in vitro assay revealed that overexpression of SIRT2 inhibited the proliferation and metastatic progression of SW480 cells while blocking of SIRT2 expression induced the proliferation and metastatic progression of HT29 cells. Shikonin inhibited the viability, migration and invasion of SW480 cells and it also inhibited the tumor growth in the nude mice model; while AGK2 (a specific inhibitor of SIRT2) reversed these effects. Epidermal growth factor (EGF, an activator of ERK) and ERK-overexpression inhibited the effects of shikonin on SIRT2 expression, proliferation and metastasis in SW480 cells. However, this proliferative effect of EGF was reversed by SIRT2 overexpression. In conclusion, these results suggest that SIRT2 is a new therapeutic target for the treatment of CRC. The antitumor effects of shikonin on CRC seem to be mediated by SIRT2 upregulation via phospho-ERK inhibition.

The role of myocardin-related transcription factor-A in Aß25-35 induced neuron apoptosis and synapse injury.

Myocardin-related transcription factor-A (MRTF-A) highly expressed in brain has been demonstrated to promote neuronal survival via regulating the transcription of related target genes as a powerful co-activator of serum response factor (SRF). However, the role of MRTF-A in Alzheimer's disease (AD) is still unclear. Here, we showed that MRTF-A was significantly downregulated in cortex of the Aß25-35-induced AD rats, which played a key role in Aß25-35 induced cerebral neuronal degeneration in vitro. Bilateral intracerebroventricular injection of Aß25-35 caused significantly MRTF-A expression decline in cortex of rats, along with significant neuron apoptosis and plasticity damage. In vitro, transfection of MRTF-A into primary cultured cortical neurons prevented Aß25-35 induced neuronal apoptosis and synapses injury. And luciferase reporter assay determined that MRTF-A could bind to and enhance the transactivity of the Mcl-1 (Myeloid cell leukemia-1) and Arc (activity-regulated cytoskeletal-associated protein) promoters by activating the key CArG box element. These data demonstrated that the decreasing of endogenous MRTF-A expression might contribute to the development of AD, whereas the upregulation MRTF-A in neurons could effectively reduce Aß25-35 induced synapse injury and cell apoptosis. And the underlying mechanism might be partially due to MRTF-A-mediated the transcription and expression of Mcl-1 and Arc by triggering the CArG box.

Baicalin inhibiting cerebral ischemia/hypoxia-induced neuronal apoptosis via MRTF-A-mediated transactivity.

Baicalin has been shown to provide the neuroprotective effect by alleviating cerebral ischemia injury. However, little's known about the underlying mechanism. Here, a cerebral artery occlusion (MACO)/reperfusion rat model and rat primary cortical neuron culture exposed to hydrogen peroxide (H2O2) were established to evaluate the effect of baicalin on ischemia-induced neuronal apoptosis. We found baicalin can significantly less neurological deficit and reduced infarct volume in vivo. And it efficiently inhibited neuronal apoptosis in vivo and vitro, which was especially characterized by the enhancing of transcription and expression of myeloid cell leukemia-1 (MCL-1) and B-cell lymphoma-2 (BCL-2) in a dose-dependent manner. Furthermore, Baicalin markedly increased myocardin-related transcription factor-A (MRTF-A) level either in ischemic hemisphere or in primary cortical neuron cultures, whiles the anti-apoptosis effect of baicalin was significantly inhibited by transfected with the small interfering RNA of MRTF-A (MRTF-A siRNA) in primary cortical neuron cultures. The luciferase assays also indicated baicalin enhanced the transactivity of MCL-1 and BCL-2 promoter by activating the key CArG box (CC [A/T] 6GG) element, which was reduced by MRTF-A siRNA, suggesting MRTF-A may participate the anti-apoptosis effect of baicalin, and MRTF-A was involved in the transcriptional activity of MCL-1 and BCL-2 that was induced by baicalin. LY294002 (phosphatidylinositol-3 kinase (PI3K) inhibitor) and PD98059 (extracellular signal regulates kinase-1/2 (ERK1/2) inhibitor) obviously reduced baicalin-induced MRTF-A expression and transactivity and expression of MCL-1 and BCL-2, which further abolished the anti-apoptotic effect of baicalin on neuronal apoptosis. Taken together, our data provided the evidence demonstrating the neuroprotective effect of baicalin partially due to MRTF-A-mediated transactivity and expression of MCL-1 and BCL-2 by triggering the CArG box, which might be controlled by the activation of PI3K and ERK1/2.

Design, synthesis and evaluation of nitric oxide releasing derivatives of 3-n-butylphthalide as antiplatelet and antithrombotic agents.

Novel nitric oxide (NO) releasing derivatives (7a-7l) of 3-n-butylphthalide (NBP) were designed and synthesized. Compound 7e inhibited the adenosine diphosphate (ADP), thrombin (TH) and arachidonic acid (AA)-induced in vitro platelet aggregation, superior to NBP and aspirin, released moderate levels of NO, and improved aqueous solubility relative to NBP. Furthermore, 7e exhibited greater antithrombotic activity than NBP and aspirin in rats, and protected against collagen and adrenaline-induced thrombosis in mice. Therefore, NO-releasing NBP derivatives possessed potent antiplatelet aggregation and antithrombotic activity. Our findings may aid in the design of new therapeutic agents for the treatment of thrombosis-related ischemic stroke.

ZJM-289, a novel nitric oxide donor, alleviates the cerebral ischaemic-reperfusion injury in rats.

1. Current studies indicate that nitric oxide (NO) plays a dual role as both a protective and pathogenic factor in focal cerebral ischaemia depending on the level, location, source and environment. The present study hypothesized that the NO donor ZJM-289 could inhibit cerebral ischaemia-reperfusion (I/R) injury and investigated the mechanism of the beneficial events. 2. Adult male rats were randomly divided into four groups: (i) sham operated; (ii) I/R (ischaemia for 90 min and reperfusion for 24 h) treated with vehicle; (iii) I/R treated with 0.1 mmol/kg body weight ZJM-289; and (iv) I/R treated with 0.2 mmol/kg body weight ZJM-289. We evaluated the changes in brain infarction, brain-water content, neurological deficits and histopathology. Western blot analysis was used to study the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the brain after I/R. The levels of NO and cyclic guanosine monophosphate (cGMP) were also determined. 3. ZJM-289 reduced infarct volume and brain-water content in ischemic brains and promoted functional recovery. Western blotting showed significant inhibition of nNOS in ZJM-289 treated rats compared with untreated rats. However, eNOS expression in the ischemic brain was enhanced in the ZJM-289 groups. The cGMP and NO levels increased in the ZJM-289 groups after I/R. The study showed that ZJM-289 could alleviate cerebral injury after I/R through inhibition of nNOS and stimulation of the NO/soluble guanylate cyclase/cGMP pathway. Therefore, a suitable NO donor might be an effective candidate for the treatment of acute stroke by neuroprotection.