Second-generation antipsychotic quetiapine blocks voltage-dependent potassium channels in coronary arterial smooth muscle cells.

Voltage-dependent K+ (Kv) channels play an important role in restoring the membrane potential to its resting state, thereby maintaining vascular tone. In this study, native smooth muscle cells from rabbit coronary arteries were used to investigate the inhibitory effect of quetiapine, an atypical antipsychotic agent, on Kv channels. Quetiapine showed a concentration-dependent inhibition of Kv channels, with an IC50 of 47.98 ± 9.46 µM. Although quetiapine (50 µM) did not alter the steady-state activation curve, it caused a negative shift in the steady-state inactivation curve. The application of 1 and 2 Hz train steps in the presence of quetiapine significantly increased the inhibition of Kv current. Moreover, the recovery time constants from inactivation were prolonged in the presence of quetiapine, suggesting that its inhibitory action on Kv channels is use (state)-dependent. The inhibitory effects of quetiapine were not significantly affected by pretreatment with Kv1.5, Kv2.1, and Kv7 subtype inhibitors. Based on these findings, we conclude that quetiapine inhibits Kv channels in both a concentration- and use (state)-dependent manner. Given the physiological significance of Kv channels, caution is advised in the use of quetiapine as an antipsychotic due to its potential side effects on cardiovascular Kv channels.

Drug Repositioning via Graph Neural Networks: Identifying Novel JAK2 Inhibitors from FDA-Approved Drugs through Molecular Docking and Biological Validation.

The increasing utilization of artificial intelligence algorithms in drug development has proven to be highly efficient and effective. One area where deep learning-based approaches have made significant contributions is in drug repositioning, enabling the identification of new therapeutic applications for existing drugs. In the present study, a trained deep-learning model was employed to screen a library of FDA-approved drugs to discover novel inhibitors targeting JAK2. To accomplish this, reference datasets containing active and decoy compounds specific to JAK2 were obtained from the DUD-E database. RDKit, a cheminformatic toolkit, was utilized to extract molecular features from the compounds. The DeepChem framework's GraphConvMol, based on graph convolutional network models, was applied to build a predictive model using the DUD-E datasets. Subsequently, the trained deep-learning model was used to predict the JAK2 inhibitory potential of FDA-approved drugs. Based on these predictions, ribociclib, topiroxostat, amodiaquine, and gefitinib were identified as potential JAK2 inhibitors. Notably, several known JAK2 inhibitors demonstrated high potential according to the prediction results, validating the reliability of our prediction model. To further validate these findings and confirm their JAK2 inhibitory activity, molecular docking experiments were conducted using tofacitinib-an FDA-approved drug for JAK2 inhibition. Experimental validation successfully confirmed our computational analysis results by demonstrating that these novel drugs exhibited comparable inhibitory activity against JAK2 compared to tofacitinib. In conclusion, our study highlights how deep learning models can significantly enhance virtual screening efforts in drug discovery by efficiently identifying potential candidates for specific targets such as JAK2. These newly discovered drugs hold promises as novel JAK2 inhibitors deserving further exploration and investigation.

Machine Learning-Based Drug Repositioning of Novel Janus Kinase 2 Inhibitors Utilizing Molecular Docking and Molecular Dynamic Simulation.

Machine learning algorithms have been increasingly applied in drug development due to their efficiency and effectiveness. Machine learning-based drug repurposing can contribute to the identification of novel therapeutic applications for drugs with other indications. The current study used a trained machine learning model to screen a vast chemical library for new JAK2 inhibitors, the biological activities of which were reported. Reference JAK2 inhibitors, comprising 1911 compounds, have experimentally determined IC50 values. To generate the input to the machine learning model, reference compounds were subjected to RDKit, a cheminformatic toolkit, to extract molecular descriptors. A Random Forest Regression model from the Scikit-learn machine learning library was applied to obtain a predictive regression model and to analyze each molecular descriptor's role in determining IC50 values in the reference data set. Then, IC50 values of the library compounds, comprised of 1,576,903 compounds, were predicted using the generated regression model. Interestingly, some compounds that exhibit high IC50 values from the prediction were reported to possess JAK inhibition activity, which indicates the limitations of the prediction model. To confirm the JAK2 inhibition activity of predicted compounds, molecular docking and molecular dynamics simulation were carried out with the JAK inhibitor reference compound, tofacitinib. The binding affinity of docked compounds in the active region of JAK2 was also analyzed by the gmxMMPBSA approach. Furthermore, experimental validation confirmed the results from the computational analysis. Results showed highly comparable outcomes concerning tofacitinib. Conclusively, the machine learning model can efficiently improve the virtual screening of drugs and drug development.

The inhibitory effects of pimozide, an antipsychotic drug, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells.

Pimozide is an antipsychotic drug used to treat chronic psychosis, such as Tourette's syndrome. Despite its widespread clinical use, pimozide can cause unexpected adverse effects, including arrhythmias. However, the adverse effects of pimozide on vascular K+ channels have not yet been determined. Therefore, we investigated the effects of pimozide on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Pimozide concentration-dependently inhibited the Kv currents with an IC50 value of 1.78 ± 0.17 µM and a Hill coefficient of 0.90 ± 0.05. The inhibitory effect on the Kv current by pimozide was highly voltage-dependent in the voltage range of Kv channel activation, and additive inhibition of the Kv current by pimozide was observed in the full activation voltage range. The decay rate of inactivation was significantly accelerated by pimozide. Pimozide shifted the inactivation curve to a more negative potential. The recovery time constant from inactivation increased in the presence of pimozide. Furthermore, pimozide-induced inhibition of the Kv current was augmented by applying train pulses. Although pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and the Kv7 subtype inhibitor linopirdine did not alter the degree of pimozide-induced inhibition of the Kv currents, pretreatment with the Kv1.5 channel inhibitor DPO-1 reduced the inhibitory effects of pimozide on Kv currents. Pimozide induced membrane depolarization. We conclude that pimozide inhibits Kv currents in voltage-, time-, and use (state)-dependent manners. Furthermore, the major Kv channel target of pimozide is the Kv1.5 channel.

EWS/FLI1 Characterization, Activation, Repression, Target Genes and Therapeutic Opportunities in Ewing Sarcoma.

Despite their clonal origins, tumors eventually develop into complex communities made up of phenotypically different cell subpopulations, according to mounting evidence. Tumor cell-intrinsic programming and signals from geographically and temporally changing microenvironments both contribute to this variability. Furthermore, the mutational load is typically lacking in childhood malignancies of adult cancers, and they still exhibit high cellular heterogeneity levels largely mediated by epigenetic mechanisms. Ewing sarcomas represent highly aggressive malignancies affecting both bone and soft tissue, primarily afflicting adolescents. Unfortunately, the outlook for patients facing relapsed or metastatic disease is grim. These tumors are primarily fueled by a distinctive fusion event involving an FET protein and an ETS family transcription factor, with the most prevalent fusion being EWS/FLI1. Despite originating from a common driver mutation, Ewing sarcoma cells display significant variations in transcriptional activity, both within and among tumors. Recent research has pinpointed distinct fusion protein activities as a principal source of this heterogeneity, resulting in markedly diverse cellular phenotypes. In this review, we aim to characterize the role of the EWS/FLI fusion protein in Ewing sarcoma by exploring its general mechanism of activation and elucidating its implications for tumor heterogeneity. Additionally, we delve into potential therapeutic opportunities to target this aberrant fusion protein in the context of Ewing sarcoma treatment.

Computational Exploration of the Effects of Mutations on GABA Aminotransferase in GABA Aminotransferase Deficiency.

Gamma-aminobutyric acid (GABA) transaminase-also called GABA aminotransferase (GABA-AT)-deficiency is a rare autosomal recessive disorder characterized by a severe neonatal-infantile epileptic encephalopathy with symptoms such as seizures, hypotonia, hyperreflexia, developmental delay, and growth acceleration. GABA transaminase deficiency is caused by mutations in GABA-AT, the enzyme responsible for the catabolism of GABA. Mutations in multiple locations on GABA-AT have been reported and their locations have been shown to influence the onset of the disease and the severity of symptoms. We examined how GABA-AT mutations influence the structural stability of the enzyme and GABA-binding affinity using computational methodologies such as molecular dynamics simulation and binding free energy calculation to understand the underlying mechanism through which GABA-AT mutations cause GABA-AT deficiency. GABA-AT 3D model depiction was carried out together with seven individual mutated models of GABA-AT. The structural stability of all the predicted models was analyzed using several tools and web servers. All models were evaluated based on their phytochemical values. Additionally, 100 ns MD simulation was carried out and the mutated models were evaluated using RMSD, RMSF, Rg, and SASA. gmxMMPBSA free energy calculation was carried out. Moreover, RMSD and free energy calculations were also compared with those obtained using online web servers. Our study demonstrates that P152S, Q296H, and R92Q play a more critical role in the structural instability of GABA-AT compared with the other mutated models: G465R, L211F, L478P, and R220K.

Discovery of GABA Aminotransferase Inhibitors via Molecular Docking, Molecular Dynamic Simulation, and Biological Evaluation.

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades γ-aminobutyric (GABA) in the brain. GABA is an important inhibitory neurotransmitter that plays important neurological roles in the brain. Therefore, GABA-AT is an important drug target that regulates GABA levels. Novel and potent drug development to inhibit GABA-AT is still a very challenging task. In this study, we aimed to devise novel and potent inhibitors against GABA-AT using computer-aided drug design (CADD) tools. Since the crystal structure of human GABA-AT was not yet available, we utilized a homologous structure derived from our previously published paper. To identify highly potent compounds relative to vigabatrin, an FDA-approved drug against human GABA-AT, we developed a pharmacophore analysis protocol for 530,000 Korea Chemical Bank (KCB) compounds and selected the top 50 compounds for further screening. Preliminary biological analysis was carried out for these 50 compounds and 16 compounds were further assessed. Subsequently, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were carried out. In the results, four predicted compounds, A07, B07, D08, and H08, were found to be highly potent and were further evaluated by a biological activity assay to confirm the results of the GABA-AT activity inhibition assay.

Investigation of Flavonoid Scaffolds as DAX1 Inhibitors against Ewing Sarcoma through Pharmacoinformatic and Dynamic Simulation Studies.

Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1) is an orphan nuclear receptor encoded by the NR0B1 gene. The functional study showed that DAX1 is a physiologically significant target for EWS/FLI1-mediated oncogenesis, particularly Ewing Sarcoma (ES). In this study, a three-dimensional DAX1 structure was modeled by employing a homology modeling approach. Furthermore, the network analysis of genes involved in Ewing Sarcoma was also carried out to evaluate the association of DAX1 and other genes with ES. Moreover, a molecular docking study was carried out to check the binding profile of screened flavonoid compounds against DAX1. Therefore, 132 flavonoids were docked in the predicted active binding pocket of DAX1. Moreover, the pharmacogenomics analysis was performed for the top ten docked compounds to evaluate the ES-related gene clusters. As a result, the five best flavonoid-docked complexes were selected and further evaluated by Molecular Dynamics (MD) simulation studies at 100 ns. The MD simulation trajectories were evaluated by generating RMSD, hydrogen bond plot analysis, and interaction energy graphs. Our results demonstrate that flavonoids showed interactive profiles in the active region of DAX1 and can be used as potential therapeutic agents against DAX1-mediated augmentation of ES after in-vitro and in-vivo evaluations.

Computational Exploration of Licorice for Lead Compounds against Plasmodium vivax Duffy Binding Protein Utilizing Molecular Docking and Molecular Dynamic Simulation.

Plasmodium vivax (P. vivax) is one of the human's most common malaria parasites. P. vivax is exceedingly difficult to control and eliminate due to the existence of extravascular reservoirs and recurring infections from latent liver stages. Traditionally, licorice compounds have been widely investigated against viral and infectious diseases and exhibit some promising results to combat these diseases. In the present study, computational approaches are utilized to study the effect of licorice compounds against P. vivax Duffy binding protein (DBP) to inhibit the malarial invasion to human red blood cells (RBCs). The main focus is to block the DBP binding site to Duffy antigen receptor chemokines (DARC) of RBC to restrict the formation of the DBP-DARC complex. A molecular docking study was performed to analyze the interaction of licorice compounds with the DARC binding site of DBP. Furthermore, the triplicates of molecular dynamic simulation studies for 100 ns were carried out to study the stability of representative docked complexes. The leading compounds such as licochalcone A, echinatin, and licochalcone B manifest competitive results against DBP. The blockage of the active region of DBP resulting from these compounds was maintained throughout the triplicates of 100 ns molecular dynamic (MD) simulation, maintaining stable hydrogen bond formation with the active site residues of DBP. Therefore, the present study suggests that licorice compounds might be good candidates for novel agents against DBP-mediated RBC invasion of P. vivax.

Exploration of Flavonoids as Lead Compounds against Ewing Sarcoma through Molecular Docking, Pharmacogenomics Analysis, and Molecular Dynamics Simulations.

Ewing sarcoma (ES) is a highly malignant carcinoma prevalent in children and most frequent in the second decade of life. It mostly occurs due to t(11;22) (q24;q12) translocation. This translocation encodes the oncogenic fusion protein EWS/FLI (Friend leukemia integration 1 transcription factor), which acts as an aberrant transcription factor to deregulate target genes essential for cancer. Traditionally, flavonoids from plants have been investigated against viral and cancerous diseases and have shown some promising results to combat these disorders. In the current study, representative flavonoid compounds from various subclasses are selected and used to disrupt the RNA-binding motif of EWS, which is required for EWS/FLI fusion. By blocking the RNA-binding motif of EWS, it might be possible to combat ES. Therefore, molecular docking experiments validated the binding interaction patterns and structural behaviors of screened flavonoid compounds within the active region of the Ewing sarcoma protein (EWS). Furthermore, pharmacogenomics analysis was used to investigate potential drug interactions with Ewing sarcoma-associated genes. Finally, molecular dynamics simulations were used to investigate the stability of the best selected docked complexes. Taken together, daidzein, kaempferol, and genistein exhibited a result comparable to ifosfamide in the proposed in silico study and can be further analyzed as possible candidate compounds in biological in vitro studies against ES.

Inhibitory effects of the atypical antipsychotic, clozapine, on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an half-inhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapine-induced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentrationand use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapine-induced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.

Inhibition of voltage-dependent K+ channels by antimuscarinic drug fesoterodine in coronary arterial smooth muscle cells.

Fesoterodine, an antimuscarinic drug, is widely used to treat overactive bladder syndrome. However, there is little information about its effects on vascular K+ channels. In this study, voltage-dependent K+ (Kv) channel inhibition by fesoterodine was investigated using the patch-clamp technique in rabbit coronary artery. In whole-cell patches, the addition of fesoterodine to the bath inhibited the Kv currents in a concentration-dependent manner, with an IC50 value of 3.19 ± 0.91 µM and a Hill coefficient of 0.56 ± 0.03. Although the drug did not alter the voltage-dependence of steady-state activation, it shifted the steady-state inactivation curve to a more negative potential, suggesting that fesoterodine affects the voltage-sensor of the Kv channel. Inhibition by fesoterodine was significantly enhanced by repetitive train pulses (1 or 2 Hz). Furthermore, it significantly increased the recovery time constant from inactivation, suggesting that the Kv channel inhibition by fesoterodine is use (state)-dependent. Its inhibitory effect disappeared by pretreatment with a Kv 1.5 inhibitor. However, pretreatment with Kv2.1 or Kv7 inhibitors did not affect the inhibitory effects on Kv channels. Based on these results, we conclude that fesoterodine inhibits vascular Kv channels (mainly the Kv1.5 subtype) in a concentration- and use (state)-dependent manner, independent of muscarinic receptor antagonism.

3,4,5-Trihydroxycinnamic Acid Inhibits LPS-Induced Inflammatory Response by Increasing SIRT1 Expression in Human Umbilical Vein Endothelial Cells.

3,4,5-Trihydroxycinnamic acid (THC) has been demonstrated to exert anti-inflammatory activities in LPS-induced RAW264.7 murine macrophage cells and in LPS-induced septic mice. However, the effect of THC on the inflammatory response in vascular endothelial cells has not been clearly examined. The goal of the present study was to elucidate the anti-inflammatory properties of THC and its underlying mechanism in LPS-challenged human umbilical vein endothelial cells (HUVECs). THC significantly suppressed LPS-induced interleukin-1ß production and intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression and significantly decreased LPS-induced nuclear factor-κB activation by attenuating p65 phosphorylation and inhibitor of kappa B degradation. To understand the underlying mechanism of the anti-inflammatory effect of THC, the involvement of the sirtuin 1 (SIRT1) signaling pathway was examined. THC resulted in increased expression of SIRT1 in LPS-challenged HUVECs. Among the downstream molecular targets of SIRT1, the level of LPS-induced acetylated p53 was significantly decreased by THC treatment, whereas no noticeable change was observed in the levels of forkhead box O3 and peroxisome proliferator activated receptor gamma coactivator 1 alpha. In conclusion, the results clearly demonstrate that THC possesses anti-inflammatory properties by increasing SIRT1 expression and subsequent suppression of p53 activation in LPS-challenged HUVECs.

Isorhamnetin-3-O-Glucuronide Suppresses JNK and p38 Activation and Increases Heme-Oxygenase-1 in Lipopolysaccharide-Challenged RAW264.7 Cells.

Preclinical Research Isorhanmetin (ISH) exhibits a wide range of biological properties including anticancer, anti-oxidant and anti-inflammatory activities. However, the pharmacological properties of isorhamnetin-3-O-glucuronide (IG), a glycoside derivative of ISH, have not been extensively examined. The objective of this study was to examine the anti-inflammatory properties of IG and its underlying mechanism in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells in comparison with its aglycone, ISH. IG suppressed LPS-induced extracellular secretion of the proinflammatory mediators, nitric oxide (NO) and PGE2 , and proinflammatory protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2. IG also increased expression of heme oxygenase-1 (HO-1). IG attenuated LPS-induced activation of c-Jun N-terminal kinase (JNK) and p38 in a concentration-dependent manner with negligible suppression of extracellular signal-regulated kinases (ERK) phosphorylation. In conclusion, this study demonstrates that IG exerts anti-inflammatory activity by increasing HO-1 expression and by suppressing JNK and p38 signaling pathways in LPS-challenged RAW264.7 macrophage cells. Drug Dev Res 77 : 143-151, 2016. © 2016 Wiley Periodicals, Inc.

Apocynin Suppresses Lipopolysaccharide-Induced Inflammatory Responses Through the Inhibition of MAP Kinase Signaling Pathway in RAW264.7 Cells.

Apocynin, an inhibitor of NADPH oxidase, exhibits anti-inflammatory properties in ulcerative colitis. However, the underlying mechanism by which apocynin exerts this effect has not been clearly demonstrated. The objective of this study was to elucidate the anti-inflammatory mechanism of apocynin in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells. Apocynin inhibited LPS-induced extracellular secretion of the pro-inflammatory mediators, nitric oxide (NO) and PGE2 and the expression of inducible nitric oxide synthase and cyclooxygenase-2. Apocynin also suppressed LPS-induced secretion of the pro-inflammatory cytokine, tumor necrosis factor-α and LPS-induced degradation of IκB, which retains NF-κB in the cytoplasm, consequently inhibiting the transcription of pro-inflammatory genes by NF-κB in the nucleus. To elucidate the underlying anti-inflammatory mechanism of apocynin, the involvement of the mitogen-activated protein (MAP) kinases, c-jun N-terminal kinase, extracellular signal-regulated kinases, and p38 was examined. Apocynin attenuated LPS-induced activation of all three MAP kinases in a concentration-dependent manner. The present study demonstrates apocynin exerts anti-inflammatory activity via the suppression of MAP kinase signaling pathways in LPS-challenged RAW264.7 macrophage cells. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc.

Sequestration of sorcin by aberrant forms of tau results in the defective calcium homeostasis.

Neurofi brillary tangles (NFTs) of microtubule-associated protein tau are a pathological hallmark of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress has been known to be involved in the pathogenesis of AD. However, the exact role of ER stress in tau pathology has not yet been clearly elucidated. In present study, the possible relationship between tau pathology and ER stress was examined in terms of sorcin, which is a calcium binding protein and plays an important role in calcium homeostasis. Our previous yeast two hybrid study showed that sorcin is a novel tau interacting protein. Caspase-3-cleaved tau (T4C3) showed significantly increased tau-sorcin interaction compared to wild type tau (T4). Thapsigargin-induced ER stress and co-expression of constitutively active GSK3ß (GSK3ß-S9A) also exhibited significantly increased tau-sorcin interactions. T4C3-expressing cells showed potentiated thapsigargin-induced apoptosis and disruption of intracellular calcium homeostasis compared to T4-expressing cells. Overexpression of sorcin signifi cantly attenuated thapsigargin-induced apoptosis and disruption of calcium homeostasis. In contrary, siRNA-mediated knock-down of sorcin showed significantly increased thapsigargin-induced apoptosis and disruption of calcium homeostasis. These data strongly suggest that sequestration of sorcin by aberrant forms of tau compromises the function of sorcin, such as calcium homeostasis and cellular resistance by ER stress, which may consequently result in the contribution to the progression of AD.

3,4,5-Trihydroxycinnamic acid increases heme-oxygenase-1 (HO-1) and decreases macrophage infiltration in LPS-induced septic kidney.

We previously demonstrated that 3,4,5-trihydorxycinnamic acid (THC), a derivative of hydroxycinnamic acids, possesses protective effect in lipopolysaccharide (LPS)-induced endotoxemia models. However, the effects of THC in LPS-induced septic kidney are still unclear. Therefore, the present study was carried out to examine the effects of THC in LPS-challenged septic kidney using mesangial cell line and Balb/c mice. THC pretreatment effectively inhibited LPS-induced macrophage infiltration and the secretion of pro-inflammatory cytokines in the kidney of LPS-challenged animals. Pretreatment of rat mesangial cells with THC significantly attenuated LPS-induced PGE2 production and COX-2 expression. THC also significantly suppressed LPS-induced expression of MCP-1 in LPS-activated septic kidney and rat mesangial cells. In addition, THC significantly attenuated LPS-induced degradation of IκB-α in LPS-induced rat mesangial cells. THC also increased the expression of heme oxygenase-1 (HO-1) in LPS-challenged septic kidney and mesangial cells. Multiple signaling pathways including p38 and AKT have been observed to be involved in the THC-induced activation of HO-1 expression. The present data clearly demonstrate that THC protects LPS-challenged septic kidney by decreasing macrophage infiltration and increasing HO-1 expression, suggesting that THC might be a valuable therapeutic agent for compromised kidney in sepsis.

3,4,5-trihydroxycinnamic acid inhibits lipopolysaccharide (LPS)-induced inflammation by Nrf2 activation in vitro and improves survival of mice in LPS-induced endotoxemia model in vivo.

NF-E2-related factor 2 (Nrf2) has been demonstrated to be a key transcription factor regulating the anti-inflammatory genes including heme oxygenase-1 (HO-1) in experimental sepsis models. Based on the fact that 3,4,5-trihydorxycinnamic acid (THC) has been reported to possess anti-inflammatory properties in BV2 microglial cells, the possible effects of THC and its underlying mechanism was examined against lipopolysaccharide (LPS)-induced RAW 264.7 cell culture and septic mouse models. Pretreatment of RAW 264.7 cells with THC significantly attenuated LPS-induced NO, PGE2 production, and expression of iNOS and COX-2. THC also significantly suppressed LPS-induced release of pro-inflammatory cytokines and degradation of IκB-α. Increased phosphorylation of Nrf2 and nuclear translocation of Nrf2 were observed with THC treatment with consequent expression of HO-1. The data demonstrated that multiple signaling pathways including Akt, p38, and PKC are involved in the THC-induced activation of Nrf2/HO-1 pathway. Treatment of THC resulted in significantly increased survival of LPS-induced septic mice. THC also significantly ameliorated LPS-induced septic features such as hypothermia and increased vascular leakage. In accordance with the data from cell culture model, THC exhibited increased expression of HO-1 in kidney and decreased serum level of pro-inflammatory mediators such as TNF-α, IL-1ß, and NO. Taken together, the present study for the first time demonstrates that THC inhibits inflammation in LPS-induced RAW264.7 cells by Nrf2 activation and improves survival of mice in LPS-induced endotoxemia model.

Phosphorylation of Akt Mediates Anti-Inflammatory Activity of 1-p-Coumaroyl ß-D-Glucoside Against Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells.

Hydroxycinnamic acids have been reported to possess numerous pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor properties. However, the biological activity of 1-p-coumaroyl ß-D-glucoside (CG), a glucose ester derivative of p-coumaric acid, has not been clearly examined. The objective of this study is to elucidate the anti-inflammatory action of CG in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In the present study, CG significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE2 and the protein expression of iNOS and COX-2. CG also inhibited LPS-induced secretion of pro-inflammatory cytokines, IL-1ß and TNF-α. In addition, CG significantly suppressed LPS-induced degradation of IκB. To elucidate the underlying mechanism by which CG exerts its anti-inflammatory action, involvement of various signaling pathways were examined. CG exhibited significantly increased Akt phosphorylation in a concentration-dependent manner, although MAPKs such as Erk, JNK, and p38 appeared not to be involved. Furthermore, inhibition of Akt/PI3K signaling pathway with wortmannin significantly, albeit not completely, abolished CG-induced Akt phosphorylation and anti-inflammatory actions. Taken together, the present study demonstrates that Akt signaling pathway might play a major role in CG-mediated anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells.

Discovery of Novel Aldose Reductase Inhibitors via the Integration of Ligand-Based and Structure-Based Virtual Screening with Experimental Validation.

Aldose reductase plays a central role in diabetes mellitus (DM) associated complications by converting glucose to sorbitol, resulting in a harmful increase of reactive oxygen species (ROS) in various tissues, such as the heart, vasculature, neurons, eyes, and kidneys. We employed a comprehensive approach, integrating both ligand- and structure-based virtual screening followed by experimental validation. Initially, candidate compounds were extracted from extensive drug and chemical libraries using the DeepChem's GraphConvMol algorithm, leveraging its capacity for robust molecular feature representation. Subsequent refinement employed molecular docking and molecular dynamics (MD) simulations, which are crucial for understanding compound-receptor interactions and dynamic behavior in a simulated physiological environment. Finally, the candidate compounds were subjected to experimental validation of their biological activity using an aldose reductase inhibitor screening kit. The comprehensive approach led to the identification of a promising compound, demonstrating significant potential as an aldose reductase inhibitor. This comprehensive approach not only yields a potential therapeutic intervention for DM-related complications but also establishes an integrated protocol for drug development, setting a new benchmark in the field.