5-LOX Protein Research Articles

Study on mechanism of Zishen Pill treating benign prostatic hyperplasia based on serum pharmacochemistry and network pharmacology

Zishen Pill (ZSP) is a traditional Chinese medicine that is frequently used to treat Benign Prostatic Hyperplasia (BPH), however its specific mechanism of action and active ingredients are yet unknown. We used a combination of serum pharmacochemistry and network pharmacology and a series of biochemical assays to explore the action mechanism of ZSP in the treatment of BPH. The BPH rat model was created using testosterone propionate, and following oral ZSP administration, the components of ZSP in rat serum were detected by UPLC-Q-Exactive Orbitrap/MS method. A "component-target-disease" network and PPI networks were constructed on this foundation. The primary mechanism of ZSP decreasing BPH in rats was studied by KEGG pathway and GO analysis. Finally, the potential pathways and key targets were further verified in vivo by molecular biology and immunological methods. 46 substances were charactered from rat serum, and 164 anti-BPH targets were screened from the database. According to network pharmacology, the primary targets were CASP3, STAT3, JUN, and PTGS2/COX2. Three related pathways (PI3K/Akt signaling pathway, AGE-RAGE signaling pathway and EGFR tyrosine kinase inhibitor resistance) were closely related to the therapeutic effects of ZSP. The findings of molecular biology demonstrated that ZSP may bring Bcl-2, BAX, CASP3, COX2, and 5LOX protein and gene expression in BPH rats appreciably closer to that of normal rats. Additionally, ZSP can lessen the expression of inflammatory cytokines in BPH rats, including VEGF, TNF-α, CCL5, and interleukin. ConclusionThe above results suggest that ZSP may reduce BPH through inflammation/immunity and apoptosis/proliferation-related pathways. This study offers a fresh approach to investigate the basic pharmacological effects and mechanism of ZSP in the treatment of BPH.

Effects of inhibition of 5-lipoxygenase and 12-lipoxygenase pathways on skeletal muscle fiber regeneration

We investigated the effect of inhibition of 5-lipoxigenase (LOX) and 12-LOX pathways on the regeneration of skeletal muscle fibers after injury induced by a myotoxin (MTX) phospholipase A2 from snake venom in an in vivo experimental model. Gastrocnemius muscles of mice injected with MTX presented an increase in 5-LOX protein expression, while 12-LOX was found to be a constitutive protein of skeletal muscle. Animals that received oral treatments with 5-LOX inhibitor MK886 or 12-LOX inhibitor baicalein 30 min and 48 h after MTX-induced muscle injury showed a reduction in the inflammatory process characterized by a significant decrease of cell influx and injured fibers in the degenerative phase (6 and 24 h after injury). In the beginning of the regeneration process (3 days), mice that received MK886 showed fewer new basophilic fibers, suggesting fewer proliferative events and myogenic cell fusion. Furthermore, in the progression of tissue regeneration (14–21 days), the mice treated with 5-LOX inhibitor presented a lower quantity of central nucleus fibers and small-caliber fibers, culminating in a muscle that is more resistant to the stimulus of fatigue during muscle regeneration with a predominance of slow fibers. In contrast, animals early treated with the 12-LOX inhibitor presented functional fibers with higher diameters, less resistant to fatigue and predominance of fast heavy-chain myosin fibers as observed in control animals. These effects were accompanied by an earlier expression of myogenic factor MyoD. Our results suggest that both 5-LOX and 12-LOX pathways represent potential therapeutic targets for muscle regeneration. It appears that inhibition of the 5-LOX pathway represses only the degenerative process by reducing tissue inflammation levels. Meanwhile, inhibition of the 12-LOX pathway also favors the anticipation of maturation and earlier recovery of muscle fiber activity function after injury.

Daidzein phosphorylates and activates 5-lipoxygenase via the MEK/ERK pathway: a mechanism for inducing the production of 5-lipoxygenase metabolite that inhibit influenza virus intracellular replication

We previously reported that the soy isoflavone daidzein (Dz) suppresses the intracellular replication of influenza virus and that arachidonic acid-derived oxidation product via lipid oxidase 5-lipoxygenase (5-LOX) is involved in its antiviral effect. The activation of 5-LOX by Dz triggers anti-influenza activity; however, the mechanism of activation of 5-LOX remains unclear. Therefore, in this study, we aimed to clarify the activation mechanism using human monocyte-derived THP-1 cells differentiated using phorbol 12-myristate 13-acetate. THP-1 cells expressed 5-LOX endogenously and Dz did not induce 5-LOX expression. However, 8 h after treatment with Dz, the amount of 5-hydroxyeicosatetraenoic acid (5-HETE), an arachidonic acid oxidation product via 5-LOX, increased significantly suggesting that the enzyme is activated regardless of changes in 5-LOX protein levels. Intracellular Ca2+ content, ATP concentration, 5-LOX protein phosphorylation, and 5-LOX intracellular localization are known 5-LOX activation factors. The intracellular Ca2+ and ATP concentrations were not affected by Dz treatment. The enzymatic activity of 5-LOX is regulated by the phosphorylation of three serine residues and four tyrosine residues. Pretreatment with inhibitors of each kinase revealed that Dz-induced 5-HETE production was suppressed by the MEK/ERK inhibitor. 5-LOX in which the Ser663 residue was phosphorylated was found to be increased in the nuclear fraction of Dz-treated THP-1 cells. Furthermore, immunocytochemistry showed that 5-LOX translocates to the nuclear envelope following Dz treatment. These results indicate that Dz activates 5-LOX by phosphorylating Ser663 via the MEK/ERK pathway. Thus, these results demonstrate that Dz exerts anti-influenza virus activity via the MEK/ERK signal transduction pathway.

5-Lipoxygenase Contributes to Benzo[a]pyrene-Induced Cytotoxicity and DNA Damage in Human Bronchial Epithelial Cells.

Metabolic activation of indirect-acting carcinogens in target organs is a recognized mechanism of carcinogenesis. This study aimed to determine the role of benzo[a]pyrene (BaP) metabolism enzymes lipoxygenase (LOX), cytochrome P4501A1 (CYP1A1), and prostaglandin synthetase (PGS) in the cytotoxicity and DNA damage induced by BaP in the human tracheobronchial epithelial cells (HBECs) using RNA interference strategy and metabolic enzyme inhibitors. Our results showed that in three epithelial cell lines (HBE, HTR-8/SVneo, and HaCat), BaP significantly upregulated 5-LOX protein expression. 15-LOX-2 expression also increased with increasing BaP concentration, but the change was less pronounced than that of 5-LOX. BaP caused significant cytotoxicity, DNA strand breaks, and 8-hydroxy-2'-deoxyguanosine formation in HBE, which was inhibited by 5-LOXshRNA, a specific inhibitor of 5-LOX (AA861), the CYP1A1 inhibitor α-naphthoflavone, and the PGS inhibitor naproxen. The protective effects of 5-LOXshRNA were stronger than AA861, naproxen and α-naphthoflavone. We conclude that BaP may be activated more by 5-LOX than by CYP1A1 and PGS to produce cytotoxicity and DNA damage in HBE.

The role of macrophages phenotypes in the activation of resolution pathways within human granulosa cells

BackgroundInflammatory state within the ovaries can disrupt normal follicular dynamics, leading to reduced oocyte quality and infertility. How the production of inflammatory mediators generated by macrophages with different gene expression profile (M1 and M2) might activate inflammatory pathways, such as cyclooxygenase-2 (COX-2) and 5-, 12-, and 15-lipoxygenase (LOX), in human granulosa cells (hGCs) remains unclear.MethodsIn this study, we evaluated how M1 and M2 macrophages found in the ovaries affect the functions of hGCs isolated from women undergoing assisted reproductive technology (ART) and human ovarian granulosa COV434 cells. For this purpose, a model of interaction between hGCs and COV434 cells and conditioned media (CMs) obtained from culture of M0, M1 and M2 macrophages was established. We used real-time PCR and western blotting to detect the expression of COX-2 and 5-, 12-, and 15-LOX as biomarkers of oocyte competence.ResultsOur data showed that M2 macrophages with anti-inflammatory characteristics were able to significantly increase the expression of COX-2 in hGCs. We also demonstrated that M1 macrophages with pro-inflammatory characteristics were able to significantly increase the expression of 12-LOX in hGCs. However, there was no observed expression of 5-LOX and no significant alteration in the expression of 15-LOX in hGCs. Regarding COV434 cells, we found that CM from M2 macrophage resulted in an increase in COX-2, 5-LOX and 15-LOX mRNA and protein levels. No expression of 12-LOX by COV434 cells was observed when exposed to CMs from M1 and M2 macrophages.ConclusionsOur research indicated that the production of pro-resolving mediators by hGCs can, at least in part, reverse the physiological inflammation present in the ovaries.

Flavonoids derived from Anemarrhenae Rhizoma ameliorate inflammation of benign prostatic hyperplasia via modulating COX/LOX pathways

Ethnopharmacological relevanceFlavonoids are the main components of the traditional Chinese medicine Anemarrhenae Rhizoma (dried rhizome of Anemarrhena asphodeloides Bge.), which has been reported to possess activity against inflammation and tumor. Aim of studyRegulation of the arachidonic acid (AA) cascade through cyclooxygenase (COX) and lipoxygenase (LOX) represent the two major pathways to treat inflammatory of benign prostatic hyperplasia (BPH). In this study, Anemarrhenae Rhizoma flavonoids and its main compounds (mangiferin, neomangiferin and isomangiferin) were investigated for effects on AA metabolism. MethodsUltra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to monitor AA metabolites in BPH rats and in PC-3 cells. COX-2 and 5-LOX protein and mRNA levels were measured by Western blot and qPCR, respectively, along with histopathological assessment of prostate tissues. ResultsTreatment with flavonoids significantly ameliorated BPH-associated prostate inflammation and inhibited the expression of COX-2 and 5-LOX at the protein and mRNA levels. Quantitative metabolomic analysis of blood plasma showed flavonoids treatment decreased AA levels and its metabolites associated with the COX and LOX pathways. Further exploration of the flavonoid compounds mangiferin, neomangiferin and isomangiferin showed they inhibited AA metabolism to varying degrees in PC-3 cell cultures. ConclusionAnemarrhenae Rhizoma flavonoids act to inhibit BPH-related inflammation in vivo and in vitro by targeting AA metabolism and interfering with COX and LOX pathways. The identification of mangiferin, neomangiferin and isomangiferin as anti-inflammatory components suggests flavonoids interventions represent a promising therapeutic approach for BPH.

The Potential of Acetylfuran and Furfural from Tamarindus indica as Lipoxygenase Inhibitor: In Silico Study

Background: Tamarindus indica is a type of plant sub-family Caesalpinioideae, which is predicted to have anti-inflammatory properties. When inflammation occurs, arachidonic acid will undergo metabolism, the LOX pathway will release 5-lipoxygenase (5-LOX). Objective: This study aimed to analyze the potential of acetylfuran and furfural compounds on LOX action. Methods: The compound Acetylfuran (CID 14505), Furfural (CID 7362) were downloaded from the PubChem database. The 5-LOX protein was obtained from PDB (6N2W), preparation by removing ligands and molecules that bind to Discovery Studio V19.1.0.18287. Compound and protein interactions have interacted with the Vina autodock software integrated into the PyRX software and analyzed by Discovery Studio V19.1.0.18287. Results: The results showed that the content of Acetylfuran and Furfural compounds in Tamarindus indica is predicted to have the potential as an inhibitor of the LOX gene on different amino acid residues, namely 3 amino acid residues and 4 amino acid residues, respectively and produce binding energy. In addition, van der Waals forces, hydrogen and hydrophobic bonds were found, giving the strength of the bonds formed. Conclusion: Bioactive acetylfuran and furfural have the potential as a drug to curve inflammation in the human body.

Structural, CSD, and computational studies of 6b, 11b-Dihydroxy-6b, 11-b-dihydro-7H-indeno[1,2-b]naptho[2,1-d]furan-7-one, a therapeutic potential small molecule

Biochemical and pharmacological investigations carried out on the small molecule 6b, 11b-Dihydroxy-6b, 11-b-dihydro-7H-indeno[1,2-b]naptho[2,1-d]furan-7-one (DHFO) has shown that it is an analgesic, anti-inflammatory agent, and a dual 5-LOX/COX-2 inhibitor. DHFO is also a free-radical scavenger, a potent antioxidant, and an inhibitor of lipid peroxidation. We studied the structural properties of DHFO using single-crystal X-ray crystallography, Hirshfeld surfaces, and energy frameworks studies. Additionally, we also examined the conformational behavior of 29 molecular analogues of DHFO retrieved from the Cambridge Structural Database (CSD), which were similar to DHFO in flexibility, the effect of the substitution, and electrostatic interaction forces. Besides, we carried out the molecular docking studies to analyze the binding efficiency of DHFO with COX-1, COX-2, 5-LOX, and Cdc25B-cd (the catalytic domain of human Cdc25B phosphatase, which is identified as an arsenic reductase) proteins co-crystallized with standard drugs such as meloxicam. DHFO showed a better affinity with COX-2 than COX-1 and can potentially bind with CdC25B-cd protein. The drug-likeness of DHFO was also evaluated using Lipinski's rule, the SwissADME database, and DHFO possessed potential drug-likeness properties. In summary, the DHFO may potentially act as a multifunctional inhibitor by binding cyclooxygenase and arsenic binding proteins.

Dual COX and 5-LOX inhibition by clerodane diterpenes from seeds of Polyalthia longifolia (Sonn.) Thwaites

Natural metabolites with their specific bioactivities are being considered as a potential source of materials for pharmacological studies. In this study, we successfully isolated and identified five known clerodane diterpenes, namely 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (1), 16-hydroxy-cleroda-3,13-dien-15-oic acid (2), 16-hydroxy-cleroda-4(18),13-dien-16,15-olide (3), 3α,16α-dihydroxy-cleroda-4(18),13(14)Z-dien-15,16-olide (4), and 16α-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (5) from the methanolic extract of seeds of Polyalthia longifolia. Initially, all the isolated metabolites were investigated for COX-1, COX-2, and 5-LOX inhibitory activities using the standard inhibitory kits. Of which, compounds 3, 4, and 5 exhibited to be potent COX-1, COX-2, and 5-LOX inhibitors with the IC50 values similar or lower to those of the reference drugs. To understand the underlying mechanism, these compounds were subjected to molecular docking on COX-1, COX-2, and 5-LOX proteins. Interestingly, the in silico study results were in high accordance with in vitro studies where compounds 3, 4, and 5 hits assumed interactions and binding pattern comparable to that of reference drugs (indomethacin and diclofenac), as a co-crystallized ligand explaining their remarkable dual (COX/LOX) inhibitor actions. Taken together, our findings demonstrated that compounds 3, 4, and 5 functioned as dual inhibitors of COX/5-LOX and can contribute to the development of novel, more effective anti-inflammatory drugs with minimal side-effects.

Study on antitumor activities of the chrysin-chromene-spirooxindole on Lewis lung carcinoma C57BL/6 mice in vivo

The our previous study synthesized the chrysin-chromene-spirooxindole hybrids 3, and further found compound 3e had good antitumor activity against A549 cells in vitro through multi-target co-regulation of the p53 signalling pathway to inhibit the proliferation of A549 cells. This study was designed to evaluate the antitumor effects of compound 3e on Lewis lung carcinoma of C57BL/6 mice in vivo. Compound 3e significantly inhibited the growth of transplanted tumors in C57BL/6 mice and induced the apoptosis of tumor cells. Further studies showed that compound 3e activates and expands the anti-cancer activity of p53 by inhibiting the expression of MDM2, Akt and 5-Lox proteins, accordingly promotes the expressions Bax and inhibit the Bcl-2 protein, the release of Cyt c as well, which resulted in the activation of apoptotic pathway in tumor cells eventually. Moreover, Compound 3e inhibited tumor metastasis by down-regulating VEGF, ICAM-1 and MMP-2 protein expression and angiogenesis. These results suggested that compound 3e exerts an effective antitumor activity in vivo through activating the p53 signaling pathway, which could be exploited as a promising candidate for the development of new anti-tumour drugs.

Characterization of spirostanol glycosides and furostanol glycosides from anemarrhenae rhizoma as dual targeted inhibitors of 5-lipoxygenase and Cyclooxygenase-2 by employing a combination of affinity ultrafiltration and HPLC/MS

Background : Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR).Methods: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies.Results: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC50 ≤ 6.07 μM.Conclusion: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.

The Investigation of Lipoxygenases as Therapeutic Targets in Malignant Pleural Mesothelioma

Advanced malignant pleural mesothelioma (MPM) has an extremely poor prognosis with limited chemotherapy options, therefore the identification of new therapeutic targets would aid in disease management. Arachidonic acid is metabolised by cyclooxygenase and lipoxygenase enzymes. The lipoxygenase isoenzymes 5-LOX and 12-LOX have been implicated in carcinogenesis. We aimed to examine 5-LOX and 12-LOX protein expression in a large retrospective series of mesothelioma samples. Further to this, the in vitro cytotoxic effects of lipoxygenase pathway inhibitors were investigated in mesothelioma cells. Archival samples from 83 patients with MPM were examined by immunohistochemistry for expression of the 5-LOX and 12-LOX proteins. The MTS assay was used to assess cell viability following 72 h treatment with the lipoxygenase pathway inhibitors baicalein, licofelone, MK-886 and zileuton in the MPM cell lines NCI-H2052, NCI-H2452 and MSTO-211H. Positive 12-LOX protein expression was recorded in 69/83 (83%) and positive 5-LOX expression was observed in 56/77 (73%) of MPM tissue samples. Co-expression of 5-LOX with 12-LOX was seen in 46/78 (58%) of MPM samples. Positive expression of 5-LOX, 12-LOX and COX-2 proteins was identified in the NCI-H2052, NCI-H2452 and MSTO-211H MPM cell lines. Baicalein (12-LOX and 15-LOX inhibitor) was effective in 3/3 MPM cell lines at low concentrations with an IC50 range of 9.6 μM to 20.7 μM. We have demonstrated that the 5-LOX and 12-LOX proteins are expressed in a significant proportion of MPM samples (73% and 83% respectively) and may represent novel therapeutic targets in this disease. We have demonstrated that the inhibition of the LOX pathway using baicalein may be effective as a novel treatment for MPM, however further human pharmacokinetic studies are required in order to establish whether the concentration used in vitro is clinically achievable.

Lipoxygenase Protein Expression and Its Effect on Oxidative Stress Caused by Benzidine in Normal Human Urothelial Cell Lines.

Metabolic activation of indirect-acting carcinogens in the target organ is an effective mechanism of carcinogenesis. Lipoxygenase (LOX) can co-oxidize the bladder carcinogen benzidine (BZ). However, it is not entirely clear whether BZ is activated and which enzyme is involved in its activation in bladder epithelial cells. Our results showed that BZ induced 5-LOX protein expression but had no significant influence on the expression of 15-LOX-2, CYP1B1, and CYP2E1 in SV-40 immortalized human uroepithelial SV-HUC-1 cells. BZ induced oxidative stress in SV-HUC-1 cells by increasing reactive oxygen species (ROS) and malondialdehyde levels significantly in the 100 and 200 μmol/L-BZ-treated groups and decreased the level of the antioxidant reduced glutathione significantly at 200 μmol/L BZ. Concurrently, the activity of catalase was increased, while the activity of superoxide dismutase was increased at 50 μmol/L BZ but gradually decreased with increasing concentrations of BZ ( P < 0.05). However, the oxidative stress and damage in SV-HUC-1 cells caused by BZ were effectively inhibited by the 5-LOX-specific inhibitor AA861 at 10 μmol/L. Thus, 5-LOX is probably the major LOX isozyme to co-oxidize exogenous chemicals in SV-HUC-1 cells. AA861 has a protective effect on the oxidative stress and damage induced by BZ in SV-HUC-1 cells. We conclude that BZ can be activated by 5-LOX to produce ROS and oxidative stress, which may be associated with bladder cancer caused by BZ.

Computational modeling and study of the anti-cancer activity of novel NSAID 1-acyl-4-cycloalkyl/arylsemicarbazide and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazide derivatives using molecular docking and molecular dynamics simulations

In this study, a series of NSAID 1-acyl-4-cycloalkyl/arylsemicarbazides and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazides possess anti-cancer activity against three human cancer cell lines L1210, CEM, and HeLa have been investigated using a combined approach including quantitative structure activity relationship (QSAR) study, molecular docking, and molecular dynamics (MD) simulations. First, different molecular descriptors were calculated for these compounds. Stepwise multiple linear regression (MLR) method was performed for selecting some common descriptors, including VEA1, hydration energy (HE), log P, and binding energy for all three cell lines. Then, other QSAR models were constructed using support vector regression (SVR) method. According to the results, SVR models were more efficient in predicting the anti-cancer activity. To better understand the mechanism of the binding interactions of NSAID 1-acyl-4-cycloalkyl/arylsemicarbazide and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazide derivatives with 5-lipoxygenase (5-LOX) protein, molecular docking studies were conducted. These studies have also been used to explore the effects of HE, log P, and binding energy on anti-cancer activity of the studied compounds. The results of molecular docking suggest that hydrophobic interactions of ligands with the active site of 5-LOX are responsible for their anti-cancer activities. Finally, molecular dynamics (MD) simulations using GROMACS package were used for evaluating the stability of 5-LOX in complex with NSAID 1-acyl-4-cycloalkyl/arylsemicarbazide and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazide derivatives. The results of MD simulations demonstrate stability of protein structure in complex with active compounds, 12 and 27.

QSAR classification-based virtual screening followed by molecular docking studies for identification of potential inhibitors of 5-lipoxygenase

Developments of novel inhibitors to prevent the function of 5-lipoxygenase (5-LOX) proteins that are responsible for a variety of inflammatory and allergic disease are a major challenge in the scientific community. In this study, robust QSAR classification models for predicting 5-LOX activity were developed using machine learning algorithms. The Support Vector Machines (SVM), Logistic Regression, k-Nearest Neighbour (NN) and Decision Trees were adopted to improve the prediction ability of the classification models. The most informative molecular descriptors that contribute to the prediction of 5-LOX activity are screened from e-Dragon, Ochem, PowerMV and Combined databases using Filter-based feature selection methods such as Correlation Feature Selection (CFS) and Information Gain (IG). Performances of the models were measured by 5-fold cross-validation and external test sets prediction. Evaluation of performance of feature selection revealed that the CFS method outperforms the IG method for all descriptor databases except for PowerMV database. The best ensemble classification model was obtained with the IG filtered ‘PowerMV’ descriptor database using kNN (k = 5) algorithm which displayed an overall accuracy of 76.6% for the training set and 77.9% for the test set. Finally, we employed this model as a virtual screening tool for identifying potential 5-LOX inhibitors from the e-Drug3D drug database and found 43 potential hit candidates. This top screened hits containing one known 5-LOX inhibitors zileuton as well as novel scaffolds. These compounds further screened by applying molecular docking simulation and identified four potential hits such as Belinostat, Masoprocol, Mefloquine and Sitagliptin having a comparable binding affinity to zileuton.

Modulating effect of inositol hexaphosphate on arachidonic acid-dependent pathways in colon cancer cells

Cyclooxygenase (COX) and lipoxygenase (LOX) are key enzymes of arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in the pathogenesis of inflammatory bowel diseases and colorectal cancer. The aim of the study was to examine the influence of inositol hexaphosphate (IP6), a naturally occurring phytochemical, on the expression of genes encoding COX and LOX isoforms and synthesis of their products (PGE2 and LTB4) in colon cancer cell line Caco-2 stimulated with pro-inflammatory agents (IL-1β/TNFα). Real-time RT-qPCR was used to validate mRNAs level of examined genes. The concentrations of COX-2 and 5-LOX proteins as well as PGE2 and LTB4 were determined by the ELISA method. Based on these studies it may be concluded that IP6 may limit inflammatory events in the colonic epithelium and prevent colon carcinomas by modulating the expression of genes encoding COX and LOX isoforms at both mRNA and protein levels as well as by affecting the synthesis and secretion of prostaglandins and leukotrienes.

Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production.

Propofol is an intravenous anesthetic that produces its anesthetic effect, largely via the GABAA receptor in the CNS, and also reduces the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophil respiratory burst. Because fMLP-stimulated neutrophils produce leukotriene (LT)B4, we examined the effect of propofol on LTB4 production in vivo and in vitro Cecal ligation and puncture surgery was performed in mice, with or without exposure to propofol. Propofol attenuated the production of 5-lipoxygenase (5-LOX)-related arachidonic acid (AA) derivatives in the peritoneal fluid. Also, in the in vitro experiments on fMLP-stimulated neutrophils and 5-LOX-transfected human embryonic kidney cells, propofol attenuated the production of 5-LOX-related AA derivatives. Based on these results, we hypothesized that propofol would directly affect 5-LOX function. Using meta-azi-propofol (AziPm), we photolabeled stable 5-LOX protein, which had been used to solve the X-ray crystallographic structure of 5-LOX, and examined the binding site(s) of propofol on 5-LOX. Two propofol binding pockets were identified near the active site of 5-LOX. Alanine scanning mutagenesis was performed for the residues of 5-LOX in the vicinity of propofol, and we evaluated the functional role of these pockets in LTB4 production. We demonstrated that these pockets were functionally important for 5-LOX activity. These two pockets can be used to explore a novel 5-LOX inhibitor in the future.-Okuno, T., Koutsogiannaki, S., Ohba, M., Chamberlain, M., Bu, W., Lin, F.-Y., Eckenhoff, R. G., Yokomizo T., Yuki, K. Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production.

Synthesis, bioactivity evaluation, and docking study of 5-aminosalicylic acid’s fatty acid derivatives

Some new oleic acid, linoleic acid, lauric acid derivatives of 5-aminosalicylic acid were produced in order to enhance bioactivity properties of 5-aminosalicylic acid. All new compounds’ structures were confirmed by spectroscopic methods. Moreover, in vitro antibacterial, anticancer, and anti-inflammatory activities of new synthesized compounds were investigated. Antibacterial activity was studied against pathogenic Gram-negative bacteria, Escherichia coli and Gram-positive bacteria, Staphylococcus aureus via disc diffusion method. Additionally, all derivatives’ anti-inflammatory activity were evaluated through NO suppression assay using interferon gamma (IFN-γ)/lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Furthermore, in vitro cytotoxicity assay was probed using MTT test against 3T3 and HT-29 cell lines. In another effort, docking studies were performed to predict the possible interactions and binding energy of new compounds against cyclooxygenase (COX-1/COX-2) and lipoxygenase (5-LOX) proteins. In conclusion, all new compounds exhibited moderate to better bioactivity improvements in comparison with parent drug. Also study of interactions between the new derivatives with active sites of proteins presented greater binding affinities than 5-aminosalicylic acid.

Low leukotriene B4 receptor 1 leads to ALOX5 downregulation at diagnosis of chronic myeloid leukemia.

ALOX5 is implicated in chronic myeloid leukemia development in mouse leukemic stem cells, but its importance in human chronic myeloid leukemia is unknown. Functional ALOX5 was assessed using an LTB4 ELISA and ALOX5, and LTB4R1 mRNA expression was determined via a TaqMan gene expression assay. LTB4R1 and 5-LOX protein levels were assessed by cell surface flow cytometry analysis. At diagnosis ALOX5 was below normal in both blood and CD34(+) stem cells in all patients. On treatment initiation, ALOX5 levels increased in all patients except those who were destined to progress subsequently to blast crisis. LTB4 levels were increased despite low ALOX5 expression, suggesting that the arachidonic acid pathway is functioning normally up to the point of LTB4 production. However, the LTB4 receptor (BLT1) protein in newly diagnosed patients was significantly lower than after a period of treatment (P<0.0001). The low level of LTB4R1 at diagnosis explains the downregulation of ALOX5. In the absence of LTB4R1, the arachidonic acid pathway intermediates (5-HEPTE and LTA4) negatively regulate ALOX5. ALOX5 regulation is aberrant in chronic myeloid leukemia patients and may not be important for the development of the disease. Our data suggest caution when extrapolating mouse model data into human chronic myeloid leukemia.

Sickle erythrocytes and platelets augment lung leukotriene synthesis with downregulation of anti-inflammatory proteins: relevance in the pathology of the acute chest syndrome.

Initiation, progression, and resolution of vaso-occlusive pain episodes in sickle cell disease (SCD) have been recognized as reperfusion injury, which provokes an inflammatory response in the pulmonary circulation. Some 5-lipoxygenase (5-lox) metabolites are potent vasoconstrictors in the pulmonary circulation. We studied stimulation of production of the inflammatory eicosanoids leukotrienes (LTs) and prostaglandin E2 (PGE2) by isolated rat lungs perfused with sickle (HbSS) erythrocytes. Our hypothesis is that HbSS erythrocytes produce more LTs than normal (HbAA) erythrocytes, which can induce vaso-occlusive episodes in SCD patients. Lung perfusates were collected at specific time points and purified by high-pressure liquid chromatography, and LTC4 and PGE2 contents were measured by enzyme-linked immunosorbent assay (ELISA). Rat lung explants were also cultured with purified HbAA and HbSS peptides, and 5-lox, cyclooxygenase 1/2, and platelet-activating factor receptor (PAFR) proteins were measured by Western blotting, while prostacyclin and LTs produced by cultured lung explants were measured by ELISA. Lung weight gain and blood gas data were not different among the groups. HbSS-perfused lungs produced more LTC4 and PGE2 than HbAA-perfused lungs: 10.40 ± 0.62 versus 0.92 ± 0.2 ng/g dry lung weight (mean ± SEM; P = 0.0001) for LTC4. Inclusion of autologous platelets (platelet-rich plasma) elevated LTC4 production to 12.6 ± 0.96 and 7 ± 0.60 ng/g dry lung weight in HbSS and HbAA perfusates, respectively. HbSS lungs also expressed more 5-lox and PAFR. The data suggest that HbSS erythrocytes and activated platelets in patient's pulmonary microcirculation will enhance the synthesis and release of the proinflammatory mediators LTC4 and PGE2, both of which may contribute to onset of the acute chest syndrome in SCD.