Identification of potent inhibitors of kynurenine-3-monooxygenase from natural products: In silico and in vitro approaches.

Existing inhibitors of kynurenine-3-monooxygenase (KMO) have side effects and poorly cross the blood-brain barrier. Therefore, the discovery of new molecules targeting KMO isnecessary.This study aims to develop a novel therapeutic drug targeting KMO using computational methods and experimental validation of natural compounds.The results of our study show that the top four compounds, namely, 3'-Hydroxy-alpha-naphthoflavone exhibited the best docking scores with KMO (-10.0 kcal/mol), followed by 3'-Hydroxy-ss-naphthoflavone (-9.9 kcal/mol), genkwanin (-9.2 kcal/mol) and apigenin(-9.1 kcal/mol) respectively. Molecular dynamics was used to assess the stability of the primary target, KMO, and inhibitor complexes. We found stable interactions of 3'-Hydroxy-ss-naphthoflavone and apigenin with KMO up to 100 ns. Further, kinetic measurements showed that 3'-Hydroxy-alpha-naphthoflavone and 3'-Hydroxy-ss-naphthoflavone induce competitive inhibition with a good IC50 activity (15.85 ± 0.98 µM and 18.71 ± 0.78, respectively), while Genkwanin and Apigenin exhibit non-competitive inhibition mechanism (21.61 ± 0.97 µM and 24.14 ± 1.00 µM, respectively).Drug-likeness features and ADME analysis features also showed that the top four compounds could be used as potential candidates to replace the synthetic KMO inhibitor drugs with known side effects and poor brain-blood barrier penetration.

Purification and Biochemical Characterization of Novel Galectin from the Black Poplar Medicinal Mushroom Cyclocybe cylindracea (Agaricomycetes) Strain MEST42 from Algeria.

In the present study, a new galectin designated Cyclocybe cylindracea lectin (CCL) was extracted from the fruiting bodies of the wild black popular mushroom C. cylindracea grown in Algeria. The protein was isolated using sepharose 4B as affinity chromatography matrix, and galactose as elutant. The purified galectin was composed of two subunits of 17.873 kDa each, with a total molecular mass of 35.6 kDa. Its agglutinant activity was impeded by galactose and its derivatives, as well as melibiose. Lactose showed the highest affinity, with a minimal inhibitory concentration of 0.0781 mM. CCL was sensitive to extreme pH conditions, and its binding function decreased when incubated with 10 mM EDTA, and it could be restored by metallic cations such as Ca2+, Mg2+, and Zn2+. CCL agglutinated human red blood cells, without any discernible specificity. Circular dichroism spectra demonstrated that its secondary structure contained ß-sheet as dominant fold. In addition, bioinformatics investigation on their peptide fingerprint obtained after MALDI-TOF/TOF ionization using mascot software confirmed that CCL was not like any previous purified lectin from mushroom: instead, it possessed an amino acid composition with high similarity to that of the putative urea carboxylase of Emericella nidulans (strain FGSC A4/ATCC 38163/CBS 112.46/NRRL 194/M139) with 44% of similarity score.

Characterization of Cytotoxic Lactose Binding Lectin from Sulphur Polypore, Laetiporus sulphureus (Agaricomycetes), from Algeria.

Mushroom lectins have important biological and biomedical applications. Most lectins purified from these organisms exhibit high toxicity in animal cells and toward microbial agents. They are able to induce cell growth inhibition and metabolism by their ability to interact with glyconjugate components (glycoproteins receptors, glycolipids) present in their membrane. After lectins bind to these membrane receptors, they induce cellular signalization chains in which gene expression is regulated and cell death programming (apoptosis) is activated. In this work, a new multimeric lectin was characterized from the rare saprobic edible mushroom, Laetiporus sulphureus strain TMES43, grown in the Algerian forest. Lectin was isolated with ammonium sulfate precipitation followed by affinity chromatography on a Sepharose 4B column, with specific activity of 1204.7 units of hemagglutination activity/mg and 35.55% yield. The protein has a tetrameric structure with a molecular weight of 36 kDa for each subunit, with a total molecular weight of approximately 140 kDa. In addition, a Mascot peptide fingerprint study on a matrix-assisted laser desorption ionization-time of flight tandem fragment showed identity with autophagy-related protein 16 from Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324; Expasy ID: ATG16_PICGU) and no sequence similarity to known mushroom lectins. L. sulphureus hemagglutination activity was reduced by 5 mM of lactose and 10 mM of EDTA incubation and was recovered by metallic cations such as CaCl2, MgCl2, and ZnCl2. L. sulphureus purified lectin had no human ABO group specificity and showed low temperature and alkaline pH stabilities. The MTT preliminary assay showed that L. sulphureus purified lectin induced high cytotoxicity for tumor cells and normal cells.

Agomelatine effects on fat-enriched diet induced neuroinflammation and depression-like behavior in rats.

Growing evidence suggests that a high fat diet (HFD) induces oxidative stress on the central nervous system (CNS), which predisposes to mood disorders and neuroinflammation. In this study we postulated that in addition to improving mood, antidepressant therapy would reverse inflammatory changes in the brain of rats exposed to a HFD. To test our hypothesis, we measured the effect of the antidepressant agomelatine (AGO) on anxiety- and depressive-like behaviors, as well as on CNS markers of inflammation in rats rendered obese. Agomelatine is an agonist of the melatonin receptors MT1 and MT2 and an antagonist of the serotonin receptors 5HT2B and 5HT2C. A subset of rats was also treated with lipopolysaccharides (LPS) to determine how additional neuroinflammation alters behavior and affects the response to the antidepressant. Specifically, rats were subjected to a 14-week HFD, during which time behavior was evaluated twice, first at the 10th week prior to LPS and/or agomelatine, and then at the 14th week after a bi-weekly exposure to LPS (250 µg/kg) and daily treatment with agomelatine (40 mg/kg). Immediately after the second behavioral testing we measured the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL-1ß), markers of oxidative stress thiobarbituric acid reactive substances (TABRS), catalase (CAT) and glutathione peroxidase (GPx), the growth factor BDNF, as well as the apoptosis marker caspase-3. Our results show that a HFD induced an anxiety-like behavior in the open field test (OFT) at the 10th week, followed by a depressive-like behavior in the forced swim test (FST) at the 14th week. In the prefrontal and hippocampal cortices of rats exposed to a HFD we noted an overproduction of TNF-α, IL-6, IL-1ß, and TABRS, together with an increase in caspase-3 activity. We also observed a decrease in BDNF, as well as reduced CAT and GPx activity in the same brain areas. Treatment with agomelatine reversed the signs of anxiety and depression, and decreased the cytokines (TNF-α, IL-6 and IL-1ß), TABRS, as well as caspase-3 activity. Agomelatine also restored BDNF levels and the activity of antioxidant enzymes CAT and GPx. Our findings suggest that the anxiolytic/antidepressant effect of agomelatine in obese rats could result from a reversal of the inflammatory and oxidative stress brought about by their diet.

The antidepressant effect of melatonin and fluoxetine in diabetic rats is associated with a reduction of the oxidative stress in the prefrontal and hippocampal cortices.

In the past few years possible mechanisms that link diabetes and depression have been found. One of these mechanisms is the increase in lipid peroxidation and decrease in antioxidant activity in the hippocampal and prefrontal cortices, which are brain areas involved in mood. The goal of the present study was to evaluate the effect of an antidepressant and of an antioxidant on behavior and oxidative activity in brains of diabetic rats. Rats rendered diabetic after a treatment with streptozotocin (STZ) (60mg/kg) were treated with fluoxetine (15mg/kg), melatonin (10mg/kg), or vehicle for 4 weeks. All animals were tested for signs of depression and anxiety using the elevated plus maze (EPM), open field test (OFT) and the forced swim test (FST). Four groups were compared: (1) normoglycemic, (2) hyperglycemic vehicle treated, and hyperglycemic (3) fluoxetine or (4) melatonin treated rats. On the last day of the study, blood samples were obtained to determine the levels of hemoglobin A1c (HbA1c). Also, brain samples were collected to measure the oxidative stress in the hippocampal and prefrontal cortices using the thiobarbituric acid reactive substances (TBARS) assay. The activity of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were also measured on the brain samples. The results show that both fluoxetine and melatonin decrease the signs of depression and anxiety in all tests. Concomitantly, the levels of HbA1c were reduced in drug treated rats, and to a greater degree in the fluoxetine group. In the cerebral cortex of diabetic rats, TBARS was increased, while the activity of CAT, GPx and GST were decreased. Fluoxetine and melatonin treatments decreased TBARS in both cortices. In the prefrontal cortex, fluoxetine and melatonin restored the activity of CAT, while only melatonin improved the activity of GPx and GST. In the hippocampus, the activity of GPx alone was restored by melatonin, while fluoxetine had no effect. These results suggest that antidepressants and antioxidants can counter the mood and oxidative disorders associated with diabetes. While these effects could result from a decreased production of reactive oxygen species (ROS) remains to be established.