Discorhabdin alkaloids from Antarctic Latrunculia spp. sponges as a new class of cholinesterase inhibitors.

The brominated pyrroloiminoquinone alkaloids discorhabdins B, L and G and 3-dihydro-7,8- dehydrodiscorhabdin C, isolated from methanol extracts of two specimens of Latrunculia sp. sponges collected near the Antarctic Peninsula, are here demonstrated for the first time to be reversible competitive inhibitors of cholinesterases. They showed Ki for electric eel acetylcholinesterase of 1.6-15.0 µM, for recombinant human acetylcholinesterase of 22.8-98.0 µM, and for horse serum butyrylcholinesterase of 5.0-76.0 µM. These values are promising when compared to the current cholinesterase inhibitors used for treatment of patients with Alzheimer's disease, to counteract the acetylcholine deficiency in the brain. Good correlation was obtained between IC50 data and results by molecular docking calculation on the binding interactions within the acetylcholinesterase active site, which also indicated the moieties in discorhabdin structures involved. To avoid unwanted peripheral side effects that can appear in patients using some acetylcholinesterase inhibitors, electrophysiological experiments were carried out on one of the most active of these compounds, discorhabdin G, which confirmed that it had no detectable undesirable effects on neuromuscular transmission and skeletal muscle function. These findings are promising for development of cholinesterase inhibitors based on the scaffold of discorhabdins, as potential new agents for treatment of patients with Alzheimer's disease.

Biological activities of organic extracts of four Aureobasidium pullulans varieties isolated from extreme marine and terrestrial habitats.

We report on the screening for biological activities of organic extracts from seven strains that represent four varieties of the fungus Aureobasidium pullulans, that is A. pullulans var. melanogenum, A. pullulans var. pullulans, A. pullulans var. subglaciale and A. pullulans var. namibiae. We monitored haemolysis, cytotoxicity, antioxidant capacity and growth inhibition against three bacterial species. The haemolytic activity of A. pullulans var. pullulans EXF-150 strain was due to five different haemolytically active fractions. Extracts from all of the other varieties contained at least one haemolytically active fraction. Short-term exposure of cell lines to these haemolytically active organic extracts resulted in more than 95% cytotoxicity. Strong antioxidant capacity, corresponding to 163.88 µg ascorbic acid equivalent per gram of total solid, was measured in the organic extract of the strain EXF-3382, obtained from A. pullulans var. melanogenum, isolated from the deep sea. Organic extracts from selected varieties of A. pullulans exhibited weak antibacterial activities.

Salt induces biosynthesis of hemolytically active compounds in the xerotolerant food-borne fungus Wallemia sebi.

Wallemia sebi is a xerotolerant, ubiquitous, food-borne, mycotoxigenic fungus. An ethanol extract of its mycelium demonstrated a strong hemolytic activity, which was further enhanced at high salt concentrations in the growth medium. Characterization of the extract using gas chromatography-mass spectrometry revealed a mixture of sterols and unsaturated fatty acids, indicating the latter as responsible for the hemolytic activity. The lytic activity of the extract is here studied using red blood cells and artificial small lipid vesicles with various lipid compositions. This shows concentration-dependent hemolysis and preferential activity toward lipid membranes with greater fluidity. The W. sebi lytic activity on mammalian erythrocytes shows its potential involvement in the formation of lesions in subcutaneous infections, in farmer's lung disease, and in consumption of food and feed that are contaminated with food-borne W. sebi.

Unsaturated fluoro-ketopyranosyl nucleosides: synthesis and biological evaluation of 3-fluoro-4-keto-beta-d-glucopyranosyl derivatives of N(4)-benzoyl cytosine and N(6)-benzoyl adenine.

The protected beta-nucleosides 1-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-beta-d-glucopyranosyl)-N(4)-benzoyl cytosine (2a) and 9-(2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-beta-d-glucopyranosyl)-N(6)-benzoyl adenine (2b), were synthesized by the coupling of peracetylated 3-deoxy-3-fluoro-d-glucopyranose (1) with silylated N(4)-benzoyl cytosine and N(6)-benzoyl adenine, respectively. The nucleosides were deacetylated and several subsequent protection and deprotection steps afforded the partially acetylated nucleosides of cytosine 7a and adenine 7b, respectively. Finally, direct oxidation of the free hydroxyl group at 4'-position of 7a and 7b, and simultaneous elimination reaction of the beta-acetoxyl group, afforded the desired unsaturated 3-fluoro-4-keto-beta-d-glucopyranosyl derivatives. These newly synthesized compounds were evaluated for their potential antitumor and antiviral activities. Compared to 5FU, the newly synthesized derivatives showed to be more efficient as antitumor growth inhibitors and they exhibited direct antiviral effect toward rotavirus.

Synthesis and molecular modelling of unsaturated exomethylene pyranonucleoside analogues with antitumor and antiviral activities.

This report describes the total and facile synthesis of the unsaturated keto and exomethylene pyranonucleoside analogues, 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-alpha-D-glycero-hex-2-enopyranosyl)uracil (10), 1-(2,3-dideoxy-alpha-D-glycero-hex-2-enopyranosyl-4-ulose)uracil (17) and 1-(2,3,4-trideoxy-4-methylene-alpha-D-glycero-hex-2-enopyranosyl)uracil (18). Commercially available 1,2,3,4,6-penta-O-acetyl-alpha-D-mannopyranose (1) was condensed with silylated uracil, deacetylated and acetalated to afford 1-(2,3-O-isopropylidene-alpha-D-mannopyranosyl)uracil (4). Two different synthetic routes were investigated for the conversion of 4 into the olefinic derivative 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-alpha-D-glycero-hex-2-enopyranosyl)uracil (10). Although the two procedures are quite similar with respect to yields and final products, the second also leads to the keto-2',3'-unsaturated analogue (17). The new analogues were evaluated for their anticancer and antiviral activities using several tumor cell lines and gastrointestinal rotavirus. All of the compounds showed direct antiviral effect against rotavirus infectivity in Caco-2 cell line. Moreover, 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-alpha-D-glycero-hex-2-enopyranosyl)uracil (10) was found to be potent in MCF-7 breast carcinoma cell line.

Exomethylene pyranonucleosides: efficient synthesis and biological evaluation of 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine.

A new series of unsaturated pyranonucleosides with an exocyclic methylene group and thymine as heterocyclic base have been designed and synthesized. d-Galactose (1) was readily transformed in three steps into the corresponding 1-(beta-d-galactopyranosyl)thymine (2). Selective protection of the primary hydroxyl group of 2 with a t-butyldimethylsilyl (TBDMS) group, followed by specific acetalation, and oxidation gave 1-(6-O-t-butyldimethylsilyl-3,4-O-isopropylidene-beta-d-lyxo-hexopyranosyl-2-ulose)thymine (5). Wittig reaction of the ketonucleoside 5, deprotection and tritylation of the 6'-hydroxyl group gave 1-(2-deoxy-2-methylene-6-O-trityl-beta-d-lyxo-hexopyranosyl)thymine (9). Exomethylene pyranonucleoside 9 was converted to the olefinic derivative 10, which after detritylation afforded the title compound 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine (11). These novel synthesized compounds were evaluated for antiviral activity against rotaviral infection and cytotoxicity in colon cancer. As compared to AZT, compounds 1-(2-deoxy-2-methylene-beta-d-lyxo-hexopyranosyl)thymine (7) and 1-(beta-d-lyxo-hexopyranosyl-2-ulose)thymine (8) showed to be more efficient, in rotavirus infections and in treatment of colon cancer.

Interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus.

Macrophages are an important cellular component of the innate immune system and are normally rapidly recruited and/or activated at the site of virus infection. They can participate in the antiviral response by killing infected cells, by producing antiviral cytokines such as nitric oxide and by producing chemokines and immunoregulatory cytokines that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions. Probiotics, as a part of the normal gut intestinal flora, are important in supporting a functional yet balanced immune system. Improving our understanding of their role in the activation of macrophages and their stimulation of proinflammatory cytokine production in early viral infection was the main goal of this study. Our in vitro model study showed that probiotic bacteria, either from the species Lactobacillus or Bifidobacteria have the ability to decrease viral infection by establishing the antiviral state in macrophages, by production of NO and inflammatory cytokines such as interleukin 6 and interferon-gamma. These effects correlated with the mitochondrial activity of infected macrophages, therefore, the measurements of mitochondrial dehydrogenases activity could be implied as the first indicator of potential inhibitory effects of the probiotics on virus replication. The interactions between probiotic bacteria, macrophages and vesicular stomatitis virus (VSV), markedly depended on the bacterial strain studied.

An efficient synthesis of 3-fluoro-5-thio-xylofuranosyl nucleosides of thymine, uracil, and 5-fluorouracil as potential antitumor or/and antiviral agents.

1,2:5,6-Di-O-isopropylidene-alpha-D-glucofuranose by the sequence of mild oxidation, reduction, fluorination, periodate oxidation, borohydride reduction, and sulfonylation gave 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-toluenesulfonyl-alpha-D-xylofuranose (5). Tosylate 5 was converted to thioacetate derivative 6, which after acetolysis gave 1,2-di-O-acetyl-5-S-acetyl-3-deoxy-3-fluoro-5-thio-D-xylofuranose (7). Condensation of 7 with silylated thymine, uracil, and 5-fluorouracil afforded nucleosides 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) thymine (8), 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) uracil (9), and 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) 5-fluorouracil (10). Compounds 8, 9, and 10 are biologically active against rotavirus infection and the growth of tumor cells.

A novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria.

As shown in many intervention studies, probiotic bacteria can have a beneficial effect on rotavirus and HIV-induced diarrhoea. In spite of that fact, antiviral effects of probiotic bacteria have not been systematically studied yet. Non-tumorigenic porcine intestinal epithelial cells (IPEC-J2) and alveolar macrophages (3D4/2) were treated in different experimental designs with probiotic and other lactic bacteria and their metabolic products. Vesicular stomatitis virus (VSV) was used in the study as a model virus. Cell survival and viral inhibition were determined by antiviral assay and confirmed by immunofluorescence. Pre-incubation of cell monolayers with probiotic bacteria reduced viral infectivity up to 60%. All bacteria used prevented VSV binding to the cell monolayers by direct binding of VSV to their surface. Probiotic and other lactic bacteria prevented viral infection also by establishment of the antiviral state in pre-treated cell monolayers. Probiotic and other lactic bacteria secreted antiviral substances during their growth, as the infectivity of the virus was diminished by 68% when bacterial supernatants were tested. It was shown for the first time that probiotic and other lactic bacteria exhibit an antiviral activity in a cell culture model. Possible mechanisms of antiviral activity include: 1) hindering the adsorption and cell internalisation of the VSV due to the direct trapping of the virus by the bacteria, 2) "cross-talk" with the cells in establishing the antiviral protection and 3) production of metabolites with a direct antiviral effect.

Fluoro-ketopyranosyl nucleosides: synthesis and biological evaluation of 3-fluoro-2-keto-beta-D-glucopyranosyl derivatives of N4-benzoyl cytosine.

1,2:5,6-Di-O-isopropylidene-alpha-d-glucofuranose on mild oxidation, reduction, fluorination, and deisopropylidenation followed by acetylation gave peracetylated 3-deoxy-3-fluoro-d-glucopyranose. This was coupled with silylated N(4)-benzoyl cytosine. The nucleoside was deacetylated and after several subsequent protection and deprotection steps afforded the desired 3-fluoro-2-keto-beta-d-glucopyranosyl derivatives. These novel synthesized compounds were evaluated for antiviral and cytotoxic activities against rotavirus, vesicular stomatitis virus, and the human colon adenocarcinoma cell line Caco-2, and have a promising potential in combating the rotaviral infections and in the treatment of colon cancer. As compared to AZT, a nucleoside analogue of reverse transcriptase inhibitor, the novel synthesized 1-(3,4-dideoxy-3-fluoro-beta-d-glycero-hex-3-enopyranosyl-2-ulose)-N(4)-benzoyl cytosine showed to be more effective at lower concentrations in inhibition of rotavirus infection as well as in the same range of antitumor activity.