Rui M.V. Abreu

Antioxidants in Wild Mushrooms

Maintenance of equilibrium between free radical production and antioxidant defences (enzymatic and non enzymatic) is an essential condition for normal organism functioning. When this equilibrium has a tendency for the production of free radicals we say that the organism is in oxidative stress. In this situation, excess free radicals may damage cellular lipids, proteins and DNA, affecting normal function and leading to various diseases. In aerobic organisms, the free radicals are constantly produced during the normal cellular metabolism, mainly in the form of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). Exposition of the organism to free radicals has led to the development of endogenous defence mechanisms to eliminate them. These defences were the response of evolution to the inevitability of ROS production in aerobic conditions. Natural products with antioxidant activity may help the endogenous defence system. In this perspective the antioxidants present in the diet assume a major importance as possible protector agents reducing oxidative damage. Particularly, the antioxidant properties of wild mushrooms have been extensively studied by our research group and by others, and many antioxidant compounds extracted from these sources have been identified, such as phenolic compounds, tocopherols, ascorbic acid, and carotenoids. We will review the compounds identified so far in mushrooms, as well as the mechanism of action involved in their antioxidant properties. Wild mushrooms might be used directly in diet and promote health, taking advantage of the additive and synergistic effects of all the bioactive compounds present.

1-Aryl-3-[4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl]ureas as VEGFR-2 Tyrosine Kinase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modelling Studies

The vascular endothelial growth factor receptor-2 (VEGFR-2) is a tyrosine kinase receptor involved in the growth and differentiation of endothelial cells that are implicated in tumor-associated angiogenesis. In this study, novel 1-aryl-3-[4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl]ureas were synthesized and evaluated for the VEGFR-2 tyrosine kinase inhibition. Three of these compounds showed good VEGFR-2 inhibition presenting low IC50 values (150–199 nM) in enzymatic assays, showing also a significant proliferation inhibition of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) at low concentrations (0.5–1 µM), using the Bromodeoxyuridine (BrdU) assay, not affecting cell viability. The determination of the total and phosphorylated (active) VEGFR-2 was performed by western blot, and it was possible to conclude that the compounds significantly inhibit the phosphorylation of the receptor at 1 µM pointing to their antiproliferative mechanism of action in HUVECs. The molecular rationale for inhibiting the tyrosine kinase domain of VEGFR-2 was also performed and discussed using molecular docking studies.

Antimicrobial activity of phenolic compounds identified in wild mushrooms, SAR analysis and docking studies

Although the antimicrobial activity of extracts from several mushroom species has been reported, studies with the individual compounds present in that extracts are scarce. Herein, the antimicrobial activity of different phenolic compounds identified and quantified in mushroom species from all over the world was evaluated. Furthermore, a structure–activity relationship (SAR) analysis and molecular docking studies were performed, in order to provide insights into the mechanism of action of potential antimicrobial drugs for resistant micro‐organisms.

Anti-hepatocellular carcinoma activity using human HepG2 cells and hepatotoxicity of 6-substituted methyl 3-aminothieno[3,2-b]pyridine-2- carboxylate derivatives: In vitro evaluation, cell cycle analysis and QSAR studies

Hepatocellular carcinoma (HCC) is a highly complex cancer, resistant to commonly used treatments and new therapeutic agents are urgently needed. A total of thirty-two thieno[3,2-b]pyridine derivatives of two series: methyl 3-amino-6-(hetero)arylthieno[3,2-b]pyridine-2-carboxylates (1a-1t) and methyl 3-amino-6-[(hetero)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates (2a-2n), previously prepared by some of us, were evaluated as new potential anti-HCC agents by studying their in vitro cell growth inhibition on human HepG2 cells and hepatotoxicity using a porcine liver primary cell culture (PLP1). The presence of amino groups linked to a benzene moiety emerges as the key element for the anti-HCC activity. The methyl 3-amino-6-[(3-aminophenyl)ethynyl]thieno[3,2-b]pyridine-2-carboxylate (2f) is the most potent compound presenting GI(50) values on HepG2 cells of 1.2 μM compared to 2.9 μM of the positive control ellipticine, with no observed hepatotoxicity (PLP1 GI(50) > 125 μM against 3.3 μM of ellipticine). Moreover this compound changes the cell cycle profile of the HepG2 cells, causing a decrease in the % of cells in the S phase and a cell cycle arrest in the G2/M phase. QSAR studies were also performed and the correlations obtained using molecular and 1D descriptors revealed the importance of the presence of amino groups and hydrogen bond donors for anti-HCC activity, and hydrogen bond acceptors for hepatotoxicity. The best correlations were obtained with 3D descriptors belonging to different subcategories for anti-HCC activity and hepatotoxicity, respectively. These results point to different molecular mechanisms of action of the compounds in anti-HCC activity and hepatotoxicity. This work presents some promising thieno[3,2-b]pyridine derivatives for potential use in the therapy of HCC. These compounds can also be used as scaffolds for further synthesis of more potent analogs.

MOLA: a bootable, self-configuring system for virtual screening using AutoDock4/Vina on computer clusters

BackgroundVirtual screening of small molecules using molecular docking has become an important tool in drug discovery. However, large scale virtual screening is time demanding and usually requires dedicated computer clusters. There are a number of software tools that perform virtual screening using AutoDock4 but they require access to dedicated Linux computer clusters. Also no software is available for performing virtual screening with Vina using computer clusters. In this paper we present MOLA, an easy-to-use graphical user interface tool that automates parallel virtual screening using AutoDock4 and/or Vina in bootable non-dedicated computer clusters.ImplementationMOLA automates several tasks including: ligand preparation, parallel AutoDock4/Vina jobs distribution and result analysis. When the virtual screening project finishes, an open-office spreadsheet file opens with the ligands ranked by binding energy and distance to the active site. All results files can automatically be recorded on an USB-flash drive or on the hard-disk drive using VirtualBox. MOLA works inside a customized Live CD GNU/Linux operating system, developed by us, that bypass the original operating system installed on the computers used in the cluster. This operating system boots from a CD on the master node and then clusters other computers as slave nodes via ethernet connections.ConclusionMOLA is an ideal virtual screening tool for non-experienced users, with a limited number of multi-platform heterogeneous computers available and no access to dedicated Linux computer clusters. When a virtual screening project finishes, the computers can just be restarted to their original operating system. The originality of MOLA lies on the fact that, any platform-independent computer available can he added to the cluster, without ever using the computer hard-disk drive and without interfering with the installed operating system. With a cluster of 10 processors, and a potential maximum speed-up of 10x, the parallel algorithm of MOLA performed with a speed-up of 8,64× using AutoDock4 and 8,60× using Vina.

A QCAR model for predicting antioxidant activity of wild mushrooms

Wild mushrooms have been described as sources of natural antioxidants, particularly phenolic compounds. However, many other compounds present in wild mushrooms can also act as antioxidants (reducers), so whole extracts from a wide range of species need to be examined. To gain further knowledge in this area, the relationship between the antioxidant potential (scavenging effect and reducing power) and chemical composition of twenty three samples from seventeen Portuguese wild mushroom species was investigated. A wide range of analytical parameters reported by our research group (including ash, carbohydrates, proteins, fat, monounsaturated fatty acids, polyunsaturated fatty acids, saturated fatty acids, phenolics, flavonoids, ascorbic acid and β-carotene) were studied and the data were analysed by partial least squares (PLS) regression analysis to allow correlation of all the parameters. Antioxidant activity correlated well with phenolic and flavonoid contents. A QCAR (Quantitative Composition-Activity Relationships) model was constructed, using the PLS method, and its robustness and predictability were verified by internal and external cross-validation methods. Finally, this model proved to be a useful tool in the prediction of mushrooms’ reducing power.

Selective flexibility of side-chain residues improves VEGFR-2 docking score using AutoDock Vina.

Selective side‐chain residue flexibility is an option available on AutoDock Vina docking software. This approach is promising as it attempts to provide a more realistic ligand–protein interaction environment without an unmanageable increase in computer processing time. However, studies validating this approach are still scarce. VEGFR‐2 (vascular endothelial growth factor receptor 2), a known protein target for anti‐angiogenic agents, was used in this study. Four residues located in the VEGFR‐2 kinase site were selected and made flexible: Lys868, Glu885, Cys919, and Asp1046. The docking scores for all possible combinations of flexible residues were compared to the docking scores using a rigid conformation. The best overall docking scores were obtained using the Glu885 flexible conformation, with Pearson and Spearman rank correlation values of 0.568 and 0.543, respectively, and a 51% increase in processing time. Using different VEGFR‐2 crystal structures, a similar trend was observed with the Glu885 flexible conformation presenting best scores. This study demonstrates that careful use of selective side‐chain residue flexibility can improve AutoDock Vina docking score accuracy, without a significant increase in processing time. This methodology can be a valuable tool in drug design projects using VEGFR‐2 but will also probably be useful if applied to other protein targets.

Synthesis, antiangiogenesis evaluation and molecular docking studies of 1-aryl-3-[(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas: Discovery of a new substitution pattern for type II VEGFR-2 Tyr kinase inhibitors.

The synthesis and biological evaluation of novel 1-aryl-3-[2-, 3- or 4-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 3, 4 and 5 as VEGFR-2 tyrosine kinase inhibitors, are reported. The 1-aryl-3-[3-(thieno[3,2-b]pyridin-7-ylthio)phenyl]ureas 4a-4h, with the arylurea in the meta position to the thioether, showed the lowest IC₅₀ values in enzymatic assays (10-206 nM), the most potent compounds 4d-4h (IC₅₀ 10-28 nM) bearing hydrophobic groups (Me, F, CF₃ and Cl) in the terminal phenyl ring. A convincing rationalization was achieved for the highest potent compounds 4 as type II VEGFR-2 inhibitors, based on the simultaneous presence of: (1) the thioether linker and (2) the arylurea moiety in the meta position. For compounds 4, significant inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) proliferation (BrdU assay), migration (wound-healing assay) and tube formation were observed at low concentrations. These compounds have also shown to increase apoptosis using the TUNEL assay. Immunostaining for total and phosphorylated (active) VEGFR-2 was performed by Western blotting. The phosphorylation of the receptor was significantly inhibited at 1.0 and 2.5 μM for the most promising compounds. Altogether, these findings point to an antiangiogenic effect in HUVECs.

New di(hetero)arylethers and di(hetero)arylamines in the thieno[3,2-b]pyridine series: Synthesis, growth inhibitory activity on human tumor cell lines and non-tumor cells, effects on cell cycle and on programmed cell death

New fluorinated and methoxylated di(hetero)arylethers and di(hetero)arylamines were prepared functionalizing the 7-position of the thieno[3,2-b]pyridine, using copper (C-O) or palladium (C-N) catalyzed couplings, respectively, of the 7-bromothieno[3,2-b]pyridine, also prepared, with ortho, meta and para fluoro or methoxy phenols and anilines. The compounds obtained were evaluated for their growth inhibitory activity on the human tumor cell lines MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HCT15 (colon carcinoma), HepG2 (hepatocellular carcinoma) and HeLa (cervical carcinoma). The most active compounds, a di(hetero)arylether with a methoxy group in the meta position relative to the ether function and two di(hetero)arylamines with a methoxy group either in the ortho or in the meta position relative to the NH, were further tested at their GI50 concentrations on NCI-H460 cells causing pronounced alterations in the cell cycle profile and a strong and significant increase in the programmed death of these cells. The fluorinated and the other methoxylated compounds did not show important activity, presenting high GI₅₀ values in all the cell lines tested. Furthermore, the hepatotoxicity of the compounds was assessed using porcine liver primary cells (PLP2), established by some of us. Results showed that one of the most active compounds was not toxic to the non-tumor cells at their GI₅₀ concentrations showing to be the most promising as antitumoral.

Aminodi(hetero)arylamines in the Thieno(3,2-b)pyridine Series: Synthesis, Effects in Human Tumor Cells Growth, Cell Cycle Analysis, Apoptosis and Evaluation of Toxicity Using Non-Tumor Cells

Three aminodi(hetero)arylamines were prepared via a palladium-catalyzed C-N Buchwald-Hartwig coupling of methyl 3-aminothieno[3,2-b]pyridine-2-carboxylate with different bromonitrobenzenes, followed by reduction of the nitro groups of the coupling products to the corresponding amino compounds. The aminodi(hetero)arylamines thus obtained were evaluated for their growth inhibitory effect on four human tumor cell lines MCF-7 (breast adenocarcinoma), A375-C5 (melanoma), NCI-H460 (non-small cell lung cancer) and HepG2 (hepatocellular carcinoma). The toxicity to non-tumor cells was also evaluated using a porcine liver primary cell culture (PLP1), established by us. The aminodi(hetero)arylamine with the NH2 group in the ortho position and an OMe group in the para position to the NH of the di(hetero)arylamine, is the most promising compound giving the lowest GI50 values (1.30–1.63 µM) in all the tested human tumor cell lines, presenting no toxicity to PLP1 at those concentrations. The effect of this compound on the cell cycle and induction of apoptosis was analyzed in the NCI-H460 cell line. It was observed that it altered the cell cycle profile causing a decrease in the percentage of cells in the G0/G1 phase and an increase of the apoptosis levels.