Hugo J.C. Froufe

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.

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.

Docking Studies in Target Proteins Involved in Antibacterial Action Mechanisms: Extending the Knowledge on Standard Antibiotics to Antimicrobial Mushroom Compounds

In the present work, the knowledge on target proteins of standard antibiotics was extended to antimicrobial mushroom compounds. Docking studies were performed for 34 compounds in order to evaluate their affinity to bacterial proteins that are known targets for some antibiotics with different mechanism of action: inhibitors of cell wall synthesis, inhibitors of protein synthesis, inhibitors of nucleic acids synthesis and antimetabolites. After validation of the molecular docking approach, virtual screening of all the compounds was performed against penicillin binding protein 1a (PBP1a), alanine racemase (Alr), d-alanyl-d-alanine synthetase (Ddl), isoleucyl-tRNA sinthetase (IARS), DNA gyrase subunit B, topoisomerase IV (TopoIV), dihydropteroate synthetase (DHPS) and dihydrofolate reductase (DHFR) using AutoDock4. Overall, it seems that for the selected mushroom compounds (namely, enokipodins, ganomycins and austrocortiluteins) the main mechanism of the action is the inhibition of cell wall synthesis, being Alr and Ddl probable protein targets.

Virtual screening of low molecular weight mushrooms compounds as potential Mdm2 inhibitors.

In some human cancer cases, the activity of p53 is inhibited by over-expressed Mdm2. The Mdm2 acts as an ubiquitin ligase, resulting in p53 ubiquitination and subsequent p53 proteasomal degradation. The disruption of the Mdm2-p53 interaction using small-molecule inhibitors is recognized as a promising strategy for anti-cancer drug design. Mushrooms are an important source of powerful compounds with anti-tumour properties. In this study, the first virtual screening of low molecular weight compounds present in mushroom is presented as potential Mdm2 inhibitors. A re-docking and cross-docking method was used to validate the virtual screening protocol. The steroids: ganoderic acids X (Ki = 16nM), Y (Ki = 22nM) and F (Ki = 69nM); 5,6-epoxy-24(R)-methylcholesta-7,22-dien-3β-ol (Ki = 74nM) and polyporenic acid C (Ki = 59nM) stand out as the top ranked potential inhibitors of Mdm2. The docking pose of the most promising compounds were carefully analysed and the information provided shows several interesting starting points for further development of Mdm2 inhibitors.

Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase

Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

Using molecular docking to investigate the anti-breast cancer activity of low molecular weight compounds present on wild mushrooms

Mushrooms represent an unlimited source of compounds with anti-tumour and immunostimulating properties, and their intake has been shown to reduce the risk of breast cancer. A large number of low molecular weight (LMW) compounds present in mushrooms have been identified, including phenolic acids, flavonoids, tocopherols, carotenoids, sugars and fatty acids. In order to evaluate which wild mushroom LMW compounds may be involved in anti-breast cancer activity we selected a representative dataset of 43 LMW compounds and performed molecular docking against three known protein targets involved in breast cancer (aromatase, estrone sulfatase and 17β-HSD-1) using AutoDock4 as docking software. The estimated inhibition constants for all LMW compounds were determined, and the potential structure–activity relationships for the compounds with the best estimated inhibition constants are discussed for each compound family. 4-O-caffeoylquinic, naringin and lycopene stand out as the top-ranked potential inhibitors for aromatase, estrone sulfatase and 17β-HSD1, respectively, and the 3-D docked conformations for these compounds are discussed in detail. This information provides several interesting starting points for further development of aromatase, estrone sulfatase and 17β-HSD1 inhibitors.