Farmakohemijski aspekti delovanja odabranih 4-fenil hidroksikumarina-integrisana in vitro i komjuterska studija

Introduction. Coumarins are widely present in the plant’s kingdom and represent a class of bioactive molecules with different functional groups attached to the basic coumarin structure which can be defined as a lactone where benzene ring is connected to α-piron. It was confirmed that molecules with hydroxyl groups attached to the basic coumarin structure exhibit antioxidant activity since they have the ability to react with free radicals. Coumarins are also considered as potential antibacterial agents which inhibit the ATPase activity of bacterial DNA gyrase. Results from a large number of studies have shown that the spectrum of biological activity of coumarins includes anticancer, anti-inflammatory, antibacterial, anticoagulant and immunomodulatory activity. One of the major goals of medicinal chemistry is to find new molecules with the antioxidant activities, which are capable of reacting with free radicals and neutralize their harmful effects. On the other hand, the morbidity and mortality caused by infectious diseases are still a major health issue. The phenomenon of bacterial resistance to existing antibiotics is one of the biggest problems of today's medicine so that rational use of antibiotics in clinical practice and the development of new antibacterial agents with a broad spectrum of activity present contemporary scientific challenges. The isolation of natural compounds and their application as a leading structure for the chemical modification and further pharmacochemical activity improvement, pharmacophore identification and the establishment of quantitative structure activity relationship (QSAR) parallel with in vitro and computer studies are the subject of modern research. Neoflavones present a group of natural compounds with the basic structure of 4- phenylcoumarin. 4-phenyl hydroxycoumarins have a phenyl group at position 4 and hydroxyl groups attached to the basic coumarin structure and coumarins selected for the study in this doctoral dissertation are: 7-Hydroxy-4-phenyl coumarin (7C), 5,7-dihydroxy-4-phenyl coumarin (5,7C), 7,8-dihydroxy-4-phenyl coumarin (7,8C). Aims of this research were to determine the antioxidant activity of selected 4-phenyl hydroxycoumarins, to establish a relationship between the structure and antioxidant activity with the application of DFT descriptors and to determine the mechanism of antioxidant action on the basis of physico-chemical parameters obtained with DFT calculations, to investigate the antibacterial activity and to establish a correlation between the potential antibacterial activity and the binding affinity of the studied molecules to the active site of bacterial enzymes with the application of computational molecular docking methods, to establish a QSAR models for the studied coumarins’ selected pharmacochemical activity and to assess the physico-chemical and molecular properties in order to predict the bioavailability of the studied 4-phenyl hydroxycoumarins. Methods. For the determination of antioxidant and antibacterial activity of selected 4-phenyl hydroxycoumarins in vitro methods have been used. DPPH•, ABTS•+, ferric thiocyanate method, FRAP and CUPRAC were applied for the determination of antioxidant activity. In addition to the studied compounds, for comparison, the antioxidant activities of commercial synthetic antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trolox, and α-tocopherol) were determined and used further as a control group. The significant differences between the antioxidant activity of studied coumarins and standard antioxidants was determined with the application of Dunett’s test (p<0.05 was considered as significant and p<0.01 as a very significant difference). Microdilution method was applied for the determination of antibacterial activity. Modern computer (in silico) methods (DFT, molecular docking, QSAR) are applied for establishing the correlation between the structure and the activity of the studied compounds and to determine the mechanisms by which this correlation is realized. In order to gain insight into the possible mechanisms of antibacterial and anti-HIV activities studied 4-phenyl hydroxycoumarins were subjected to docking simulations. S. aureus tyrosyl-tRNA synthetase, E. coli topoisomerase II DNA gyrase β and HIV-1 integrase have been chosen as target enzymes for the study of inhibitory activity. Results. 7,8C showed significantly higher antioxidant activity (p <0.01) in comparison to standard antioxidants determined with DPPH• and ABTS•+ method, while 7C and 5,7C didn’t show that activity. All tested molecules showed inhibitory effect on lipid peroxidation. After 60 h reaction time the percentage of the inhibition of lipid peroxidation (with 95% confidence interval) was: 56.61 (52.67 to 60.55)% for 7C, 82.48 (75.24 to 89.72)% for 5,7C and 90.939 (88.542 to 93.324)% for 7,8C. Of all the studied coumarin molecules only 7C did not show reducing power determined by FRAP and CUPRAC methods. The slope values (with a confidence interval of 95% and a correlation coefficient r2), which are directly proportional to reducing power were 0.0068 (0.00578-0.00777), 0.929 for 5,7C and 0.0269 (0.0235-0.0303), 0.985 for 7,8C determined with FRAP method and 0.0314 (0.0275-0.0354), 0.994 for 5,7C and 0.059 (0.0548-0.06316), 0.99924 for 7,8C determined with CUPRAC method. The calculated values for the DFT descriptors (HOMO, LUMO, the energy difference, IP, ΔEiso and dipole moment) for the studied coumarins in vacuum, n-octanol, ethanol and water show that of all studied 4-phenyl hydroxycoumarins 7,8C has the highest antioxidant activity, which is in agreement with obtained experimental data. The analysis of the parameters obtained with computational calculations indicate that the HAT is thermodynamically dominant mechanism in the gas phase, while in other studied systems, the most likely mechanism is SPLET. The obtained results for 7,8C show that the thermodynamically favored position of the hydroxyl group in the reactions involving BDE, PDE and ETE parameters is 8 and in the reactions which include the PA parameter is position 7. For 5,7C in the gas-phase favored position of the hydroxyl group in all mechanisms is position 5. In other studied systems, in the reaction mechanisms involving thermodynamic parameters BDE, PDE and ETE position 5 is favored, while in the mechanism that include parameter PA the position 7 is favored. It was determined that studied coumarins show better inhibitory effect against Gram (+) bacteria (MIC values range was from 0.01 to 2.50 mg/mL) in comparison to the Gram (-) (MIC values range was 0.16-10 0 mg/mL). 5,7 C and 7,8C show the weakest inhibitory effect against P. aeruginosa and P. mirabilis, while 7C even at the highest tested concentration of 10 mg/mL didn’t show inhibitory effect. All studied coumarins had the strongest inhibitory effect on the growth of strains of S. aureus, L. monocytogenes, L. innocua and M. luteus. The lowest activity among studied coumarin had 7C. 5,7C showed better inhibitory effect on the majority of the tested strains in comparison to 7,8C. For the interpretation of these results, data obtained by molecular docking studies of the studied compounds for the active site of characteristic enzymes were used. MolDock score values for the binding of the studied 4-phenyl hydroxycoumarins to the active site of tyrosyl tRNA synthetase demonstrated that 5,7C has the highest binding affinity, while 7,8C showed the highest binding affinity to the active site of topoisomerase II DNA gyrase β. Hbond values for the tyrosyl tRNA synthetase showed that 7,8C has the strongest interaction, whereas for the topoisomerase II DNA gyrase β 5,7C has strongest interaction. According to all Score values 7C shows the weakest interactions with the enzymes. Best QSAR models for the inhibition of HIV-1 integrase by a coumarin molecules have the following statistical parameters - for the 3' Processing activity: R2 = 0.9980 and Q2 = 0.9977 for the training set, and R2 = 0.9788 for the test set and for the Integration activity: R2 = 0.9999, and Q2 = 0.9998 for training set and R2 = 0.9213 for the test set. QSAR models are applied to the studied 4- phenyl hydroxycoumarins for calculation and evaluation of their inhibition activities on HIV- 1 integrase. All the obtained score values for the studied coumarins binding to the active site of HIV-1 integrase have shown that 7,8C has the highest binding affinity. Calculated physico-chemical and molecular characteristics showed that the investigated 4-phenyl hydroxycoumarins obey Lipinski rules. Conclusion. 7,8C showed a much more pronounced antioxidant activity in comparison to 7C and 5,7C which indicates that the ortho position of the hydroxyl groups in 4-phenyl hydroxycoumarins significantly contributes to their antioxidant activities. HOMO and LUMO energies and their energy difference of 4-phenyl hydroxycoumarins examined in this study also indicate that 7,8C has the highest antioxidant activity. Between 5,7C and 7C better antioxidant activity has 5,7C. ΔEiso values indicate that 7,8C has the greatest ability to capture free radicals. Total spin density indicates that for all examined hydroxyl groups least favored position in the basic structure of the 4-phenylcoumarins for free-radical reactions is position 7. DFT descriptors as well as obtained experimental data confirm that 7,8C has the highest antioxidative activity in comparison to other studied coumarins. HAT is thermodynamically dominant mechanism in the gas phase, while in other studied systems, the most likely antioxidative mechanism of action of studied coumarins is SPLET. The antibacterial activity of the compounds can be summarized in the following sequence: 7C<7,8C<5.7C. The molecular docking was applied for the evaluation of antibacterial activity on the basis of binding affinity of the studied coumarins in the active sites of tyrosyl tRNA synthetase (S. aureus) and topoisomerase II DNA gyrase β (E.coli). Based on the score values obtained with molecular docking, the binding affinity of the investigated molecules was determined and these results are in agreement with the results of their antibacterial activity. QSAR method was successfully applied for building of a mathematical model for calculating the anti-HIV activity of coumarin molecules on the basis of inhibition of HIV-1 integrase. All studied 4-phenyl hydroxycoumarins obey the Lipinski rules indicating their satisfactory pharmacokinetic profile

Nano-QSAR: Model of mutagenicity of fullerene as a mathematical function of different conditions

The experimental data on the bacterial reverse mutation test (under various conditions) on C60 nanoparticles for the cases (i) TA100, and (ii) WP2uvrA/pkM101 are examined as endpoints. By means of the optimal descriptors calculated with the Monte Carlo method a mathematical model of these endpoints has been built up. The models are a mathematical function of eclectic data such as (i) dose (g/plate); (ii) metabolic activation (i.e. with mix S9 or without mix S9); and (iii) illumination (i.e. darkness or irradiation). The eclectic data on different conditions were represented by so-called quasi-SMILES. In contrast to the traditional SMILES which are representation of molecular structure, the quasi-SMILES are representation of conditions by sequence of symbols. The calculations were carried out with the CORAL software, available on the Internet at http://www.insilico.eu/coral. The main idea of the suggested descriptors is the accumulation of all available eclectic information in the role of logical and digital basis for building up a model. The computational experiments have shown that the described approach can be a tool to build up models of mutagenicity of fullerene under different conditions

QSAR modeling of dihydrofolate reductase inhibitors as a therapeutic target for multiresistant bacteria

Antibacterial resistance is a growing public health threat of major concern around the world so development of new therapeutic approaches to prevent bacterial multidrug resistance has become a primary consideration for medicinal chemistry research. QSAR models for the dihydrofolate reductase inhibition with 2,4-diamino-5-(substituted-benzyle)-pyramidine derivatives were developed with further computer-aided design of new derivatives with desired activity. The Monte Carlo method has been used as a computational tool for QSAR modeling. For the representation of molecular structure and optimal descriptor calculation, the simplified molecular input line entry system (SMILES) together with the molecular graph (hydrogen-suppressed graph-HSG, hydrogen-filled graph-HFG, and the graph of atomic orbitals-GAO) was used. One-variable models have been calculated for one data split into training, test, and validation set. The impact of Morgan's extended connectivity index on built QSAR models and outliers was determined. Statistical parameters for the best QSAR model are satisfying. Structural indicators (molecular fragments) responsible for the increase and the decrease of the stated activity are defined, and with the application of defined structural alerts, the computer-aided design of new derivatives with desired activity is presented. Computational experiments presented and applied in this research can satisfactorily predict desired endpoint and can be used further for computer-aided antibacterial drug design

RNA-targeting low-molecular-weight fluorophores for nucleoli staining: synthesis, in silico modelling and cellular imaging

Herein we present our work on the synthesis, investigation of the photophysical properties, interactions with nucleic acids, molecular docking, and imaging application of three carbocyanine dyes. The described low-molecular-weight compounds were found to exhibit high resistance against photobleaching and showed promising optical properties as fluorescent labeling agents for ribonucleic acids. They form strong biocomplexes (log K-s = 6.11-7.84) and revealed remarkable sensitivity towards RNA, reaching up to a 379 times increase of the emission signal when bound to AU-rich sequences. According to the score values obtained from the molecular docking studies, the compounds show strong binding affinity towards RNA macromolecules. All fluorophores exhibit significant cell tolerance since they were found to be 16 to 60 times less toxic against MRC5 cells (healthy human fibroblasts) compared to the conventional Thiazole Orange - TO. The IC50 concentrations for the compounds were calculated up to 40 mu M in human fibroblasts MRC5, A549 adenocarcinomic human alveolar basal epithelial cells, the HCT116 human colon cancer cell line, and MDA-MB-231 adenocarcinoma cells. Analyzing the dyes for preferential cytotoxicity towards cancer cell lines in comparison to the normal human fibroblasts, we found a candidate exhibiting promising anticancer potential. Based on the selectivity (Si) towards cancer cells and more specifically against difficult to treat colon HCT116 carcinoma, we can suggest these small molecules as an interesting platform for further development. We have also demonstrated the efficiency of the carbocyanines as staining agents for in vivo labeling of human cells as well as microbial and eukaryotic cells

Potent anti-melanogenic activity and favorable toxicity profile of selected 4-phenyl hydroxycoumarins in the zebrafish model and the computational molecular modeling studies

7-Hydroxy-4-phenylcoumarin (7C) and 5,7-dihydroxy-4-phenylcoumarin (5,7C) have been evaluated as potential anti-melanogenic agents in the zebrafish (Danio rerio) model in comparison to commercially utilized depigmenting agents hydroquinone and kojic acid. 7C and 5,7C decreased the body pigmentation at 5 mu g/mL, while did not affect the embryos development and survival at doses lt = 50 mu g/mL and lt = 25 mu g/mL. Unlike hydroquinone and kojic acid, 4-phenyl hydroxycoumarins were no melanocytotoxic, showed no cardiotoxic side effects, neither caused neutropenia in zebrafish embryos, suggesting these compounds may present novel skin-whitening agents with improved pharmacological properties. Inhibition of tyrosinase was identified as the possible mode of anti-melanogenic action. Molecular docking studies using the homology model of human tyrosinase as well as adenylate cyclase revealed excellent correlation with experimentally obtained results

Selected 4-phenyl hydroxycoumarins: In vitro cytotoxicity, teratogenic effect on zebrafish (Danio rerio) embryos and molecular docking study

A study of structure cytotoxic-activity relationship of three hydroxy 4-phenyl-coumarins and basic coumarin molecule against two human cell lines (MRC5 fibroblasts and A375 melanoma cells) is presented. Of all investigated compounds the highest cytotoxic activity in both cell lines was determined for 7,8-dihydroxy-4-phenyl coumarin. SAR studies revealed the influence of phenyl group and hydroxyl group's number and position on cytotoxic activity. In addition, to get an insight about their binding preferences at the active site of the receptor (catalytic subunit of cAMP-dependent protein kinase) molecular docking studies were performed. Docking studies suggest that 4-phenyl hydroxycoumarins are potent cAMP-dependent protein kinase inhibitors, better than their analogs without phenyl group. The teratogenic potential was assessed in zebrafish embryo toxicity test and results showed that 4-phenyl dihydroxycoumarins were more while 7-hydroxy-4-phenyl coumarin was less embryo toxic in comparison to coumarin. In order to examine selected 4-phenyl hydroxycoumarins as a new lead compounds the druglikeness of selected 4-phenyl hydroxycoumarins was estimated by using Lipinski's "rule of five". All selected 4-phenyl hydroxycoumarins proved to have satisfying pharmacokinetic profile

Biological activity of Pinus nigra terpenes-Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity

In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50=25.08 mg/mL and VitC=0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity. (C) 2014 Elsevier Ltd. All rights reserved.Ministry of Education and Science of the Republic of Serbia [173029

Biological activity of Pinus nigra terpenes-Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity

In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50=25.08 mg/mL and VitC=0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity. (C) 2014 Elsevier Ltd. All rights reserved.Ministry of Education and Science of the Republic of Serbia [173029

Nano-QSAR: Model of mutagenicity of fullerene as a mathematical function of different conditions

The experimental data on the bacterial reverse mutation test (under various conditions) on C60 nanoparticles for the cases (i) TA100, and (ii) WP2uvrA/pkM101 are examined as endpoints. By means of the optimal descriptors calculated with the Monte Carlo method a mathematical model of these endpoints has been built up. The models are a mathematical function of eclectic data such as (i) dose (g/plate); (ii) metabolic activation (i.e. with mix S9 or without mix S9); and (iii) illumination (i.e. darkness or irradiation). The eclectic data on different conditions were represented by so-called quasi-SMILES. In contrast to the traditional SMILES which are representation of molecular structure, the quasi-SMILES are representation of conditions by sequence of symbols. The calculations were carried out with the CORAL software, available on the Internet at http://www.insilico.eu/coral. The main idea of the suggested descriptors is the accumulation of all available eclectic information in the role of logical and digital basis for building up a model. The computational experiments have shown that the described approach can be a tool to build up models of mutagenicity of fullerene under different conditions