Bioconcentration factor (BCF) describes the behaviour of a chemical in terms of its likelihood of concentrating in organisms in the environment. It is a fundamental property in recent regulations, such as the European Community Regulation on chemicals and their safe use or the Globally Harmonized System for classification, labelling and packaging. These new regulations consider the possibility of reducing or waiving animal tests using alternative methods, such as in silico methods. This study assessed and validated the CAESAR predictive model for BCF in fish.
Results
To validate the model, new experimental data were collected and used to create an external set, as a second validation set (a first validation exercise had been done just after model development). The performance of the model was compared with BCFBAF v3.00. For continuous values and for classification purposes the CAESAR BCF model gave better results than BCFBAF v3.00 for the chemicals in the applicability domain of the model. R2 and Q2 were good and accuracy in classification higher than 90%. Applying an offset of 0.5 to the compounds predicted with BCF close to the thresholds, the number of false negatives (the most dangerous errors) dropped considerably (less than 0.6% of chemicals).
Conclusions
The CAESAR model for BCF is useful for regulatory purposes because it is robust, reliable and predictive. It is also fully transparent and documented and has a well-defined applicability domain, as required by REACH. The model is freely available on the CAESAR web site and easy to use. The reliability of the model reporting the six most similar compounds found in the CAESAR dataset, and their experimental and predicted values, can be evaluated.
The new REACH legislation requires assessment of a large number of chemicals in the European market for several endpoints. Developmental toxicity is one of the most difficult endpoints to assess, on account of the complexity, length and costs of experiments. Following the encouragement of QSAR (in silico) methods provided in the REACH itself, the CAESAR project has developed several models.
Results
Two QSAR models for developmental toxicity have been developed, using different statistical/mathematical methods. Both models performed well. The first makes a classification based on a random forest algorithm, while the second is based on an adaptive fuzzy partition algorithm. The first model has been implemented and inserted into the CAESAR on-line application, which is java-based software that allows everyone to freely use the models.
Conclusions
The CAESAR QSAR models have been developed with the aim to minimize false negatives in order to make them more usable for REACH. The CAESAR on-line application ensures that both industry and regulators can easily access and use the developmental toxicity model (as well as the models for the other four endpoints).
The new European Regulation on chemical safety, REACH, (Registration, Evaluation, Authorisation and Restriction of CHemical substances), is in the process of being implemented. Many chemicals used in industry require additional testing to comply with the REACH regulations. At the same time EU member states are attempting to reduce the number of animals used in experiments under the 3 Rs policy, (refining, reducing, and replacing the use of animals in laboratory procedures). Computational techniques such as QSAR have the potential to offer an alternative for generating REACH data. The FP6 project CAESAR was aimed at developing QSAR models for 5 key toxicological endpoints of which skin sensitisation was one.
Results
This paper reports the development of two global QSAR models using two different computational approaches, which contribute to the hybrid model freely available online.
Conclusions
The QSAR models for assessing skin sensitisation have been developed and tested under stringent quality criteria to fulfil the principles laid down by the OECD. The final models, accessible from CAESAR website, offer a robust and reliable method of assessing skin sensitisation for regulatory use.
Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,β-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum.
Methods
In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations.
Results
In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed β-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation.
Conclusions
The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme’s molecular environment on substrate specificities for future investigation.
Novel six organic donor-π-acceptor molecules (D-π-A) used for Bulk Heterojunction organic solar cells (BHJ), based on thienopyrazine were studied by density functional theory (DFT) and time-dependent DFT (TD-DFT) approaches, to shed light on how the π-conjugation order influence the performance of the solar cells. The electron acceptor group was 2-cyanoacrylic for all compounds, whereas the electron donor unit was varied and the influence was investigated.
Methods
The TD-DFT method, combined with a hybrid exchange-correlation functional using the Coulomb-attenuating method (CAM-B3LYP) in conjunction with a polarizable continuum model of salvation (PCM) together with a 6-31G(d,p) basis set, was used to predict the excitation energies, the absorption and the emission spectra of all molecules.
Results
The trend of the calculated HOMO–LUMO gaps nicely compares with the spectral data. In addition, the estimated values of the open-circuit photovoltage (Voc) for these compounds were presented in two cases/PC60BM and/PC71BM.
Conclusion
The study of structural, electronics and optical properties for these compounds could help to design more efficient functional photovoltaic organic materials.
Nitroaromatic and chloronitroaromatic compounds have been a subject of great interest in industry and recently in medical-pharmaceutic field. 2-Chloro-4-nitro/2-chloro-5-nitrobenzoic acids and 4-nitrobenzoic acid are promising new agents for the treatment of main infectious killing diseases in the world: immunodeficiency diseases and tuberculosis.
Results
New ethanolamine nitro/chloronitrobenzoates were synthesized and characterized by X-ray crystallography, UV–vis, FT-IR and elementary analysis techniques. The toxicity of the compounds prepared and correspondent components was evaluated using Hydractinia echinata as test system. A significant lower toxicity was observed for nitro-derivative compared with chloronitro-derivatives and individual components. Crystallographic studies, together with the chemical reactivity and stability profiles resulted from density functional theory and ab initio molecular orbital calculations, explain the particular behavior of ethanolamine 4-nitrobenzoate in biological test.
Conclusions
The experimental and theoretical data reveal the potential of these compounds to contribute to the design of new active pharmaceutical ingredients with lower toxicity.
Mutagenicity is the capability of a substance to cause genetic mutations. This property is of high public concern because it has a close relationship with carcinogenicity and potentially with reproductive toxicity. Experimentally, mutagenicity can be assessed by the Ames test on Salmonella with an estimated experimental reproducibility of 85%; this intrinsic limitation of the in vitro test, along with the need for faster and cheaper alternatives, opens the road to other types of assessment methods, such as in silico structure-activity prediction models.A widely used method checks for the presence of known structural alerts for mutagenicity. However the presence of such alerts alone is not a definitive method to prove the mutagenicity of a compound towards Salmonella, since other parts of the molecule can influence and potentially change the classification. Hence statistically based methods will be proposed, with the final objective to obtain a cascade of modeling steps with custom-made properties, such as the reduction of false negatives.
Results
A cascade model has been developed and validated on a large public set of molecular structures and their associated Salmonella mutagenicity outcome. The first step consists in the derivation of a statistical model and mutagenicity prediction, followed by further checks for specific structural alerts in the "safe" subset of the prediction outcome space. In terms of accuracy (i.e., overall correct predictions of both negative and positives), the obtained model approached the 85% reproducibility of the experimental mutagenicity Ames test.
Conclusions
The model and the documentation for regulatory purposes are freely available on the CAESAR website. The input is simply a file of molecular structures and the output is the classification result.
The potential for a compound to cause hepatotoxicity and nephrotoxicity is a matter of extreme interest for human health risk assessment. To assess liver and kidney toxicity, repeated-dose toxicity (RDT) studies are conducted mainly on rodents. However, these tests are expensive, time-consuming and require large numbers of animals. For early toxicity screening, in silico models can be applied, reducing the costs, time and animals used. Among in silico approaches, structure–activity relationship (SAR) methods, based on the identification of chemical substructures (structural alerts, SAs) related to a particular activity (toxicity), are widely employed.
Results
We identified and evaluated some SAs related to liver and kidney toxicity, using RDT data on rats taken from the hazard evaluation support system (HESS) database. We considered only SAs that gave the best percentages of true positives (TP).
Conclusions
It was not possible to assign an unambiguous mode of action for all the SAs, but a mechanistic explanation is provided for some of them. Such achievements may help in the early identification of liver and renal toxicity of substances.
Colony stimulating factors (CSFs) are endogenous cytokines that have key roles in proliferation and differentiation of hematopoietic progenitor cells and in regulation of mature blood cells performance. The CSFs families members are widely used for therapeutic purposes in many field include microbial infections, in cancer chemotherapy, alzheimer disease, hematopoiesis process, and for some neutropenia- related pathologies. Crown ethers are chemical compounds with therapeutic application that can affect the colony formation in vitro. The primary objective of the present study is to evaluate the effect of TDN (novel crown ether) on colony formation of red bone marrow cells in incubation with lung tissues cells.
Method
In this study, bone marrow cells and lung tissue cells of Balb/C were used as a source of hematopoietic stem cells and a source to production colony-stimulating factors, respectively. These cells were incubated with TDN separately and together.
Results
Briefly, the results of this study show that the effects of TDN has excitatory in concentrations lower than 50 µg/ml on colony formation and greater than 50 µg/ml is toxic to cells and it was inhibited the colony formation. Maximum stimulatory and inhibitory effects are shown in 50 and 400 µg/ml of crown ether and no colony was observed in the latter concentration.
Conclusion
The results from this study indicate that TDN significantly able to stimulate the colon formation while increased concentrations of TDN is inhibited colony formation by induction toxic effects due to excessive production of free radicals.
The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. This regulation occurs during the direct synthesis of fatty acids and triacylglycerols (TAGs), as well as during other controlling processes in the main carbon metabolic pathway.
Results
In this study, the mRNA levels of Chlamydomonas citrate synthase (CrCIS) were found to decrease under nitrogen-limited conditions, which suggests suppressed gene expression. Gene silencing by RNA interference (RNAi) was conducted to determine whether CrCIS suppression affected the carbon flux in TAG biosynthesis. Results showed that the TAG level increased by 169.5%, whereas the CrCIS activities in the corresponding transgenic algae decreased by 16.7% to 37.7%. Moreover, the decrease in CrCIS expression led to the increased expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, overexpression of CrCIS gene decreased the TAG level by 45% but increased CrCIS activity by 209% to 266% in transgenic algae.
Conclusions
The regulation of CrCIS gene can indirectly control the lipid content of algal cells. Our findings propose that increasing oil by suppressing CrCIS expression in microalgae is feasible.
Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown.
Methods
The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown.
Results
We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What’s more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer.
Conclusion
This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.
Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.
Methods
Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.
Results
In this work we used, after several optimization reactions, creatine kinase isoforms as well as ?NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method.
Conclusion
With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.
An increased incidence of fungal infections, both invasive and superficial, has been witnessed over the last two decades. Candida species seem to be the main etiology of nosocomial fungal infections worldwide with Candida albicans, which is commensal in healthy individuals, accounting for the majority of invasive Candida infections with about 30-40% of mortality.
Results
New aromatic and heterocyclic esters 5a-k of 1-aryl-3-(1H-imidazol-1-yl)propan-1-ols 4a-d were successfully synthesized and evaluated for their anti-Candida potential. Compound 5a emerged as the most active congener among the newly synthesized compounds 5a-k with MIC value of 0.0833 μmol/mL as compared with fluconazole (MIC value >1.6325 μmol/mL). Additionally, molecular modeling studies were conducted on a set of anti-Candida albicans compounds.
Conclusion
The newly synthesized esters 5a-k showed more potent anti-Candida activities than fluconazole. Compounds 7 and 8 revealed significant anti-Candida albicans activity and were able to effectively satisfy the proposed pharmacophore geometry, using the energy accessible conformers (Econf?<?20 kcal/mol).
The aim of the current work was to determine thermo dynamical properties of 5(2-nitro phenyl)-furan-2-carbaldehyde, 5(3-nitro phenyl)-furan-2-carbaldehyde and 5(4-nitro phenyl)-furan-2-carbaldehyde.
Results
The temperature dependence of saturated vapor pressure of 5(2-nitro phenyl)-furan-2-carbaldehyde, 5(3-nitro phenyl)-furan-2-carbaldehyde and 5(4-nitro phenyl)-furan-2-carbaldehyde was determined by Knudsen’s effusion method. The results are presented by the Clapeyron–Clausius equation in linear form, and via this form, the standard enthalpies, entropies and Gibbs energies of sublimation and evaporation of compounds were calculated at 298.15 K. The standard molar formation enthalpies of compounds in crystalline state at 298.15 K were determined indirectly by the corresponding standard molar combustion enthalpy, obtained using bomb calorimetry combustion.
Conclusions
Determination of the thermodynamic properties for these compounds may contribute to solving practical problems pertaining optimization processes of their synthesis, purification and application and it will also provide a more thorough insight regarding the theoretical knowledge of their nature.
One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface.
Results
In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion.
Conclusion
Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.
In drug discovery research, cell-based phenotypic screening is an essential method for obtaining potential drug candidates. Revealing the mechanism of action is a key step on the path to drug discovery. However, elucidating the target molecules of hit compounds from phenotypic screening campaigns remains a difficult and troublesome process. Simple and efficient methods for identifying the target molecules are essential.
Results
2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) was identified as a senescence inducer from a phenotypic screening campaign. The compound is widely used as a Wnt agonist, although its target molecules remain to be clarified. To identify its target proteins, we compared a series of cellular assay results for the compound with our pathway profiling database. The database comprises the activities of compounds from simple assays of cellular reporter genes and cellular proliferations. In this database, compounds were classified on the basis of statistical analysis of their activities, which corresponded to a mechanism of action by the representative compounds. In addition, the mechanisms of action of the compounds of interest could be predicted using the database. Based on our database analysis, the compound was anticipated to be a tubulin disruptor, which was subsequently confirmed by its inhibitory activity of tubulin polymerization.
Conclusion
These results demonstrate that tubulin is identified for the first time as a target molecule of the Wnt-activating small molecule and that this might have misled the conclusions of some previous studies. Moreover, the present study also emphasizes that our pathway profiling database is a simple and potent tool for revealing the mechanisms of action of hit compounds obtained from phenotypic screenings and off targets of chemical probes.
The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated.
Results
Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in KD value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium.
Conclusions
Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.
The compounds 1,4-napthoquinone (1,4-NQ), bis-(2,4-dinitrophenyl)sulfide (2,4-DNPS), 4-nitrobenzothiadiazole (4-NBT), 3-dimethylaminopropiophenone (3-DAP) and menadione (MD) were tested for antimalarial activity against both chloroquine (CQ)-sensitive (D6) and chloroquine (CQ)-resistant (W2) strains of Plasmodium falciparum through an in vitro assay and also for analysis of non-covalent interactions with P. falciparum thioredoxin reductase (PfTrxR) through in silico docking studies.
Results
The inhibitors of PfTrxR namely, 1,4-NQ, 4-NBT and MD displayed significant antimalarial activity with IC50 values of?<?20 μM and toxicity against 3T3 cell line. 2,4-DNPS was only moderately active. In silico docking analysis of these compounds with PfTrxR revealed that 2,4-DNPS, 4-NBT and MD interact non-covalently with the intersubunit region of the enzyme.
Conclusions
In this study, tools for the identification of PfTrxR inhibitors using phenotyphic screening and docking studies have been validated for their potential use for antimalarial drug discovery project.
