Chemoprotection mediated by açaí berry (Euterpe oleracea) in white shrimp Litopenaeus vannamei exposed to the cyanotoxin saxitoxin analyzed by in vivo assays and docking modeling.

Saxitoxin (STX) is a neurotoxic cyanotoxin that also generate reactive oxygen species, leading to a situation of oxidative stress and altered metabolism. The Amazonian fruit açaí Euterpe oleracea possesses a high concentration of antioxidant molecules, a fact that prompted us to evaluate its chemoprotection activity against STX toxicity (obtained from samples of Trichodesmium sp. collected in the environment) in the shrimp Litopenaeus vannamei. For 30 days, shrimps were maintained in 16 aquaria containing 10 shrimps (15% salinity, pH 8.0, 24 °C, 12C/12D photoperiod) and fed twice daily with a diet supplemented with lyophilized açaí pulp (10%), in addition to the control diet. After, shrimps (7.21 ± 0.04 g) were exposed to the toxin added to the feed for 96 h. Four treatments were defined: CTR (control diet), T (lyophilized powder of Trichodesmium sp. 0.8 µg/g), A (10% of açaí) and the combination T + A. HPLC analysis showed predominance of gonyautoxin-1 concentrations (GTX-1) and gonyautoxin-4 concentrations (GTX-4). The results of molecular docking simulations indicated that all variants of STX, including GTX-1, can be a substrate of isoform mu of the glutathione-S-transferase (GST) enzyme since these molecules obtained similar values of estimated Free Energy of Binding (FEB), as well as similar final positions on the binding site. GSH levels were reduced in muscle tissues of shrimp in the T, A, and T + A treatments. Increased GST activity was observed in shrimp hepatopancreas of the T treatment and the gills of the A and T + A treatments. A decrease of protein sulfhydryl groups (P-SH) was observed in gills of shrimps from T + A treatment. A reduction in malondialdehyde (MDA) levels was registered in the hepatopancreas of the T + A treatment in respect to the Control, T, and A treatments. The use of açaí supplements in L. vannamei feed was able to partially mitigate the toxic effects caused by Trichodesmium sp. extracts, and points to mu GST isoform as a key enzyme for saxitoxin detoxification in L. vannamei, an issue that deserves further investigation.

E-Volve: understanding the impact of mutations in SARS-CoV-2 variants spike protein on antibodies and ACE2 affinity through patterns of chemical interactions at protein interfaces.

Background: The SARS-CoV-2 pandemic reverberated, posing health and social hygiene obstacles throughout the globe. Mutant lineages of the virus have concerned scientists because of convergent amino acid alterations, mainly on the viral spike protein. Studies have shown that mutants have diminished activity of neutralizing antibodies and enhanced affinity with its human cell receptor, the ACE2 protein. Methods: Hence, for real-time measuring of the impacts caused by variant strains in such complexes, we implemented E-Volve, a tool designed to model a structure with a list of mutations requested by users and return analyses of the variant protein. As a proof of concept, we scrutinized the spike-antibody and spike-ACE2 complexes formed in the variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma), by using contact maps depicting the interactions made amid them, along with heat maps to quantify these major interactions. Results: The results found in this study depict the highly frequent interface changes made by the entire set of mutations, mainly conducted by N501Y and E484K. In the spike-Antibody complex, we have noticed alterations concerning electrostatic surface complementarity, breaching essential sites in the P17 and BD-368-2 antibodies. Alongside, the spike-ACE2 complex has presented new hydrophobic bonds. Discussion: Molecular dynamics simulations followed by Poisson-Boltzmann calculations corroborate the higher complementarity to the receptor and lower to the antibodies for the K417T/E484K/N501Y (Gamma) mutant compared to the wild-type strain, as pointed by E-Volve, as well as an intensification of this effect by changes at the protein conformational equilibrium in solution. A local disorder of the loop α1'/ß1', as well its possible effects on the affinity to the BD-368-2 antibody were also incorporated to the final conclusions after this analysis. Moreover, E-Volve can depict the main alterations in important biological structures, as shown in the SARS-CoV-2 complexes, marking a major step in the real-time tracking of the virus mutant lineages. E-Volve is available at http://bioinfo.dcc.ufmg.br/evolve.

Whole-genome sequencing as a tool for studying the microevolution of drug-resistant serial Mycobacterium tuberculosis isolates.

Treatment of drug-resistant tuberculosis requires extended use of more toxic and less effective drugs and may result in retreatment cases due to failure, abandonment or disease recurrence. It is therefore important to understand the evolutionary process of drug resistance in Mycobacterium tuberculosis. We here in describe the microevolution of drug resistance in serial isolates from six previously treated patients. Drug resistance was initially investigated through phenotypic methods, followed by genotypic approaches. The use of whole-genome sequencing allowed the identification of mutations in the katG, rpsL and rpoB genes associated with drug resistance, including the detection of rare mutations in katG and mixed populations of strains. Molecular docking simulation studies of the impact of observed mutations on isoniazid binding were also performed. Whole-genome sequencing detected 266 single nucleotide polymorphisms between two isolates obtained from one patient, suggesting a case of exogenous reinfection. In conclusion, sequencing technologies can detect rare mutations related to drug resistance, identify subpopulations of resistant strains, and identify diverse populations of strains due to exogenous reinfection, thus improving tuberculosis control by guiding early implementation of appropriate clinical and therapeutic interventions.

Relation between ABCB1 overexpression and COX2 and ALOX5 genes in human erythroleukemia cell lines.

This study aimed to characterize the relationship between the COX2 and ALOX5 genes, as well as their link with the multidrug resistance (MDR) phenotype in sensitive (K562) and MDR (K562-Lucena and FEPS) erythroleukemia cells. For this, the inhibitors of 5-LOX (zileuton) and COX-2 (acetylsalicylic acid-ASA) and cells with the silenced ABCB1 gene were used. The treatment with ASA caused an increase in the gene expression of COX2 and ABCB1 in both MDR cell lines, and a decrease in the expression of ALOX5 in the FEPS cells. Silencing the ABCB1 gene induced a decrease in COX2 expression and an increase in the ALOX5 gene. Treatment with zileuton did not alter the expression of COX2 and ABCB1. Cytometry data showed that there was an increase in ABCB1 protein expression after exposure to ASA. In addition, the increased activity of ABCB1 in the K562-Lucena cell line indicates that ASA may be a substrate for this efflux pump, corroborating the molecular docking that showed that ASA can bind to ABCB1. Regardless of the genetic alteration in COX2 and ABCB1, the direct relationship between these genes and the inverse relationship with ALOX5 remained in the MDR cell lines. We assume that ABCB1 can play a regulatory role in COX2 and ALOX5 during the transformation of the parental cell line K562, explaining the increased gene expression of COX2 and decreased ALOX5 in the MDR cell lines.

KORP-PL: a coarse-grained knowledge-based scoring function for protein-ligand interactions.

MOTIVATION: Despite the progress made in studying protein-ligand interactions and the widespread application of docking and affinity prediction tools, improving their precision and efficiency still remains a challenge. Computational approaches based on the scoring of docking conformations with statistical potentials constitute a popular alternative to more accurate but costly physics-based thermodynamic sampling methods. In this context, a minimalist and fast sidechain-free knowledge-based potential with a high docking and screening power can be very useful when screening a big number of putative docking conformations. RESULTS: Here, we present a novel coarse-grained potential defined by a 3D joint probability distribution function that only depends on the pairwise orientation and position between protein backbone and ligand atoms. Despite its extreme simplicity, our approach yields very competitive results with the state-of-the-art scoring functions, especially in docking and screening tasks. For example, we observed a twofold improvement in the median 5% enrichment factor on the DUD-E benchmark compared to Autodock Vina results. Moreover, our results prove that a coarse sidechain-free potential is sufficient for a very successful docking pose prediction. AVAILABILITYAND IMPLEMENTATION: The standalone version of KORP-PL with the corresponding tests and benchmarks are available at https://team.inria.fr/nano-d/korp-pl/ and https://chaconlab.org/modeling/korp-pl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Long-chain fatty dihydropyridines: Docking calcium channel studies and antihypertensive activity.

AIMS: From the synthesis of 43 lipophilic dihydropyridines, the aim of this study was to verify whether the new dihydropyridines have calcium channel affinity using coupling studies and to determine antihypertensive and antioxidant properties, as well as toxicology and toxicity nifedipine and three new compounds, were chosen from the previous results. MATERIALS AND METHODS: The animals were treated for 56 days, 28 days with N (ω) -nitro-L-arginine methyl ester to induce hypertension, and then treated for another 28 days with the new di- hydropyridine and the standard drug nifedipine. Throughout the treatment the animals had their blood pressure measured and their heart rate checked by pletysmography. After treatment the animals were euthanised, blood samples were collected for creatine kinase and urea analysis, and the brain, heart and liver were collected for oxidative status analysis (quantification of reactive oxygen species, total antioxidant capacity, and lipid peroxidation). KEY FINDINGS: Compounds 2c, and 9a, and nifedipine significantly reduced blood pressure to control group levels. The tachycardia caused by the induction of hypertension was reversed by 2c and 9a compounds. Regarding oxidative stress analyzes, the compounds that had the best performances were also 2c and 9a. Overall the results demonstrate that two of the three new dihydropyridines tested demonstrated performance equal to or superior to the standard drug nifedipine. SIGNIFICANCE: In this study, for the first time, docking was applied to analyse 43 fatty dihydropyridines regarding their calcium channel binding. Afterwards, three fatty dihydropyridines were chosen and their antihypertensive and antioxidant properties.

In Vitro Effects of 2-{4-[Methylthio(methylsulfonyl)]phenyl}-3-substitutedthiazolidin-4-ones on the Acetylcholinesterase Activity in Rat Brain and Lymphocytes: Isoform Selectivity, Kinetic Analysis, and Molecular Docking.

This work evaluated the in vitro effect of thiazolidin-4-ones on the activity of AChE (total and isoforms) isolated from the cerebral cortex, hippocampus, and lymphocytes. Kinetic parameters were evaluated and molecular docking was performed. Our results showed that thiazolidinones derived from 4-(methylthio)benzaldehyde (1) and from 4-(methylsulfonyl)benzaldehyde (2) were capable of inhibiting the AChE activity in vitro. Three compounds, two with a propylpiperidine (1b and 2b) moiety and one with a 3-(diethylamino)propyl (1c) moiety showed IC50 values of 13.81 µM, and 3.13 µM (1b), 55.36 µM and 44.33 µM (1c) for cerebral cortex and hippocampus, respectively, and 3.11 µM for both (2b). Enzyme kinetics revealed that the type of AChE inhibition was mixed. Compound 1b inhibited the G1 and G4 AChE isoforms, while compounds 1c and 2b selectively inhibited the G4 isoform. Molecular docking showed a possible three-dimensional fit into the enzyme. Our findings showed that these thiazolidin-4-ones, especially those containing the propylpiperidine core, have a potential cholinesterase inhibitory activity and can be considered good candidates for future Alzheimer's therapy.

A Computational Method to Propose Mutations in Enzymes Based on Structural Signature Variation (SSV).

With the use of genetic engineering, modified and sometimes more efficient enzymes can be created for different purposes, including industrial applications. However, building modified enzymes depends on several in vitro experiments, which may result in the process being expensive and time-consuming. Therefore, computational approaches could reduce costs and accelerate the discovery of new technological products. In this study, we present a method, called structural signature variation (SSV), to propose mutations for improving enzymes' activity. SSV uses the structural signature variation between target enzymes and template enzymes (obtained from the literature) to determine if randomly suggested mutations may provide some benefit for an enzyme, such as improvement of catalytic activity, half-life, and thermostability, or resistance to inhibition. To evaluate SSV, we carried out a case study that suggested mutations in ß-glucosidases: Essential enzymes used in biofuel production that suffer inhibition by their product. We collected 27 mutations described in the literature, and manually classified them as beneficial or not. SSV was able to classify the mutations with values of 0.89 and 0.92 for precision and specificity, respectively. Then, we used SSV to propose mutations for Bgl1B, a low-performance ß-glucosidase. We detected 15 mutations that could be beneficial. Three of these mutations (H228C, H228T, and H228V) have been related in the literature to the mechanism of glucose tolerance and stimulation in GH1 ß-glucosidase. Hence, SSV was capable of detecting promising mutations, already validated by in vitro experiments, that improved the inhibition resistance of a ß-glucosidase and, consequently, its catalytic activity. SSV might be useful for the engineering of enzymes used in biofuel production or other industrial applications.

Molecular modelling and competitive inhibition of a Mycobacterium tuberculosis multidrug-resistance efflux pump.

Tuberculosis is a major cause of mortality and morbidity in developing countries, and the emergency of multidrug and extensive drug resistance cases is an utmost issue on the control of the disease. Despite the efforts on the development of new antibiotics, eventually there will be strains resistant to them as well. Efflux plays an important role in the evolution of resistance in Mycobacterium tuberculosis. Tap is an important efflux pump associated with tuberculosis resistant to isoniazid, rifampicine and ofloxacin and with multidrug resistance. The development of efflux inhibitors for Tap could raise the effectiveness of second line drugs and reduce the duration of the current treatment. Therefore the objective of this study is to build a reliable molecular model of Tap efflux pump and test the possible competitive inhibition between efflux inhibitors and antibiotics in the optimized structure. We built twenty five Tap models with molecular modelling to elect the best according to the results of the validation analysis. The elected model went through to a 100 ns molecular dynamics simulation in a lipid bilayer, and the resulting optimized structure was used in docking studies to test if the used efflux inhibitors may act via competitive inhibition on antibiotics. The validation results pointed the model built by Phyre2 as the closest to a possible native Tap structure, and therefore it was the elected model. RSMD analysis revealed the model is stable, where the predicted binding site stabilized between 15 and 20 ns, maintaining the RMSD at around 0.35 Šthroughout the molecular dynamics simulation in a lipid bilayer. Therefore this model is reliable and can also be used for further studies. The docking studies showed a possibility of competitive inhibition by NUNL02 on ofloxacin and bedaquiline, and by verapamil on ofloxacin and rifampicin. This presents the possibility that NUNL02 and verapamil are possible inhibitors of Tap efflux and highlights the importance of including efflux inhibitors as adjuvants to the tuberculosis therapy, as it indicates a possible extrusion of ofloxacin, rifampicin and bedaquilin by Tap.