Laccase catalytic activity shielded by SiO2 nanostructured materials: an in vitro and in silico approach.

This study investigates the enhancement of enzymatic catalytic performance by immobilizing laccase on various nanostructured mesoporous silica materials (SBA-15, MCF, and MSU-F). The activity of immobilized laccase was evaluated under different hydrothermal, pH, and solvent conditions, with laccase@MSU-F showing a three-fold increase in stability. Laccase immobilized on these materials demonstrated stability in a pH range of 4.5 to 10.0, while free laccase was inactivated at pH higher than 7. Molecular dynamics simulations revealed that electrostatic interactions and protective confinement effects contribute to the improved stability of immobilized laccase. Overall, the findings suggest that nanomaterials can enhance the operational stability and recovery of enzymes.Communicated by Ramaswamy H. Sarma.

Molecular dynamics and free energy calculations of clozapine bound to D2 and H1 receptors reveal a cardiometabolic mitigated derivative.

Most atypical antipsychotics derive from a high dropout of drug treatments due to adverse cardiometabolic side effects. These side effects are caused, in part, by the H1 receptor blockade. The current work sought a clozapine derivative with a reduced affinity for the H1 receptor while maintaining its therapeutic effect linked to D2 receptor binding. Explicit molecular dynamics simulations and end-point free energy calculations of clozapine in complex with the D2 and H1 receptors embedded in cholesterol-rich lipid bilayers were performed to analyze the intermolecular interactions and address the relevance of clozapine-functional groups. Based on that, free energy perturbation calculations were performed to measure the change in free energy of clozapine structural modifications. Our results indicate the best clozapine derivative is the iodine atom substitution for chlorine. The latter is mainly due to electrostatic interaction loss for the H1 receptor, while the halogen orientation out of the D2 active site reduces the impact on the affinity.Communicated by Ramaswamy H. Sarma.

Synthesis and optimization of photothermal properties of NIR emitting LiGa5O8: Cr3+ and gold nanorods as hybrid materials for theranostic applications.

The increase in incidence of degenerative diseases has fueled the development of novel materials, mostly focused on reducing adverse effects caused by current medical therapies. Theranostic materials represent an alternative to treat degenerative diseases, since they combine diagnostic properties and localized therapy within the same material. This work presents the synthesis and characterization of hybrid materials designed for theranostic purposes. The hybrid materials were composed of LiGa5O8:Cr3+ (LGO) with emission lines in the near infrared (NIR), hence providing an excellent diagnostic ability. As for the therapy part, the hybrid nanomaterials contained gold nanorods (AuR) with localized surface plasmon resonance (LSPR). Once AuR are excited, plasmonic processes are triggered at their surface resulting in increased localized temperature capable of inducing irreversible damage to the cells. A detailed characterization of the hybrid materials confirmed proper assembly of LGO and AuR. Moreover, these nanocomposites preserved their luminescent properties and LSPR. Finally, the cytotoxic potential of the hybrid material was evaluated in different cell lines by cell viability colorimetric assays to determine its possible use as theranostic agent. The success in the synthesis of hybrid materials based on LGO with emission in the NIR coupled with AuR, provides a new perspective for the design of hybrid materials with improved properties to be used in biomedical fields.

Influence of the fluoroethylene carbonate on the electrochemical behavior of Bi3Ge4O12 as Lithium-ion anode.

Systematic ex-situ X-ray diffraction (XRD) characterization and electrochemical study revealed the key roles that the cut-off voltage and fluoroethylene carbonate (FEC) additive play on improving electrochemical performance of the Bi3Ge4O12-based (BGO) electrode. The ex-situ XRD analysis revealed that BGO particles suffer multiphase transitions during the (dis)charge reactions, being observed some phases as Bi2O2.33, BiLi3, Li2O, Ge4Li15, Ge2Li7, Ge3Li7, Ge5Li22, Ge4Li9, Bi2O3 and GeO2. The electrochemical evaluation exhibited that the addition of 5 v/v% of FEC in 1.0 M lithium hexafluorophosphate (LiPF6) in ethylene carbonate and diethyl carbonate (EC: DEC) at an applied cut-off voltage (1.5 V vs Li/Li+) improves the specific capacity (29%, delivering 479 mAh g-1), capacity retention (12%) and rate capability (369 mAh g-1 at 1000 mA g-1) of the BGO-based electrode. Also, FEC promotes the formation of a stable solid-electrolyte interface (SEI) layer on the anode at a cut-off voltage of 1.5 V vs Li/Li+. It displays the lowest values of SEI and charge transfer (CT) resistances, and electrode polarization, improving the reversibility of the alloying reactions related to Ge-Li and Bi-Li and maintaining their redox activity after 100 cycles, according to dQ dV-1 data.

Improving Structure-Based Virtual Screening with Ensemble Docking and Machine Learning.

One of the main challenges of structure-based virtual screening (SBVS) is the incorporation of the receptor's flexibility, as its explicit representation in every docking run implies a high computational cost. Therefore, a common alternative to include the receptor's flexibility is the approach known as ensemble docking. Ensemble docking consists of using a set of receptor conformations and performing the docking assays over each of them. However, there is still no agreement on how to combine the ensemble docking results to obtain the final ligand ranking. A common choice is to use consensus strategies to aggregate the ensemble docking scores, but these strategies exhibit slight improvement regarding the single-structure approach. Here, we claim that using machine learning (ML) methodologies over the ensemble docking results could improve the predictive power of SBVS. To test this hypothesis, four proteins were selected as study cases: CDK2, FXa, EGFR, and HSP90. Protein conformational ensembles were built from crystallographic structures, whereas the evaluated compound library comprised up to three benchmarking data sets (DUD, DEKOIS 2.0, and CSAR-2012) and cocrystallized molecules. Ensemble docking results were processed through 30 repetitions of 4-fold cross-validation to train and validate two ML classifiers: logistic regression and gradient boosting trees. Our results indicate that the ML classifiers significantly outperform traditional consensus strategies and even the best performance case achieved with single-structure docking. We provide statistical evidence that supports the effectiveness of ML to improve the ensemble docking performance.

Structure-Based Virtual Screening of Sterols and Triterpenoids Isolated from Ganoderma (Agaricomycetes) Medicinal Mushrooms Shows Differences in Their Affinity for Human Glucocorticoid and Mineralocorticoid Receptors.

An extensive database of sterols and triterpenoids isolated from Ganoderma mushrooms was evaluated by in silico structure-based virtual screening to determine their respective ligand affinities for the glucocorticoid or mineralocorticoid receptor (GCR or MNR). The main ligands for GCR in our database were ergosta-7,22-dien-3-one (compound 1) and ganodermaside B (compound 2), while the best ligands for MNR were 2ß,3α,9α-trihydroxyergosta-7,22-diene (compound 8) and 5α-ergosta-7,22-dien-3ß-ol (compound 3). The binding free energy (BFE) values calculated for such metabolites were similar to those of the natural ligands for each receptor (i.e., dexamethasone for GCR and aldosterone for MNR). Moreover, the differences between mean BFE values calculated for both receptors suggest that ergosta-7,22-dien-3-one (compound 1), ganodermaside B (compound 2), fungisterol (compound 5), ganoderic acid Ma (compound 9), and cerevisterol (compound 10) might be used as specific ligands for GCR, with a significantly lower affinity for MNR. Finally, it is worth noting that even though this work is exclusively theoretical, the reported bioactivities (either pro- or anti-inflammatory) for those metabolites that were previously studied are consistent with our findings, suggesting that the well-known immunomodulatory effect of Ganoderma triterpenoids and sterols might be attributed, at least partially, to their ability to act as specific GCR ligands.

In Silico Design of Novel Mutant Anti-MUC1 Aptamers for Targeted Cancer Therapy.

The transmembrane glycoprotein mucin 1 (MUC1) is an attractive tumor marker for cancer therapy and diagnosis. The nine amino acid extracellular epitope APDTRPAPG of this protein is selectively recognized by the S2.2 single-stranded DNA anti-MUC1 aptamer, which has emerged as a promising template for designing novel targeting agents for MUC1-directed therapy. In this work, 100 ns molecular dynamics (MD) simulations, MM/GBSA binding free energy calculations, and conformational analysis were employed to propose a novel prospective anti-MUC1 aptamer with increased affinity toward the MUC1 epitope resulting from the double mutation of the T11 and T12 residues with PSU and U nucleosides, respectively. The double mutant aptamer exhibits a tight interaction with the MUC1 epitope and adopts a groove conformation that structurally favors the intermolecular contact with the epitope through the intermediate T11-A18 region leaving the 3' and 5' ends free for further chemical conjugation with a nanocarrier or pharmaceutical. These results are valuable to gain understanding about the molecular features governing aptamer-epitope interactions and constitute a first key step for the design of novel aptamer-based nanocarriers for MUC1-targeted cancer therapy.

Molecular modeling simulation studies reveal new potential inhibitors against HPV E6 protein.

High-risk strains of human papillomavirus (HPV) have been identified as the etiologic agent of some anogenital tract, head, and neck cancers. Although prophylactic HPV vaccines have been approved; it is still necessary a drug-based treatment against the infection and its oncogenic effects. The E6 oncoprotein is one of the most studied therapeutic targets of HPV, it has been identified as a key factor in cell immortalization and tumor progression in HPV-positive cells. E6 can promote the degradation of p53, a tumor suppressor protein, through the interaction with the cellular ubiquitin ligase E6AP. Therefore, preventing the formation of the E6-E6AP complex is one of the main strategies to inhibit the viability and proliferation of infected cells. Herein, we propose an in silico pipeline to identify small-molecule inhibitors of the E6-E6AP interaction. Virtual screening was carried out by predicting the ADME properties of the molecules and performing ensemble-based docking simulations to E6 protein followed by binding free energy estimation through MM/PB(GB)SA methods. Finally, the top-three compounds were selected, and their stability in the E6 docked complex and their effect in the inhibition of the E6-E6AP interaction was corroborated by molecular dynamics simulation. Therefore, this pipeline and the identified molecules represent a new starting point in the development of anti-HPV drugs.

Bismuth germanate (Bi4Ge3O12), a promising high-capacity lithium-ion battery anode.

An unexplored promising lithiation-host anode material, Bi4Ge3O12, delivers a reversible specific discharge capacity of ∼586 mA h g-1 at 200 mA g-1 after 500 cycles with a coulombic efficiency of ∼99.8%. DFT calculations detected distorted [BiO6]9- octahedra, and the band structure of BGO revealed an indirect gap of 3.50 eV. A plausible reaction mechanism of storing lithium is proposed.

Enhanced Degradation of Pesticide Dichlorophen by Laccase Immobilized on Nanoporous Materials: A Cytotoxic and Molecular Simulation Investigation.

Use of pesticides is usually related to overproduction of crops in order to overcome worldwide demand of food and alimentary safety. Nevertheless, pesticides are environmental persistent molecules, such as the organochlorine pesticides, which are often found in undesired places. In this work, we show that a hybrid nanomaterial (laccase-MSU-F) readily oxidizes the pesticide dichlorophen, reducing its acute genotoxicity and apoptotic effects. In order to predict chronic alterations related to endocrine disruption, we compared the calculated affinity of dichlorophen oxidized subproducts to steroid hormone nuclear receptors (NRs), using molecular simulation methods. We found a reduction in theoretical affinity of subproducts of oxidized dichlorophen for the ligand-binding pocket of NRs (∼5 kcal/mol), likewise of changes in binding modes, that suggests a reduction in binding events (RMSD values < 10 Å).

Scoring of Side-Chain Packings: An Analysis of Weight Factors and Molecular Dynamics Structures.

The protein side-chain packing problem (PSCPP) is a central task in computational protein design. The problem is usually modeled as a combinatorial optimization problem, which consists of searching for a set of rotamers, from a given rotamer library, that minimizes a scoring function (SF). The SF is a weighted sum of terms, that can be decomposed in physics-based and knowledge-based terms. Although there are many methods to obtain approximate solutions for this problem, all of them have similar performances and there has not been a significant improvement in recent years. Studies on protein structure prediction and protein design revealed the limitations of current SFs to achieve further improvements for these two problems. In the same line, a recent work reported a similar result for the PSCPP. In this work, we ask whether or not this negative result regarding further improvements in performance is due to (i) an incorrect weighting of the SFs terms or (ii) the constrained conformation resulting from the protein crystallization process. To analyze these questions, we (i) model the PSCPP as a bi-objective combinatorial optimization problem, optimizing, at the same time, the two most important terms of two SFs of state-of-the-art algorithms and (ii) performed a preprocessing relaxation of the crystal structure through molecular dynamics to simulate the protein in the solvent and evaluated the performance of these two state-of-the-art SFs under these conditions. Our results indicate that (i) no matter what combination of weight factors we use the current SFs will not lead to better performances and (ii) the evaluated SFs will not be able to improve performance on relaxed structures. Furthermore, the experiments revealed that the SFs and the methods are biased toward crystallized structures.

A workflow for in silico design of hIL-10 and ebvIL-10 inhibitors using well-known miniprotein scaffolds.

The over-expression of immune-suppressors such as IL-10 is a crucial landmark in both tumor progression, and latent viral and parasite infection. IL-10 is a multifunctional protein. Besides its immune-cell suppressive function, it also promotes B-cell tumorigenesis of lymphomas and melanoma. Human pathogens like unicellular parasites and viruses that remain latent inside B cells promote the over-expression of hIL-10 upon infection, which inhibits cell-mediated immune surveillance, and at the same time mediates B cell proliferation. The B-cell specific oncogenic latent virus Epstein-Barr virus (EBV) encodes a viral homologue of hIL-10 (ebvIL-10), expressed during lytic viral proliferation. Once expressed, ebvIL-10 inhibits cell-mediated immune surveillance, assuring EBV re-infection. During long-term latency, EBV-infected B cells over-express hIL-10 to assure B-cell proliferation, occasionally inducing EBV-mediated lymphomas. The amino acid sequences of hIL-10 and ebvIL-10 are more than 80% identical and thus have a very similar tridimensional structure. Based on their published crystallographic structures bound to their human receptor IL10R1, we report a structure-based design of hIL-10 and ebvIL-10 inhibitors based on 3 loops from IL10R1 that establish specific hydrogen bonds with the two IL10s. We have grafted these loops onto a permissible loop in three well-known miniprotein scaffolds-the Conus snail toxin MVIIA, the plant-derived trypsin inhibitor EETI, and the human appetite modulator AgRP. Our computational workflow described in detail below was invigorated by the negative and positive controls implemented, and therefore paves the way for future in vitro and in vivo validation assays of the IL-10 inhibitors engineered.

A biosensor based on Coriolopsis gallica laccase immobilized on nitrogen-doped multiwalled carbon nanotubes and graphene oxide for polyphenol detection.

The use of nanomaterials allows the design of ultrasensitive biosensors with advantages in the detection of organic molecules. Catechol and catechin are molecules that occur naturally in fruits, and their presence in products like dyes and wines affects quality standards. In this study, catechol and catechin were measured at the nanoscale by means of cyclic voltammetry. The oxidation of Coriolopsis gallica laccase immobilized on nitrogen-doped multiwalled carbon nanotubes (Lac/CN x -MWCNT) and on graphene oxide (Lac/GO) was used to measure the concentrations of catechol and catechin. Nitrogen-doped multiwalled carbon nanotubes (CN x -MWCNT) were synthesized by spray pyrolysis and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and x-ray photoelectron spectroscopy (XPS). Covalently bonded hybrids with laccase (Lac/CN x -MWCNT and Lac/GO) were generated. Catalytic activity of free enzymes determined with syringaldazine yielded 14 584 UmL-1. With Lac/CN x -MWCNT at concentrations of 6.4 mmol L-1 activity was 9326 U mL-1, while enzyme activity measured with Lac/GO at concentration of 6.4 mmol L-1 was 9 234 U mL-1. The Lac/CN x -MWCNT hybrid showed higher stability than Lac/GO at different ethyl alcohol concentrations. The Lac/CN x -MWCNT hybrid can measure concentrations, not previously reported, as low as 1 × 10-8 mol L-1 by measuring the electric current responses.

Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes.

A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250 µA cm(-2) was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 µW cm(-2), while with ethanol 40 µW cm(-2) was obtained.

Strategies for specifically directing metal functionalization of protein nanotubes: constructing protein coated silver nanowires.

Biological molecules that self-assemble in the nanoscale range are useful multifunctional materials. Rotavirus VP6 protein self-assembles into tubular structures in the absence of other rotavirus proteins. Here, we present strategies for selectively directing metal functionalization to the lumen of VP6 nanotubes. The specific in situ metal reduction in the inner surface of nanotube walls was achieved by the simple modification of a method previously reported to functionalize the nanotube outer surface. Silver nanorods and nanowires as long as 1.5 µm were formed inside the nanotubes by coalescence of nanoparticles. Such one-dimensional structures were longer than others previously obtained using bioscaffolds. The interactions between silver ions and the nanotube were simulated to understand the conditions that allowed nanowire formation. Molecular docking showed that a naturally occurring arrangement of aspartate residues enabled the stabilization of silver ions on the internal surface of the VP6 nanotubes. This is the first time that such a spatial arrangement has been proposed for the nucleation of silver nanoparticles, opening the possibility of using such an array to direct functionalization of other biomolecules. These results demonstrate the natural capabilities of VP6 nanotubes to function as a versatile biotemplate for nanomaterials.

Peroxidase activity stabilization of cytochrome P450(BM3) by rational analysis of intramolecular electron transfer.

Combined quantum mechanical and molecular mechanical (QM/MM) calculations were used to explore the electron pathway involved in the suicide inactivation of cytochrome P450BM3 from Bacillus megaterium. The suicide inactivation is a common phenomenon observed for heme peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by highly oxidizing enzyme intermediates formed during the catalytic cycle. The selected model was a mutant comprising only the heme domain (CYPBM3 21B3) that had been previously evolved to efficiently catalyze hydroxylation reactions with hydrogen peroxide (H2O2) as electron acceptor. An extensive mapping of residues involved in electron transfer routes was obtained from density functional calculations on activated heme (i.e. Compound I) and selected amino acid residues. Identification of oxidizable residues (electron donors) was performed by selectively activating/deactivating different quantum regions. This method allowed a rational identification of key oxidizable targets in order to replace them for less oxidizable residues by site-directed mutagenesis. The residues W96 and F405 were consistently predicted by the QM/MM electron pathway to hold high spin density; single and double mutants of P450BM3 on these positions (W96A, F405L, W96A/F405L) resulted in a more stable variants in the presence of hydrogen peroxide, displaying a similar reaction rate than P450BM3 21B3. Furthermore, mass spectrometry confirmed these oxidation sites and corroborated the possible routes described by QM/MM electron transfer (ET) pathways.

Prediction model based on decision tree analysis for laccase mediators.

A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (ɛ°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (Δ(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.

Substitution of the catalytic metal and protein PEGylation enhances activity and stability of bacterial phosphotriesterase.

Phosphotriesterase, a pesticide-degrading enzyme, from Flavobacterium sp. was cloned and expressed in Escherichia coli. The catalytic zinc ions were replaced by cobalt atoms increasing the catalytic activity of phosphotriesterase on different pesticides. This metal substitution increased the catalytic activity from 1.4 times to 4 times according to the pesticide. In order to explain this catalytic increase, QM/MM calculations were performed. Accordingly, the HOMO energy of the substrate is closer to the LUMO energy of the cobalt-substituted enzyme. The chemical modification of the enzyme surface with poly(ethylene glycol) increased the thermostability and stability against metal chelating agents of both metal phosphotriesterase preparations.

Bioreduction of some common carbonylic compounds mediated by yeasts.

Bioreduction of several prochiral carbonylic compounds such as acetophenone (1), ethyl acetoacetate (2) and ethyl phenylpropionate (3) to the corresponding optically active sec-alcohols 1a-3a was performed using wild-type strains of Pichia pastoris UBB 1500, Rhodotorula sp., and Saccharomyces cerevisiae. The reductions showed moderate to excellent conversion and high enantiomeric excess, in an extremely mild and environmentally benign manner in aqueous medium, using glucose as cofactor regeneration system. The obtained alcohols follow Prelog's rule, but in the reduction of 1 with P. pastoris UBB 1500 the anti-Prelog enantiopreference was observed.

Biotransformation of indole derivatives by mycelial cultures.

Biotransformation of tryptophan to tryptamine and 3-methyl-indole by Psilocybe coprophila was performed. On the other hand, Aspergillus niger was able to transform tryptophan to 5-hydroxy-tryptophan. P. coprophila biotransformed 5-hydroxy-tryptophan to 5-hydroxytryptamine. These results prove once more that fungi are good tools to establish hydroxyindole derivatives.