Metabolic Processing of Selenium-Based Bioisosteres of meso-Diaminopimelic Acid in Live Bacteria.

A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is composed of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are targets of potent antibiotics as proper assembly of the PG is essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study the bacterial cell structure, growth, and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to access synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of m-DAP (selenolanthionine) and show that it is installed within the PG of live bacteria by the native cell wall crosslinking machinery in mycobacterial species. This PG probe has an orthogonal release mechanism that could be important for downstream proteomics studies. Finally, we describe a bead-based assay that is compatible with high-throughput screening of cell wall enzymes. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP-containing organisms.

Facile Synthesis and Metabolic Incorporation of m-DAP Bioisosteres Into Cell Walls of Live Bacteria.

Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes cross-linked by cell wall transpeptidases (TP). While synthetic PG fragments containing l-lysine in the third position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on widely available building blocks to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAP and evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. Additionally, we have used hybrid quantum mechanical/molecular mechanical (QM/MM) and molecular dynamics (MD) simulations to explore the influence of m-DAP analogs on the PG cross-linking. The results showed that the cross-linking mechanism of transpeptidases occurred through a concerted process. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.

Exploring Chloride Selectivity and Halogenase Regioselectivity of the SalL Enzyme through Quantum Mechanical/Molecular Mechanical Modeling.

The catalytic mechanism of SalL chlorinase has been investigated by combining quantum mechanical/molecular mechanical (QM/MM) techniques and umbrella sampling simulations to compute free energy profiles. Our results shed light on the interesting fact that the substitution of chloride with fluorine in SalL chlorinase leads to a loss of halogenase activity. The potential of mean force based on DFTB3/MM analysis shows that fluorination corresponds to a barrier 13.5 kcal·mol-1 higher than chlorination. Additionally, our results present a molecular description of SalL acting as a chlorinase instead of a methyl-halide transferase.

Computational study of conformational changes in human 3-hydroxy-3-methylglutaryl coenzyme reductase induced by substrate binding.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR (hHMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of hHMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo hHMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of hHMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of hHMGR. Communicated by Ramaswamy H. Sarma.

Computational analyses of interactions between ALK-5 and bioactive ligands: insights for the design of potential anticancer agents.

Activin Receptor-Like Kinase 5 (ALK-5) is related to some types of cancer, such as breast, lung, and pancreas. In this study, we have used molecular docking, molecular dynamics simulations, and free energy calculations in order to explore key interactions between ALK-5 and six bioactive ligands with different ranges of biological activity. The motivation of this work is the lack of crystal structure for inhibitor-protein complexes for this set of ligands. The understanding of the molecular structure and the protein-ligand interaction could give support for the development of new drugs against cancer. The results show that the calculated binding free energy using MM-GBSA, MM-PBSA, and SIE is correlated with experimental data with r2 = 0.88, 0.80, and 0.94, respectively, which indicates that the calculated binding free energy is in excellent agreement with experimental data. In addition, the results demonstrate that H bonds with Lys232, Glu245, Tyr249, His283, Asp351, and one structural water molecule play an important role for the inhibition of ALK-5. Overall, we discussed the main interactions between ALK-5 and six inhibitors that may be used as starting points for designing new molecules to the treatment of cancer.

Inhibition of tyrosinase by 4H-chromene analogs: Synthesis, kinetic studies, and computational analysis.

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a Ki value of 4 µm, comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site.

Binding Free Energy Calculations of Nine FDA-approved Protease Inhibitors Against HIV-1 Subtype C I36T↑T Containing 100 Amino Acids Per Monomer.

In this work, have investigated the binding affinities of nine FDA-approved protease inhibitor drugs against a new HIV-1 subtype C mutated protease, I36T↑T. Without an X-ray crystal structure, homology modelling was used to generate a three-dimensional model of the protease. This and the inhibitor models were employed to generate the inhibitor/I36T↑T complexes, with the relative positions of the inhibitors being superimposed and aligned using the X-ray crystal structures of the inhibitors/HIV-1 subtype B complexes as a reference. Molecular dynamics simulations were carried out on the complexes to calculate the average binding free energies for each inhibitor using the molecular mechanics generalized Born surface area (MM-GBSA) method. When compared to the binding free energies of the HIV-1 subtype B and subtype C proteases (calculated previously by our group using the same method), it was clear that the I36T↑T proteases mutations and insertion had a significant negative effect on the binding energies of the non-pepditic inhibitors nelfinavir, darunavir and tipranavir. On the other hand, ritonavir, amprenavir and indinavir show improved calculated binding energies in comparison with the corresponding data for wild-type C-SA protease. The computational model used in this study can be used to investigate new mutations of the HIV protease and help in establishing effective HIV drug regimes and may also aid in future protease drug design.

A comparative modeling and molecular docking study on Mycobacterium tuberculosis targets involved in peptidoglycan biosynthesis.

An alarming rise of multidrug-resistant Mycobacterium tuberculosis strains and the continuous high global morbidity of tuberculosis have reinvigorated the need to identify novel targets to combat the disease. The enzymes that catalyze the biosynthesis of peptidoglycan in M. tuberculosis are essential and noteworthy therapeutic targets. In this study, the biochemical function and homology modeling of MurI, MurG, MraY, DapE, DapA, Alr, and Ddl enzymes of the CDC1551 M. tuberculosis strain involved in the biosynthesis of peptidoglycan cell wall are reported. Generation of the 3D structures was achieved with Modeller 9.13. To assess the structural quality of the obtained homology modeled targets, the models were validated using PROCHECK, PDBsum, QMEAN, and ERRAT scores. Molecular dynamics simulations were performed to calculate root mean square deviation (RMSD) and radius of gyration (Rg) of MurI and MurG target proteins and their corresponding templates. For further model validation, RMSD and Rg for selected targets/templates were investigated to compare the close proximity of their dynamic behavior in terms of protein stability and average distances. To identify the potential binding mode required for molecular docking, binding site information of all modeled targets was obtained using two prediction algorithms. A docking study was performed for MurI to determine the potential mode of interaction between the inhibitor and the active site residues. This study presents the first accounts of the 3D structural information for the selected M. tuberculosis targets involved in peptidoglycan biosynthesis.

Targeting the cell wall of Mycobacterium tuberculosis: a molecular modeling investigation of the interaction of imipenem and meropenem with L,D-transpeptidase 2.

The single crystal X-ray structure of the extracellular portion of the L,D-transpeptidase (ex-LdtMt2 - residues 120-408) enzyme was recently reported. It was observed that imipenem and meropenem inhibit activity of this enzyme, responsible for generating L,D-transpeptide linkages in the peptidoglycan layer of Mycobacterium tuberculosis. Imipenem is more active and isothermal titration calorimetry experiments revealed that meropenem is subjected to an entropy penalty upon binding to the enzyme. Herein, we report a molecular modeling approach to obtain a molecular view of the inhibitor/enzyme interactions. The average binding free energies for nine commercially available inhibitors were calculated using MM/GBSA and Solvation Interaction Energy (SIE) approaches and the calculated energies corresponded well with the available experimentally observed results. The method reproduces the same order of binding energies as experimentally observed for imipenem and meropenem. We have also demonstrated that SIE is a reasonably accurate and cost-effective free energy method, which can be used to predict carbapenem affinities for this enzyme. A theoretical explanation was offered for the experimental entropy penalty observed for meropenem, creating optimism that this computational model can serve as a potential computational model for other researchers in the field.

Antifungal activity and computational study of constituents from Piper divaricatum essential oil against Fusarium infection in black pepper.

Fusarium disease causes considerable losses in the cultivation of Piper nigrum, the black pepper used in the culinary world. Brazil was the largest producer of black pepper, but in recent years has lost this hegemony, with a significant reduction in its production, due to the ravages produced by the Fusarium solani f. sp. piperis, the fungus which causes this disease. Scientific research seeks new alternatives for the control and the existence of other Piper species in the Brazilian Amazon, resistant to disease, are being considered in this context. The main constituents of the oil of Piper divaricatum are methyleugenol (75.0%) and eugenol (10.0%). The oil and these two main constituents were tested individually at concentrations of 0.25 to 2.5 mg/mL against F. solani f. sp. piperis, exhibiting strong antifungal index, from 18.0% to 100.0%. The 3D structure of the ß-glucosidase from Fusarium solani f. sp. piperis, obtained by homology modeling, was used for molecular docking and molecular electrostatic potential calculations in order to determine the binding energy of the natural substrates glucose, methyleugenol and eugenol. The results showed that ß-glucosidase (Asp45, Arg113, Lys146, Tyr193, Asp225, Trp226 and Leu99) residues play an important role in the interactions that occur between the protein-substrate and the engenol and methyleugenol inhibitors, justifying the antifungal action of these two phenylpropenes against Fusarium solani f. sp. piperis.

Combined kinetic studies and computational analysis on kojic acid analogous as tyrosinase inhibitors.

Tyrosinase is a key enzyme in melanin synthesis and widely distributed in plants and animals tissues. In mammals, this enzyme is related to pigment production, involved in wound healing, primary immune response and it can also contribute to catecholamines synthesis in the brain. Consequently, tyrosinase enzyme represents an attractive and selective target in the field of the medicine, cosmetics and bio-insecticides. In this paper, experimental kinetics and computational analysis were used to study the inhibition of tyrosinase by analogous of Kojic acid. The main interactions occurring between inhibitors-tyrosinase complexes and the influence of divalent cation (Cu2+) in enzymatic inhibition were investigated by using molecular docking, molecular dynamic simulations and electrostatic binding free energy by using the Linear Interaction Energy (LIE) method. The results showed that the electrostatic binding free energy are correlated with values of constant inhibition (r2 = 0.97).Thus, the model obtained here could contribute to future studies of this important system and, therefore, eventually facilitate development of tyrosinase inhibitors.

Acetylcholinesterase inhibitory activity and molecular docking study of 1-nitro-2-phenylethane, the main constituent of Aniba canelilla essential oil.

The odoriferous principle of Aniba canelilla (H.B.K.) Mez is due 1-nitro-2-phenylethane, the main constituent of its essential oil and also responsible for the plant's cinnamon scent. This nitroderivative was previously reported by their antioxidant, antinociception, cardiovascular, and vasorelaxant properties, and now it was tested as the inhibitor of acetylcholinesterase using bioautography on TLC plates. The oil and a purified fraction containing 1-nitro-2-phenylethane were analyzed by GC and GC-MS. The percentage content of 1-nitro-2-phenylethane in the oil and after fractionation was 70.2% and 98.0%, respectively. The results showed that the oil and 1-nitro-2-phenylethane are strong acetylcholinesterase inhibitors with the detection limit of 0.01 ng, equivalent to physostigmine used as the positive control. A molecular docking study was used to determine the position and conformation of the 1-nitro-2-phenylethane inhibitor in the receptor-binding pocket of the acetylcholinesterase enzyme. The nitrogroup of 1-nitro-2-phenylethane was positioned near of the catalytic serine residue of acetylcholinesterase, forming strong hydrogen bond with its hydroxyl group. Therefore, the electronegative character of 1-nitro-2-phenylethane may explain the interaction that occurs with the catalytic serine residue and its significant inhibitory activity of acetylcholinesterase.

Design and evaluation of 4-aminophenol and salicylate derivatives as free-radical scavenger.

This theoretical and experimental study describes the design and evaluation of the free-radical scavenging effect for the molecular association of 4-aminophenol and salicylate derivatives. For this purpose, we employed theoretical methods for the selection of antioxidant drugs and the rapid methods of evaluation: the 1,1-diphenyl-2-picrylhydrazyl radical and the thiobarbituric acid reactive substances in the lipid peroxidation initiated by Fe(2+) and ascorbic acid in human erythrocytes. The associate derivatives exhibited a more potent inhibition than the salicylic acid, while the benzoyl compound exhibited a more potent inhibition than paracetamol. The molecular parameters related to the electron distribution and structure (ionization potential and energy of the highest occupied molecular orbital) correlated very well with the antioxidant action of the compounds studied here in different tests.

Molecular modeling of T. rangeli, T. brucei gambiense, and T. evansi sialidases in complex with the DANA inhibitor.

Trypanosomal (trans-) sialidases are enzymes that catalyze the transfer of sialic acid residues between host and parasite glycoconjugates. Herein, we have used homology modeling to construct the 3D structures of sialidases from Trypanosoma brucei and Trypanosoma evansi. Hybrid quantum mechanical/molecular mechanical molecular dynamics simulations were used to determine the interaction energy between the 2-deoxy-2,3-didehydro-N-acetylneuraminic acid inhibitor and the three sialidases studied here. Our results suggest that the two constructed enzymes share the same basic fold motive of the Trypanosoma rangeli crystallographic structure. In addition, quantum mechanical/molecular mechanical molecular dynamics simulations show that the 2-deoxy-2,3-didehydro-N-acetylneuraminic acid inhibitor forms a stronger complex with Trypanosoma rangeli than with Trypanosoma brucei and Trypanosoma evansi sialidases. Finally, the interaction energy by residues shows that the arginine triad plays a decisive role to complex 2-deoxy-2,3-didehydro-N-acetylneuraminic acid with the enzyme through hydrogen bonding.

Structure modeling of a metalloendopeptidase from Corynebacterium pseudotuberculosis.

Metalloendopeptidases are zinc-dependent hydrolases enzymes with many different roles in biological systems, ranging from remodeling conjunctive tissue to removing signaling sequences from nascent proteins. Here, we describe the three-dimensional structure of the metalloendopeptidase from Corynebacterium pseudotuberculosis generated by homology modeling and molecular dynamics. Analysis of key distances shows that His-132, Asp-136, His-211, Leu-212 and one molecule of water play an important role in the protein-Zn(2+) ion interaction. The model obtained may provide structural insights into this enzyme and can be useful for the design of new caseous lymphadenitis vaccines based on genetic attenuation from key point mutation.

Structure of dihydrochalcones and related derivatives and their scavenging and antioxidant activity against oxygen and nitrogen radical species.

Quantum mechanical calculations at B3LYP/6-31G** level of theory were employed to obtain energy (E), ionization potential (IP), bond dissociation enthalpy (O-H BDE) and stabilization energies (DE(iso)) in order to infer the scavenging activity of dihydrochalcones (DHC) and structurally related compounds. Spin density calculations were also performed for the proposed antioxidant activity mechanism of 2,4,6-trihydroxyacetophenone (2,4,6-THA). The unpaired electron formed by the hydrogen abstraction from the phenolic hydroxyl group of 2,4,6-THA is localized on the phenolic oxygen at 2, 6, and 4 positions, the C3 and C6 carbon atoms at ortho positions, and the C5 carbon atom at para position. The lowest phenolic oxygen contribution corresponded to the highest scavenging activity value. It was found that antioxidant activity depends on the presence of a hydroxyl at the C2 and C4 positions and that there is a correlation between IP and O-H BDE and peroxynitrite scavenging activity and lipid peroxidation. These results identified the pharmacophore group for DHC.

Kojic acid, a secondary metabolite from Aspergillus sp., acts as an inducer of macrophage activation.

KA (kojic acid) is a secondary metabolite isolated from Aspergillus fungi that has demonstrated skin whitening, antioxidant and antitumour properties among others. However, limited information is available regarding its effects on macrophages, the major cell involved in cell defence. The aim of the present study was to analyse whether KA affects functional properties related to macrophage activation, such as phagocytosis and spreading ability over a substrate. Treatment of resident macrophages with 50 µg/ml KA for 1 h induced both morphological and physiological alterations in cells. Immunofluorescence microscopy revealed enhanced cell spreading and an increase in cell surface exposure, associated with a rearrangement of microtubules, actin filaments and intermediate filaments. KA also potentiated phagocytosis by macrophages, as demonstrated by the increase in phagocytic activity towards yeast, when compared to untreated cells. KA increased the production of ROS (reactive oxygen species), but not NO (nitric oxide) production. Three tests were used to assess cell viability; MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], NR (neutral red) uptake and PI (propidium iodide) exclusion test, which showed that macrophages maintain their viability following KA treatment. Results indicate that KA can modulate macrophage activation through cytoskeleton rearrangement, increase cell surface exposure, enhance the phagocytic process and ROS production. The study demonstrates a new role for KA as a macrophage activator.

Identification of (-)(E)-N-[2(S)-Hydroxy-2-(4-hydroxyphenyl) ethyl]ferulamide, a natural product isolated from Croton pullei: theoretical and experimental analysis.

Ferulic acid (FA) and its derivatives (FADs) are known for a variety of biological activities, such as photo-protective agent, antioxidant, antiatherogenic and antiplasmodial activities. During structural definition of a FAD isolated from Croton pullei, the possibility of a heterologous series made this definition difficult. In this regard, computational simulations were performed using theoretical calculations at DFT level to predict Infrared (IR) and Nuclear Magnetic Resonance (NMR) data. The IR and NMR (13)C and (1)H data were compared with the theoretical calculations performed for three structural possibilities of a heterologous series. The theoretical results were compared with the experimental data through linear regression in order to define the most probable structure and showed satisfactory values.

Biotransformation of sucrose into 5-hydroxy-2-hydroxymethyl-γ-pirone by Aspergillus flavus

The sucrose hydrolysis and the preference of consumption of glucose instead of fructose were investigated for the production of 5-hydroxy-2-hydroxymethyl-γ-pyrone (HHMP) in the presence of Aspergillus flavus IOC 3974 cultivated in liquid Czapeck medium. Standardized 0.5g of pellets were transferred as inoculum into twelve conical flasks of 250 ml containing 100 ml of medium with different sucrose concentration, which was kept at 120 rpm and 28"C for 16 days without pH adjustment. Aliquots of 500μl of the broth culture were withdrawn at 24 h intervals and analyzed. The major yield of HHMP was 26g l-1 in 120g l-1 of sucrose. At these conditions, A. flavus produced an invertase capable of hydrolyzing 65 percent of total sucrose concentration in 24h, and an isomerase capable of converting fructose into glucose. In this work, it focused the preference for glucose and, then, of fructose by A. flavus and the strategy used to produce HHMP.

Foram investigadas a hidrólise da sacarose e a preferência pela glicose frente à frutose no processo de produção do 5-hidroxi-2-hidroximetil-γ-pirona (HHMP) na presença de Aspergillus flavus IOC 3974 cultivado em meio líquido Czapeck. Quantidades de 0,5g de pelletes foram utilizadas como inóculo. Doze frascos cônicos de 250 ml contendo 100 ml de meio de culturacom diferentes concentrações de sacarose foram utilizados.Os microrganismos foram cultivados a 120 rpm e 28"C por 16 dias sem ajuste do pH. O maior rendimento do HHMP foi 26g l-1em 120g l-1de sacarose. Nestas condições, A. flavus, foi capaz de produzir uma invertase possibilitando a hidrólise de 65 por cento da concentração total de sacarose em 24 horas, conjuntamente com a produção de uma isomerase que foi capaz de converter a frutose em glicose. Este trabalho está focalizado preferencialmente no consumo da glicose frente à frutose por A. flavus e na estratégia de produção do HHMP.

Biotransformation of sucrose into 5-hydroxy-2-hydroxymethyl-γ-pirone by Aspergillus flavus.

The sucrose hydrolysis and the preference of consumption of glucose instead of fructose were investigated for the production of 5-hydroxy-2-hydroxymethyl-γ-pyrone (HHMP) in the presence of Aspergillus flavus IOC 3974 cultivated in liquid Czapeck medium. Standardized 0.5g of pellets were transferred as inoculum into twelve conical flasks of 250 ml containing 100 ml of medium with different sucrose concentration, which was kept at 120 rpm and 28"C for 16 days without pH adjustment. Aliquots of 500 µl of the broth culture were withdrawn at 24 h intervals and analyzed. The major yield of HHMP was 26g l(-1) in 120g l(-1) of sucrose. At these conditions, A. flavus produced an invertase capable of hydrolyzing 65% of total sucrose concentration in 24h, and an isomerase capable of converting fructose into glucose. In this work, it focused the preference for glucose and, then, of fructose by A. flavus and the strategy used to produce HHMP.