BrainPepPass: A Framework Based on Supervised Dimensionality Reduction for Predicting Blood-Brain Barrier-Penetrating Peptides.

Peptides that pass through the blood-brain barrier (BBB) not only are implicated in brain-related pathologies but also are promising therapeutic tools for treating brain diseases, e.g., as shuttles carrying active medicines across the BBB. Computational prediction of BBB-penetrating peptides (B3PPs) has emerged as an interesting approach because of its ability to screen large peptide libraries in a cost-effective manner. In this study, we present BrainPepPass, a machine learning (ML) framework that utilizes supervised manifold dimensionality reduction and extreme gradient boosting (XGB) algorithms to predict natural and chemically modified B3PPs. The results indicate that the proposed tool outperforms other classifiers, with average accuracies exceeding 94% and 98% in 10-fold cross-validation and leave-one-out cross-validation (LOOCV), respectively. In addition, accuracy values ranging from 45% to 97.05% were achieved in the independent tests. The BrainPepPass tool is available in a public repository for academic use (https://github.com/ewerton-cristhian/BrainPepPass).

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.

Removal of Organic Pollutants Benzene and Phenol Using Nanofiltration: A Molecular Dynamics Study.

Population growth has resulted in an increased demand for clean water. It is known that chemical pollutants such as phenol and benzene often make water unfit for consumption, and can be responsible for the appearance of diseases such as cancer. In this sense, studies aimed at decontaminating water are still necessary. In this study, molecular dynamics simulations were performed to evaluate the abilities of activated charcoal structures to adsorb benzene and phenol; the results of which were evaluated on the basis of root mean square deviations for all systems. The data were collected from the molecular dynamics (MD) trajectories and edited with the grace plotting tool. Visual molecular dynamics software was used to visualize the MD paths, and images were created using the UCSF chimera software. The results show that activated charcoal are viable alternatives for water decontamination by nanofiltration.

Catalysis by solvation rather than the desolvation effect: exploring the catalytic efficiency of SAM-dependent chlorinase.

Chlorinase SalL halogenate S-adenosyl-l-methionine (SAM) reacts with chloride to generate 5'-chloro-5'-deoxyadenosine and l-methionine through a nucleophilic substitution mechanism. Although it is known that chlorinase enhances the rate of reaction by a factor of 1.2 × 1017 fold, it is not entirely clear how this is accomplished. The search for the origin of the catalysis of chlorinase and other enzymes has led to a desolvation hypothesis. In the present work, we have used well defined computational simulations in order to evaluate the origin of the catalytic efficiency of chlorinase. The results demonstrate that the catalytic effect of chlorinase is associated with the fact that Cl- is "solvated" by the protein more than by the reference solution reaction, which is not in accordance with proposed catalysis by desolvation. It is found that chlorinase SalL active sites provide electrostatic stabilization of the transition state which is the origin of its catalytic effect.

Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Molecular characterization and in silico evaluation of surfactins produced by endophytic bacteria from Phanera splendens.

The Phanera splendens (Kunth) Vaz. is a medicinal plant that is used in traditional medicine for the treatment of various diseases, such as malaria. This plant presents highly efficient endophytic bacterial isolates with biocontrol properties. Bacillus sp. is responsible for the production of a variety of non-ribosomal synthesized cyclic lipopeptides which highlight the surfactins. Surfactins have a wide range of antimicrobial activity, including antiplasmodial activity. There is scientific evidence that surfactin structure 2d-01 can be a potent inhibitor against a Plasmodium falciparum sirtuin (Sir2) by acting on the Sir2A protein as the target. The Pf genome encodes two known sirtuins, PfSir2A and PfSir2B, where PfSir2A is a regulator of asexual growth and var gene expression. Herein, we have identified six surfactins produced by endophytic bacteria and performed in silico analysis to elucidate the binding mode of surfactins at the active site of the PfSir2A enzyme. Among the characterized surfactins, 1d-02 showed the highest affinity for the PfSir2A enzyme, with binding energy values equal to -45.08 ± 6.0 and -11.95 ± 0.8 kcal/mol, using MM/GBSA and SIE methods, respectively. We hope that the information about the surfactin structures obtained in this work, as well as the potential binding affinity with an important enzyme from P. falciparum, could contribute to the design of new compounds with antimalarial activity.

Biological Membrane-Penetrating Peptides: Computational Prediction and Applications.

Peptides comprise a versatile class of biomolecules that present a unique chemical space with diverse physicochemical and structural properties. Some classes of peptides are able to naturally cross the biological membranes, such as cell membrane and blood-brain barrier (BBB). Cell-penetrating peptides (CPPs) and blood-brain barrier-penetrating peptides (B3PPs) have been explored by the biotechnological and pharmaceutical industries to develop new therapeutic molecules and carrier systems. The computational prediction of peptides' penetration into biological membranes has been emerged as an interesting strategy due to their high throughput and low-cost screening of large chemical libraries. Structure- and sequence-based information of peptides, as well as atomistic biophysical models, have been explored in computer-assisted discovery strategies to classify and identify new structures with pharmacokinetic properties related to the translocation through biomembranes. Computational strategies to predict the permeability into biomembranes include cheminformatic filters, molecular dynamics simulations, artificial intelligence algorithms, and statistical models, and the choice of the most adequate method depends on the purposes of the computational investigation. Here, we exhibit and discuss some principles and applications of these computational methods widely used to predict the permeability of peptides into biomembranes, exhibiting some of their pharmaceutical and biotechnological applications.

Advances in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Covalent Inhibition.

Peptidoglycan is a cross-linked polymer responsible for maintaining the bacterial cell wall integrity and morphology in Gram-negative and Gram-positive bacteria. The peptidoglycan pathway consists of the enzymatic reactions held in three steps: cytoplasmic, membrane-associated, and periplasmic. The Mur enzymes (MurA-MurF) are involved in a cytoplasmic stage. The UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme is responsible for transferring the enolpyruvate group from phosphoenolpyruvate (PEP) to UDP-N-acetylglucosamine (UNAG) to form UDP-N-acetylglucosamine enolpyruvate (EP-UNAG). Fosfomycin is a natural product analogous to PEP that acts on the MurA target enzyme via binding covalently to the key cysteine residue in the active site. Similar to fosfomycin, other MurA covalent inhibitors have been described with a warhead in their structure that forms a covalent bond with the molecular target. In MurA, the nucleophilic thiolate of Cys115 is pointed as the main group involved in the warhead binding. Thus, in this minireview, we briefly describe the main recent advances in the design of MurA covalent inhibitors.

Virtual screening of natural products against 5-enolpyruvylshikimate-3-phosphate synthase using the Anagreen herbicide-like natural compound library.

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a reaction involved in the production of amino acids essential for plant growth and survival. EPSPS is the main target of glyphosate, a broad-spectrum herbicide that acts as a competitive inhibitor concerning phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. In the present study, we introduce a natural compound library, named Anagreen, which is a compendium of herbicide-like compounds obtained from different natural product databases. Herein, we combined the structure- and ligand-based virtual screening strategies to explore Anagreen against EPSPS using the structure of glyphosate complexed with a T102I/P106S mutant of EPSPS from Eleusine indica (EiEPSPS) as a starting point. First, ligand-based pharmacophore screening was performed to select compounds with a similar pharmacophore to glyphosate. Then, structure-based pharmacophore modeling was applied to build a model which represents the molecular features of glyphosate. Then, consensus docking was performed to rank the best poses of the natural compounds against the PEP binding site, and then molecular dynamics simulations were performed to analyze the stability of EPSPS complexed with the selected ligands. Finally, we have investigated the binding affinity of the complexes using free energy calculations. The selected hit compound, namely AG332841, showed a stable conformation and binding affinity to the EPSPS structure and showed no structural similarity to the already known weed EPSPS inhibitors. Our computational study aims to clarify the inhibition of the mutant EiEPSPS, which is resistant to glyphosate, and identify new potential herbicides from natural products.

A molecular model to study FosA enzyme inhibition.

Fosfomycin resistance protein (FosA) is a metalloenzyme known for catalyzing a nucleophilic addition reaction of glutathione to the epoxide ring of Fosfomycin, a broad-spectrum antibiotic used to combat Gram-positive pathogens. The reaction leads fosfomycin to lose its pharmacological effect, thus promotes antibiotic resistance. A small-molecule FosA inhibitor has been discovered. ANY1 (3-bromo-6-[3-(3-bromo-2-oxo-1H-pyrazolo[1,5-a]pyrimidin-6-yl)-4-nitro-1H-pyrazol-5-yl]-1H-pyrazolo[1,5-a]pyrimidin-2-one) is competitive with the antibiotic for binding the active site of the enzyme. Through Molecular Mechanics methods, using the AMBER force field, we carry out molecular dynamics simulations and binding free energy calculations to investigate the most important interactions between the enzyme and inhibitor. Our results were able to reproduce the trend of experimental data with R2 of 77.51%. Furthermore, we have shown that electrostatic and van der Waals interactions, as well as cavitation energies, are favorable for maintaining the enzyme-inhibitor complex, while reactive field energies and non-polar interactions act in an unfavorable way for interactions between FosA and ANY1.

QM/MM Study of the Fosfomycin Resistance Mechanism Involving FosB Enzyme.

Multidrug-resistant organisms contain antibiotic-modifying enzymes that facilitate resistance to a variety of antimicrobial compounds. Particularly, the fosfomycin (FOF) drug can be structurally modified by several FOF-modifying enzymes before it reaches the biological target. Among them, FosB is an enzyme that utilizes l-cysteine or bacillithiol in the presence of a divalent metal to open the epoxide ring of FOF and, consequently, inactivate the drug. Here, we have used hybrid quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of the reaction involving FosB and FOF. The calculated free-energy profiles show that the cost to open the epoxide ring of FOF at the C2 atom is ∼3.0 kcal/mol higher than that at the C1 atom. Besides, our QM/MM MD results revealed the critical role of conformation change of Cys9 and Asn50 to release the drug from the active site. Overall, the present study provides insights into the mechanism of FOF-resistant proteins.

Structural, energetic and lipophilic analysis of SARS-CoV-2 non-structural protein 9 (NSP9).

In SARS-CoV-2 replication complex, the Non-structural protein 9 (Nsp9) is an important RNA binding subunit in the RNA-synthesizing machinery. The dimeric forms of coronavirus Nsp9 increase their nucleic acid binding affinity and the N-finger motif appears to play a critical role in dimerization. Here, we present a structural, lipophilic and energetic study about the Nsp9 dimer of SARS-CoV-2 through computational methods that complement hydrophobicity scales of amino acids with molecular dynamics simulations. Additionally, we presented a virtual N-finger mutation to investigate whether this motif contributes to dimer stability. The results reveal for the native dimer that the N-finger contributes favorably through hydrogen bond interactions and two amino acids bellowing to the hydrophobic region, Leu45 and Leu106, are crucial in the formation of the cavity for potential drug binding. On the other hand, Gly100 and Gly104, are responsible for stabilizing the α-helices and making the dimer interface remain stable in both, native and mutant (without N-finger motif) systems. Besides, clustering results for the native dimer showed accessible cavities to drugs. In addition, the energetic and lipophilic analysis reveal that the higher binding energy in the native dimer can be deduced since it is more lipophilic than the mutant one, increasing non-polar interactions, which is in line with the result of MM-GBSA and SIE approaches where the van der Waals energy term has the greatest weight in the stability of the native dimer. Overall, we provide a detailed study on the Nsp9 dimer of SARS-CoV-2 that may aid in the development of new strategies for the treatment and prevention of COVID-19.

Constituents of Chamaecrista diphylla (L.) Greene Leaves with Potent Antioxidant Capacity: A Feature-Based Molecular Network Dereplication Approach.

Chamaecrista diphylla (L.) Greene (Fabaceae/Caesalpiniaceae) is a herbaceous plant that is widely distributed throughout the Americas. Plants from this genus have been used in traditional medicine as a laxative, to heal wounds, and to treat ulcers, snake and scorpion bites. In the present study, we investigated the chemical composition of Chamaecrista diphylla leaves through a mass spectrometry molecular network approach. The oxygen radical absorbance capacity (ORAC) for the ethanolic extract, enriched fractions and isolated compounds was assessed. Overall, thirty-five compounds were annotated for the first time in C. diphylla. Thirty-two of them were reported for the first time in the genus. The isolated compounds 9, 12, 24 and 33 showed an excellent antioxidant capacity, superior to the extract and enriched fractions. Bond dissociation energy calculations were performed to explain and sustain the antioxidant capacity found. According to our results, the leaves of C. diphylla represent a promising source of potent antioxidant compounds.

Investigation of the target-site resistance of EPSP synthase mutants P106T and T102I/P106S against glyphosate.

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) catalyzes the reaction involved in the production of amino acids essential for plant growth and survival. Thus, EPSPS is the main target of various herbicides, including glyphosate, a broad-spectrum herbicide that acts as a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. However, punctual mutations in the EPSPS gene have led to glyphosate resistance in some plants. Here, we investigated the mechanism of EPSPS resistance to glyphosate in mutants of two weed species, Conyza sumatrensis (mutant, P106T) and Eleusine indica (mutant, T102I/P106S), both of which have an economic impact on industrial crops. Molecular dynamics (MD) simulations and binding free energy calculations revealed the influence of the mutations on the affinity of glyphosate in the PEP-binding site. The amino acid residues of the EPSPS protein in both species involved in glyphosate resistance were elucidated as well as other residues that could be useful for protein engineering. In addition, during MD simulations, we identified conformational changes in glyphosate when complexed with resistant EPSPS, related to loss of herbicide activity and binding affinity. Our computational findings are consistent with previous experimental results and clarify the inhibitory activity of glyphosate as well as the structural target-site resistance of EPSPS against glyphosate.

Targeting shikimate pathway: In silico analysis of phosphoenolpyruvate derivatives as inhibitors of EPSP synthase and DAHP synthase.

The shikimate pathway consists of seven enzymatic steps involved in the conversion of erythrose-4-phosphate and phosphoenolpyruvate to chorismate and also responsible to the production of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan which are essential to the bacterial metabolism. The 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) catalyze important steps in the shikimate pathway using as substrate the phosphoenolpyruvate (PEP). Due to the importance of PEP in shikimate pathway, its structure has been investigated to develop new bioinspired competitive inhibitors against DAHPS and EPSPS. In the present study, we perform a literature survey of 28 PEP derivatives, then we analyzed the selectivity and affinity of these compounds against the EPSPS and DAHPS structures using consensual molecular docking, pharmacophore prediction, molecular dynamics (MD) simulations, and binding free energy calculations. Here, we propose consistent binding modes of the selected ligands and indicate that their structures show interesting pharmacophoric properties related to multi-targets inhibitors for both enzymes. Our computational results are supported by previous experimental findings related to the interactions of PEP derivatives with DAHPS and EPSPS structures.

Computed insight into a peptide inhibitor preventing the induced fit mechanism of MurA enzyme from Pseudomonas aeruginosa.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the key enzymes involved in peptidoglycan biosynthesis. The peptide HESFWYLPHQSY (called PEP 1354) is an inhibitor of MurA with an IC50 value of 200 µm. In this article, we have used the FlexPepDock ab-initio protocol from the Rosetta program homology modeling and molecular dynamics simulations to analyze, for the first time, the interaction of the PEP 1354 peptide with MurA enzyme from Pseudomonas aeruginosa (MurA-PA). Our modeling results suggest that the peptide binds to the same active site as the natural substrate UDP-N-acetylglucosamine (UNAG). Additionally, the MurA-peptide complex revealed that the peptide seems to prevent the closure of the Pro114-123 loop and, consequently, the open-closed transition of the MurA structure.

Unraveling the Addition-Elimination Mechanism of EPSP Synthase through Computer Modeling.

Enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the hydroxyl group of shikimate-5-OH-3-phosphate (S3P) is catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase in a reaction that involves breaking the C-O bond of PEP. Catalysis involves an addition-elimination mechanism with the formation of a tetrahedral intermediate (THI). Experiments have elucidated the mechanism of THI formation and breakdown. However, the catalytic action of EPSP synthase and the individual roles of catalytic residues Asp313 and Glu341 remains unclear. We have used a hybrid quantum mechanical/molecular mechanical (QM/MM) approach to explore the free energy surface in a reaction catalyzed by EPSP synthase. The Glu341 was the most favorable acid/base catalyst. Our results indicate that the protonation of PEP C3 precedes the nucleophilic attack on PEP C2 in the addition mechanism. Also, the breaking of the C-O bond of THI to form an EPSP cation intermediate must occur before proton transfer from PEP C3 to Glu341 in the elimination mechanism. Analysis of the FES supports cationic intermediate formation during the reaction catalyzed by EPSP synthase. Finally, the computational model indicates a proton transfer shift (Hammond shift) from Glu341 to C3 for an enzyme-based reaction with the shifted transition state, earlier than in the reference reaction in water.

Protein-ligand interaction study of CpOGA in complex with GlcNAcstatin.

The GlcNAcstatin is a potent inhibitor of O-glycoprotein 2-acetamino-2-deoxy-ß-D-glucopyranosidase, which has been related with type II diabetes and neurodegenerative disorders. Herein, hybrid quantum mechanics/molecular mechanics, molecular dynamics simulations, and potential of mean force were employed to study the interactions established between GlcNAcstatin and a bacterial O-GlcNAcase enzyme from Clostridium perfringens. The results reveal that the imidazole nitrogen atom of GlcNAcstatin has shown a better interaction with the active site of Clostridium perfringens in its protonated form, which is compatible with a substrate-assisted reaction mechanism involving two conserved aspartate residues (297 and 298). Furthermore, the quantum mechanics/molecular mechanics-molecular dynamics simulations appointed a strong interaction between Asp401, Asp298, and Asp297 residues and the GlcNAcstatin inhibitor, which is in accordance with experimental data. Lastly, these results may contribute to understand the molecular mechanism of inhibition of Clostridium perfringens by GlcNAcstatin inhibitor and, consequently, this study might be useful to design new molecules with more interesting inhibitory activity.

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.