Bioassay-guided identification of antithrombotic compounds from Cnidoscolus aconitifolius (Mill.) I. M. Jhonst.: molecular docking, bioavailability, and toxicity prediction.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Conventional antithrombotic therapy has reported hemorrhagic accidents. Ethnobotanical and scientific reports point to Cnidoscolus aconitifolius as an antithrombotic adjuvant. Previously, C. aconitifolius leaves ethanolic extract displayed antiplatelet, anticoagulant, and fibrinolytic activities. This work aimed to identify compounds from C. aconitifolius with in vitro antithrombotic activity through a bioassay-guided study. Antiplatelet, anticoagulant, and fibrinolytic tests guided the fractionation. Ethanolic extract was subjected to a liquid-liquid partitioning, followed by vacuum liquid, and size exclusion chromatography to obtain the bioactive JP10B fraction. The compounds were identified through UHPLC-QTOF-MS, and their molecular docking, bioavailability, and toxicological parameters were determined computationally. Kaempferol-3-O-glucorhamnoside and 15(S)-HPETE were identified; both showed affinity for antithrombotic targets, low absorption, and safety for human consumption. Further in vitro and in vivo evaluations will better understand their antithrombotic mechanism. This bioassay-guided fractionation demonstrated that C. aconitifolius ethanolic extract has antithrombotic compounds.Communicated by Ramaswamy H. Sarma.

Application of Molecular Dynamics Simulations to Determine Interactions between Canary Seed (Phalaris canariensis L.) Bioactive Peptides and Skin-Aging Enzymes.

Food bioactive peptides are well recognized for their health benefits such as antimicrobial, antioxidant, and antihypertensive benefits, among others. Their drug-like behavior has led to their potential use in targeting skin-related aging factors like the inhibition of enzymes related with the skin-aging process. In this study, canary seed peptides (CSP) after simulated gastrointestinal digestion (<3 kDa) were fractioned by RP-HPLC and their enzyme-inhibition activity towards elastase and tyrosinase was evaluated in vitro. CSP inhibited elastase (IC50 = 6.2 mg/mL) and tyrosinase (IC50 = 6.1 mg/mL), while the hydrophobic fraction-VI (0.2 mg/mL) showed the highest inhibition towards elastase (93%) and tyrosinase (67%). The peptide fraction with the highest inhibition was further characterized by a multilevel in silico workflow, including physicochemical descriptor calculations, antioxidant activity predictions, and molecular dynamics-ensemble docking towards elastase and tyrosinase. To gain insights into the skin permeation process during molecular dynamics simulations, based on their docking scores, five peptides (GGWH, VPPH, EGLEPNHRVE, FLPH, and RPVNKYTPPQ) were identified to have favorable intermolecular interactions, such as hydrogen bonding of polar residues (W, H, and K) to lipid polar groups and 2-3 Å van der Waals close contact of hydrophobic aliphatic residues (P, V, and L). These interactions can play a critical role for the passive insertion of peptides into stratum corneum model skin-membranes, suggesting a promising application of CSP for skin-aging treatments.

An Overall View of the Functional and Structural Characterization of Glucose-6-Phosphate Dehydrogenase Variants in the Mexican Population.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting an estimated 500 million people worldwide, is a genetic disorder that causes human enzymopathies. Biochemical and genetic studies have identified several variants that produce different ranges of phenotypes; thus, depending on its severity, this enzymopathy is classified from the mildest (Class IV) to the most severe (Class I). Therefore, understanding the correlation between the mutation sites of G6PD and the resulting phenotype greatly enhances the current knowledge of enzymopathies' phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments for patients with G6PD deficiency. In this review, we analyzed and compared the structural and functional data from 21 characterized G6PD variants found in the Mexican population that we previously characterized. In order to contribute to the knowledge regarding the function and structure of the variants associated with G6PD deficiency, this review aimed to determine the molecular basis of G6PD and identify how these mutations could impact the structure, stability, and function of the enzyme and its relation with the clinical manifestations of this disease.

Nitazoxanide Inhibits the Bifunctional Enzyme GlG6PD::6PGL of Giardia lamblia: Biochemical and In Silico Characterization of a New Druggable Target.

Giardiasis, which is caused by Giardia lamblia infection, is a relevant cause of morbidity and mortality worldwide. Because no vaccines are currently available to treat giardiasis, chemotherapeutic drugs are the main options for controlling infection. Evidence has shown that the nitro drug nitazoxanide (NTZ) is a commonly prescribed treatment for giardiasis; however, the mechanisms underlying NTZ's antigiardial activity are not well-understood. Herein, we identified the glucose-6-phosphate::6-phosphogluconate dehydrogenase (GlG6PD::6PGL) fused enzyme as a nitazoxanide target, as NTZ behaves as a GlG6PD::6PGL catalytic inhibitor. Furthermore, fluorescence assays suggest alterations in the stability of GlG6PD::6PGL protein, whereas the results indicate a loss of catalytic activity due to conformational and folding changes. Molecular docking and dynamic simulation studies suggest a model of NTZ binding on the active site of the G6PD domain and near the structural NADP+ binding site. The findings of this study provide a novel mechanistic basis and strategy for the antigiardial activity of the NTZ drug.

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.

In-depth peptidomic profile and molecular simulation studies on ACE-inhibitory peptides derived from probiotic fermented milk of different farm animals.

Investigations into ACE inhibitory properties of probiotic fermented bovine, camel, goat, and sheep milk were performed and studied for two weeks of refrigerated storage. Results from the degree of proteolysis suggested higher susceptibility of goat milk proteins, followed by sheep and camel milk proteins, to the probiotic-mediated proteolysis. ACE-inhibitory properties displayed continuous decline in ACE-IC50 values for two weeks of refrigerated storage. Overall, goat milk fermented with Pediococcus pentosaceus caused maximum ACE inhibition (IC50: 262.7 µg/mL protein equivalent), followed by camel milk (IC50: 290.9 µg/mL protein equivalent). Studies related to peptide identification and in silico analysis using HPEPDOCK score revealed presence of 11, 13, 9 and 9 peptides in fermented bovine, goat, sheep, and camel milk, respectively, with potent antihypertensive potential. The results obtained suggest that the goat and camel milk proteins demonstrated higher potential for generating antihypertensive peptides via fermentation when compared to bovine and sheep milk.

The ovaries of ivermectin-resistant Rhipicephalus microplus strains display proteomic adaptations involving the induction of xenobiotic detoxification and structural remodeling mechanisms.

Controlling Rhipicephalus microplus is among the most significant challenges for livestock production worldwide. The indiscriminate use of acaricides stimulates the selection of resistant tick populations and is therefore ineffective. Understanding the molecular foundations of resistance could help inform the search for new alternatives for tick control. Although the ovary has been suggested as a relevant target organ for tick control, there are few existing studies that focus on tick ovarian tissue. Therefore, we conducted a comparative proteomic analysis on ovaries of R. microplus strains with differential resistance to ivermectin. In resistant ticks, we observed the over-accumulation of proteins involved in several biological processes, including translation, proteolysis, transport, cellular organization, differentiation, and xenobiotic detoxification. We also observed the accumulation of many structural and extracellular proteins such as papilin-like protein, which glycosylation increase its stability-based molecular modeling. Therefore, we propose that ovaries of ivermectin-resistant ticks overcome the negative impact of ivermectin through the activation of detoxification mechanisms and structural proteins associated with the remodeling of the ovary's extracellular matrix. SIGNIFICANCE: Understanding the molecular foundation of ivermectin resistance in Rhipicephalus microplus represents an essential step in cattle farming, which could provide clues and alternatives for tick control. Excessive use of chemicals like ivermectin allows the generation of resistant tick strains in different countries. However, limited molecular information is available concerning the tick's resistance to ivermectin. Detailed proteomics scrutiny in various tick organs will provide more comprehensive molecular information. Thus, we conducted an ovary comparative proteomic-based TMT-SPS-MS3 approach. We highlight in ivermectin-resistant ticks the over-accumulation of structural proteins and enzymes connected to detoxification mechanisms.

Synthesis, insecticidal activity, and ensembled docking of nitroguanidines bearing S- and R-proline.

BACKGROUND: The amino acids R- and S-proline were used to synthesize novel neonicotinoid derivatives that, after being characterized by 1 H, DEPTQ 135, and HRMS-QTOF, were evaluated for use as insecticides against Galleria mellonella (caterpillar), Sitophilus zeamais, Xylosandrus morigerus, Xyleborus affinis, and Xyleborus ferrugineus. RESULTS: Comparisons of biological activity and absolute configuration showed that the R enantiomer had excellent and outstanding insecticidal activity against the insects tested, with up to 100% mortality after 12 h compared with dinotefuran at the same concentration. CONCLUSIONS: The results suggest that compound R6 is an excellent lead enantiopure insecticide for future development in the field of crop protection. Furthermore, intermolecular interactions between nicotinic acetylcholine receptors and the R enantiomer displays a lower score which mean a higher affinity to the nAChR receptor and the π-π interactions are more stable than the S derivative. © 2023 Society of Chemical Industry.

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.

Multifunctional Analysis of Chia Seed (Salvia hispanica L.) Bioactive Peptides Using Peptidomics and Molecular Dynamics Simulations Approaches.

Chia seed peptides (CSP) can be a source of multifunctional biopeptides to treat non-communicable diseases. However, interactions and binding affinity involved in targeting specific receptors remains unexplored. In this study, molecular simulation techniques were used as virtual screening of CSP to determine drug-like candidates using a multi-target-directed ligand approach. CSP fraction with the best bioactivities in vitro was sequenced. Then, a prediction model was built using physicochemical descriptors (hydrophobicity, hydrophilicity, intestinal stability, antiangiogenic, antihypertensive, and anti-inflammatory) to calculate potential scores and rank possible biopeptides. Furthermore, molecular dynamics simulations (MDS) and ensemble molecular docking analysis were carried out using four human protein targets (ACE, angiotensin converting enzyme; VEGF, vascular endothelial growth factor; GLUC, glucocorticoid and MINC, mineralocorticoid receptors). Five known-sequence peptides (NNVFYPF, FNIVFPG, SRPWPIDY, QLQRWFR, GSRFDWTR) and five de novo peptides (DFKF, DLRF, FKAF, FRSF, QFRF) had the lowest energy score and higher affinity for ACE and VEGF. The therapeutic effects of these selected peptides can be related to the inhibition of the enzymes involved in angiogenesis and hypertension, due to formation of stable complexes with VEGF and ACE binding sites, respectively. The application of MDS is a good resource for identifying bioactive peptides for future experimental validation.

Integration of Molecular Docking Analysis and Molecular Dynamics Simulations for Studying Food Proteins and Bioactive Peptides.

In silico tools, such as molecular docking, are widely applied to study interactions and binding affinity of biological activity of proteins and peptides. However, restricted sampling of both ligand and receptor conformations and use of approximated scoring functions can produce results that do not correlate with actual experimental binding affinities. Molecular dynamics simulations (MDS) can provide valuable information in deciphering functional mechanisms of proteins/peptides and other biomolecules, overcoming the rigid sampling limitations in docking analysis. This review will discuss the information related to the traditional use of in silico models, such as molecular docking, and its application for studying food proteins and bioactive peptides, followed by an in-depth introduction to the theory of MDS and description of why these molecular simulation techniques are important in the theoretical prediction of structural and functional dynamics of food proteins and bioactive peptides. Applications, limitations, and future prospects of MDS will also be discussed.

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.

Ergosterol exerts a differential effect on AR-dependent LNCaP and AR-independent DU-145 cancer cells.

Androgen-dependent LNCaP and androgen-independent DU-145 cells, were treated with different concentrations of ergosterol (15 µM and 25 µM) and its respective cell viability was measured by MTT bioassay. While ergosterol showed an antiproliferative effect on LNCaP, on DU-145 promoted cell proliferation. This differential effect suggests that the effect of ergosterol might be related to its ability to act as an Androgen Receptor ligand. In silico Molecular Dynamics simulations were performed to analyze the interaction mechanism between androgen receptor and ergosterol, in comparison with natural ligands, 5α-dihydrotestosterone and testosterone. Our model suggests that the binding of androgen receptor with ergosterol is thermodinamically feasible, which is concordant with our experimental results.

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.

Insights into Ergosterol Peroxide's Trypanocidal Activity.

Trypanosoma cruzi, which causes Chagas disease, is a significant health threat in many countries and affects millions of people. Given the magnitude of this disease, a broader understanding of trypanocidal mechanisms is needed to prevent and treat infection. Natural endoperoxides, such as ergosterol peroxide, have been shown to be toxic to parasites without causing harm to human cells or tissues. Although prior studies have demonstrated the trypanocidal activity of ergosterol peroxide, the cellular and molecular mechanisms remain unknown. The results of this study indicate that a free-radical reaction occurs in T. cruzi following ergosterol peroxide exposure, leading to cell death. Using a combination of biochemical, microscopic and in silico experimental approaches, we have identified, for the first time, the cellular and molecular cytotoxic mechanism of an ergosterol peroxide obtained from Pleurotus ostreatus (Jacq) P. Kumm. f. sp. Florida.

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.

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).

Molecular Dynamics and Virtual Screening Analysis of Lanosterol Derivatives from Ganoderma Medicinal Mushrooms (Agaricomycetes) as Selective Ligands of Human Androgen Receptor.

Male sex hormones such as testosterone and dihydrotestosterone play important roles in several physiological and pathological processes. The biological activities of the aforementioned metabolites are mediated by the multidomain androgen receptor (AR), which is therefore a well-studied drug target. Ganoderma mushroom lanostanoid extracts have previously been shown to exert antiandrogenic activity; therefore, this work aims to identify which lanostane derivatives might act as selective ligands for AR. Because protein flexibility is of paramount importance for ligand binding, different conformations of AR were sampled to account for binding modes within a ligand binding site, then subjected to virtual screening against a metabolite library. Fifteen Ganoderma lanostanoids were selected as AR ligands, according to their calculated binding affinity to this nuclear receptor. The results show the relevance of certain structural and chemical aspects of our ligands, such as the presence of a ketonic group on C-3, which influences the process through which they bind to AR.

In Silico Analysis of Lanostanoids Characterized in Ganoderma Mushrooms (Agaricomycetes) as Potential Ligands of the Vitamin D Receptor.

The metabolism of vitamin D is a very important pathway involved in the regulation of sterols and maintenance of cell health. The physiological activity of the human hormone 1α,25-dihydroxyvitamin D3, or calcitriol, is mediated by the vitamin D receptor (VDR), an endocrine member of the nuclear receptor superfamily that inhibits cell growth and stimulates cell differentiation, suggesting a potential application in cancer chemoprevention. Since nonpolar extracts obtained from Ganoderma mushrooms have also been shown to exert an antiproliferative effect on several cancer cell lines, it was suggested that at least part of its activity might be mediated by VDR. The aim of this work was to identify possible VDR ligands from an extensive library of lanostanoids isolated from several Ganoderma mushrooms. Using an in silico approach, 30 lanostanoids were found to interact with the VDR ligand-binding pocket in the same way as calcitriol. The possible implications of using these compounds are discussed here.

Simple approach for ranking structure determining residues.

Mutating residues has been a common task in order to study structural properties of the protein of interest. Here, we propose and validate a simple method that allows the identification of structural determinants; i.e., residues essential for preservation of the stability of global structure, regardless of the protein topology. This method evaluates all of the residues in a 3D structure of a given globular protein by ranking them according to their connectivity and movement restrictions without topology constraints. Our results matched up with sequence-based predictors that look up for intrinsically disordered segments, suggesting that protein disorder can also be described with the proposed methodology.