Identification of 1,2,3-triazole-phthalimide derivatives as potential drugs against COVID-19: a virtual screening, docking and molecular dynamic study.

In this work we aimed to perform an in silico predictive screening, docking and molecular dynamic study to identify 1,2,3-triazole-phthalimide derivatives as drug candidates against SARS-CoV-2. The in silico prediction of pharmacokinetic and toxicological properties of hundred one 1,2,3-triazole-phtalimide derivatives, obtained from SciFinder® library, were investigated. Compounds that did not show good gastrointestinal absorption, violated the Lipinski's rules, proved to be positive for the AMES test, and showed to be hepatotoxic or immunotoxic in our ADMET analysis, were filtered out of our study. The hit compounds were further subjected to molecular docking on SARS-CoV-2 target proteins. The ADMET analysis revealed that 43 derivatives violated the Lipinski's rules and 51 other compounds showed to be positive for the toxicity test. Seven 1,2,3-triazole-phthalimide derivatives (A7, A8, B05, E35, E38, E39, and E40) were selected for molecular docking and MFCC-ab initio analysis. The results of molecular docking pointed the derivative E40 as a promising compound interacting with multiple target proteins of SARS-CoV-2. The complex E40-Mpro was found to have minimum binding energy of -10.26 kcal/mol and a general energy balance, calculated by the quantum mechanical analysis, of -8.63 eV. MD simulation and MMGBSA calculations confirmed that the derivatives E38 and E40 have high binding energies of -63.47 ± 3 and -63.31 ± 7 kcal/mol against SARS-CoV-2 main protease. In addition, the derivative E40 exhibited excellent interaction values and inhibitory potential against SAR-Cov-2 main protease and viral nucleocapsid proteins, suggesting this derivative as a potent antiviral for the treatment and/or prophylaxis of COVID-19.Communicated by Ramaswamy H. Sarma.

A new biocompatible silver/polypyrrole composite with in vitro antitumor activity.

We used an in situ chemical oxidation method to prepare a new composite of silver nanoparticles (AgNPs) with polypyrrole (PPy), whose properties were optimized through a 23-factorial design of the synthesis conditions. The successful formation of the AgNPs/PPy composite was confirmed by UV-Visible and FTIR spectroscopies. Transmission electron microscopy revealed the presence of AgNPs smaller than 100 nm, dispersed into the PPy matrix. This hybrid composite exhibits a blue fluorescence emission after excitation in the ultraviolet region. In MTT assays, the AgNPs/PPy composite exhibited low cytotoxicity toward non-tumoral cell lines (fibroblast, Vero, and macrophages) and selectively inhibited the viability of HeLa cells. The AgNPs/PPy composite induces ultrastructural changes in HeLa cells that are consistent with the noticeable selectivity exhibited toward them when compared to its action against non-tumoral cell lineages. Also, the AgNPs/PPy exhibited a hemolytic activity below 14% for all blood groups tested, at concentrations up to 125 µg/mL. These results suggest that the AgNPs/PPy composite has a promising potential for use as an antitumoral agent.

Antileishmanial activity of 4-phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) derivative on Leishmania amazonensis and Leishmania braziliensis: In silico ADMET, in vitro activity, docking and molecular dynamic simulations.

Organic compounds obtained by click chemistry reactions have demonstrated a broad spectrum of biological activities being widely applied for the development of molecules against pathogens of medical and veterinary importance. Cutaneous leishmaniasis (CL), caused by intracellular protozoa parasite of genus Leishmania, comprises a complex of clinical manifestations that affect the skin and mucous membranes. The available drugs for the treatment are toxic and costly, with long periods of treatment, and the emergence of resistant strains has been reported. In this study we investigated the in vitro effects of a phthalimide-1,2,3-triazole derivative, the 4-Phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole (PT4) obtained by click chemistry, on mammalian cells and on L. amazonensis and L. braziliensis, the causative agents of CL in Brazil. In silico ADMET evaluation of PT4 showed that this molecule has good pharmacokinetic properties with no violation of Lipinski's rules. The in vitro assays showed that PT4 was more selective for both Leishmania species than to mammalian cells. This compound also presented low cytotoxicity to mammalian cells with CC50 > 500 µM. Treatment of promastigote forms with different concentrations of PT4 resulted in ultrastructural alterations, such as plasma membrane wrinkling, shortening of cell body, increased cell volume and cell rupture. The molecular dynamic simulations showed that PT4 interacts with Lanosterol 14 α-demethylase from Leishmania, an essential enzyme of lipid synthesis pathway in this parasite. Our results demonstrated PT4 was effective against both species of Leishmania. PT4 caused a decrease of mitochondrial membrane potential and increased production of reactive oxygen species, which may lead to parasite death. Taken together, our results pointed PT4 as promissing therapeutic agent against CL.

A C-type lectin from Bothrops leucurus snake venom forms amyloid-like aggregates in RPMI medium and are efficiently phagocytosed by peritoneal macrophages.

Lectins are carbohydrate-binding proteins that play important roles in the immune system. Under specific conditions, lectins can form amyloids, proteinaceous aggregates rich in cross ß-strand structures. A Ca++-dependent lectin, isolated from Bothrops leucurus snake venom (BLL) has demonstrated relevant biological activities such as antibacterial and antitumor activity. In this work, we aimed to study the interaction of BLL with macrophages. The formation of amyloid structures by BLL in a cell culture medium, the effects of the lectin on macrophage morphology and cytokine production were investigated. BLL amyloid-like fibrils in RMPI medium, pH 7.2, at 37 °C was confirmed by binding of Congo Red, Thioflavin T and electron microscopy. Neither binding of amyloid markers nor fibrillar structures were found when the lectin was incubated in RPMI plus galactose, the specific BLL-binding carbohydrate. Several phagocytic compartments containing fibrillar structures were observed in BLL-treated macrophages in RPMI medium for 24 h; these compartments showed an apple-green birefringence after Congo Red staining and were positive for thioflavin S and anti-amyloid antibody, indicating the presence of amyloid-like fibrils. No fibrillar material and no labeling were observed when the macrophages were treated with BLL plus galactose or cytochalasin B, an inhibitor of phagocytosis. BLL did not affect the viability of the cells. A significant release of proinflammatory (TNF-α, IL-6, INF-ϒ and IL-1ß) and regulatory (IL-10) cytokines was observed in BLL-treated macrophages. Taken together, our results shed light on the structural organization of BLL, improving knowledge about the interaction of lectin with macrophages. The phagocytosis of amyloid-like aggregates together with the proinflammatory response induced by BLL may open new perspectives for the use of this lectin as an interesting model to study cytokines and the production of other mediators as well as understand the mechanisms occurring in human immune cells during amyloid protein deposition.

Antimicrobial activity of Buchenavia tetraphylla against Candida albicans strains isolated from vaginal secretions.

CONTEXT: Buchenavia tetraphylla (Aubl.) RA Howard (Combretaceae: Combretoideae) is an ethnomedicinal plant with reported antifungal action. OBJECTIVE: This study evaluates the antimicrobial activity of B. tetraphylla leaf extracts against clinical isolates of Candida albicans. The morphological alterations, combinatory effects with fluconazole and the cytotoxicity of the active extract were analyzed. MATERIALS AND METHODS: Extracts were obtained using different solvents (hexane: BTHE; chloroform: BTCE; ethyl acetate: BTEE; and methanol: BTME). Antimicrobial activity was determined by the broth microdilution method using nine strains of C. albicans isolated from vaginal secretions and one standard strain (UFPEDA 1007). RESULTS: All extracts showed anti-C. albicans activity, including against the azole-resistant strains. The MIC values ranged from 156 to 2500 µg/mL for the BTHE; 156 to 1250 µg/mL for the BTCE; 625 to 1250 µg/mL for the BTME and 625 µg/mL to 2500 µg/mL for the BTEE. BTME showed the best anti-C. albicans activity. This extract demonstrated additive/synergistic interactions with fluconazole. Scanning electron microscopy analysis suggested that the BTME interferes with the cell division and development of C. albicans. BTME showed IC50 values of 981 and 3935 µg/mL, against J774 macrophages and human erythrocytes, respectively. This extract also enhanced the production of nitric oxide by J774 macrophages. DISCUSSION AND CONCLUSION: Buchenavia tetraphylla methanolic extract (BTME) is a great source of antimicrobial compounds that are able to enhance the action of fluconazole against different C. albicans strains; this action seems related to inhibition of cell division.

Diversity of polymyxin resistance mechanisms among Acinetobacter baumannii clinical isolates.

Polymyxins have become drugs of last resort for treatment of multi-drug resistant (MDR) Gram-negative infections. However, the mechanisms of resistance to this compound have not been completely elucidated. In this study, we evaluated the mechanisms of resistance to this antimicrobial in two A. baumannii clinical isolates, respectively, susceptible (A027) and resistant (A009) to polymyxin B before and after polymyxin B exposure (A027ind and A009ind). The pmrAB and lpxACD were sequenced and their transcriptional levels were analyzed by qRT-PCR. The bacterial cell morphology was evaluated by transmission electronic microscopy (TEM) and the membrane potential was measured using Zeta-potential analyzer. The virulence of strains was studied using a Caenorhabditis elegans model. Both clinical isolates exhibited an elevation of the polymyxin B MIC after exposure to this compound. On the other hand, A027ind showed decreased values of MIC for ß-lactams, aminoglycosides, vancomycin, teicoplanin, oxacillin and erythromycin. A027ind harbored two mutations in pmrB and the ISAba125 disrupting the lpxA. In contrast, A009ind strain exhibited increase of pmrB transcriptional level, after polymyxin B exposure, despite the absence of mutations in the pmrAB genes. The TEM images revealed a thicker and more electron-dense peptidoglycan layer for A009 than that of A027. The exposure to polymyxin B induced a strong condensation and darkening of intracellular material, mainly in A009ind. In addition, the surface charge of A009 was significantly less negative than the one of A027. Using the C. elegans model, only A027ind strain showed a reduction on virulence. The diversity of polymyxin B resistance mechanisms among A. baumannii strains evaluated in this study confirms the complexity of these mechanisms, which may vary depending of the background of each strain.

In vitro cell-based assays for evaluation of antioxidant potential of plant-derived products.

Several plant-derived compounds have been screened by antioxidant assays, but many of these results are questionable, since they do not evaluate the pharmacologic parameters. In fact, the development of better antioxidants stills a great challenge. In vitro cell-based assays have been employed to assess the antioxidant effect of various compounds at subcellular level. Cell-based assays can also reveal compounds able to enhance the antioxidant pathways, but without direct radical scavenging action (which could not be detected by traditional assays). These methodologies are general of easy implementation and reproducible making them suitable for the early stages of drug discovery. Hydrogen peroxide, a nonradical derivative of oxygen, can be employed as an oxidative agent in these assays due its biochemical properties (presence of all biological systems, solubility) and capacity to induce cell death. Truthfully, if their limitations are understood (such as difference on cell metabolism when in in vitro conditions), these cell-based assays can provide useful information about the pathways involved in the protective effects of phytochemicals against cell death induced by oxidative stress, which can be exploited to develop new therapeutic approaches.

The cytotoxic effect of essential oils from Origanum vulgare L. and/or Rosmarinus officinalis L. on Aeromonas hydrophila.

This study aimed to evaluate the antibacterial activities of the essential oils from Origanum vulgare L. (OV) and Rosmarinus officinalis L. (RO), both singly and in combination at sub-inhibitory concentrations (» MIC + » MIC), against Aeromonas hydrophila and to investigate the possible mechanisms underlying these activities. Used singly (OV: 2.5 µL/mL; RO: 20 µL/mL) or in a mixture (OV: 0.625 µL/mL + RO: 5 µL/L), these essential oils led to a significant decrease (p<0.01) in bacterial viability after 24 h of exposure. A decrease in glucose consumption by A. hydrophila and release of cellular material were observed immediately after the addition of the essential oils, both singly and as a mixture, and continued for up to 6 h. Electron microscopy of cells exposed to the essential oils revealed severe changes in the plasma membrane, cytoplasmic appearance, and cell shape during the 6-h exposure period. OV and RO essential oils combined at sub-inhibitory concentrations could be rationally applied to inhibit the growth of A. hydrophila in food products, particularly minimally processed vegetables.