Alpha-Naphthoflavone as a Novel Scaffold for the Design of Potential Inhibitors of the APH(3')-IIIa Nucleotide-Binding Site of Enterococcus faecalis.

The spread of nosocomial infections caused by antibiotic-resistant Enterococcus faecalis is one of the major threats to global health at present. While aminoglycosides are often used to combat these infections, their effectiveness is reduced by various resistance mechanisms, including aminoglycoside modifying enzymes, and there are currently no drugs to inhibit these enzymes. To address this issue, this study was conducted to identify potential aminoglycoside adjuvants from a database of 462 flavones. The affinity of these molecules with the nucleotide-binding site (NBS) of aminoglycoside phosphotransferase type IIIa of E. faecalis (EfAPH(3')-IIIa) was evaluated, and the five molecules with the highest binding energies were identified. Of these, four were naphthoflavones, suggesting that their backbone could be useful in designing potential inhibitors. The highest-ranked naphthoflavone, 2-phenyl-4H-benzo[h]chromen-4-one, was modified to generate two new derivatives (ANF2OHC and ANF2OHCC) to interact with the NBS similarly to adenine in ATP. These derivatives showed higher binding free energies, better stability in molecular dynamics analysis and superior pharmacokinetic and toxicological profiles compared to the parent molecule. These findings suggest that these alpha-naphthoflavone derivatives are potential inhibitors of EfAPH(3')-IIIa and that this core may be a promising scaffold for developing adjuvants that restore the sensitivity of aminoglycosides.

An In-Silico Evaluation of Anthraquinones as Potential Inhibitors of DNA Gyrase B of Mycobacterium tuberculosis.

The World Health Organization reported that tuberculosis remains on the list of the top ten threats to public health worldwide. Among the main causes is the limited effectiveness of treatments due to the emergence of resistant strains of Mycobacterium tuberculosis. One of the main drug targets studied to combat M. tuberculosis is DNA gyrase, the only enzyme responsible for regulating DNA topology in this specie and considered essential in all bacteria. In this context, the present work tested the ability of 2824 anthraquinones retrieved from the PubChem database to act as competitive inhibitors through interaction with the ATP-binding pocket of DNA gyrase B of M. tuberculosis. Virtual screening results based on molecular docking identified 7122772 (N-(2-hydroxyethyl)-9,10-dioxoanthracene-2-sulfonamide) as the best-scored ligand. From this anthraquinone, a new derivative was designed harbouring an aminotriazole moiety, which exhibited higher binding energy calculated by molecular docking scoring and free energy calculation from molecular dynamics simulations. In addition, in these last analyses, this ligand showed to be stable in complex with the enzyme and further predictions indicated a low probability of cytotoxic and off-target effects, as well as an acceptable pharmacokinetic profile. Taken together, the presented results show a new synthetically accessible anthraquinone with promising potential to inhibit the GyrB of M. tuberculosis.

Virtual screening, optimization and molecular dynamics analyses highlighting a pyrrolo[1,2-a]quinazoline derivative as a potential inhibitor of DNA gyrase B of Mycobacterium tuberculosis.

Tuberculosis is a disease that remains a significant threat to public health worldwide, and this is mainly due to the selection of strains increasingly resistant to Mycobacterium tuberculosis, its causative agent. One of the validated targets for the development of new antibiotics is DNA gyrase. This enzyme is a type II topoisomerase responsible for regulating DNA topology and, as it is essential in bacteria. Thus, to contribute to the search for new molecules with potential to act as competitive inhibitors at the active site of M. tuberculosis DNA gyrase B, the present work explored a dataset of 20,098 natural products that were filtered using the FAF-Drugs4 server to obtain a total of 5462 structures that were subsequently used in virtual screenings. The consensus score analysis between LeDock and Auto-Dock Vina software showed that ZINC000040309506 (pyrrolo[1,2-a]quinazoline derivative) exhibit the best binding energy with the enzyme. In addition, its subsequent optimization generated the derivative described as PQPNN, which show better binding energy in docking analysis, more stability in molecular dynamics simulations and improved pharmacokinetic and toxicological profiles, compared to the parent compound. Taken together, the pyrrolo[1,2-a]quinazoline derivative described for the first time in the present work shows promising potential to inhibit DNA gyrase B of M. tuberculosis.

An in-silico analysis reveals 7,7'-bializarin as a promising DNA gyrase B inhibitor on Gram-positive and Gram-negative bacteria.

Bacterial diseases are considered by the World Health Organization to be one of the greatest threats to public health worldwide, mainly due to the increasingly frequent resistance to traditional antibiotics. Estimates from the World Bank indicate that the annual global economic impacts of antibiotic resistance will reach US$1.0-3.4 trillion by 2030. With this, the demand for studies aiming at the discovery of new antibiotics or molecules that may play a synergistic role within the spectrum of drug-resistant bacteria is of fundamental importance. In this in silico study, ligands generated from anthraquinones with established antibacterial activity were evaluated as potential inhibitors of the DNA gyrase subunit B of two species of Gram-positive and two Gram-negative bacteria. The main result of molecular docking-based virtual screening reveals several anthraquinones with remarkable binding energies, of which 7,7'-bializarin (ZINC000004783172) exhibited the highest value for all DNA gyrases subunit B studied and formed stable complexes, as evidenced by molecular dynamics simulations. Collectively, the results presented here reveal the potential of this molecule to bind tightly to the active site of DNA gyrases subunit B of Escherichia coli, Salmonella enterica (subtype typhi), Enterococcus faecalis, and Staphylococcus aureus, and therefore represents a promising candidate for further in vitro testing aimed at evaluating its antibacterial effect.

Genome-Wide Proteomics and Phosphoproteomics Analysis of Trypanosoma cruzi During Differentiation.

Trypanosoma cruzi is a pathogenic protozoan that still has an impact on public health, despite the decrease in the number of infection cases along the years. T. cruzi possesses an heteroxenic life cycle in which it differentiates in at least four forms. Among the differentiation processes, metacyclogenesis has been exploited in different views by researchers. An intriguing question that rises is how metacyclogenesis is triggered and controlled by cell signaling and which are the differentially expressed proteins and posttranslational modifications involved in this process. An important cell signaling pathway is the protein phosphorylation, and it is reinforced in T. cruzi in which the gene expression control occurs almost exclusively posttranscriptionally. Additionally, the number of protein kinases in T. cruzi is relatively high compared to other organisms. A way to approach these questions is evaluating the cells through phosphoproteomics and proteomics. In this chapter, we will describe the steps from the cell protein extraction, digestion and fractionation, phosphopeptide enrichment, to LC-MS/MS analysis as well as a brief overview on peptide identification. In addition, a published method for in vitro metacyclogenesis will be detailed.

Quantitative phosphoproteome and proteome analyses emphasize the influence of phosphorylation events during the nutritional stress of Trypanosoma cruzi: the initial moments of in vitro metacyclogenesis.

Phosphorylation is an important event in cell signaling that is modulated by kinases and phosphatases. In Trypanosoma cruzi, the etiological agent of Chagas disease, approximately 2% of the protein-coding genes encode for protein kinases. This parasite has a heteroxenic life cycle with four different development stages. In the midgut of invertebrate vector, epimastigotes differentiate into metacyclic trypomastigotes in a process known as metacyclogenesis. This process can be reproduced in vitro by submitting parasites to nutritional stress (NS). Aiming to contribute to the elucidation of mechanisms that trigger metacyclogenesis, we applied super-SILAC (super-stable isotope labeling by amino acids in cell culture) and LC-MS/MS to analyze different points during NS. This analysis resulted in the identification of 4205 protein groups and 3643 phosphopeptides with the location of 4846 phosphorylation sites. Several phosphosites were considered modulated along NS and are present in proteins associated with various functions, such as fatty acid synthesis and the regulation of protein expression, reinforcing the importance of phosphorylation and signaling events to the parasite. These modulated sites may be triggers of metacyclogenesis.

Macrophage activation and leishmanicidal activity by galactomannan and its oxovanadium (IV/V) complex in vitro.

Compounds that activate macrophage antimicrobial activity are potential targets for treatment of leishmaniasis. The present study investigated the in vitro immunomodulatory effects of a galactomannan (GALMAN-A) isolated from seeds of Mimosa scabrella and its oxovanadium (IV/V) complex (GALMAN-A:VO(2+)/VO(3+)) on macrophage activity. GALMAN-A increased nitric oxide levels by ~33% at a concentration of 250µg/ml, while GALMAN-A:VO(2+)/VO(3+) decreased nitric oxide levels by ~33% at a concentration of 50µg/ml. Furthermore, GALMAN-A increased interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) levels by 5.5 and 2.3 times, respectively, at a concentration of 25µg/ml; at the same concentration, GALMAN-A:VO(2+)/VO(3+) promoted an increase in IL-1ß and IL-6 production by 8 and 5.5 times, respectively. However, neither GALMAN-A nor GALMAN-A:VO(2+)/VO(3+) affected tumor necrosis factor alpha (TNF-α) or interleukin-10 (IL-10) levels. Importantly, both GALMAN-A and GALMAN-A:VO(2+)/VO(3+) exhibited leishmanicidal activity on amastigotes of Leishmania (L.) amazonensis, reaching ~60% activity at concentrations of 100 and 25µg/ml, respectively. These results indicate that GALMAN-A is three times more potent and its oxovanadium complex is twelve times more potent than Glucantime (300µg/ml), which is the drug of choice in leishmaniasis treatment. The IC50 value for GALMAN-A:VO(2+)/VO(3+) was 74.4µg/ml (0.58µg/ml of vanadium). Thus, the significant activation of macrophages and the noted leishmanicidal effect demonstrate the need for further studies to clarify the mechanisms of action of these compounds.