Nifuroxazide as JAK2 inhibitor: A binding mode proposal and Hel cell proliferation assay.

Nifuroxazide has been employed as an anti-diarrheic agent since 1966, but in the last decade has brought to the research spotlight again due to its recently described antitumoral activity through the JAK2 inhibitory potential. Since 2008, more than 70 papers have been published about the issue and more are expected to the following years. Herein we discuss the findings of molecular modelling studies which were performed to elucidate the potential binding mode of this drug into the JAK2 ATP recognition site and also into the allosteric region near the catalytic site. Molecular modelling followed by dynamics simulations indicated the NFZ could bind at both sites, such as a Type II kinase inhibitor since residues from both ATP and modulatory site would exhibit contacts with the drug when in a stable complex. Synthesis of NFZ and its sulfur bioisosteric analogue GPQF-63 were performed and experimental assays against HEL cells indicate the potential of NFZ and, mainly of its analogue GPQF-63 in acting as inhibitors of cell growth. HEL-cells present the JAK2 V617F mutation which leads to an enhanced JAK/STAT pathway and they have never been tested by the NFZ activity before. A mechanistic approach was also performed and revealed that both compounds induce cell apoptosis.Taken together, both the theoretical and experimental approaches point out the N-acylhydrazones as good starting points in the search for JAK2 modulatory small molecules which could then, be studied as promising leads toward new alternatives to control the JAK-STAT pathway related pathologies. This is the first study, as far as we have known, to propose a potential binding mode for NFZ as well as reporting the activity of this drug against HEL cells, which are a usual cellular model to human erythroleukemia and other myeloproliferative diseases.

Exposure to sub-inhibitory ciprofloxacin and nitrofurantoin concentrations increases recA gene expression in uropathogenic Escherichia coli: The role of RecA protein as a drug target.

Sub-inhibitory concentrations (sub-MIC) of antimicrobial agents can lead to genetic changes in bacteria, modulating the expression of genes related to bacterial stress and leading to drug resistance. Herein we describe the impact of sub-MIC of ciprofloxacin and nitrofurantoin on three uropathogenic Escherichia coli strains. Disk-diffusion assays with different antimicrobial agents were tested to detect phenotype alterations, and quantitative real-time PCR (qRT-PCR) was performed to analyze the expression of ompF and recA genes. Significant reduction on the susceptibility to ciprofloxacin and nitrofurantoin was detected on disk diffusion test. The qRT-PCR results revealed a 1.2-4.7 increase in recA expression in all E. coli studied, while the ompF expression varied. Because RecA was pointed as an important component to the development of drug resistance, molecular docking studies were performed with three experimentally known inhibitors of this enzyme. These studies aimed to understand the inhibitory binding mode of such compounds. The results confirmed the ADP/ATP binding site as a potential site of inhibitor recognition and a binding mode based on π-stacking interactions with Tyr103 and hydrogen bonds with Tyr264. These findings can be useful for guiding the search and design of new antimicrobial agents, mainly concerning the treatment of infections with resistant bacterial strains.

N-Myristoyltransferases as antileishmanial targets: a piggyback approach with benzoheterocyclic analogues

Leishmaniasis is one of the neglected diseases that remain in need for pharmacological alternatives. In this context, N-Myristoyltransferases (NMT) arise as interesting targets to explore since they are involved in the co/post-translational processing of peptides which are responsible for host cell invasion. Studies that consider these enzymes as targets point out the potential of benzoheterocyclic compounds as inhibitors of Candida albicans's N-myristoyltransferase. Here we applied a combination of comparative binding site analysis and molecular docking studies based on a Piggyback approach in the search for new Leishmania major NMT ligands. Our results revealed that NMT enzymes from both pathogens present enough structural similarity to allow extrapolation of the knowledge available from C. albicans studies to develop new L. major NMT inhibitors. Molecular docking studies with benzoheterocyclic analogues indicate the potential of benzothiazole derivatives as L. major NMT ligands, giving rise to a completely new class of chemical compounds to be explored in the development of antileishmanial drugs.

4-Phenyl-1,3-thiazole-2-amines as scaffolds for new antileishmanial agents.

BACKGROUND: There is still a need for new alternatives in pharmacological therapy for neglected diseases, as the drugs available show high toxicity and parenteral administration. That is the case for the treatment of leishmaniasis, particularly to the cutaneous clinical form of the disease. In this study, we present the synthesis and biological screening of eight 4-phenyl-1,3-thiazol-2-amines assayed against Leishmania amazonensis. Herein we propose that these compounds are good starting points for the search of new antileishmanial drugs by demonstrating some of the structural aspects which could interfere with the observed activity, as well as suggesting potential macromolecular targets. METHODS: The compounds were easily synthesized by the methodology of Hantzsch and Weber, had their purities determined by Gas Chromatography-Mass spectrometry and assayed against the promastigote forms of Leishmania amazonensis as well as against two white cell lines (L929 and THP-1) and the monkey's kidney Vero cells. PrestoBlue® and MTT viability assays were the methodologies applied to measure the antileishmanial and cytotoxic activities, respectively. A molecular modeling target fishing study was performed aiming to propose potential macromolecular targets which could explain the observed biological behavior. RESULTS: Four out of the eight compounds tested exhibited important anti-promastigote activity associated with good selectivity indexes when considering Vero cells. For the most promising compound, compound 6, IC50 against promastigotes was 20.78 while SI was 5.69. Compounds 3 (IC50: 46.63 µM; SI: 26.11) and 4 (IC50: 53.12 µM; SI: 4.80) also presented important biological behavior. A target fishing study suggested that S-methyl-5-thioadenosine phosphorylase is a potential target to these compounds, which could be explored to enhance activity and decrease the potential toxic side effects. CONCLUSIONS: This study shows that 4-phenyl-1,3-thiazol-2-amines could be good scaffolds to the development of new antileishmanial agents. The S-methyl-5-thioadenosine phosphorylase could be one of the macromolecular targets involved in the action.

Flavones-bound in benzodiazepine site on GABAA receptor: Concomitant anxiolytic-like and cognitive-enhancing effects produced by Isovitexin and 6-C-glycoside-Diosmetin.

Increasing evidence suggests that flavones can modulate memory and anxiety-like behaviour. However, these therapeutic effects are inconsistent and induce of adverse effects, which have been associated with interactions at the Benzodiazepine (BZ)-binding site. To improve our understanding of flavone effects on memory and anxiety, we employed a plus-maze discriminative avoidance task. Furthermore, we evaluated the potential of the compounds in modulating GABAA receptors via BZ-binding site using molecular modelling studies. Adult male Wistar rats were treated 30 min before training session with Vicenin-2 (0.1 and 0.25 mg/kg), Vitexin (0.1 and 0.25 mg/kg), Isovitexin (0.1 and 0.25 mg/kg) and 0.1 mg/kg 6-C-glycoside-Diosmetin, vehicle and a GABAA receptor agonist. The analysis of the time spent in the non-aversive vs aversive enclosed arms during the test session and percentage of time in the open arms within the training session revealed that treatment with Isovitexin and 6-C-glycoside-Diosmetin had memory-enhancing and anxiolytic-like effects (P < 0.001). In contrast, treatment with a higher dose of Diazepam impaired short-and long-term memory when it alleviated anxiety level. Docking studies revealed that flavones docked in a very similar way to that observed to the Diazepam, except by a lack of interaction in residue α1His101 in the BZ-binding site on GABAA receptors, which may be related to memory-enhancing effect. The occurrence of the α1His101 interaction could justify the memory-impairing observed following Diazepam treatment. These findings provide the first evidence that Isovitexin and 6-C-glycoside-Diosmetin could exert their memory-enhancing and anxiolytic-like effects via GABAA receptor modulation, which likely occurs via their benzodiazepine-binding site.

4-Phenyl-1, 3-thiazole-2-amines as scaffolds for new antileishmanial agents

There is still a need for new alternatives in pharmacological therapy for neglected diseases, as the drugs available show high toxicity and parenteral administration. That is the case for the treatment of leishmaniasis, particularly to the cutaneous clinical form of the disease. In this study, we present the synthesis and biological screening of eight 4-phenyl-1,3-thiazol-2-amines assayed against Leishmania amazonensis. Herein we propose that these compounds are good starting points for the search of new antileishmanial drugs by demonstrating some of the structural aspects which could interfere with the observed activity, as well as suggesting potential macromolecular targets. Methods: The compounds were easily synthesized by the methodology of Hantzsch and Weber, had their purities determined by Gas Chromatography-Mass spectrometry and assayed against the promastigote forms of Leishmania amazonensis as well as against two white cell lines (L929 and THP-1) and the monkey's kidney Vero cells. PrestoBlue® and MTT viability assays were the methodologies applied to measure the antileishmanial and cytotoxic activities, respectively. A molecular modeling target fishing study was performed aiming to propose potential macromolecular targets which could explain the observed biological behavior. Results: Four out of the eight compounds tested exhibited important anti-promastigote activity associated with good selectivity indexes when considering Vero cells. For the most promising compound, compound 6, IC50 against promastigotes was 20.78 while SI was 5.69. Compounds 3 (IC50: 46.63 µM; SI: 26.11) and 4 (IC50: 53.12 µM; SI: 4.80) also presented important biological behavior. A target fishing study suggested that S-methyl-5-thioadenosine phosphorylase is a potential target to these compounds, which could be explored to enhance activity and decrease the potential toxic side effects. Conclusions: This study shows that 4-phenyl-1,3-thiazol-2-amines could be good scaffolds to the development of new antileishmanial agents. The S-methyl-5-thioadenosine phosphorylase could be one of the macromolecular targets involved in the action.(AU)