Structure⁻Activity Relationship of Piplartine and Synthetic Analogues against Schistosoma mansoni and Cytotoxicity to Mammalian Cells.

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious disease mainly associated with poverty that affects millions of people worldwide. Since treatment for this disease relies only on the use of praziquantel, there is an urgent need to identify new antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae) that exhibits antischistosomal properties. The aim of this study was to evaluate the structure­function relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory calculations and in silico analysis were used to predict physicochemical and toxicity parameters. Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5⁻10 µM), but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at 785 µM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations. This study demonstrated that piplartine analogues showed less activity against S. mansoni but presented lower toxicity than piplartine.

Antischistosomal activity of the terpene nerolidol.

Schistosomiasis is a neglected tropical disease that affects hundreds of millions of people worldwide. Since the treatment of this disease currently relies on a single drug, praziquantel, new and safe schistosomicidal agents are urgently required. Nerolidol, a sesquiterpene present in the essential oils of several plants, is found in many foods and was approved by the U.S. Food and Drug Administration. In this study we analysed the in vitro antiparasitic effect of nerolidol on Schistosoma mansoni adult worms. Nerolidol at concentrations of 31.2 and 62.5 µM reduced the worm motor activity and caused the death of all male and female schistosomes, respectively. In addition, confocal laser scanning microscopy revealed morphological alterations on the tegument of worms such as disintegration, sloughing and erosion of the surface, and a correlation between viability and tegumental damage was observed. In conclusion, nerolidol may be a promising lead compound for the development of antischistosomal natural agents.

Vanillin-Related N-Acylhydrazones: Synthesis, Antischistosomal Properties and Target Fishing Studies.

BACKGROUND: Schistosomiasis is a neglected disease, which affects millions of people in developing countries. Its treatment relies on a single therapeutic alternative, the praziquantel. This situation may lead to drug resistance which, in turn, made urgent the need for new antischistosomal agents. Nacylhydrazones are usually explored as good antimicrobial agents, but the vanillin-related N-acylhydrazones have never been tested by their antiparasitic potential. OBJECTIVE: Herein, we report the synthesis of seven analogues, three of them unpublished, their biological investigation against Schistosoma mansoni and Target Fishing studies. METHODS: The compounds were synthesized following classical synthetical approaches. The anthelmintic potential was assessed as well as their cytotoxicity profile. Confocal laser scanning microscopy and target fishing study were performed to better understand the observed antischistosomal activity. RESULTS: Compound GPQF-407 exhibited good antischistosomal activity (47.91 µM) with suitable selectivity index (4.14). Confocal laser scanning microscopy revealed that it triggered severe tegumental destruction and tubercle disintegration. Target fishing studies pointed out some probable targets, such as the serine-threonine kinases, dihydroorotate dehydrogenases and carbonic anhydrase II. CONCLUSION: The GPQF-407 was revealed to be a promising antischistosomal agent which, besides presenting the N-acylhydrazone privileged scaffold, also could be easily synthesized on large scales from commercially available materials.

Antiparasitic, structural, pharmacokinetic, and toxicological properties of riparin derivatives.

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is one of the most important parasitic diseases in the world, affecting over 200 million people in developing countries. Riparins are natural alkamides found in Aniba riparia (Lauraceae) fruits that possess several pharmacological properties. In this study, we reported the synthesis, characterization and structural analysis of six riparin derivatives (A-F), as well as their schistosomicidal activity against S. mansoni worms together with a biological, pharmacokinetic and toxicological in silico evaluation. Firstly, these compounds were synthesized, purified and characterized by elemental analysis, FT-IR spectroscopy, X-ray diffraction and theoretical calculations to evaluate their stability and conformation. Next, the schistosomicidal activity of the riparins was tested against S. mansoni worms. Bioassays revealed that Riparins E and F were the most active compounds, showing half-maximum inhibitory concentration at low micromolar ranges (IC50 values ~10 µM). Also, confocal laser scanning microscopy studies revealed tegumental damage in parasites after exposition with Riparins B, E and F. Additionally, based on MTT assay, all tested riparins showed no cytotoxic potential toward mammalian cells. Finally, in silico analyses were used to predict the absorption, distribution, metabolism, elimination and toxicity (ADMET) of the compounds. Taken together, the results revealed a promising ADMET profile and suggested that riparins could be starting points for lead optimization programs for natural products with antischistosomal properties.

Cyclohexene-fused 1,3-oxazines with selective antibacterial and antiparasitic action and low cytotoxic effects.

Oxazine derivatives, a class of heterocyclic compounds, exhibit a variety of biological properties, such as anticonvulsant and antitumor activities. In this study, we evaluated the effect of two cyclohexene-fused 1,3-oxazines (cis­1-benzyl-N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-imine (1) and trans­N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-imine (2)) in cultures of Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Serratia marcescens, Shigella flexneri and Staphylococcus aureus by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Additionally, the ex vivo antiparasitic activity of oxazines was assessed against Schistosoma mansoni, a helminth that is one of the major agents of the disease schistosomiasis Also, oxazines were evaluated on three tumor cell lines, NCI-H292 (human lung carcinoma), MCF-7 (human breast adenocarcinoma) and HEp-2 (human cervix carcinoma), and two normal cell lines (Vero and red blood cells). Bioassays revealed that oxazine 2 is more effective against bacteria than oxazine 1, with the lowest MIC and MBC values of 3.91 and 32.5µg/mL, respectively. Similarly, compound 2 demonstrated higher antiparasitic activity than 1, and scanning electron microscopy analysis showed several morphological alterations in the tegument of worms in a concentration-dependent manner. In contrast, both oxazines exhibited low cytotoxic effects on cancer and normal cell lines. These results indicated that oxazines exerted direct effects on bacteria and parasite schistosomes. More importantly, since schistosomiasis control programs rely on one drug, praziquantel, oxazines may have the potential to become new antischistosomal agents.

Synergistic effects of in vitro combinations of piplartine, epiisopiloturine and praziquantel against Schistosoma mansoni.

Schistosomiasis is a world health problem, and praziquantel is the only drug currently used for the treatment. There is some evidence that extensive monotherapy of praziquantel may be leading to drug resistance in the parasite. In order to find alternative treatments, the effects of the combination of epiisopiloturine (EPI), piplartine (PPT) and praziquantel (PZQ) were evaluated. Similarity analysis of these compounds was performed using optimized molecular structures to compare the shape and the charge modeling of combinations between PZQ and EPI or PPT. Supported by this data, in vitro association of PZQ-PPT, PZQ-EPI, and EPI-PPT was carried out, and the activity of these combinations against Schistosoma mansoni was assessed. The results showed synergistic activity with a combination index (CI) of 0.42 for the treatment with PZQ-PPT. Both PZQ-EPI and EPI-PPT combinations also showed synergistic effects, with CI values of 0.86 and 0.61, respectively. Surface alterations in the tegument of adult schistosomes after the treatments were observed using laser confocal microscopy and scanning electron microscopy. Additionally, the association of EPI-PPT decreased the cytotoxicity when compared with both isolated compounds in three different lines of mammalian cells. Thus, synergistic combinations of PZQ-PPT, PZQ-EPI, and EPI-PPT create the possibility of reduced doses to be used against Schistosoma mansoni.

Structural parameters, molecular properties, and biological evaluation of some terpenes targeting Schistosoma mansoni parasite.

The use of natural products has a long tradition in medicine, and they have proven to be an important source of lead compounds in the development of new drugs. Among the natural compounds, terpenoids present broad-spectrum activity against infective agents such as viruses, bacteria, fungi, protozoan and helminth parasites. In this study, we report a biological screening of 38 chemically characterized terpenes from different classes, which have a hydroxyl group connected by hydrophobic chain or an acceptor site, against the blood fluke Schistosoma mansoni, the parasite responsible for schistosomiasis mansoni. In vitro bioassays revealed that 3,7-dimethyl-1-octanol (dihydrocitronellol) (10) was the most active terpene (IC50 values of 13-52 µM) and, thus, we investigated its antischistosomal activity in greater detail. Confocal laser scanning microscopy revealed that compound 10 induced severe tegumental damage in adult schistosomes and a correlation between viability and tegumental changes was observed. Furthermore, we compared all the inactive compounds with dihydrocitronellol structurally by using shape and charge modeling. Lipophilicity (miLogP) and other molecular properties (e.g. molecular polar surface area, molecular electrostatic potential) were also calculated. From the 38 terpenes studied, compound 10 is the one with the greatest flexibility, with a sufficient apolar region by which it may interact in a hydrophobic active site. In conclusion, the integration of biological and chemical analysis indicates the potential of the terpene dihydrocitronellol as an antiparasitic agent.

Effect of neem (Azadirachta indica A. Juss) leaf extract on resistant Staphylococcus aureus biofilm formation and Schistosoma mansoni worms.

ETHNOPHARMACOLOGICAL RELEVANCE: There are ethnopharmacological reports supporting the use of neem (Azadirachta indica A. Juss) leaf against bacterial and worm infections. However there is a lack of studies about its effect on bacterial biofilm formation and Schistosoma mansoni worms. This study reports the in vitro effects of neem leaf ethanolic extract (Neem EE) on Methicillin-resistant Staphylococcus aureus (MRSA) biofilm and planktonic aggregation formation, and against S. mansoni worms. MATERIALS AND METHODS: Quantification of the Azadirachtin (AZA), thought to be one of their main compounds related to biological effects, was performed. The effect of sub-inhibitory concentrations of Neem EE on biofilm formation and planktonic aggregates of S. aureus was tested using the crystal violet dye method and atomic force microscopy (AFM) analysis, respectively. Changes in S. mansoni motor activity and death of worms were analyzed in vitro after exposition to the extract. Treated schistosomes were also examined using confocal laser scanning microscopy. RESULTS: It was observed the presence of AZA in the extract (0.14 ± 0.02 mg/L). Testing Neem EE sub-inhibitory concentrations, a significant biofilm adherence inhibition from 62.5 µg/mL for a sensitive S. aureus and 125 µg/mL for two MRSA strains was observed. AFM images revealed that as the Neem EE concentration increases (from 250 to 1000 µg/mL) decreased ability of a chosen MRSA strain to form large aggregates. In relation of anti-schistosoma assay, the extract caused 100% mortality of female worms at a concentration of 50 µg/mL at 72 h of incubation, while 300 µg/mL at 24h of incubation was required to achieve 100% mortality of male worms. The extract also caused significant motor activity reduction in S. mansoni. For instance, at 96 h of incubation with 100 µg/mL, 80% of the worms presented significant motor activity reduction. By the confocal microscopy analysis, the dorsal surface of the tegument of worms exposed to 300 µg/mL (male) and 100 µg/mL (female) of the extract showed severe morphological changes after 24h of treatment. CONCLUSIONS: Neem leaf ethanolic extract presented inhibitory effect on MRSA biofilm and planktonic aggregation formation, and anthelmintic activity against S. mansoni worms.