3-H-[1,2]Dithiole as a New Anti-Trypanosoma cruzi Chemotype: Biological and Mechanism of Action Studies.

The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM). Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.

Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation and lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).

Altered lipid metabolism has been shown in fish fed plant protein sources. The present study aimed to gain further insights into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal (PM) and soya-saponin (SA) inclusion in salmon feed. Post-smolt Atlantic salmon were fed for 10 weeks one of four diets based on fishmeal or PM, with or without 10 g/kg SA. PM inclusion resulted in decreased growth performance, excessive lipid droplet accumulation in the pyloric caeca and liver, and reduced plasma cholesterol levels. Intestinal and hepatic gene expression profiling revealed an up-regulation of the expression of genes involved in lipid absorption and lipoprotein (LP) synthesis (apo, fatty acid transporters, microsomal TAG transfer protein, acyl-CoA cholesterol acyltransferase, choline kinase and choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase) and associated transcription factors (sterol regulatory element-binding protein 2 and PPARγ). SA inclusion resulted in reduced body pools of cholesterol and bile salts. The hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cytochrome P450 7A1 (cyp7a1)) as well as the transcription factor liver X receptor and the bile acid transporter abcb11 (ATP-binding cassette B11) was down-regulated by SA inclusion. A significant interaction was observed between PM inclusion and SA inclusion for plasma cholesterol levels. In conclusion, gene expression profiling suggested that the capacity for LP assembly and cholesterol synthesis was up-regulated by PM exposure, probably as a compensatory mechanism for excessive lipid droplet accumulation and reduced plasma cholesterol levels. SA inclusion had hypocholesterolaemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.

Effect of miconazole and terbinafine on artemisinin content of shooty teratoma of Artemisia annua.

The effects produced by the addition of sterol synthesis inhibitors on the artemisinin content of the transgenic organ culture (A. tumefaciens ATCC 33970 or 15955) of Artemisia annua are presented. The transgenic tissue produced 3-4 fold higher levels of artemisinin 0.84% (56.3 mg/L) within a short culture period compared with field grown plants (0.23%). The addition of the sterol synthesis inhibitors, miconazole and terbinafine, to these transgenic cultures resulted in enhanced artemisinin content up to 1.15% and 1.44%, respectively. Further enhancement of artemisinin content was achieved by varying the addition time of the sterol synthesis inhibitor to the cultures. The best artemisinin content (2.62%) was observed after terbinafine (10 mg/L) addition on the sixteenth day of the culture period.

Effects of sterols on the development and aging of Caenorhabditis elegans.

Although Caenorhabditis elegans lacks several components of the de novo sterol biosynthetic pathway, it requires sterols as essential nutrients. Supplemental cholesterol undergoes extensive enzymatic modification in C. elegans to form certain sterols of unknown function. Since sterol metabolism in C. elegans differs from that in other species, such as mammals and yeast, it is important to examine how sterols regulate worm physiology. To examine the functions of sterols in C. elegans, a sterol-feeding experiment was carried out and several critical parameters, such as brood size, growth rate, and life span, were measured. In addition, the change in lipid distribution in C. elegans can be both qualitatively and quantitatively determined by various methods, including staining and chromatographic techniques. Taken together, the effects of sterols on C. elegans are very prominent and can be easily assessed using the techniques described here.

Trypanosoma cruzi targets for new chemotherapeutic approaches.

BACKGROUND: Strategies for development of anti-parasite chemotherapy involve identification of active principles of plants, investigation of drugs already licensed for other pathologies, or validation of specific targets identified within key metabolic pathways. OBJECTIVE: To review the state of the art of drug targets against Trypanosoma cruzi with emphasis on sterol metabolism, kinetoplast DNA (kDNA) sites, trypanothione reductase, cysteine proteinase, hypoxanthine-guanine phosphoribosyltransferase, dihydrofolate reductase and glyceraldehyde-3-phosphate dehydrogenase. METHODS: Current knowledge, accumulated over the last three decades, on targets for design and development of new trypanocidal compounds is described. RESULTS/CONCLUSION: There is an urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox only allopurinol and a few sterol inhibitors have moved to clinical trials, despite the long list of natural and synthetic compounds assayed against T. cruzi. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity associated with a lack of definitive preclinical evidence of parasitological cure in animal models.

Meiosis activating sterol (MAS) regulate FSH-induced meiotic resumption of cumulus cell-enclosed porcine oocytes via PKC pathway.

Meiosis activating sterol (MAS) have been found to be able to promote oocytes meiotic maturation of small animals in vitro, such as mouse, rat and rabbit. But in large animals, whether MAS play the same function, especially the physiological mechanisms of MAS on oocytes maturation are not clear. To our knowledge, this is the first time to investigate the role and signal pathway of MAS on FSH-induced porcine oocytes meiotic resumption. Porcine cumulus-enclosed oocytes (CEOs) isolated from 3 to 5mm follicles were cultured in the FSH-medium for 24h supplemented with 0-50 microM RS21745 or 0-100 microM RS21607 (two specific inhibitors of lanosterol 14alpha-demethylase that converts lanosterol to FF-MAS), or cultured in FSH-medium with 25 microM RS21745 for 0-24h firstly, then transferred into a new FSH-medium (the total culture time is 24h). The results revealed that RS21745 or RS21607 could inhibit FSH-induced porcine CEOs meiotic resumption in a dose and time-dependent manner. Meanwhile, FSH-induced cumulus expansion could also be inhibited dose-dependently by RS21745 or RS21607. Otherwise, AY9944-A-7, an inhibitor of Delta14-reductase which promotes cholesterol accumulation from FF-MAS, had no effect on both denuded oocytes (DOs) cultured for 24 or 44 h and CEOs cultured for 24h meiotic resumption, but it could promote CEOs meiotic resumption after 44 h culture. In addition, we got that 10(-8) to 10(-6)M PMA, an activator of PKC pathway, could reverse the inhibiting effect of RS21745 on FSH-induced CEOs meiotic resumption and enhance the rate of germinal vesicle breakdown (GVBD) of CEOs cultured in medium with hypoxanthine (HX). Moreover, 5-10 microM chelerythrine chloride, an inhibitor of PKC, could enhance the inhibitory effect of RS21745 on FSH-induced porcine oocytes resumption of meiosis. All the data of this study support that endogenous FF-MAS takes part in the FSH-induced porcine oocytes meiotic resumption and might play an active role via PKC signal pathway.

Cholesterol import by Aspergillus fumigatus and its influence on antifungal potency of sterol biosynthesis inhibitors.

High mortality rates from invasive aspergillosis in immunocompromised patients are prompting research toward improved antifungal therapy and better understanding of fungal physiology. Herein we show that Aspergillus fumigatus, the major pathogen in aspergillosis, imports exogenous cholesterol under aerobic conditions and thus compromises the antifungal potency of sterol biosynthesis inhibitors. Adding serum to RPMI medium led to enhanced growth of A. fumigatus and extensive import of cholesterol, most of which was stored as ester. Growth enhancement and sterol import also occurred when the medium was supplemented with purified cholesterol instead of serum. Cells cultured in RPMI medium with the sterol biosynthesis inhibitors itraconazole or voriconazole showed retarded growth, a dose-dependent decrease in ergosterol levels, and accumulation of aberrant sterol intermediates. Adding serum or cholesterol to the medium partially rescued the cells from the drug-induced growth inhibition. We conclude that cholesterol import attenuates the potency of sterol biosynthesis inhibitors, perhaps in part by providing a substitute for membrane ergosterol. Our findings establish significant differences in sterol homeostasis between filamentous fungi and yeast. These differences indicate the potential value of screening aspergillosis antifungal agents in serum or other cholesterol-containing medium. Our results also suggest an explanation for the antagonism between itraconazole and amphotericin B, the potential use of Aspergillus as a model for sterol trafficking, and new insights for antifungal drug development.

[Screening of natural compounds with hypolipidemic activity]. / Skrining prirodnykh soedinenii s gipolipidemicheskoi aktivnost'iu.

In the screening programme for natural hypolipidemic compounds 702 strains of soil microorganisms were tested and 25 of them were selected because of their ability to produce compounds inhibiting sterol synthesis in Hep G2 hepatoma cells. The compounds were estimated in the microbiological model with Tolypocladium inflatum 106 as the test microbe. The 2nd stage of the screening resulted in isolation of 13 strains producing compounds with high hypolipidemic activity, analogous to or higher than the activity of lovastatin in the experimental models.

[Screening of natural immunosuppressors by their ability to modify steroid synthesis in hepatocytes]. / Otbor prirodnykh immunosupressorov po sposobnosti k modifikatsii sinteza sterolov kletkami gepatotsitov.

In the programme for screening sterol synthesis inhibitors with the use of actinomycetes and fungi 702 strains were tested. The effect of alcohol extracts of the mycelium of fungi and actinomycetes at a dilution of 1/10(3) on sterol synthesis by the Hep G2 hepatome cells was determined by incorporation of 3H acetate into sterols and proteins. Lovastatin (200 pg/ml) was used as the control: the sterol synthesis was decreased by 49 +/- 4% without inhibiting the protein synthesis. A number of the cultures produced compounds inhibiting under the experimental conditions the synthesis of sterols by 70 to 80% with simultaneous inhibition of the protein synthesis at least by 60 to 70%. Three compounds from that group produced by streptomycetes were subjected to a more detailed investigation. The compounds were demonstrated to be active antifungal antibiotics (MIC 0.1-1 mcg/ml). In a dose of 0.1-1 mcg/ml they showed high immunosuppressive activity in models of lymphocyte transformation in mice, whereas cyclosporin was active in a dose of 1 mcg/ml. Therefore, the model for screening hypolipidemic compounds could be considered useful for screening promising natural immunosuppressors.

Impairment of sterol biosynthesis leads to phosphorus and calcium accumulation in Leishmania acidocalcisomes.

The induction of the formation of inclusion vesicles in Leishmania amazonensis by the sterol biosynthesis inhibitors (SBI) ketoconazole and terbinafine has been reported previously. These compartments were recently identified as acidocalcisomes. By the use of electron spectroscopic imaging and energy loss spectroscopy, the presence of calcium, phosphorus and oxygen in the electron-dense inclusions located within the acidocalcisomes has been demonstrated. Endoplasmic reticulum cisternae formed membrane whorls which enclosed large portions of the cytoplasm and sometimes circumscribed acidocalcisomes. In addition, acid phosphatase activity, as well as the endocytic tracers horseradish peroxidase and gold-labelled transferrin and cystatin C were detected within these organelles in both SBI-treated and untreated parasites. These data suggest that impairment of sterol biosynthesis induces the biogenesis of acidocalcisomes and triggers an autophagic process that leads to intersection of the endosomal/lysosomal system with the acidocalcisomes.