Primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi.

BACKGROUND: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PURPOSE: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. METHODS: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. RESULTS: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. CONCLUSION: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Antileishmanial Activity of Ezetimibe: Inhibition of Sterol Biosynthesis, In Vitro Synergy with Azoles, and Efficacy in Experimental Cutaneous Leishmaniasis.

Leishmaniasis affects mainly low-income populations in tropical regions. Radical innovation in drug discovery is time-consuming and expensive, imposing severe restrictions on the ability to launch new chemical entities for the treatment of neglected diseases. Drug repositioning is an attractive strategy for addressing a specific demand more easily. In this project, we have evaluated the antileishmanial activities of 30 drugs currently in clinical use for various morbidities. Ezetimibe, clinically used to reduce intestinal cholesterol absorption in dyslipidemic patients, killed Leishmania amazonensis promastigotes with a 50% inhibitory concentration (IC50) of 30 µM. Morphological analysis revealed that ezetimibe caused the parasites to become rounded, with multiple nuclei and flagella. Analysis by gas chromatography (GC)-mass spectrometry (MS) showed that promastigotes treated with ezetimibe had smaller amounts of C-14-demethylated sterols, and accumulated more cholesterol and lanosterol, than untreated promastigotes. We then evaluated the combination of ezetimibe with well-known antileishmanial azoles. The fractional inhibitory concentration index (FICI) indicated synergy when ezetimibe was combined with ketoconazole or miconazole. The activity of ezetimibe against intracellular amastigotes was confirmed, with an IC50 of 20 µM, and ezetimibe reduced the IC90s of ketoconazole and miconazole from 11.3 and 11.5 µM to 4.14 and 8.25 µM, respectively. Subsequently, we confirmed the activity of ezetimibe in vivo, showing that it decreased lesion development and parasite loads in murine cutaneous leishmaniasis. We concluded that ezetimibe has promising antileishmanial activity and should be considered in combination with azoles in further preclinical and clinical studies.

Stereoselective Metabolism of the Sterol Biosynthesis Inhibitor Fungicides Fenpropidin, Fenpropimorph, and Spiroxamine in Grapes, Sugar Beets, and Wheat.

Metabolism of chiral pesticides in crops is typically studied using achiral analytical methods and, consequently, the stereoisomer composition of residues is unknown. In this study, we developed an enantioselective GC-MS/MS method to quantify residues of the fungicides fenpropidin, fenpropimorph, and spiroxamine in plant matrices. In field trials, the fungicides were applied to grapevines, sugar beets, or wheat. Fenpropidin was metabolized with no or only weak enantioselectivity. For fenpropimorph, slightly enantioselective metabolism was observed in wheat but more pronounced in sugar beets. This enantioselectivity was due to different rates of metabolism and not due to interconversion of enantiomers. The four stereoisomers of spiroxamine were also metabolized at different rates, but selectivity was only found between diastereomers and not between enantiomers. trans-Spiroxamine was preferentially degraded in grapes and cis-spiroxamine in wheat. These findings may affect the consumer dietary risk assessment because toxicological end points were determined using racemic test substances.

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.

In vivo anti-Trypanosoma cruzi activity of hydro-ethanolic extract and isolated active principles from Aristeguietia glutinosa and mechanism of action studies.

The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Attempting to find alternative drugs isolated from Nature as agents against Trypanosoma cruzi has been our goal. Recently, we have demonstrated the in vitro anti-T. cruzi activities of two secondary metabolites isolated from the hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa (Lam.), (family Asteraceae). These active principles displayed poor hemolytic activity, low toxicity against murine macrophages, and absence of mutagenicity. Herein, proof of concept in vivo studies of the whole hydro-ethanolic extract of the aerial parts of Aristeguietia glutinosa and of the most active component isolated from the hydro-ethanolic extract, i.e., (+)-15-hydroxy-7-labden-17-al, was done in a murine acute model of Chagas disease. Both treatments caused a decrease in the animals' parasitemia. Metabolomic mechanism of action studies were done by 1H-NMR, both on the extract and on the active compounds, examining the effects of the metabolites both on membrane sterol biosynthesis and mitochondrial dehydrogenases, whereby we found that one of the metabolites inhibited the activity of the parasite mitochondrial dehydrogenases and the other inhibited the biosynthesis of parasite membrane sterols. The results are interesting in the context of popular use of plants for the treatment of Chagas disease.

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism.

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid ß-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase ß-independent and liver kinase ß 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.

Bioactive sterols from marine resources and their potential benefits for human health.

Bioactive agents from marine resources have shown their valuable health beneficial effects. Therefore, increase knowledge on novel functional ingredients with biological activities from marine animal and microbe has gained much attention. Sterols are recognized as potential in development functional food ingredients and pharmaceutical agents. Marine resources, with a great diversity, can be a very interesting natural resource of sterols. This chapter focuses on biological activities of marine animal and microbe sterols with potential health beneficial applications in functional foods and pharmaceuticals.

Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures.

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D(3) metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 µM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3'-diaminobenzidine (DAB) for hydrogen peroxide (H(2)O(2)) detection and phloroglucinol for lignin staining showed that BTH causes H(2)O(2) accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (ß-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D(3), and the hydroxylated metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D(3), though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D(3) and 1α,25(OH)(2)D(3) metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol.

Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism.

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and ß forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.

Spice oil cinnamaldehyde exhibits potent anticandidal activity against fluconazole resistant clinical isolates.

Fluconazole resistance is becoming an important clinical concern. We studied the in vitro effects of cinnamaldehyde against 18 fluconazole-resistant Candida isolates. MIC(90) of cinnamaldehyde against different Candida isolates ranged 100-500 µg/ml. Growth and sensitivity of the organisms were significantly affected by cinnamaldehyde at different concentrations. The rapid irreversible action of this compound on fungal cells suggested membrane-located targets for its action. Insight studies to mechanism suggested that cinnamaldehyde exerts its antifungal activity by targeting sterol biosynthesis and plasma membrane ATPase activity. Inhibition of H(+) (-)ATPase leads to intracellular acidification and cell death. Toxicity against H9c2 rat cardiac myoblasts was studied to exclude the possibility of further associated cytotoxicity. The observed selectively fungicidal characteristics against fluconazole-resistant Candida isolates signify a promising candidature of this essential oil as an antifungal agent in treatments for candidosis.

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.

Perkinsus marinus, a protozoan parasite of the eastern oyster, has a requirement for dietary sterols.

Perkinsus marinus, a protozoan parasite of the eastern oyster, Crassostrea virginica, causes high mortality in its host along the Atlantic and Gulf coasts of North America. P. marinus meronts cultured in vitro in medium containing complete lipid supplement (cod liver oil, cholesterol and alpha tocopherol acetate in detergent) are able to synthesize a wide variety of lipids, yet cultures cannot be maintained in lipid-free medium. To determine P. marinus lipid requirements meronts were inoculated into media containing different combinations of lipid components in detergent. Treatments included complete lipid supplement (positive control), detergent only (negative control), cholesterol in detergent, alpha tocopherol acetate in detergent and cholesterol+alpha tocopherol acetate in detergent. Meronts proliferated in the positive control medium and media containing cholesterol or cholesterol+alpha tocopherol acetate, but failed to proliferate in the negative control medium and the medium containing just alpha tocopherol acetate. Gas chromatography analysis of P. marinus meronts grown in medium with added (13)C sodium acetate (0.5 mg mL(-1)) revealed the presence of fatty acids containing (13)C, but the only sterol present was cholesterol containing no (13)C. These results suggest that P. marinus cannot synthesize sterols and must sequester them from its host.

A mini-review of recent W.O. patents (2004-2005) of novel anti-fungal compounds in the field of anti-infective drug targets.

Anti-infective agents are used to treat disorders caused by bacteria, viruses, protozoa, worms, and fungi (including yeast). They are substances used on humans, animals and plants that destroy harmful microorganisms or inhibit their activity. In this mini-review, we are focusing on novel development of anti-fungal agents that have been published during the past two years. Fungi are eukaryotic microorganisms comprising over 100,000 species. Nearly 40% of all deaths from hospital-acquired infections were caused by fungi over the past 20 years. An effective anti-fungal agent is toxic to the pathogenic fungi, but not to the host. Treatment of fungi diseases, however, is often limited because anti-fungal agents are often toxic to the mammalian or plant host. In this review, 11 patents were chronologically and unbiasedly selected from 454 that fit the criteria out of a 4,716,037 patents search. These patents highlight the novel approaches that have been used to tackle difficult fungi. The strategies include targeting key fungal structures and metabolic pathways, developing manufacturing processes of anti-fungal reagents, unique membrane interfering fungicides, and plant and insect defensins.

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.