Sterol Biosynthesis Inhibitors Research Articles

Ultrastructural alterations induced by Δ24(25)-sterol methyltransferase inhibitors on Trichomonas vaginalis

Trichomonas vaginalis is an important human parasite that causes trichomoniasis, a cosmopolitan sexually transmitted disease. Currently, the treatment of choice for T. vaginalis infections is metronidazole. The increase in metronidazole-resistant parasites and undesirable side effects of this drug make the search for alternative chemotherapeutic approaches a priority for the management of trichomoniasis. Here, the antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were investigated. It was found that 22,26-azasterol (5 μM) and 24(R,S),25-epiminolanosterol (10 μM), known inhibitors of Δ(24(25))-sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations were (1) membrane blebbing and disruption, (2) wrinkled cells and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines Madin-Darby canine kidney cells showed no effect of the azasterols on the viability and proliferation of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis.

Effect of sterol biosynthesis-inhibiting (SBI) fungicides on the fermentation rate and quality of young ale beer

The influence of five sterol biosynthesis-inhibiting (SBI) fungicides (cyproconazole, diniconazole, epoxiconazole, flutriafol, and tebuconazole) on the fermentation and quality of young ale beer was assessed. Fungicides were individually supplied to the brewer wort to obtain a concentration of 0.2μg/ml. A noticeable influence of the fungicide residues on the fermentation rate was observed in all cases. From the third day onwards, the fermentation rate was low in all treated samples. At the end of fermentation (8days), statistically significant differences (p<0.05) were found for the extract and attenuation values, which were higher for all the samples treated with fungicides. As a consequence, a higher amount of residual sugars (mainly maltose and maltotriose) was found in the beer. No significant differences were found for the pH and the polyphenol content after fermentation among the blank and the treated samples, while the values of colour intensity (lower) and tint (higher) in the beer were statistically different.

Fenpropimorph and fenhexamid impact phosphorus translocation by arbuscular mycorrhizal fungi

Fenpropimorph and fenhexamid are sterol biosynthesis inhibitor (SBI) molecules widely used to control diseases in agriculture. Both molecules, at increasing concentrations, have been shown to impact on the non-target arbuscular mycorrhizal (AM) fungi. Root colonization, spore production and mycelium architecture, including the branched absorbing structures which are thought to be involved in phosphorus (P) uptake, were affected. In the present study, we investigated the capacity of Glomus sp. MUCL 43204 to take up, transfer and translocate labelled P to Medicago truncatula in the presence of these SBI molecules. We used a strict in vitro cultivation system associating an autotrophic plant of M. truncatula with the AM fungus. In addition, the effects of both SBI molecules on the proportion of hyphae with alkaline phosphatases (ALP), succinate dehydrogenase (SDH) activity and on the expression of the mycorrhiza-specific plant phosphate transporter MtPT4 gene were examined. We demonstrated that the two SBI molecules impacted the AM fungus. This was particularly evidenced for fenpropimorph. A decrease in P transport and ALP and SDH activities associated with the extraradical mycelium and MtPT4 expression level was noted. These three factors were closely related to the development of the AM fungus, suggesting a direct impact not only on the AM fungal growth but also on the physiology and metabolic activities of the AM fungus. These results further emphasized the interest on the autotrophic in vitro culture system as an alternative to pot experiments to investigate the mechanisms behind the impact of disease control molecules on the non-target AM fungal symbionts.

ChemInform Abstract: Naftifine‐Analogues as anti‐Trypanosoma cruzi Agents.

AbstractThe naftifine analogues are designed as potential T.cruzi membrane sterol biosynthesis inhibitors.

Fate of triazole fungicide residues during malting, mashing and boiling stages of beermaking

The fate and behaviour of five sterol biosynthesis-inhibiting (SBI) fungicides (cyproconazole, diniconazole, epoxiconazole, flutriafol, and tebuconazole) during different stages of beermaking (malting, mashing and boiling) on laboratory scale was assessed. A significant correlation ( r > 0.93, P < 0.05) was observed between amounts removed after steeping and the octanol/water partition coefficient (as log K OW) of the fungicides. Although in smaller proportion than steeping, germination and kilning were significant stages on the fungicide dissipation. The carryover after malting ranged from 30% to 40% of the initial residues on barley. After mashing, a substantial fraction was removed with the spent grains in all cases (36–49%) whereas a weak fall after boiling was observed for the fungicide residues (⩽2.5%). The calculated transfer factors (TFs) do not indicate a concentration effect after processing in any case (TFs < 1). Finally, after the storage of malt and spent grains (3 months) a slow fall in the residual content was observed.

ChemInform Abstract: Synthesis of the Optical Isomers of 4-[1-(4-tert-Butylphenyl)-2-oxo-pyrrolidine-4-yl]methyloxybenzoic Acid (S-2) and Their Biological Evaluation as Antilipidemic Agent.

The enantiomers of (+/-)-4-[1-(4-tert-butylphenyl)-2-oxo-pyrrolidine- 4-yl]methyloxybenzoic acid (S-2), a new antilipidemic agent having dual action on the plasma triglyceride (TG) and cholesterol (Cho) lowering effects, were prepared via separation by Chiralcel OJ column chromatography of their methyl ester and also by the same method as the described racemate's synthesis from optically active 1-(4-tert-butylphenyl)-2-oxo-pyrrolidine-4-carboxylic acid respectively. These optically active carboxylic acids were prepared by the resolution of diastereomeric N-[(S)-(-)-[4-methyl-(alpha-methyl)benzyl]]-1-(4-tert-butylphenyl)-2-oxo - pyrrolidine-4-carboxyamide using silica gel column chromatography, followed by deamination with N2O4. The absolute configurations for the enantiomers of S-2 were indirectly determined using X-ray analysis of the 4-bromo-2-fluorobenzamide of the (+)-4-[1-(4-tert-butylphenyl)-2- oxo-pyrrolidine-4-yl]-methyloxybenzoic acid. S-2 and its enantiomers showed an essentially equipotent activity on the fatty acid- and sterol-biosynthesis inhibition in vitro. On the other hand, in the in vivo activity, (S)-(+)-4-[1-(4-tert-butylphenyl)-2-oxo-pyrrolidine- 4-yl]methyloxybenzoic acid (S-2E) was superior in the lowering abilities of the plasma TG and phospholipid(PL) and was chosen as a candidate for a novel antilipidemic agent. The difference in the in vivo activity among S-2 and its enantiomers was explained from the pharmacokinetics after administration p.o.

Characterization of a CuZn superoxide dismutase gene in the arbuscular mycorrhizal fungus Glomus intraradices

To gain further insights into the mechanisms of redox homeostasis in arbuscular mycorrhizal fungi, we characterized a Glomus intraradices gene (GintSOD1) showing high similarity to previously described genes encoding CuZn superoxide dismutases (SODs). The GintSOD1 gene consists of an open reading frame of 471 bp, predicted to encode a protein of 157 amino acids with an estimated molecular mass of 16.3 kDa. Functional complementation assays in a CuZnSOD-defective yeast mutant showed that GintSOD1 protects the yeast cells from oxygen toxicity and that it, therefore, encodes a protein that scavenges reactive oxygen species (ROS). GintSOD1 transcripts differentially accumulate during the fungal life cycle, reaching the highest expression levels in the intraradical mycelium. GintSOD1 expression is induced by the well known ROS-inducing agents paraquat and copper, and also by fenpropimorph, a sterol biosynthesis inhibitor (SBI) fungicide. These results suggest that GintSOD1 is involved in the detoxification of ROS generated from metabolic processes and by external agents. In particular, our data indicate that the antifungal effects of fenpropimorph might not be only due to the interference with sterol metabolism but also to the perturbation of other biological processes and that ROS production and scavenging systems are involved in the response to SBI fungicides.

Naftifine-analogues as anti- Trypanosoma cruzi agents

Chagas disease represents a relevant health problem in Central and South America. The first line of treatment is Nifurtimox and Benznidazole which have a great deal of disadvantages that demands the rapid generation of therapeutic alternatives. Based in our research on aza-thiaheterocycles as anti- Trypanosoma cruzi agents we identified pharmacophores that act through oxidative stress. Here, we describe the synthesis and the activity of new containing bioactive-heterocycles analogues of naftifine as potential T. cruzi membrane sterol biosynthesis inhibitors. Benzimidazole 1,3-dioxides ( 11 and 13) and quinoxaline 1,4-dioxides ( 22 and 23) displayed excellent parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated with the compounds showed that any of them are able to accumulate squalene suggesting that in the anti- T. cruzi mechanism of action is not involved the inhibition of sterol biosynthesis. Some derivatives were also tested as antifungal agents. The results obtained in the present work open potential therapeutic possibilities of new compounds for these infectious diseases.

Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants

The replication of plus-strand RNA viruses depends on subcellular membranes. Recent genome-wide screens have revealed that the sterol biosynthesis genes ERG25 and ERG4 affected the replication of Tomato bushy stunt virus (TBSV) in a yeast model host. To further our understanding of the role of sterols in TBSV replication, we demonstrate that the downregulation of ERG25 or the inhibition of the activity of Erg25p with an inhibitor (6-amino-2-n-pentylthiobenzothiazole; APB) leads to a 3- to 5-fold reduction in TBSV replication in yeast. In addition, the sterol biosynthesis inhibitor lovastatin reduced TBSV replication by 4-fold, confirming the importance of sterols in viral replication. We also show reduced stability for the p92(pol) viral replication protein as well as a decrease in the in vitro activity of the tombusvirus replicase when isolated from APB-treated yeast. Moreover, APB treatment inhibits TBSV RNA accumulation in plant protoplasts and in Nicotiana benthamiana leaves. The inhibitory effect of APB on TBSV replication can be complemented by exogenous stigmasterol, the main plant sterol, suggesting that sterols are required for TBSV replication. The silencing of SMO1 and SMO2 genes, which are orthologs of ERG25, in N. benthamiana reduced TBSV RNA accumulation but had a lesser inhibitory effect on the unrelated Tobacco mosaic virus, suggesting that various viruses show different levels of dependence on sterol biosynthesis for their replication.

Arbuscular mycorrhiza partially protect chicory roots against oxidative stress induced by two fungicides, fenpropimorph and fenhexamid.

The present work examined the oxidative stress induced by different concentrations (0.02 and 0.2 mg l-1) of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) in non-target chicory root colonised or not by Glomus intraradices in a monoxenic system. The fungicides were found to cause oxidative damage by increasing lipid peroxidation measured by malondialdehyde production in non-colonised roots. Detoxification of the H(2)O(2) product was measured at 0.2 mg l-1 of fenpropimorph by an increase in peroxidase activities suggesting an antioxidant capacity in these roots. Moreover, this study pointed out the ability of arbuscular mycorrhiza to alleviate partially the oxidative stress in chicory roots, probably by lowering reactive oxygen species concentrations, resulting from increases in antioxidant defences. Our results suggest that the enhanced fungicide tolerance in the AM symbiosis could be related to less cell membrane damage.

Regulation of HMGCoA Reductase Activity by Policosanol and Octacosadienol, a New Synthetic Analogue of Octacosanol

Octacosa-10,19-dien-1-ol is a newly synthesized long-chain alcohol, an unsaturated analogue of 1-octacosanol, the major component of policosanol, the purified natural mixture of different higher aliphatic alcohols obtained from sugarcane wax. Our efficient synthetic protocol (five steps with 50% overall yield) is well suited for gram scale preparations and a rapid generation of analogues with different degrees of unsaturation. Beneficial effects of policosanol in the prevention of atherosclerosis and thromboembolic disorders have been reported and related to the inhibition of sterol biosynthesis possibly by the regulation of the activity of HMGCoA reductase mediated by AMP-dependent kinase AMPK. We have compared the effect of octacosadienol and policosanol on the regulation of HMGCoA reductase in HUVEC and HepG2 human hepatoma cells. Octacosadienol was as effective as policosanol in inhibiting the upregulation of HMGCoA reductase, in inducing the phosphorylation of AMPK and in downregulating the HMGCoA reductase mRNA.

Sterol Biosynthesis Inhibition by Paclobutrazol Induces Greater Aluminum (Al) Sensitivity in Al-Tolerant Rice

Sterol Biosynthesis Inhibition by Paclobutrazol Induces Greater Aluminum (Al) Sensitivity in Al-Tolerant Rice

Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-Δ24-reductase-deficient cell line

Cholesterol homoeostasis is critical for cell viability and proliferation. The SREBP (sterol regulatory element-binding protein) pathway is crucial for the maintenance of cholesterol homoeostasis. This pathway is controlled by cholesterol and cholesterol-derived oxysterols. J774 cells cannot convert desmosterol into cholesterol, a defect resulting from the absence of mRNA for sterol-Delta24-reductase. Using J774 cells, we addressed the capacity of desmosterol to replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway. J774 cells were able to grow indefinitely after the virtually total replacement of cholesterol by desmosterol (J774-D cells). Inhibition of sterol biosynthesis with lovastatin suppressed J774-D cell proliferation. Desmosterol prevented this effect, but its analogue, cholest-5,22-trans-dien-3beta-ol, did not. Addition of desmosterol inhibited processing of SREBP-1 and -2 and also reduced the expression of SREBP-targeted genes. As occurs in cholesterol-containing cells, 25-hydroxycholesterol was more potent than desmosterol or cholesterol in suppressing these processes. Moreover, desmosterol addition enhanced the expression of Abca1 and Srebf1c, two LXR (liver X receptor)-targeted genes. To test the ability of endogenously produced desmosterol to regulate gene expression, J774-D cells were pretreated with lovastatin to inhibit sterol biosynthesis. After removal of the inhibitor the expression of SREBP-targeted genes decreased and that of an LXR-targeted gene increased, reaching control levels. Our results demonstrate that the virtually complete replacement of cholesterol by desmosterol is compatible with cell growth and the functioning of the SREBP pathway. In these cells, desmosterol suppresses SREBP processing and targeted gene expression, and it is especially effective activating LXR-targeted genes.

First occurrence of resistance to strobilurin fungicides in Microdochium nivale and Microdochium majus from French naturally infected wheat grains

Microdochium nivale (Fr.) Samuels & Hallet and Microdochium majus (Wollenweber) belong to the Fusarium ear blight (FEB) fungal complex affecting cereals. In 2007 and 2008, major Microdochium sp. infestations were observed in France, and the efficacy of strobilurins was found to be altered in some field trials. The aim of this study was to determine the sensitivity to strobilurins of French isolates of Microdochium and to characterise the possible mechanisms of resistance. Half of the strains collected in 2007 were resistant to strobilurins, and most also displayed strong resistance to benzimidazoles. Strobilurin resistance was found mostly in M. majus isolates. Positive cross-resistance was observed between all strobilurins tested, but not with the phenylpyrrole derivative fludioxonil and the various classes of sterol biosynthesis inhibitors (SBIs). In most strains, resistance was correlated with the G143A substitution in cytochrome b, the molecular target of strobilurins. Two other mechanisms were also detected at lower frequencies. This is the first report of strobilurin resistance in Microdochium. Several resistance mechanisms have evolved independently in populations and may have different impacts on field efficacy. This makes the accurate detection and quantification of QoI resistance difficult. The management of field resistance and efficacy must be adapted to take these findings into account.

Indomethacin Amides as a Novel Molecular Scaffold for Targeting Trypanosoma cruzi Sterol 14α-Demethylase

Trypanosoma cruzi (TC) causes Chagas disease, which in its chronic stage remains incurable. We have shown recently that specific inhibition of TC sterol 14alpha-demethylase (TCCYP51) with imidazole derivatives is effective in killing both extracellular and intracellular human stages of TC. An alternative set of TCCYP51 inhibitors has been identified using optical high throughput screening followed by web-database search for similar structures. The best TCCYP51 inhibitor from this search was found to have structural similarity to a class of cyclooxygenase-2-selective inhibitors, the indomethacin-amides. A number of indomethacin-amides were found to bind to TCCYP51, inhibit its activity in vitro, and produce strong antiparasitic effects in the cultured TC cells. Analysis of TC sterol composition indicated that the mode of action of the compounds is by inhibition of sterol biosynthesis in the parasite.

Functional characterization of a C-4 sterol methyl oxidase from the endomycorrhizal fungus Glomus intraradices

Sterols are crucial components of eukaryotic membranes that control membrane fluidity and permeability. They play an important role in cell signaling, polarity and sorting. Since many steps in the pathway are essential, sterol biosynthesis inhibitors (SBI) are widely used as antifungal agents. This work reports the identification and the characterization of a C-4 sterol methyl oxidase (SMO), the first gene involved in the sterol biosynthetic pathway, so far described from an arbuscular mycorrhizal fungus. The sequence, called GintSMO, shows a primary structure, a hydrophobicity profile and a pattern of histidine-rich motifs which are typical of C-4 methyl sterol oxidases. The complementation assay in a Saccharomyces cerevisiae mutant strain demonstrates that GintSMO encodes a functional SMO. Changes in GintSMO transcript levels and in the amount of the sterol precursor squalene were observed in in vitro grown extraradical structures exposed to the fenpropimorph SBI fungicide.

Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots

Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2 mg l −1) and fenhexamid (0.02; 0.2; 2; 20 mg l −1), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Δ 5-sterols were replaced by unusual compounds such as 9β,19-cyclopropylsterols (24-methylpollinastanol), Δ 8,14-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Δ 8-sterols (Δ 8 sitosterol) and Δ 7-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected. Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization.

ChemInform Abstract: Inhibitors of Sterol Biosynthesis as Staphylococcus aureus Antibiotics

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Effect of the new pyrazole carboxamide fungicide penthiopyrad on late leaf spot and stem rot of peanut

Management of early leaf spot (Cercospora arachidicola Hori.), late leaf spot [Cercosporidium personatum (Berk. & MA Curtis) Deighton] and stem rot (Sclerotium rolfsii Sacc.) of peanut (Arachis hypogaea L.) in the southeastern USA is heavily dependent upon sterol biosynthesis inhibitor (SBI) and quinone outside inhibitor (QoI) fungicides. Effective new fungicides with different modes of action could improve overall disease control and extend the utility of the current fungicides. Penthiopryad is a pyrazole carboxamide fungicide being evaluated for use on peanut. Field experiments were conducted from 2004 to 2007 to determine the effect of a range of rates (0-0.36 kg AI ha(-1)) of penthiopyrad on leaf spot and stem rot and the relative efficacy of penthiopyrad and current fungicide standards chlorothalonil, tebuconazole and azoxystrobin. Leaf spot control in plots treated with penthiopyrad at 0.20 kg AI ha(-1) or higher was similar to or better than that for the chlorothalonil standard. The incidence of stem rot for all penthiopyrad treatments was usually less than that for the tebuconazole or azoxystrobin standard treatments. Pod yields for all penthiopyrad treatments were similar to or higher than those for the respective standards. Penthiopyrad has excellent potential for management of late leaf spot and stem rot of peanut, and may complement current SBI and QoI fungicides.

Differential interaction of branch-specific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), which catalyzes the formation of mevalonic acid (MVA), can be specifically blocked by mevinolin. Inhibition of HMGR in vivo leads to an arrest in cell cycle progression in tobacco (Nicotiana tabacum L. Bright Yellow 2) cells. As MVA in plants is the common precursor of a myriad of isoprenoid products synthesized in the cytosol and mitochondria, it is difficult to identify among such MVA-dependent molecules those whose lack may lead to cell cycle arrest. In an attempt to do so, branch-specific inhibitors of the cytosolic isoprenoid pathway downstream from MVA were used to study their capacity to block cell cycle progression. The effects of squalestatin (sterol biosynthesis inhibitor), chaetomellic acid A and patulin (protein prenyltransferase (PT) inhibitors) and tunicamycin (inhibitor of dolichol-dependent protein glycosyl transferase, thus mimicking the effect of an absence of dolichol) were compared to those induced by mevinolin. In this way, squalestatin and chaetomellic acid were identified as behaving like true cell cycle inhibitors, in that they led to a specific arrest in the cell cycle. However, they did not exactly mimic the mevinolin-induced effects. Patulin proved to be of high general toxicity, which suggests that it may affect other reactions besides blockage of protein isoprenylation. Finally, tunicamycin efficiently blocked growth of cell suspension cultures, but did not arrest the cells in a specific phase of the cell cycle. Results are discussed in the context of a better understanding of the essential implication of isoprenoids in plant cell cycle progression.