Ergosterol Biosynthesis Inhibitors Research Articles

Effects of sterol biosynthesis inhibitor fungicides in the phytopathogenic fungus,Nectria haematococca:ergosterol depletion versus precursor or abnormal sterol accumulation as the mechanism of fungitoxicity

The relative importance of the depletion of ergosterol versus the accumulation of precursor or abnormal sterols in the mechanism of fungal growth inhibition by sterol biosynthesis inhibitor fungicides is incompletely understood. In order to investigate this problem further, the degree of inhibition of the growth of Nectria haematococca by fungicides with different enzymatic targets in the sterol biosynthetic pathway was determined and compared with their effects on the sterol profile. The sensitivity of N. haematococca was highest towards fenpropimorph, followed by tebuconazole, terbinafine, fenpropidin and tridemorph. Terbinafine, a squalene epoxidase inhibitor, induced a very large accumulation of squalene without very significant inhibition of ergosterol biosynthesis and growth. The fungus appeared able to tolerate large amounts of squalene. In the case of tebuconazole, a sterol 14α-demethylase inhibitor, it seemed that the accumulation of C4 mono- and dimethyl sterols was responsible for fungitoxicity. Fenpropimorph and fenpropidin seemed to be good inhibitors of both sterol Δ14-reductase and Δ8Δ7-isomerase, whereas tridemorph was a better inhibitor of Δ8Δ7-isomerase than of the Δ14-reductase. Large accumulations of Δ8,14- or Δ8-sterols and correspondingly large decreases in the ergosterol content are both implicated in the fungitoxicity of these compounds in N. haematococca. © 1998 Society of Chemical Industry

Synthesis and Antifungal Activity of Metal Complexes ContainingDichloro‐Tetramorpholino‐Cyclophosphazatriene

Metal complexes of dichloro-tetramorpholino-cyclophosphazatriene containing divalent cations such as Ni(II), Co(II), and Mn(II) have been prepared and characterised by standard physico-chemical procedures (elemental chemical analysis, IR and UV-VIS spectra, conductimetric measurement). The newly synthesised compounds possessed antifungal activity against Aspergillus and Candida spp., some of them showing effects comparable to ketoconazole (with minimum inhibitory concentrations in the range of 2- 30 μg/mL) but being generally less active as compared to the azole. Best activity was detected against C. albicans, and worst activity against A. niger. The mechanism of action of these compounds probably involves inhibition of ergosterol biosynthesis, and interaction with lanosterol-14-α-demethylase (CYP51A1), since reduced amounts of ergosterol were evidenced by means of HPLC in cultures of the sensitive strain A. niger treated with some of these inhibitors.

Stereoselective interaction of the azole antifungal agent SCH39304 with the cytochrome P-450 monooxygenase system isolated from Cryptococcus neoformans

We investigated the stereoselective inhibition of growth and ergosterol biosynthesis by SCH39304 in the pathogenic fungus Cryptococcus neoformans obtained from four AIDS patients who failed fluconazole therapy and compared the results to those obtained with a wild-type strain. For all strains, the MICs of the RR isomer were approximately half those of the racemate, with the SS enantiomer showing no inhibitory activity. The 50% inhibitory concentrations for in vitro ergosterol biosynthesis correlated with the MIC data, indicating stereoselective inhibition of their target P-450 enzyme, sterol 14alpha-demethylase, as the cause of this difference. The RR enantiomer produced classical type II spectra on addition to microsomal extracts of the strains, whereas the SS enantiomer showed an absence of binding. Stereo- and regio-specific localization of N-1 substituent groups of SCH39304 within the active site of the enzyme determined the unique discrimination between its two enantiomers, and the inability to bind to sterol 14alpha-demethylase is also true of other P-450 enzymes contained in the microsomal fraction. As previously observed for other antifungal azoles, isolates obtained following failure of fluconazole therapy showed resistance to SCH39304 and its RR enantiomer. This resistance could be associated with an alteration in the sensitivity of ergosterol biosynthesis in vitro. These alterations did not cause any changes allowing the SS enantiomer to bind to the P-450 mediating sterol 14alpha-demethylation.

In-vitro activity of D0870, a new triazole antifungal drug, in comparison with fluconazole and itraconazole against Aspergillus fumigatus and Candida krusei.

The activity of the new triazole antifungal D0870 was compared with those of itraconazole and fluconazole against Candida krusei and Aspergillus fumigatus, two fungi showing inherent tolerance of fluconazole. The activity of D0870 resembled that of itraconazole against whole cells of C. krusei, but it was less effective against A. fumigatus. However, the effect on sterol biosynthesis, in terms of the sterol type accumulating and IC50 for in-vitro biosynthesis, appeared similar in both species. The superior antifungal effect of D0870 over fluconazole appeared related to better inhibition of ergosterol biosynthesis in A. fumigatus, but in C. krusei this did not account for the entire difference which may result mainly from reduced efflux of drug.

Inhibition of ergosterol biosynthesis is not accompanied by a change in fatty acid composition in Saccharomyces cerevisiae treated with the antifungal agent 6-amino-2- n-pentylthiobenzothiazole

The antifungal agent 6-amino-2- n-pentylthiobenzothiazole at a concentration of 40 μM lowered the specific growth rate of exponentially growing cultures of Saccharomyces cerevisiae by 36%. Treatment with 6-amino-2- n-pentylthiobenzothiazole inhibited the biosynthesis of ergosterol and caused an accumulation of the methylated sterol precursors ergosta-5,7-dienol and squalene, but had no significant effect on the composition and the rate of biosynthesis of fatty acids. The results indicate that neither the inhibition of ergosterol biosynthesis, nor the slowing-down of culture growth by this antifungal agent, led to a compensatory alteration in the pattern of fatty-acyl chains in membrane lipids. This finding contradicts the accepted wisdom for the action of a number of antifungal agents.

Inhibitors of Ergosterol Biosynthesis as Antifungal Agents

Abstract: The rise in the incidence of fungal infections over the past two to three decades, particularly those caused by opportunistic pathogens in immune-compromised patients, has strengthened the need for new antifungal drugs. Up to only 30 years ago the choice of systemically available antimycotics was between two drugs, amphotericin B and 5-flurocytosine, neither of which was satisfactory. Then a series of inhibitors of the biosynthesis of ergosterol, the major sterol of the fungal cell membrane, were found to have excellent antifungal activity, improved safety, and some were also active after oral or parenteral application. Starting with miconazole and ketoconazole, and improving through fluconazole and itraconazole, the imidazole and triazole inhibitors of lanosterol Cl4u-demethylation have been the most successful. Other steps in the linear sterol biosynthesis pathway are inhibited by the allylamines naftifine and terbinafine, and the morpholine derivative amorolphine, which have also improved the therapy of superficial mycoses. The discovery, mode of action, current clinical application and future possibilities for these drugs is reviewed. The status and potential of SBI antifungals in development, exclusively azoles, is discussed. Attempts to discover non-azole inhbitors of lanosterol C14u-demethylase are mentioned, as well as current work on other targets in the ergosterol biosynthetic pathway. Particular attention is paid to the oxidsqualene cyclase, which has been extensively studied. The current knowledge about resistance to antifungal agents is reviewed.

Evidence for and characterization of cytochrome P‐450 in Neurospora crassa

Cytochrome P-450 was detected in microsomes and presumably in cytosol of Neurospora crassa, and was found to be inducible by progesterone. In the microsomal fraction cytochrome b5 and NADPH-cytochrome c reductase activities were measurable, too. Cytochrome P-450 of Neurospora crassa is inhibited by SKF-525 A and by inhibitors of ergosterol biosynthesis. After induction of cytochrome P-450 with progesterone 11 alpha-hydroxyprogesterone as one metabolite of progesterone was detected in the culture media as well as in the mycelia. After 42 hours about 70% of progesterone were metabolized.

5527687 Enzyme induction screen for ergosterol biosynthesis inhibitors

5527687 Enzyme induction screen for ergosterol biosynthesis inhibitors

Effects of two ergosterol-inhibiting fungicides on soil ergosterol and microbial biomass

The effects of two ergosterol biosynthesis-inhibiting fungicides, epoxiconazole and triadimefon (at 2 and 20 ×, and 1 and 10 × field rate, respectively), and of straw amendment on ergosterol and microbial biomass C in a sandy loam soil, were investigated. Initial soil ergosterol and microbial biomass C contents were about 1.6 μg g −1 and 330 μg g −1 soil, respectively. Both concentrations of the fungicides decreased soil ergosterol content by about 30% after 7 days incubation in unamended soil, following which contents were broadly similar to the control soil. Microbial biomass C remained largely unaffected in the unamended soil, except for a temporary inhibition of about 12% caused by triadimefon after 13 days incubation. After amendment of the soil with wheat straw, the inhibition of ergosterol biosynthesis reappeared, ranging from 4 to 34%, with epoxiconazole having a greater effect than triadimefon, both compared to ergosterol biosynthesis in soils given straw without the biocides. This inhibition was also transient, but was longer-lasting than in the unamended soil. A decrease in soil microbial biomass C, ranging from 5 to 12%, also occurred, in contrast to the unamended treatments. This indicates that the fungicides were more active against the newly synthesized microbial biomass than ainst the original population. Soil ergosterol content was a more sensitive measure of pesticide side-effects than microbial biomass C, and could be a useful method in this respect.

Sensitivity of South African Ramulispora herpotrichoides isolates to carbendazim and ergosterol biosynthesis inhibitors

In a survey conducted during 1991–1992, single‐spored isolates of the eyespot fungus from the Swartland area were characterized and tested for sensitivity to carbendazim and ergosterol inhibiting fungicides. The 100 isolates tested were all fast growing, even marginate, and designated as Ramulispora herpotrichoides. Fungal growth was completely inhibit on PDA amended with carbendazim (1 μg/ml), indicating that the local population of the fungus is still at baseline sensitivity to benzimidazoles. The mean concentration of prochloraz calculated to inhibited growth by 50% (IC50 value) was 0.043 ± 0.029 μg/ml, which is comparable with the baseline sensitivity reported for European isolates. Of the 36 representative isolates screened against 2 μg/ml triadimenol, 44% were sensitive, while 36% were resistant. The triadimenol‐resistant isolates were sensitive to propiconazole and flusilazole. However, four of the triadimenol‐resistant isolates were also resistant to tebuconazole. These results indicate that South African isolates of R. herpotrichoides are sensitive towards carbendazim, prochloraz, propiconazole and flusilazole. They were found to differ, however, in sensitivity towards triadimenol and tebuconazole, where some isolates had an IC50 value greater than 2 μg/ml.

Disease control and yield improvement obtained with fungicide applications to spring wheat in South Africa

Foliar sprays containing benzimidazole and/or compounds inhibiting ergosterol biosynthesis in fungi were tested for their effects on spring wheat at different localities in the Western Cape Province, South Africa. Fungicide treatments caused significant yield increases ( P > 0.05) at most of the localities where plants were sprayed at Zadok's growth stages 31 and 39. Plants were affected by physiological leaf spot, which occurred in all localities. At one locality good control of leaf rust ( Pucdnia recondita f.sp. tritici) was obtained with triazole-containing sprays, but not with imidazole- and benzimidazole-containing sprays. Lodging due to eyespot ( Tapesia yallundae) occurred at another locality where it was partially controlled by benzimidazole-containing fungicide mixtures. Fungicides had little or no effect on yield when planting date was delayed or when the rate of soil-applied nitrogen on fallow land was increased. The study indicates that in the Western Cape Province growth regulating effects, rather than fungicidal effects of fungicides, are often responsible for yield increases of wheat.

Effects of prochloraz and malathion on the red-legged partridge: A semi-natural field study

A semi-natural field study was carried out to assess the likelihood of a potentiation of toxicity between the ergosterol biosynthesis inhibiting (EBI) fungicide, prochloraz, and the organophosphorus (OP) insecticide, malathion, in the red-legged partridge ( Alectoris rufa). Groups of partridges kept in four large grassland enclosures were exposed to either prochloraz-treated or control wheat for 7 days after which two of the enclosures were sprayed with malathion whilst the remaining two were sham-sprayed. Cytochrome P-450, aldrin epoxidase and 7-ethoxyresorufin- O-deethylase (EROD) activities were found to be significantly higher in the group exposed to prochloraz alone compared to controls, suggesting that induction of the hepatic microsomal monooxygenase system had occurred by ingestion of prochloraz-treated wheat. However, the level of induction produced was not sufficient to cause a potentiation of malathion toxicity. There was evidence for induction of several forms of P-450 recognised by antibodies raised against 1A1, 2C6 and 4A1 in the prochloraz-exposed partridges.

Ergosterol biosynthesis inhibition: a target for antifungal agents.

The isoprenoid sterols play a crucial role in the viability of all fungi; those unable to synthesise ergosterol because of inhibition, growth conditions or mutation must take it up from the environment. A range of compound types have been discovered which interfere with the biosynthetic pathway from acetate to ergosterol and these compounds have antifungal actions. Inhibition of several of the steps has yielded agents which have been used with great success as medical and agrochemical agents. The most important biosynthetic steps that have been exploited are inhibition of squalene epoxidase, (the allylamines and tolnaftate) C14 demethylation (the azoles), delta 7,8 isomerase and delta 14 reductase which are inhibited by the morpholines. Recent research has shown that inhibition of C24 methyltransferase and C4 demethylation also yield antifungal agents. Combination studies demonstrate that synergy between agents of different types can be measured. Fungicidal effects were observed when a combination of two fungistatic agents was used.

The study of interactive effects of pollutants: a biomarker approach

Biochemical biomarkers, such as inhibition of serum butyryl cholinesterase (BuChE) and brain acetyl cholinesterase (AChE), have been useful in studies of interactive effects of pesticides in birds. Examples of interactions due to increased activation or decreased detoxication are reviewed. Studies have shown that hybrid red-legged partridges ( Alectoris rufa cross) pretreated with the inducing ergosterol biosynthesis inhibiting (EBI) fungicide, prochloraz, were more sensitive to the toxic effects of the organophosphorous (OP) insecticide, malathion, than controls. A dose of 90 mg/kg prochloraz produced greater inhibition at 1, 4 and 24 h following oral administration of 50 mg/kg malathion, compared to corn oil controls. Pigeons ( Columba livia) given 180 or 90 mg/kg prochloraz showed greater inhibition of BuChE activity following malathion administration than did control birds. Starlings ( Sturnus vulgaris), however, appeared not to be induced by 180 or 300 mg/kg prochloraz, and no difference in BuChE activity following dosing with malathion was apparent in comparison with controls. Other EBIs and OP combinations have been investigated in the partridge. Birds pretreated with prochloraz showed a trend towards greater inhibition of serum BuChE activity at most time points following dosing with the OPs dimethoate and chlorpyriphos. Birds pretreated with the EBI penconazole showed significantly greater inhibition of serum BuChE activity at 1, 4 and 24 h after malathion administration than did controls. The mechanism of increased activation of malathion due to induction of cytochrome P-450 by prochloraz is reviewed. In the case of interactions due to inhibition of detoxication, inhibition of brain AChE activity was a useful biochemical biomarker. An increased sensitivity of partridges to the carbamate pesticide, carbaryl, following pretreatment with malathion was attributed mainly to suicide inhibition (by malathion) of cytochrome P-450, resulting in an inhibition of carbaryl metabolism. The enhancement (or potentiation) of toxicity found in these studies was the consequence of modulations of cytochrome P-450-induction (by EBIs) or inhibition (by malathion). More generally, interactions of this kind may also occur between other environmental chemicals, for example, as the consequence of inductions caused by persistent compounds such as PCBs. The biomarker assays for monooxygenases based upon cytochrome P-450 employed in the present study (measurement of enzyme activies and Western blotting) can be used to measure these changes in vertebrates in the field.

Characterization of a new gene family developing pleiotropic phenotypes upon mutation in Saccharomyces cerevisiae

The aim of this paper is to gather and complete data about four members of a new gene family. Mutation in SUR4 gene was originally selected as a suppressor of defects caused by mutations in RVS161 or RVS167 genes. Cloning and sequencing of the SUR4 gene were performed. The deduced protein contains six putative transmembrane domains. Sequence comparison revealed that two yeast genes, FEN1 and JO343, shared significant similarities with SUR4. Mutants for SUR4 and FEN1 have the same pleiotropic phenotype, including bud localization defects, resistance to an immunosuppressor and resistance to ergosterol biosynthesis inhibitors. The double inactivation of SUR4 and FEN1 genes is lethal. These data and other aspects implicating SUR4 in glucose metabolism, suggest an involvement of these genes in the dynamics of cortical actin cytoskeleton in response to nutrient availability. Moreover, the existence of a fourth homologous gene in C. elegans extends the family to pluricellular organisms.

ChemInform Abstract: Synthesis of New Substituted Quinolizidines as Potential Inhibitors of Ergosterol Biosynthesis.

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Guanidinium and amidinium fungicides: A new class of carbocation mimetic ergosterol biosynthesis inhibitors

AbstractA novel class of chemical has been designed with the aim of inhibiting the Δ14‐reductase and Δ8‐Δ7‐isomerase enzymes in the ergosterol biosynthesis pathway in fungi. Use was made of knowledge about the mechanisms of both enzymes and the mode of action of known, fungicidal inhibitors of these enzymes. Pioneer examples have been synthesised and have been demonstrated to be potent inhibitors of ergosterol biosynthesis in Ustilago maydis (DC) Corda, acting in the same manner as the commercial fungicide fenpropimorph. They also showed excellent fungicidal activity against Erysiphe graminis DC f. sp. hordei Marchal (powdery mildew of barley) and Puccinia recondita Rob. ex Desm. (wheat leaf rust) in in‐vivo glasshouse tests. Using these compounds as a starting point, systematic structural variation has been carried out. Testing of a wide range of analogues at high volume confirms the potential of this class of compound to control mildew and rust pathogens at levels comparable to those of the standards. Correlation of in‐vivo and enzymatic data is good and the structure‐activity relationship developed for this series of compounds closely parallels that found for the morpholine/piperidine class of fungicides, suggesting a common mode of action.

Alterations induced by the antifungal compounds ketoconazole and terbinafine in Leishmania.

The antiproliferative effects and ultrastructural alterations induced in vitro by two antifungal compounds, the azole ketoconazole and the allylamine terbinafine on Leishmania amazonensis are reported. Promastigotes treatment with ketoconazole and terbinafine induced growth arrest and cell lysis in 72 hours. Combination of the two agents produced additive effects on promastigote axenic growth and synergistic effects on intracellular amastigote proliferation. The amastigotes, either axenically grown or infecting murine macrophages, were about 100-fold more sensitive to the drugs. These compounds induced the appearance of large multivesicular bodies, especially after ketoconazole treatment, increased amount of lipid inclusions as well as numerous, polymorphic volutin granules, particularly in terbinafine-treated cells. Multivesicular bodies were observed in close apposition with organelles such as mitochondria, which also showed alterations in the distribution and appearance of cristae, and the formation of paracrystalline arrays within the matrix. Some cells presented large portions of cytoplasm wrapped by endoplasmic reticulum and many parasites also presented myelin-like endoplasmic reticulum profiles. Such alterations together with the strong acid phosphatase activity observed in the multivesicular bodies and volutin granules may indicate the existence of an unusual autophagic process in cells treated with ergosterol biosynthesis inhibitors.

Developmental toxicity of cyproconazole, an inhibitor of fungal ergosterol biosynthesis, in the rat

Developmental toxicity of cyproconazole, an inhibitor of fungal ergosterol biosynthesis, in the rat

Synthesis of new substituted quinolizidines as potential inhibitors of ergosterol biosynthesis

Carbocationic species (High Energy Intermediates) have been postulated as intermediates in the course of enzymatic synthesis of ergosterol. Protonated aza-analogues of the sterol are thus potential inhibitors. The synthesis of substituted quinolizidines ( 3) is reported. 4-Methoxy-2,3-dimethylpyridine ( 4) was metalated and reacted with substituted 3-chloropropanal to build the 1-methyl-2-quinolizidinone. The lateral chain was prepared via a Wittig-Horner reaction.