Accumulation of ergosta‐8,14‐dien‐3β‐ol by Saccharomyces cerevisiae cultured with an azasterol antimycotic agent

Eight antifungal agents were examined for effects on lipid biosynthesis and membrane integrity in Candida albicans. Lipids were labeled in vivo or in vitro with [14C]acetate and analyzed by thin-layer and gas chromatography. Membrane integrity was measured by a recently developed ['4C]aminoisobutyric acid radiolabel release assay. The imidazole antifungal agents miconazole, econazole, clotrimazole, and ketoconazole, at concentrations inhibiting ergosterol biosynthesis (0.1 ,uM), decreased the ratio of unsaturated to saturated fatty acids in vivo but not in vitro. Similarly, naftifine, tolnaftate, and the azasterol A25822B, at concentrations inhibiting ergosterol biosynthesis (10, 100, and 1 ,uM, respectively), decreased the ratio of unsaturated to saturated fatty acids in vivo only. This suggests that the effect on fatty acids observed with ergosterol biosynthesis inhibitors may be secondary to the effect on ergosterol. With imidazoles, oleic acid antagonized inhibition of cell growth but not inhibition of ergosterol. This suggests that, with the C-14 demethylase inhibitors, decreased unsaturated fatty acids, rather than decreased ergosterol, are responsible for growth inhibition. Cerulenin, previously reported to be a potent inhibitor of both fatty acid and ergosterol biosynthesis, was found in the present study to inhibit the former (at 5 ,uM) but not the latter (up to 100 ,uM). Of the antifungal agents tested, econazole and miconazole (at 100 ,uM) produced complete release of[14C]aminoisobutyric acid, which is consistent with membrane damage.

Section: Resultssupporting

confidence: 86%

Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candida albicans

Georgopapadakou¹,

Dix²,

Smith³

et al. 1987

“…At a lower concentration (0.18 ,uM), an accumulation of ergosta-8,14-dien-3,8-ol (Fig. 1B) was observed in the treated fungus, indicating that the sterol C-14(15) reductase enzyme was inhibited (11,12 Sterol analysis. Free sterols isolated from TLC were analyzed by gas-liquid chromatography on a Glowall Chromalab 301 (with argon ionization detector) using a glass column (183 cm by 3.4 mm) packed with 100/120-mesh Gas-Chrom Q coated with 3% SE-30 (Applied Science Laboratories) and a column temperature of 250 to 2590C.…”

mentioning

confidence: 96%

“…The effect of A25822B on sterol metabolism in Saccharomyces cerevisiae was studied by investigators at Oregon State University (2,11,12). Using high concentrations (20 and 122 ,uM), they initially observed competitive inhibition of S-adenosylmethionine:Al24-sterol methyl transferase in vitro and a reduction of ergosterol in treated cells (2).…”

mentioning

confidence: 99%

Mode of Action of the Azasteroid Antibiotic 15-Aza-24-Methylene- d -Homocholesta-8,14-Dien-3β-ol in Ustilago maydis

Woloshuk

Sisler

Dutky

1979

Ustilago maydis sporidia treated with 0.1 μg of azasterol (15-aza-24-methylene- d -homocholesta-8,14-dien-3β-ol) per ml appeared branched and vacuolated after 6 h of incubation. Sporidial multiplication, dry weight increase, and synthesis of protein, deoxyribonucleic acid, and ribonucleic acid were only slightly or moderately inhibited during the initial 3 h of incubation. An increase of free fatty acids was observed in lipid extracts of treated sporidia after incubation for 3 h or more. Ergosterol synthesis was completely inhibited within 1 h and there was a gradual decline of ergosterol content during 6 h which was accompanied by an accumulation of the sterol intermediate ergosta-8,14-dien-3β-ol. The results indicate that toxicity of the azasterol results from specific inhibition of the reduction of the sterol C-14(15) double bond. A triarimol-tolerant strain of Cladosporium cucumerinum was tolerant to the azasterol, but an imazalil-tolerant strain of Aspergillus nidulans was not.

“…We have presented studies on another compound, 15-aza-24-methylene-8,14-cholestadien- 33-ol (15-azasterol), with strong antifungal activity (13,14,23). This compound inhibits a step in C-14 demethylation (4) which leads to the accumulation of A8,14-ergostadien-3,3-ol (ignosterol).…”

mentioning

confidence: 99%

Relationship between antifungal activity and inhibition of sterol biosynthesis in miconazole, clotrimazole, and 15-azasterol

Taylor¹,

Rodriguez²,

Parks³

1983

The availability of Saccharomyces cerevisiae mutants which are defective in sterol biosynthesis makes it possible to determine whether the ability of several antifungal agents to inhibit cell growth is due to their effect on sterol production. 15-Aza-24-methylene-8,14-cholestadien-3 beta-ol (15-azasterol) is known to block the reduction of the sterol delta 14 bond following C-14 demethylation. This agent inhibits the growth of wild-type S. cerevisiae but does not inhibit the growth of a strain that is defective in the removal of the C-14 methyl group of lanosterol and in the introduction of the 5,6 double bond. 15-Azasterol does not inhibit the growth of a sterol auxotrophic strain growing on an exogenous supply of sterol. Therefore, the effect of 15-azasterol on sterol biosynthesis is clearly the cause of its ability to inhibit growth. On the other hand, growth inhibition by two imidazole antifungal agents, clotrimazole and miconazole, cannot be ascribed to their ability to prevent the removal of the C-14 methyl group of lanosterol, because they inhibit the growth of the sterol auxotrophic strain as well as that of the demethylase mutant.