Yeast mutants blocked in removing the methyl group of lanosterol at C-14. Separation of sterols by high-pressure liquid chromatography

The effects of 6-amino-2-n-pentylthiobenzothiazole (APB), a new antifungal agent, on ergosterol biosynthesis in Candida albicans and Saccharomyces cerevisiae were studied, using [ 14 C]acetate incorporation. In C. albicans, the inhibition of growth was accompanied by a marked inhibition of acetate incorporation in 4-desmethylsterols, with a significant portion of the radiolabel being incorporated in 4,4-dimethylsterols, lanosterol, and 4,4-dimethylzymosterol and minor amounts being incorporated in 4-methylsterols and squalene. The data are interpreted as evidence of a block of the ergosterol biosynthesis pathway at the level of 4-demethylation of 4,4-dimethylzymosterol, with partial inhibition of lanosterol 14-demethylation and squalene epoxidation also being possible. In 6-amino-2-n-pentylthiobenzothiazole-treated S. cerevisiae, a significant amount of the radiolabel was incorporated also in 4-methylsterols, 4-methylzymosterol, and 4-methylfecosterol, indicating that in this microorganism there are different sensitivities of the two 4-demethylations and that the pathway is blocked at the level of 4-demethylation of 4-methylsterols.Growth of the population with altered immunity, which has taken place during the last 15 years, has led to an increase in the incidence of invasive and systemic mycoses. In spite of recent advances in the development of antimycotic agents, antifungal chemotherapy remains in many cases problematic and the search for new antifungal agents continues (16).A promising new antifungal agent is 6-amino-2-n-pentylthiobenzothiazole (APB) (Fig. 1). It has been shown to be active against various Candida species in vitro, to inhibit the yeastmycelium conversion in Candida albicans, and to be active in vivo, curing systemic candidosis in mice (4,5,12).The mechanism of action of APB in Saccharomyces cerevisiae was studied (11): there was a reduction in the cellular content of ergosterol, which was accompanied by an accumulation of squalene and 4-methylated sterols, following APB treatment. These effects differed from those caused by other antifungal drugs, such as azoles, which are established ergosterol biosynthesis inhibitors. It was previously suggested (11) that APB was an inhibitor of a new target site in the ergosterol biosynthesis pathway, namely, sterol 4-demethylation.In this paper, we report the effects of APB on growth and on ergosterol biosynthesis in a clinically important yeast-like microorganism, C. albicans, and present evidence that the antifungal agent inhibits ergosterol biosynthesis at the level of 4-demethylation. So as to better understand the mechanism of action of the compound, the effects on C. albicans are compared with those observed with S. cerevisiae. MATERIALS AND METHODSAntifungal agent. APB (Fig. 1) was provided by E. Sidóová, Chemical Institute, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia. Solutions of APB were freshly prepared in dimethyl sulfoxide when required.The amount of the solvent in the culture media did not exceed 0.1% (vol/vol) a...

Section: -⌬supporting

confidence: 62%

Inhibition of sterol 4-demethylation in Candida albicans by 6-amino-2-n-pentylthiobenzothiazole, a novel mechanism of action for an antifungal agent

Kuchta

Léka

Farkaš

et al. 1995

“…Our data indicate that once the C-14 methyl is removed, C-4 demethylation proceeds rapidly even though the resulting C-14(15) double bond is not reduced. This is in contrast to the slow removal of the C-4 methyl groups when C-14 demethylation is inhibited (16,20,23). It is evident, therefore, either that C-14 methyl sterols are not good substrates for C-4 demethylation or that C-14 demethylation inhibitors also inhibit C-4 demethylation.…”

Section: Discussionmentioning

confidence: 95%

“…The failure to remove the C-14 methyl or to reduce the C-14(15) double bond does not affect the C-24 alkylation (11,17,23). This is surprising in the case of S. cerevisiae, because this reaction normally occurs late in the ergosterol biosynthetic pathway (9).…”

Section: Discussionmentioning

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.

“…Because the concentration dependence of growth inhibition by one of these compounds parallels the degree of inhibition of demethylation, it has been suggested that the presence of the unusual sterol biosynthetic intermediate is the cause of the antifungal properties (33). This conclusion may be questioned, since it has been found that some C-14 methyl sterols will support the growth of yeast cells (6,21,32).…”

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.