Ketoconazole-mediated growth inhibition in Botrytis cinerea and Saccharomyces cerevisiae

Quail, Michael A.; Arnold, Anna C.; Moore, Deborah J.; Goosey, Michael W.; Kelley, Steven L.

doi:10.1016/s0031-9422(00)94980-x

Cited by 30 publications

(18 citation statements)

References 14 publications

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“…Sterol 18, containing a C7(8) double bond in ring B, was not considered in Fig. 2 because the enzyme sterol ⌬ 8/7 -isomerase does not accept 4,4-dimethyl sterols such as eburicol (compound 2) or lanosterol (compound 1) as substrates (16,40,(45)(46)(47). Structure 15 was excluded on the basis of chromatographic data (see below).…”

Section: Resultsmentioning

confidence: 99%

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols

Keller

Müller

Engelhardt

et al. 2015

Antimicrob Agents Chemother

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Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3␤,6␣-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3␤,6␣-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.

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Section: Resultsmentioning

confidence: 99%

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols

Keller

Müller

Engelhardt

et al. 2015

Antimicrob Agents Chemother

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“…Inhibition of P-450 14␣-dm by azole resulted in the accumulation of 14␣-methyl sterols, predominantly 14␣-methyl-3,6-diol (8). C. neoformans growth inhibition by the azole drug fluconazole was examined to compare the response of this pathogen with that of S. cerevisiae.…”

Section: Resultsmentioning

confidence: 99%

“…Electron ionization mass spectra were obtained at 70 eV. Peak analysis and identification were realized by referring to the current literature (4,5,8) for relative retention times and fragmentation ion patterns. Representative data from triplicate analyses are provided.…”

mentioning

confidence: 99%

Cross-resistance to polyene and azole drugs in Cryptococcus neoformans

Joseph‐Horne

Hollomon

Loeffler

et al. 1995

Antimicrob Agents Chemother

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Fluconazole was observed to inhibit sterol 14␣-demethylase in the human pathogen Cryptococcus neoformans, and accumulation of a ketosteroid product was associated with growth arrest. A novel mechanism(s) of azole and amphotericin B cross-resistance was identified, unrelated to changes in sterol biosynthesis, as previously identified in Saccharomyces cerevisiae. Reduced cellular content of drug could account for the resistance phenotype, indicating the possible involvement of a mechanism similar to multidrug resistance observed in higher eukaryotes. Infection withCryptococcus neoformans leading to cryptococcal meningitis has been associated with up to 8% of all AIDS cases (7). Treatment failures and recurrence of infection in AIDS patients on current chemotherapeutic programs involving the use of azole antifungal agents or the polyene antibiotic amphotericin B are increasing (1,3,12,21).Ergosterol is the principal sterol in most fungi (for a review, see reference 9). Amphotericin B has been shown to bind ergosterol in the membrane, with resistance occurring through mutation in the biosynthetic pathway (3). Mode-of-action studies have shown that azole antifungal agents bind to cytochrome P-450 (P-450 14␣-dm ), preventing sterol 14␣-demethylation (20). Subsequent 4-demethylation may still occur, with formation of abnormal 14␣-methyl sterols such as obtusifoliol, 14␣-methylfecosterol, and the presumed product of attempted ⌬ 5(6) desaturation of 14␣-methylfecosterol, 14␣-methyl-3,6-diol (17). The accumulation of this last sterol has been shown genetically and biochemically to be associated with the arrest of growth in azole-treated Saccharomyces cerevisiae (17).Lesions in P-450 14␣-dm confer resistance to polyene antibiotics by preventing the formation of ergosterol, which is the target molecule for polyene action. For viability, these strains require a second defect in sterol ⌬ 5(6) -desaturase which prevents the formation of 14␣-methyl-3,6-diol (14). Azole-resistant mutants generated directly in S. cerevisiae are also defective in sterol ⌬ 5(6) -desaturase (18); these mutants do not accumulate ergosterol and are also cross-resistant to polyene antifungal agents.In the study described in this report we examined the potential for cross-resistance between azole and polyene antifungal agents in the fungal pathogen C. neoformans. A different pattern of sterol accumulation was observed following azole treatment of C. neoformans compared with that observed in S. cerevisiae, suggesting that the mechanisms for avoiding the formation of 14␣-methyl-3,6-diol were not applicable (17). However, mutants which were cross-resistant to the azoles and amphotericin B were isolated, indicating an important consideration for future antifungal therapy. MATERIALS AND METHODSCulture conditions. Two strains of the human pathogen C. neoformans (B4476 and B4500) were obtained from K. J. Kwon-Chung, National Institutes of Health, Bethesda, Md. Growth was supported on 2% (wt/vol) glucose, 2% (wt/vol) Difco peptone, and 1% (wt/vol) Difco yeast e...

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“…The silyl sterols were analyzed by gas chromatography/mass spectrometry (VG 12-250; VG BIOTECH) by using split injections with a split ratio of 20:1. Sterol identification was by reference to relative retention times and mass spectra reported previously (19,20).…”

Section: Methodsmentioning

confidence: 99%

The N-Terminal Membrane Domain of Yeast NADPH-Cytochrome P450 (CYP) Oxidoreductase Is Not Required for Catalytic Activity in Sterol Biosynthesis or in Reconstitution of CYP Activity

Kanamarlapudi¹,

Lamb²,

Kelly³

et al. 1998

Journal of Biological Chemistry

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The disruption of Saccharomyces cerevisiae NADPHcytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent. The associated phenotypes could be reversed in transformants after expression of native CPR and a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol. This indicated availability of the CPR in each case to function with the monooxygenases squalene epoxidase, CYP51, and CYP61 in the ergosterol biosynthesis pathway. Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system, as well as to allow reconstitution of activity with purified CYP61, sterol 22-desaturase. This was also observed for purified Candida albicans and human CYP51 in reconstituted systems. The ability of the yeast enzyme to function in a soluble form differed from human CPR, which is shown to be inactive in reconstituting CYP activity.

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Ketoconazole-mediated growth inhibition in Botrytis cinerea and Saccharomyces cerevisiae

Cited by 30 publications

References 14 publications

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols

Cross-resistance to polyene and azole drugs in Cryptococcus neoformans

The N-Terminal Membrane Domain of Yeast NADPH-Cytochrome P450 (CYP) Oxidoreductase Is Not Required for Catalytic Activity in Sterol Biosynthesis or in Reconstitution of CYP Activity

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