Effects of itraconazole on cytochrome P-450-dependent sterol 14 alpha-demethylation and reduction of 3-ketosteroids in Cryptococcus neoformans

Abstract

As in other pathogenic fungi, the major sterol synthesized by Cryptococcus neoformans var. neoformans is ergosterol. This yeast also shares with most pathogenic fungi a susceptibility of its cytochrome P-450-dependent ergosterol synthesis to nanomolar concentrations of itraconazole. Fifty percent inhibition of ergosterol synthesis was reached after 16 h of growth in the presence of 6.0 +/- 4.7 nM itraconazole, and complete inhibition was reached at approximately 100 nM itraconazole. This inhibition coincided with the accumulation of mainly eburicol and the 3-ketosteroid obtusifolione. The radioactivity incorporated from [14C]acetate in both compounds represents 64.2% +/- 12.9% of the radioactivity incorporated into the sterols plus squalene extracted from cells incubated in the presence of 10 nM itraconazole. The accumulation of obtusifolione as well as eburicol indicates that itraconazole inhibits not only the 14 alpha-demethylase but also (directly or indirectly) the NADPH-dependent 3-ketosteroid reductase, i.e., the enzyme catalyzing the last step in the demethylation at C-4. This latter inhibition obviates the synthesis of 4,4-demethylated 14 alpha-methylsterols that may function at least partly as surrogates of ergosterol. Eburicol and obtusifolione are unable to support cell growth, and the 3-ketosteroid has been shown to disturb membranes. The complete inhibition of ergosterol synthesis and the accumulation of the 4,4,14-trimethylsterol and of the 3-ketosteroid together with the absence of sterols, such as 14 alpha-methylfecosterol and lanosterol, which can partly fulfill some functions of ergosterol, are at the origin of the high activity of itraconazole against C. neoformans. Fifty percent inhibition of growth achieved after 16 h of incubation in the presence of 3.2 +/- 2.6 nM itraconazole.