EFFECTS OF ITRACONAZOLE ON CYTOCHROME P-450-DEPENDENT STEROL 14-ALPHA-DEMETHYLATION AND REDUCTION OF 3-KETOSTEROIDS IN CRYPTOCOCCUS-NEOFORMANS

As in other pathogenic fungi, the major sterol synthesized by Cryptoco ccus neoformans var. neoformans is ergosterol. This yeast also shares with most pathogenic fungi a susceptibility of its cytochrome P-450-de pendent ergosterol synthesis to nanomolar concentrations of itraconazo le. Fifty percent inhibition of ergosterol synthesis was reached after 16 h of growth in the presence of 6.0 +/- 4.7 nM itraconazole, and co mplete inhibition was reached at approximately 100 nM itraconazole. Th is inhibition coincided with the accumulation of mainly eburicol and t he 3-ketosteroid obtusifolione. The radioactivity incorporated from [C -14]acetate in both compounds represents 64.2% +/- 12.9% of the radioa ctivity incorporated into the sterols plus squalene extracted from cel ls incubated in the presence of 10 nM itraconazole. The accumulation o f obtusifolione as well as eburicol indicates that itraconazole inhibi ts not only the 14alpha-demethylase but also (directly or indirectly) the NADPH-dependent 3-ketosteroid reductase, i.e., the enzyme catalyzi ng the last step in the demethylation at C-4. This latter inhibition o bviates the synthesis of 4,4-demethylated 14alpha-methylsterols that m ay 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 ergos terol 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 f unctions of ergosterol, are at the origin of the high activity of itra conazole against C. neoformans. Fifty percent inhibition of growth was achieved after 16 h of incubation in the presence of 3.2 +/- 2.6 nM i traconazole.