Jos Gorrens

Biochemical Approaches to Selective Antifungal Activity. Focus on Azole Antifungals

Summary: Azole antifungals (e.g. the imida‐zoles: miconazole, clotrimazole, bifona‐zole, imazalil, ketoconazole, and the tria‐zoles: diniconazole, triadimenol, propico‐nazole, fluconazole and itraconazole) inhibit in fungal cells the 14α‐demethylation of lanosterol or 24–methylenedihydro‐lanosterol. The consequent inhibition of ergosterol synthesis originates from binding of the unsubstituted nitrogen (N‐3 or N‐4) of their imidazole or triazole moiety to the heme iron and from binding of their N‐1 substituent to the apoprotein of a cytochrome P‐450 (P‐45014DM) of the endo‐plasmic reticulum.

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 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.

Origin of differences in susceptibility of Candida krusei to azole antifungal agents.

Summary. Two Candida krusei isolates were used to compare the effects of fluconazole, ketoconazole and itraconazole on growth and ergosterol synthesis, and to measure intracellular drug contents. Fifty per cent inhibition (IC50) of growth was achieved at 0.05–0.08 μM itraconazole and 0.56–1.2 μM ketoconazole, whereas 91‐ > 100 μM fluconazole was needed to reach the IC50 value. Similar differences in sensitivity to these azole antifungal agents were seen when their effects on ergosterol synthesis from [14C]acetate were measured after 4 h and 24 h of growth. However, when the effects of the azoles on ergosterol synthesis from [14C]mevalonate by subcellular fractions were measured, fluconazole was only 2.3–6.1 times less active than itraconazole, and the IC50 values for ketoconazole were almost similar to those obtained with itraconazole. These results indicate that differences in susceptibility to itraconazole and ketoconazole are unrelated to differences in affinity for the C. krusei cytochrome P450. The much lower growth‐inhibitory effects of fluconazole can also be explained partly only by a lower affinity for the P450‐dependent 14α‐demethylase. The differences in sensitivity of both C. krusei isolates appeared to arise from differences in the intracellular itraconazole, ketoconazole and fluconazole contents. Depending on the experimental conditions, these isolates accumulated 6–41 times more itraconazole than ketoconazole and the intracellular ketoconazole content was 3.0–19.0 times higher than that of fluconazole.

Saperconazole: a selective inhibitor of the cytochrome P-450-dependent ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes.

The N‐1‐substituted triazole antifungal, saperconazole, is a potent inhibitor of ergosterol synthesis in Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes. Fifty % inhibition is already achieved at nanomolar concentrations. The saperconazole‐induced inhibition of ergosterol synthesis coincides with an accumulation of 14‐methylated sterols, such as 24‐methylenedihydrolanosterol, lanosterol, obtusifoliol, 14α‐methylfecosterol, 14α‐methylergosta‐8,24(28)‐dien‐3,β‐6α‐diol and 14α‐methylergosta‐5,7,22,24(28)‐tetraenol. This indicates that saperconazole interferes with the cytochrome P‐450 (P‐450)‐dependent 14α‐demethylation of lanosterol and/or 24‐methylenedihydrolanosterol. Saperconazole forms stable drug‐P‐450‐complexes by binding via its free triazole nitrogen to the heme iron and via its N‐1 substituent to the apoprotein moiety. The triazole derivative is a highly selective inhibitor of the 14α‐demethylase in fungal cells. It is a poor inhibitor of the 14α‐demethylation of lanosterol in rat and human liver cells. Saperconazole is, at concentrations as high as 10 µM, devoid of effects on the P‐450‐dependent cholesterol side‐chain cleavage and 11β‐hydroxylase, 17,20‐lyase, 21‐hydroxylase and aromatase. Saperconazole does not interfere with the 2α, 6α‐, 6β‐ and 7α‐hydroxylations of testosterone in microsomes from male rat liver. At high concentrations (> 5 µM) an inhibition of the 16β‐+hydroxylations is seen.

Mode of Action of Antifungals of Use in Immunocompromised Patients. Focus on Candida Glabrata and Histoplasma Capsulatum

During recent years considerable advances have been made in the identification of potential targets for antifungal agents. The most important antifungals for use in immunocompromised patients interfere with targets in the plasma membrane (polyenes), nucleus (5-fluorocytosine) or smooth endoplasmic reticulum (allylamines, morpholines, azole derivatives).

Effects of ketoconazole and itraconazole on growth and sterol synthesis in Pityrosporum ovale

The effects of ketoconazole and itraconazole on growth and sterolsynthesis in Pityrosporum ovale was studied. Itraconazole was at least 10 times more active than ketoconazole. Sterol synthesis was inhibited more rapidly than growth, suggesting that the antifungal activity of both azoles originates from an effect on the 14 alpha demethylase system, as seen in other species.