The cytochrome P450 14α-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G465S). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the Cterminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

Yeast and plant membranes contain rather small amounts of cytochrome P–450 as compared with membrane fractions prepared from bovine adrenal cortex, piglet testis and rabbit liver. The agricultural fungicides azaconazole, penconazole, propiconazole and imazalil showed a much greater affinity for microsomal cytochrome P–450 isozymes of Saccharomyces cerevisiae and Candida albicans (ATCC 28516) than for cytochrome P–450 in microsomal fractions prepared from Jerusalem artichoke tubers, maize shoots, pea seedlings or tulip bulbs and for cytochrome P–450 isozymes in mitochondrial or microsomal fractions from rabbit liver, piglet testis and bovine adrenals. The medicinal azole antifungals miconazole, clotrimazole, ketoconazole, fluconazole and itraconazole also interacted at much lower concentrations with the microsomal cytochrome P–450 isozymes from S. cerevisiae and C. albicans (ATCC‐28516, ATCC 44859, B 34226/1) than with those in mammalian membranes. Itraconazole showed the highest selectivity; bifonazole was much less selective. The microsomal fraction prepared from C. albicans (isolate B 41628) contained cytochrome P–450 isozymes with a lower affinity than the microsomal fractions from other isolates for miconazole, ketoconazole, fluconazole and itraconazole. However, itraconazole showed still high affinity for these cytochrome P–450 isozymes. In animal models this C. albicans isolate was less pathogenic and was shown to be less sensitive to azole antifungals both in vitro and in vivo. Azole antifungals inhibited ergosterol synthesis at nanomolar concentrations whereas almost micromolor concentrations were needed to obtain a similar inhibition of cholesterol or phytosterol synthesis. This inhibition coincided with the accumulation of 14α‐methylsterols such as 14‐methyl‐fecosterol, 14‐methyl‐24‐methylene‐ergosterol, 14‐methyl‐ergosta‐8, 24 (28)‐dien‐3β, 6α‐diol, obtusifoliol, lanosterol and 24‐methylenedihydro‐lanosterol. In 24‐h‐old cultures of C. albicans the 3β, 6α‐diol was the major sterol. It is speculated that by making the 14α‐methylsterols less lipophilic the cells are trying to eliminate these membrane‐disturbing compounds. This suggests that the azole‐induced ergosterol depletion might represent a greater contribution to their fungicidal activity than the accumulation of 14α‐methylsterols.

Cytochrome P-450: Target for itraconazole. Drug Dev. Res. 8:287-298, 1986.The N-substituted triazole, itraconazole, has high affinity for the cytochrome P-450 (cyt. P-450) isozyme involved in the 14a-demethylation of lanosterol in Candida albicans microsomes. Fifty per cent inhibition was already observed at itraconazole concentations < 5x lo-' M. Higher concentrations (> lo-' M) of this antifungal are needed to interfere with the 14 a-demethylation in mammalian cells. Unlike ketoconazole, itraconazole does not significantly affect in vitro androgen, gluco-and mineralocorticoid steroidogenesis. ltraconazole also does not affect the cyt. P-450-dependent 19-hydroxylation of testosterone, a step in the conversion of androgens to estrogens. The l-hydroxylation of testosterone by pig testes microscomes is only slightly inhibited. It is hypothesized that itraconazole's selective activity on ergosterol biosynthesis is due to its high affinity for the apoprotein of the C. albicans cyt. P-450 involved in the 1401-demethylation of lanosterol.

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Summary 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.