Interaction of azole derivatives with cytochrome P‐450 isozymes in yeast, fungi, plants and mammalian cells

Abstract

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.

Characterization of an azole-resistant Candida glabrata isolate

“…Cells were grown for 24 h at 30°C, and 30 ml of the cell suspension (containing 7 x 108 cells) was used to inoculate 5 liters of PYG-2 medium. Cells were grown for 16 h at 30°C on a magnetic stirrer (36).…”

“…Sterols were eluted with a VOL. 36,1992 on methanol-water mixture (95:5) for 25 min, after which the column was eluted with pure methanol. Sterols were identified according to their retention times relative to those of standards and/or by gas chromatographic-mass spectrometric analysis as described previously (38).…”

Molecular diversity of sterol 14α‐demethylase substrates in plants, fungi and humans

“…1). This step is considered to be one of the key enzymes of sterol biosynthesis of these organisms and is the target enzyme for the azole antifungals, which are thought to selectively inhibit the yeast and fungal forms over their plant and mammalian counterparts [3].…”

Altered P450 activity associated with direct selection for fungal azole resistance

“…The azoles inhibit fungal CYP51A 1 selectively, binding to the haem as a sixth ligand with the N-1 substituent group interacting with the apoprotein [5]. The interaction with the apoprotein determines the selectivity, although these compounds can inhibit other CYPs at higher concentrations including plant and animal sterol 14a-demethylases [6].…”