Cytochrome P-450 reductase screen for ergosterol biosynthesis inhibitors

A method for screening for the presence or absence of inhibition of cytochrome P450 or cytochrome P450 related enzymes or electron-transfer proteins by a chemical or biochemical test sample which comprises:

• (a) preparing, in a culture plate, a solidified culture of a Saccharomyces cerevisiae strain containing a disrupted cytochrome P450 reductase gene;
• wherein said cytochrome P450 reductase gene is involved in the ergosterol synthetic pathway
• (b) preparing, in another culture plate, a solidified culture of a Saccharomyces cerevisiae strain having an erg6 mutation;
• (c) introducing into both said cultures identical chemical or biochemical test samples in a well or on a disk;
• (d) introducing into said cultures, in a well or on a disk, a positive control selected from the group consisting of ketoconazole, miconazole, dinaconazole, and enconzole;
• (e) incubating said cultures for such time under such conditions sufficient to observe yeast cell growth in the absence of test samples;
• (f) comparing the extent of growth in the cultures of both strains in the vicinity of test samples and in the vicinity of the positive control with the extent of growth in the rest of the culture;
• and
• (g) determining the presence of inhibition of said cytochrome P450 or related proteins or enzymes by observation of whether growth inhibition in the vicinity of test sample in the disrupted reductase gene culture exceeds inhibition in the erg6 culture.

A method for screening for the presence or absence of cytochrome P450 or cytochrome P450 reductase inhibition by a test sample which comprises:

• (a) adding said sample to a well in, or a disk on, a solidified culture of a Saccharomyces cerevisiae strain containing a disrupted cytochrome-P450 reductase gene;
• wherein said cytochrome P450 reductase gene is involved in the ergosterol synthetic pathway
• (b) adding said sample to a well in, or a disk on, a solidified culture of a Saccharomyces cerevisiae strain having an erg6 mutation;
• (c) incubating said cultures for such time under such conditions sufficient to observe yeast cell growth in the absence of test sample;
• (d) comparing the extent of growth in the cultures of both strains in the vicinity of test samples;
• and,
• (e) determining the presence of inhibition of said cytochrome P450 or cytochrome P450 reductase by observation that growth inhibition in the vicinity of test sample in the disrupted reductase gene culture exceeds inhibition in the erg6 culture by at least about 8 mm.