title: A novel method for studying ergosterol biosynthesis by a cell-free preparation of Aspergillus fumigatus and its inhibition by azole antifungal agents.
abstract: A novel procedure has been developed for measuring ergosterol biosynthesis from [14C]mevalonate in a cell-free extract prepared from Aspergillus fumigatus. Ergosterol accounted for approximately 60% of the 4, 14-desmethylated sterol fraction which in turn totalled 13.2% of the non-saponifiable lipid produced. The other major sterol fractions were 4, 4-dimethylated sterols and 4-monomethylated sterols which accounted for 30.8% and 20.1% respectively of non-saponifiable lipid. The cell-free system had a narrow pH optimum of 7.2-7.4 for desmethylated sterol biosynthesis. Activity decreased by 94% at pH 6.5. Fluconazole (10(-4) M), ketoconazole (10(-6) M) and itraconazole (10(-6) M) inhibited formation of desmethylated sterols by greater than 85%, while 4-monomethylated sterols and 4, 4-dimethylated sterols were increased. The IC50s for inhibition of desmethylated sterol biosynthesis were 1.4 x 10(-6) M for fluconazole, 4.0 x 10(-8) M for ketoconazole, and 3.3 x 10(-8) M for itraconazole. The difference in intrinsic potency between fluconazole and ketoconazole is particularly interesting in view of the fact that fluconazole is a more effective agent than ketoconazole in an animal infection model of systemic aspergillosis.
authors.affiliation: Department of Discovery Biology, Pfizer Central Research, Sandwich, Kent, UK.
authors.forename: S A
authors.initials: SA
authors.lastname: Ballard
authors.forename: S W
authors.initials: SW
authors.lastname: Ellis
authors.forename: S L
authors.initials: SL
authors.lastname: Kelly
authors.forename: P F
authors.initials: PF
authors.lastname: Troke
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000378:metabolism*; D001345:Autoradiography; D001393:Azoles / Q000494:pharmacology*; D002855:Chromatography, Thin Layer; D004875:Ergosterol / Q000096:biosynthesis*; D015725:Fluconazole / Q000494:pharmacology; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D017964:Itraconazole; D007654:Ketoconazole / Q000031:analogs & derivatives / Q000494:pharmacology; D015394:Molecular Structure
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D017964:Itraconazole; D015725:Fluconazole; D007654:Ketoconazole; D004875:Ergosterol