Enzyme induction screen for ergosterol biosynthesis inhibitors

摘要

A method for screening for sterol biosynthesis inhibitors of potential use as fungicides or antihypercholesterolemic agents identifies agents by the induction of lanosterol 14- alpha -demethylase, an enzyme in the biosynthetic pathway of ergosterol and cholesterol, in cultures containing the agents. In one screening test, test samples are incubated in a culture of a Saccharomyces cerevisiae strain sensitive to ergosterol biosynthesis and containing a gene fusion of a lanosterol 14- alpha -demethylase clone with a gene for bacterial beta -galactosidase. After incubation of the culture, an increase in lancsterol 14- alpha -demethylase activity is determined indirectly by measuring beta -galactosidase activity. The culture media contains a chromogenic substrate of beta -galactosidase such as orthonitrophenyl- beta -D-galactoside or 5-bromo-4-chloro-3-indoyl- beta -D-galactoside, so that active samples are identified by the production of colored product. For comparison purposes, screening tests may employ a lanosterol 14- alpha -demethylase inhibitor such as dinaconazole as a positive control.