A convenient cellular assay for the identification of the molecular target of ergosterol biosynthesis inhibitors and quantification of their effects on total ergosterol biosynthesis

Abstract

Increasing resistance of clinically relevant fungi is causing major problems in anti-mycotic therapy. Particularly for immunosuppressed patients fungal infections are of concern and increasing resistance against clinically used antimycotic drugs is hampering successful treatment. In the search for new antifungals ergosterol biosynthesis still is the most prominent target. However, several pitfalls in the bioactivity testing of such substances remain. Two of the major drawbacks certainly are the membrane association of most enzymes participating in ergosterol biosynthesis, and the difficulty to selectively associate growth inhibitory effects with the target pathway (ergosterol biosynthesis). Here we describe a GC-MS based cellular assay for target identification and selective potency determination of test components. In the qualitative part of the assay GC-MS analysis of cell lysates allows target identification by analysis of the changes in the sterol pattern. The quantitative part of the assay makes use of 13C-acetate feeding combined with GC-MS analysis allowing the selective quantification of a compound's effect on total ergosterol biosynthesis. The described cellular assay was analytically and biologically validated and used to characterize the novel ergosterol biosynthesis inhibitor JK-250.