Quantifying Isoprenoids in the Ergosterol Biosynthesis by Gas Chromatography-Mass Spectrometry.

The ergosterol pathway is a promising target for the development of new antifungals since its enzymes are essential for fungal cell growth. Appropriate screening assays are therefore needed that allow the identification of potential inhibitors. We developed a whole-cell screening method, which can be used to identify compounds interacting with the enzymes of isoprenoid biosynthesis, an important part of the ergosterol biosynthesis pathway. The method was validated according to the EMEA guideline on bioanalytical method validation. hyphae and cells were lysed mechanically in an aqueous buffer optimized for the enzymatic deconjugation of isoprenoid pyrophosphates. The residual alcohols were extracted, silylated and analyzed by GC-MS. The obtained isoprenoid pattern provides an indication of the inhibited enzyme, due to the accumulation of specific substrates. By analyzing terbinafine-treated and mutant strains containing tunable gene copies of or , respectively, the method was verified. Downregulation of resulted in a high accumulation of intracellular farnesol as well as elevated levels of geranylgeraniol and isoprenol. The decreased expression of as well as terbinafine treatment led to an increased squalene content. Additional analysis of growth medium revealed high farnesyl pyrophosphate levels extruded during downregulation.