Membrane sterol and the sterol biosynthetic pathway have long been target sites for antifungal drugs. Two of the most widely used antifungal classes, the polyenes (amphotericin B) target plasma membrane-bound sterols and the new classes of azoles (ketoconazole, fluconazole, and itraconazole) target the cytochrome P450-mediated step, lanosterol C-14 demethylation. Saccharomyces cerevisiae contains the best characterized sterol pathway at the genetic and molecular levels. One of the goal of this proposal is to understand and evaluate the C-4 demethylation reactions as new possible target sites for antifungal therapy. Mutations in every step of the ergosterol pathway from lanosterol (the first sterol in the pathway ) to ergosterol have been isolated with the exception of the C-4 demethylase genes. We propose to study the genes involved in C-4 demethylation to assess whether the inability to remove two bulky sterol methyl groups results in loss of membrane sterol function. Secondly, we intend to investigate the gene(s) involved in sterol esterification (yeast ACAT). Sterol molecules are stored in cytoplasmic microlipid droplets as fatty acid esters and accessed during times of rapid growth by deestericiation. Drugs which interrupt this cycle may have unrecognized potential as antifungals since unlike animal cells, yeast and fungi are physiologically unable to take up sterols from the growth medium. Thus inhibition of sterol synthesis or inhibition of the conversion of esterified to free sterols cannot be compensated by uptake of cholesterol from the host. If the yeast ACAT enzyme and/or the sterol ester hydrolase play an essential role in modulating sterol synthesis, deregulation by mutation will have deleterious results. These sites would become good targets for antifungal drugs. In order to understand why interruption of a sterol biosynthetic site would make a good antifungal target site, we will address the question of suppressor mutations which restore viability to strains containing disruptions in """"""""essential sterol genes such as the cytochrome P450 (C-14 demethylation) and the C-14 reductase genes. While suppression is not necessarily a clinical concern, the elucidation of the mechanisms by which this happens have important implications for the design of additional target sites for antifungal drugs.
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