Target-specific hypersusceptible strains of Saccharomyces cerevisiae were used to screen antifungal compounds. Two novel Erg7p inhibitors were identified, providing proof of principle of the approach taken. However, observed hypersensitivities to antifungals acting via other targets imply that use of this tool to identify the mode of action requires significant deconvolution.Overexpression of a target gene can decrease the cell's susceptibility to inhibitors acting via that target (28, 30). Conversely, underexpression of a target can lead to an increased susceptibility to its inhibitors (2, 5). Here we describe how the well-studied GAL system of Saccharomyces cerevisiae (11) can be used for the identification of target-inhibitor pairs using target-specific, hypersusceptible strains of S. cerevisiae.Genes encoding putative targets that have been shown to be essential for growth of S. cerevisiae were selected (18, 26). Target-specific hypersensitive, or "switch-down," strains were constructed as previously described (17) using S. cerevisiae MEY121 (MATa his3 leu2-3,112 ura3-52 trp1 rme1) (derived from JK9-3da [14]) and a HIS3-GAL1 promoter integration cassette; correct integration was verified by a number of diagnostic PCRs. As a result, when a switch-down strain was grown at 30°C in YP (1)-2% galactose medium and transferred to YP-2% glucose medium, growth continued unabated for a number of generations until the cellular pool of target protein was presumed depleted. Since this number was constant for each target, the final density of the culture was proportional to its initial density.When wild-type strains of S. cerevisiae were incubated with growth inhibitors at sub-MICs, the growth rates of the strains were lowered but the final optical density at 600 nm was unchanged. Switch-down strains growing in the presence of glucose were expected to behave identically except when they were incubated with a compound that mediated its antifungal effect via the down-regulated target. At its 50% inhibitory concentration (IC 50 ), a target-specific compound would decrease the cellular target activity by 50% and the number of doublings the culture could undergo before the growth arrest would be one fewer. This would result in a 50% lower final optical density at 600 nm compared to growth arrest in the absence of the compound. (8), and staurosporine (31). Invariably, down-regulation of a target led to hypersusceptibility to its genuine inhibitor (Table 1), thus providing proof of principle of the approach taken. However, in some cases hypersensitivity to other compounds resulted as well (Table 1); the most striking example is the ERG1 switch-down strain that was hypersensitive to both terbinafine and fluconazole but not to any other control compound.In order to assess the utility of switch-down strains in cellbased screening of antifungal compounds, a set of 34 switchdown strains was tested against a library of 2,500 antifungal compounds with IC 50 s below 400 M against wild-type S. cerevisiae and Candida albicans. Dose respo...