A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.Defining the mechanism of action of small molecules in higher eukaryotes is hindered by their complexity, limited genetic methods, and protracted life cycles. In contrast, model eukaryotes are amenable to extensive genetic analyses in relatively short time frames. For example, unicellular eukaryotes, such as Saccharomyces cerevisiae and Aspergillus nidulans, have been used to rapidly elucidate the mechanism of action of many compounds, including drugs that are relevant to human therapeutics. The targets of the immunosuppressive compounds cyclosporine, FK506, and rapamycin were discovered in studies with yeast (9, 24). Resistance to cerulenin in S. cerevisiae was mapped to FAS2, and inhibition of the encoded enzyme, fatty acid synthase, was demonstrated biochemically (21). Similarly, isolation of an A. nidulans mutant resistant to a novel agricultural antifungal compound led to identification of dihydroorotate dehydrogenase as the target (15).In the present report, we extend the utility of S. cerevisiae for discovering and characterizing antifungal compounds. Our initial studies focused on UK-118005, a compound that has broad-spectrum antifungal activity. Using classical genetics, molecular biology, and biochemistry, we show that UK-118005 inhibits RNA polymerase (Pol) III. Although natural products have been identified that inhibit different RNA Pols, such as ␣-amanitin (32, 33) and tagetitoxin (34,35), this is the first example of a synthetic small molecule inhibiting RNA Pol. This finding demonstrates that cell-based screening can be a powerful method for identifying novel druggable targets.Additional antifungal structural analogs of UK-118005 were identified and further characterized. These results showed that whereas some analogs inhibit RNA Pol III as expected, others caused growth inhibition by an entirely different mechanism. Thus, yeast can be us...