Novel small-molecule inhibitors of RNA polymerase III

Abstract

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.

FIG. 1.

Transcript profiling reveals that UK-118005 treatment induces a GCN4 response. (A and B) Transcript profiling of cultures of MMB1489 (GCN4⁺) (A) and MMB1576 (gcn4Δ) (B). In each panel, normalized intensity values from untreated cultures (x axis) are compared to treated samples (y axis). Only genes whose normalized intensity is >0.1 are shown, since this value represents the approximate intensity at which the signal-to-noise ratio decreases sharply. (A) A set of known Gcn4p target genes (blue squares) (28) involved in amino acid biosynthesis is significantly upregulated in UK-118005-treated (3 μg/ml) cells. These target genes comprise the bulk of the highly induced genes in this data set. (B) In gcn4Δ cells, a similar treatment (6 μg/ml) produces few highly induced genes. For comparison, the set of genes encoding the subunits of the 26S proteasome (red triangles) (12) are unchanged by the treatment in either genetic background. For the relevance of this finding, please refer to Fig. 8.

FIG. 2.

Growth inhibition of S. cerevisiae with UK-118005 treatment. Growth inhibition by UK-118005 was assayed as described in Materials and Methods. The two mutant strains (•) are more resistant than the wild-type cells (▪), as indicated by at least a fourfold increase in IC₅₀ after 48 h of treatment with UK-118005. (Top panel) diploid strains; (bottom panel) haploid strains. The IC₅₀ is the concentration of compound that inhibits growth by 50%.

FIG. 3.

Cloned mutant allele of RPO31 confers resistance to UK-118005. MIC determination for the wild-type haploid strain BY4741 containing either pBM601 (vector, no insert [♦]), pRPO31-G1101S (▴), or pRPO31 (▪) after 48 h of treatment with serial dilutions of UK-118005.

FIG. 4.

(A) Site of mutation in Rpo31-G1101S mutant protein. A schematic diagram showing the eight conserved domains of Rpo31 protein and the single amino acid change from glycine (G) to serine (S) at amino acid residue 1101 in the g domain of the Rpo31 protein is shown. (B) Comparisons of homologous amino acid sequence motif g of analyzed RNA Pol large subunits from S. cerevisiae (yeast), human, mouse, Caenorhabditis elegans (worm), and Arabidopsis thaliana. The Pols from different organisms are indicated by the names of the organisms followed by 1 (Pol I), 2 (Pol II), and 3 (Pol III). Amino acid positions are given at the beginning and end of each sequence. Identical residues with the RPO31p g motif sequence (RNA Pol III) are indicated by points. The G1101S mutation site is indicated by an arrow, and its conserved surrounding amino acid residues (discussed in the text) are overlined and displayed above the overline.

FIG. 5.

Inhibition of tRNA transcription in vivo by UK-118005. A Northern blot shows a time course of treatment of wild-type BY4741 cells (top panel) and rpo31 mutant MMB2404 cells (bottom panel) either untreated (lanes 0) or treated with 15 μM or 30 μM UK-118005 as indicated (see Materials and Methods for details). The migration of the precursor and mature is indicated.

FIG. 6.

Comparison of growth inhibition by UK-118005. Growth inhibition by UK-115005 in wild-type BY4741 (A) and the resistant mutant MMB2404 (B) was measured by taking OD₆₀₀ readings of the same cultures as analyzed by Northern in Fig. 5 at the times indicated. Strains were treated with 15 μM (▪) or 30 μM (▴) UK-118005 or no UK-118005, i.e., the untreated control (♦).

FIG. 7.

Inhibition of tRNA transcription in vitro by UK-118005. (A) In vitro transcription of SUP53 (left panel) and SUP4 (right panel) in the presence or absence of UK-118005 for 1 h; (B) quantitation of dose-dependent inhibition of SUP4 in vitro transcription by UK-118005. Quantitation of radiolabeled tRNA is expressed in photostimulated luminescence (PSL) units. (Top panel) transcription with wild-type RNA Pol III nuclear extract; (bottom panel) transcription with RPO31-G1101S mutant RNA Pol III nuclear extract.

FIG. 8.

Transcript profiling reveals that ML-22952 treatment induces proteasome subunit genes. Transcript profiling of ML-22952-treated cells. Scatter plot data are presented as in Fig. 1, as are the genes encoding the subunits of the 26S proteasome (red triangles) and a set of known Gcn4p target genes (blue squares). Although the upregulation of proteasome subunits is not high, this increase is physiologically relevant (12).