Novel antifungal drug discovery based on targeting pathways regulating the fungus-conserved Upc2 transcription factor

Abstract

Infections by Candida albicans and related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level and UPC2-LacZ gene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expression in vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.

FIG 1

(A) Schematic depicting the yeast ergosterol pathway. The arrows represent individual enzymatic steps in the biosynthetic pathway. Steps inhibited by an antifungal compound are indicated. (B) Schematic of the critical path used for identifying compound inhibitors of Upc2. All compounds isolated that were positive in the UPC-LacZ assay for inhibition were tested in the PGK1-LacZ assay. The compounds that had positive results in the whole-cell assay that did not inhibit PGK1-LacZ activity were screened in the AlphaScreen assay. Compounds carried forward were assayed for inhibiting Upc2-dependent transcription factor activity. Positive compounds remaining were tested for inhibitory synergy with fluconazole. (C) Schematic of the UPC2 promoter indicating the location of each SRE. WT, wild type; MUT, mutant.

FIG 2

Cells expressing an UPC2-LacZ promoter fusion show a dose-dependent increase in β-galactosidase activity in response to increasing concentrations of fluconazole (in micrograms per milliliter). Cells were diluted 1:20 into the final assay volume of 4 μl. Test compounds in wells of solid white 1,536-well microtiter plates were redissolved in medium containing cells. Increasing concentrations of fluconazole were then added in a final assay volume of 4 μl, and cells were incubated for 4 h at 30°C. β-Galactosidase activity was performed using BetaGlo (Promega, Madison, WI, USA). β-Galactosidase activity of the UPC2-LacZ wild-type strain (solid black circles) and ecm22 upc2 UPC2-LacZ strain (white circles) are shown.

FIG 3

Molecular structures of the identified compounds The molecular structures and VB numbers of compounds 1 to 8 are shown. The compounds were isolated using the commercial small-molecule collection.

FIG 4

Upc2 DNA binding in the AlphaScreen assay shows a dose-dependent increase in activity in response to an increasing concentration of protein. Increasing concentrations of fusion proteins were diluted in assay buffer. Protein was combined with an equal volume of acceptor beads, which had been diluted in assay buffer. Part of the mixture (4 μl/well) was transferred to a white, 1,536-well assay plate. The plate was incubated for 30 min at room temperature. Biotinylated, double-stranded DNA was diluted in assay buffer and combined with an equal volume of donor beads in assay buffer. Four microliters of this mixture was added to each well, and the plate was incubated for an additional hour at room temperature in the dark. The signal was detected using a PerkinElmer EnVision microplate reader. GST-CaUpc2, glutathione S-transferase (GST) fused to the C. albicans Upc2 DNA binding domain; GST-ScUpc2, S. cerevisiae Upc2 DNA binding domain; GST-CgUpc2, C. glabrata Upc2 DNA binding domain.

FIG 5

Compounds 1, 2, and 3 inhibit fluconazole-induced Upc2-dependent activity. Cells were grown to exponential phase in the absence (−) and presence (+) of fluconazole (FLC) and each compound (compound 1, 2, or 3) for 6 h at 30°C. Total RNA was extracted, and the mRNA expression levels of ERG11, ERG25, UPC2, and PGK1 from S. cerevisiae (Sc) were determined by qRT-PCR.

FIG 6

Compounds 1, 2, and 3 inhibit fluconazole-induced CgUpc2-dependent activity. Cells were grown to exponential phase in the absence (−) and presence (+) of fluconazole and each compound (compound 1, 2, or 3) for 6 h at 30°C. Total RNA was extracted, and the mRNA expression levels of ERG11, ERG25, UPC2, and PGK1 from C. glabrata (Cg) were determined by qRT-PCR.

FIG 7

Compounds 1, 2, and 3 decrease Upc2 protein expression in the presence of fluconazole. Wild-type S. cerevisiae cells were grown to exponential phase in the absence and presence of fluconazole (FLC) and each compound (compound 1, 2, or 3) for 6 h at 30°C (con, control). Cell lysates were then prepared, and proteins were resolved by SDS-PAGE. The protein level of ScUpc2 was visualized by Western blot analysis using rabbit anti-ScUpc2 polyclonal antibodies and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG polyclonal antibodies.

FIG 8

Compounds 1, 2, and 3 decrease Upc2 SRE binding in the presence of fluconazole. Wild-type S. cerevisiae cells were grown to exponential phase in the absence and presence of fluconazole and each compound for 6 h at 30°C. ScUpc2 was immunoprecipitated from cross-linked cells using rabbit anti-ScUpc2 polyclonal antibodies. Cross-linked ScUpc2-bound DNA was isolated and quantitated by qRT-PCR using primers specific for SRE1 or SRE2.