Conserved Fungal Genes as Potential Targets for Broad-Spectrum Antifungal Drug Discovery

Abstract

The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.

Ability to grow on lipids accounts for the fully virulent phenotype in neutropenic mice of Aspergillus fumigatus null mutants in the key glyoxylate cycle enzymes

“…For instance, a list of predicted genes involved in pathogenicity, allergy, secondary metabolism or cell viability are already accessible (Nierman et al, 2005). This wealth of information permits the development of numerous research programmes on the pathobiology of A. fumigatus that will undoubtedly lead to the identification of novel antifungal targets for therapy of invasive aspergillosis to be added to those currently utilized, the fungal cell membrane and cell wall (Liu et al, 2006).…”

Effects of Depleting the Essential Central Metabolic Enzyme Fructose-1,6-Bisphosphate Aldolase on the Growth and Viability of Candida albicans : Implications for Antifungal Drug Target Discovery

“…Instead, we constructed conditional FBA1 mutants to test the essentiality of this gene in C. albicans. We used the C. albicans MET3 promoter, which is tightly repressed by methionine and cysteine (7) and which was used very recently in an effective screen of conserved fungal genes for potential antifungal targets (26). Using a PCR-amplified URA3-MET3-FBA1 cassette, the first FBA1 allele was placed under the control of this MET3 promoter.…”

“…An antifungal drug inhibits its target within a significantly shorter time scale than the depletion of the target following transcriptional repression. A classic approach to the identification of novel antifungal targets involves the screening of libraries of conditional mutants in which specific genes have been placed under the control of a regulatable promoter (26,40). This type of screen has great potential for the identification of novel essential functions that are expressed at low abundance and/or rapidly turned over in C. albicans.…”

“…This represents an effective half-life of about 45 min, which is close to the doubling time of the cells (about 50 min). We used the MET3 promoter because it is rapidly and effectively repressed by methionine and cysteine (7,25,26). However, once MET3-FBA1 transcription has been repressed, Fba1p depletion is dependent upon the turnover of FBA1 mRNA and Fba1p protein as well as the dilution rate during cell doubling.…”

Two-Component Signal Transduction Proteins as Potential Drug Targets in Medically Important Fungi

“…A desirable outcome of such a screen is a compound that has reasonable activity against a wild-type strain (compared to current antifungals) but has increased activity inhibitory to an HK or RR mutant. In diploid pathogens such as C. albicans, strains lacking one or both functional alleles have been constructed using several approaches such as gene replacement and conditional expression (42,57) and haploinsufficiency (HI) (66,73; see references 8 and 68 for reviews) to identify growthessential genes and screen for drug-target pairs against compound libraries. A similar approach has been used to identify growth-essential genes in A. fumigatus (31).…”