title: 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.
authors.affiliation: The Institute of Metabolic Disorders, Genesis Biotechnology Group, Hamilton, New Jersey, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Gallo-Ebert
authors.forename: Melissa
authors.initials: M
authors.lastname: Donigan
authors.forename: Ilana L
authors.initials: IL
authors.lastname: Stroke
authors.forename: Robert N
authors.initials: RN
authors.lastname: Swanson
authors.forename: Melissa T
authors.initials: MT
authors.lastname: Manners
authors.forename: Jamie
authors.initials: J
authors.lastname: Francisco
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Toner
authors.forename: Denise
authors.initials: D
authors.lastname: Gallagher
authors.forename: Chia-Yu
authors.initials: CY
authors.lastname: Huang
authors.forename: Scott E
authors.initials: SE
authors.lastname: Gygax
authors.forename: Maria
authors.initials: M
authors.lastname: Webb
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Nickels
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D014157:Transcription Factors