Objectives:
Candida glabrata is a pathogenic yeast associated with a high rate of morbidity and mortality in immunocompromised patients. An alarming rise in drug-resistant strains over the past decade indicates an urgent need for the development of novel therapeutic strategies. We propose a novel antifungal strategy by targeting the protein Bdf1. Bdf1 is a member of the BET (Bromodomain and Extra-Terminal) family of proteins that regulate transcription and chromatin remodelling. BET proteins associate with chromatin through their two bromodomains (BD1 and BD2), epigenetic "reader" modules which recognize acetylated lysine residues within the N-terminal tails of histones. The overall goal of this project is to validate Bdf1 bromodomains as an antifungal target in C. glabrata. Specific objectives are to: (i) verify that Bdf1 bromodomain function is essential in C. glabrata; (ii) investigate the ligand-binding activity and atomic structure of the two Bdf1 bromodomains; and (iii) identify compounds that selectively inhibit Bdf1 bromodomains for translational development as novel antifungal therapeutics.
Methods:
The project uses genetic approaches and a mouse infection model to verify whether the two bromodomains of Bdf1 are required for C. glabrata growth and infection. Biochemical and crystallographic studies are performed to investigate the structure and ligand-binding activity of these two domains. A high-throughput assay has been developed to screen chemical libraries for Bdf1 bromodomain inhibitors, and subsequently these compounds will be characterized for the ability to inhibit fungal growth in vitro.
Results:
We have shown that BDF1 is essential for the growth of C. glabrata in vitro. Deletion or point mutations inactivating both Bdf1 bromodomains also severely inhibit yeast growth. The high-resolution crystal structure of the Bdf1 BD2 domain reveals that its ligand binding pocket is stereochemically distinct from those of human BET bromodomains. We anticipate a similar conclusion for the BD1 domain following the determination of its structure, currently in progress. We have developed a homogeneous time-resolved FRET (HTRF) assay to identify Bdf1 bromodomain inhibitors and used it to screen a chemical library of over 100,000 compounds, resulting in several promising hit compounds. Current work is aimed at characterizing hit compounds in binding and inhibition assays and in fungal growth assays to assess their effect on the viability of C. glabrata. Our most promising inhibitors will be then verified for their antifungal effect in a mouse model of invasive candidiasis.
Conclusion:
Bdf1 functionality is essential in C. glabrata and its bromodomains are druggable. Structural differences between the binding pockets of C. glabrata and human BET bromodomains support the feasibility of identifying small-molecule compounds which selectively inhibit the fungal but not the human target. We anticipate that the combined results of these studies will validate Bdf1 as a potential antifungal target in C. glabrata.

Developing a novel anti-fungal strategy by targeting epigenetic reader domains in Candida glabrata