Fungi are difficult to treat therapeutically because of several factors. First, fungi are eukaryotes, and many of their Dabrafenib Raf inhibitor biologically crucial genes are also conserved in humans. Therefore, it has proven difficult to find fungi-specific therapeutic targets that minimize toxicity to humans. Second, fungi can develop resistance to most drugs currently used to treat patients. Third, early detection and diagnosis of fungal infections can be difficult in clinical settings. Finally, fungal pathogenesis is governed by complicated host-pathogen interactions. Therefore, there is a clinical need for novel and efficacious antifungal treatments. Inhibiting Rtt109-catalyzed histone acetylation may be clinically relevant for antifungal purposes. While Rtt109 is highly conserved in fungal species, it exhibits no obvious sequence homology to mammalian KATs. Additionally, it appears that Rtt109 utilizes a different catalytic mechanism than p300/CBP, the potential functional homolog of Rtt109. Inhibitors of p300 such as Lys-CoA have not shown activity versus Rtt109. Furthermore, others have shown that deletion of rtt109 in Candida albicans reduces fungal virulence in mouse models. Our group has also shown Pneumocystis carinii expresses an active Rtt109 KAT. These results support the idea that Rtt109 is an attractive antifungal therapeutic target and that compounds that inhibit Rtt109-catalyzed histone acetylation may serve as potential antifungal agents. To date, only one small-molecule has been reported to inhibit Rtt109-catalyzed histone acetylation but not other KATs like GCN5 and p300. This particular molecule did not affect cellular levels of H3K56ac or sensitivity to a genotoxic agent in either C. albicans or S. cerevisiae, but this lack of observed in vivo activity could be due to a variety of factors such as drug metabolism, cell permeability or degradation. Therefore, XL880 purchase methods must still be developed and optimized to identify compounds capable of inhibiting Rtt109-catalyzed histone acetylation, both in vitro and in vivo. In this work, we report the development and optimization of a high-throughput screen for small-molecule inhibitors of Rtt109-catalyzed histone acetylation using the well-characterized S. cerevisiae Rtt109�CVps75-Asf1 proteins. Importantly, our assays take into account the complex regulation of Rtt109 in vitro and in vivo by using purified Rtt109-Vps75 as the enzyme and Asf1 bound to full-length H3-H4 as the substrate. Given the susceptibility of this assay format to false-positives, appropriate follow-up assays and consideration of assay-specific artifacts and promiscuous inhibitors are also discussed herein.