However, Rtt109 activity is significantly attenuated in the absence of its known chaperones Asf1 and Vps75 in vitro. In contrast to most KATs that utilize free histones or nucleosomal histones as substrates, the ideal substrate for Rtt109 is the heterotrimeric complex of Asf1-dH3-H4 in vitro and most likely in cells, as Asf1 is essential for H3K56ac in vivo. Vps75 and Asf1 also direct the substrate specificity of Rtt109 differently. Vps75 forms a complex with Rtt109 in vivo, and in the absence of Vps75, Rtt109 is sensitive to proteolysis. The choice of substrate is also a nontrivial matter. Histone substrates can vary considerably across KAT assays, including histone peptides, biotinylated histone peptides and fulllength histones. Histone precipitation can be problematic, and one solution is to use histone peptides in place of full-length histone proteins. In an effort to make the assay more physiologically relevant, we were able to successfully incorporate full-length histone proteins complexed with the histone chaperone Asf1 into this assay. This is in contrast to the method used to identify a reported Rtt109 inhibitor, which utilized histone peptides in the primary HTS. In principle, the use of both chaperones and full-length histones could allow the identification of multiple classes of inhibitors. This includes compounds that can directly target Rtt109, or disrupt the interactions between Rtt109 and Vps75, Rtt109-Vps75 and its substrate Asf1-dH3-H4, as well as those disrupting interactions between Asf1 and dH3-H4. While this assay used the yeast Rtt109-Vps75-Asf1 system, in principle the HTS could be adapted to screen clinically relevant pathogenic fungal species such as C. albicans or P. carinii. A potential advantage of using yeast Rtt109 and its chaperones is the abundance of structural information already available. This suggests that Rtt109 from these fungal species have similar functions and that small-molecule scRtt109 inhibitors could inhibit Rtt109 from other species, depending on the nature of the binding site and potentially other species-specific factors. Our assay production is also notable because of the decision to include WZ8040 EGFR/HER2 inhibitor detergent mid-assay. We decided not to re-screen the compounds assayed under non-detergent conditions based on two primary factors: resource conservation and the availability of several available SAR131675 follow-up assays to identify promiscuous aggregators. Interestingly, the mean percent inhibition, the standard deviation and the percentages of primary screen hits were lower for the detergent-containing production run compared to the detergent-free run.