The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z ’ factor of 0.71. Based on a 3s cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number o
To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.