A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

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 3σ 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 of confirmed actives with IC50 values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.

赖氨酸乙酰转移酶（lysine acetyltransferase, KAT）Rtt109与Vps75形成复合物，并可催化Asf1-H3-H4复合物中组蛋白H3赖氨酸56的乙酰化修饰（H3K56ac）。在酵母以及白色念珠菌（Candida albicans）等致病真菌物种中，Rtt109与H3K56ac对于复制偶联核小体组装及遗传毒性抗性至关重要。值得注意的是，人类体内不存在Rtt109的序列同源物，因此Rtt109抑制剂被认为是具备潜在低毒性的抗真菌剂。
本文报道了针对Rtt109催化组蛋白乙酰化的小分子抑制剂的无细胞荧光高通量筛选（cell-free fluorometric high-throughput screen, HTS）体系的开发与优化过程。该检测体系的KAT组分由酵母Rtt109-Vps75复合物构成，组蛋白底物复合物则为与酵母Asf1结合的全长果蝇（Drosophila）组蛋白H3-H4。
对药理学活性化合物库（LOPAC）的重复检测结果显示，该体系具备良好的批间与板间重复性。我们以384孔板形式完成了约225,000种化合物的筛选，平均Z'因子为0.71。基于3σ截断准则，初筛中共鉴定出1587种活性化合物（占比0.7%）。
该检测方法可有效识别此前已报道的KAT抑制剂藤黄菌素（garcinol）。我们还发现了数类典型的泛assay干扰化合物，包括曼尼希碱、儿茶酚类及对羟基芳基磺酰胺类。多数初筛得到的活性化合物存在检测信号干扰，但通过一系列简便的对照筛选与正交实验，可高效去除大部分检测假阳性。
高通量筛选后的分流验证显示，仅存在少量经确认的活性化合物，其半数抑制浓度（IC50）处于低微摩尔级别。本检测体系利用了体内参与H3K56乙酰化的5种无标记蛋白，理论上可识别通过不同机制抑制Rtt109催化的H3K56乙酰化的化合物。通过该筛选体系或其衍生方法发现的化合物，可作为靶向表观遗传酶的新型抗真菌剂的化学探针或先导化合物。