Development of an HTS assay for ySas2/hMOF histone acetyltransferase inhibitors

描述（由申请方提供）：本项目的总体目标是开发一系列简便、灵敏和可重现的高通量筛选试验，用于鉴定组蛋白乙酰转移酶（HAT）活性的强效、选择性抑制剂，并将这些方案应用于强效、选择性ySas 2/hMOF HAT抑制剂的初步开发。目前至少有四个家族，包括Gcn 5/PCAF、p300/CBP、MYST和Rtt 109。HAT蛋白使用乙酰辅酶A辅因子乙酰化核心组蛋白N-末端尾部内的特定赖氨酸残基的6个氮，并与其他组蛋白修饰酶协同工作，以提供染色质改变和基因活化之间的机制联系;并调节参与几种不同生物过程的基因，包括细胞周期进展、剂量补偿和激素信号传导。异常的HAT功能也与几种人类疾病相关，包括各种癌症（白血病、黑色素瘤、结肠直肠癌、胃癌和肺癌）、心血管疾病、肥胖症、HIV病毒感染和药物成瘾。由于HAT介导的有趣的生物学以及它们与人类疾病的关系，开发新的小分子抑制剂可能用作生物探针以及药物开发的先导分子是令人感兴趣的。对于该应用，我们将分别将我们的筛选努力集中在来自酵母和人的MYST HAT直系同源物ySas 2和hMOF上，尽管该测定法应该普遍适用于其他HAT。对ySas 2/hMOF的生物学兴趣源于它在乙酰化组蛋白H4的赖氨酸16中的进化保守作用以及这种活性与年龄相关病理学的相关性。该提案的具体目的是（1）开发一种高通量筛选ySas 2/hMOF抑制剂的方法，该方法可以自动化以在384孔板格式中工作，（2）定义一种正交二级测定法，以快速定量高通量筛选中鉴定的候选命中化合物的效力，以及（3）开发具有代表性HAT家族蛋白的反筛选测定法，以评估化合物的选择性。我们希望这些研究能够定义基于生化和细胞的测定，以筛选大的小分子文库，从而鉴定ySas 2/hMOF HAT活性的有效和选择性抑制剂，并完全适用于筛选其他HAT的抑制剂。我们还预计，这些研究将导致鉴定有效和选择性的第一代ySas 2/hMOF HAT抑制剂，用作化学探针或进一步开发用于治疗应用。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop a series of facile, sensitive and reproducible high-throughput screening assays ready to identify potent, selective inhibitors of histone acetyltransferase (HAT) activity and apply these schemes to the initial development of potent and selective ySas2/hMOF HAT inhibitors. There are at least four families including Gcn5/PCAF, p300/CBP, MYST and Rtt109. HAT proteins use the acetyl-CoA cofactor to acetylate the 6 nitrogen of specific lysine residues within the N-terminal tails of core histones and work in concert with other histone modification enzymes to provide a mechanistic link between chromatin alteration and gene activation; and regulate genes involved in several different biological processes including cell cycle progression, dosage compensation and hormone signaling. Aberrant HAT function has also been correlated with several human diseases including various cancers (leukemia, melanoma, colorectal, gastric and lung), cardiovascular disease, obesity, HIV viral infection and drug addiction. Because of the interesting biology that is mediated by HATs and their relationship to human disease, it is of interest to develop novel small molecule inhibitors that might serve as biological probes as well as lead molecules for drug development. For this application, we will focus our screening efforts on the MYST HAT orthologs form yeast and human, ySas2 and hMOF, respectively, although the assay should be generally applicable to other HATs. The biological interest in ySas2/hMOF stems from it's evolutionarily conserved role in acetylating lysine 16 of histone H4 and the correlation of this activity with age-associated pathologies. The specific aims of the proposal are to (1) Develop a high-throughput screen for ySas2/hMOF inhibitors that can be automated to work in a 384-well plate format, (2) Define an orthogonal secondary assay to rapidly quantify the potency of candidate hit compounds identified in high-throughput screens, and (3) Develop counter-screening assays with representative HAT family proteins to assess compound selectivity. We expect these studies to define biochemical and cell based assays to screen large libraries of small molecules to identify potent and selective inhibitors of ySas2/hMOF HAT activity, and to be completely adaptable to screens for inhibitors of other HATs. We also anticipate that these studies will result in the identification of potent and selective first-generation ySas2/hMOF HAT inhibitors for use as chemical probes or further developed for therapeutic applications.