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

项目描述（由申请人提供）：该项目的总体目标是开发一系列简便、敏感和可重复的高通量筛选试验，以确定有效的、选择性的组蛋白乙酰转移酶（HAT）活性抑制剂，并将这些方案应用于有效的、选择性的ySas2/hMOF HAT抑制剂的初步开发。至少有Gcn5/PCAF、p300/CBP、MYST和Rtt109四个家族。HAT蛋白使用乙酰辅酶a辅助因子乙酰化核心组蛋白n端末端特定赖氨酸残基的6氮，并与其他组蛋白修饰酶协同工作，在染色质改变和基因激活之间提供机制联系；并调节涉及几个不同生物过程的基因，包括细胞周期进程，剂量补偿和激素信号。HAT功能异常还与多种人类疾病相关，包括各种癌症（白血病、黑色素瘤、结直肠癌、胃癌和肺癌）、心血管疾病、肥胖、HIV病毒感染和吸毒成瘾。由于hat介导的有趣生物学及其与人类疾病的关系，开发新的小分子抑制剂可能作为生物探针和药物开发的先导分子是人们感兴趣的。对于这项应用，我们将重点筛选酵母和人类的MYST HAT同源物，ySas2和hMOF，尽管该试验应该普遍适用于其他HAT。ySas2/hMOF的生物学意义源于它在组蛋白H4赖氨酸16乙酰化中的进化保守作用，以及这种活性与年龄相关病理的相关性。该提案的具体目标是：(1)开发ySas2/hMOF抑制剂的高通量筛选，可以在384孔板格式下自动工作；(2)定义正交二级分析，快速量化高通量筛选中确定的候选命中化合物的效力；(3)开发具有代表性的HAT家族蛋白的反筛选分析，以评估化合物的选择性。我们希望这些研究能够定义生化和基于细胞的检测方法来筛选大的小分子文库，以鉴定ySas2/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ySas2/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.