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

描述(由申请人提供)：本项目的总体目标是开发一系列简便、灵敏和可重复性的高通量筛选试验，以准备识别有效的、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和选择性的ySas2/hMOF HAT抑制剂的初步开发。至少有四个家族，包括Gcn5/Pcaf、p300/CBP、Myst和Rtt109。HAT蛋白利用乙酰辅酶A辅助因子乙酰化核心组蛋白N端特定赖氨酸残基的6个氮，并与其他组蛋白修饰酶协同工作，提供染色质改变和基因激活之间的机制联系；并调节参与几个不同生物学过程的基因，包括细胞周期进展、剂量补偿和激素信号。HAT功能的异常还与几种人类疾病有关，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、艾滋病毒感染和药物成瘾。由于HATS介导的有趣的生物学及其与人类疾病的关系，开发新型的小分子抑制剂成为人们感兴趣的事情，这些小分子抑制剂既可以作为生物探针，也可以作为药物开发的先导分子。对于这个应用，我们将重点筛选来自酵母和人类的神秘HAT同源物，分别是ySas2和hMOF，尽管该方法应该普遍适用于其他HAT。对ySas2/hMOF的生物学兴趣源于它在进化上对组蛋白H4的赖氨酸16乙酰化的保守作用，以及这种活性与年龄相关的病理学的相关性。该建议的具体目标是(1)开发一种高通量的ySas2/hMOF抑制剂筛选方法，该筛选方法可以自动化地以384孔板的形式工作，(2)定义一种正交二次试验来快速量化在高通量筛选中确定的候选命中化合物的效力，以及(3)开发具有代表性的HAT家族蛋白的反筛选方法来评估化合物的选择性。我们希望这些研究定义生化和基于细胞的分析来筛选大的小分子文库，以确定ySas2/hMOF HAT活性的有效和选择性抑制剂，并完全适应于筛选其他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，用于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.