Identification of Inhibitors of the Tip60 Histone Acetyltransferase

描述(申请人提供)：组蛋白乙酰转移酶对赖氨酸残基的乙酰化作用调节蛋白质功能。细胞含有多个不同的组蛋白乙酰转移酶家族，包括Tip60。Tip60与多种人类疾病的发病过程有关，包括癌症进展、神经退行性疾病和病毒复制。Tip60在这些不同的疾病过程中的参与反映了Tip60的S调节转录、病毒复制和细胞对DNA损伤的反应的能力。Tip60通过乙酰化和激活ATM蛋白激酶的能力来调节细胞对遗传毒性应激的反应。ATM功能的丧失与癌症发病率的增加、对电离辐射的敏感性和小脑神经变性有关。因此，Tip60是细胞修复和存活DNA损伤能力的关键调节因子。放射治疗是人类癌症的一种主要治疗方式。然而，许多肿瘤类型对放射治疗的细胞毒作用具有相对的抵抗力。因此，Tip60的选择性药理抑制为提高放化疗治疗人类肿瘤的疗效提供了一个潜在的强大的治疗靶点。目前，还没有发现组蛋白乙酰转移酶的选择性抑制剂，尤其是Tip60的选择性抑制剂。因此，在这项提案中，我们建议开发一种高通量的检测方法，以确定Tip60组蛋白乙酰转移酶的新的、特异的抑制剂。将采取2个具体目标。在具体目标1中，将利用重组Tip60对固定化多肽进行乙酰化的能力，建立一种新的高通量ELISA法。在具体目标2中，分析将被格式化为384孔格式，并将进行活性文库化合物的初步筛选。Tip60代表了一个靶点，针对这个靶点，存在着一系列不充分的选择性和有效的小分子抑制剂；筛选和鉴定这些抑制剂将对研究Tip60和相关的HATS在人类疾病进展中的作用产生重大影响。这项提议将开发Tip60 HAT的特定抑制剂，可用于扰乱细胞蛋白质乙酰化水平，目的是改善人类肿瘤的治疗。这些抑制剂可用于动物和临床研究，以测试Tip60 In激活使人类肿瘤对放射治疗和化疗敏感。此外，Tip60抑制剂可以用来探索Tip60发挥生物学效应的基本信号机制，并寻找新的Tip60底物。最后，Tip60抑制剂可以用于复杂的生物，如小鼠，以检查Tip60在癌症进展、神经退行性疾病和遗传毒性应激等疾病过程中的作用。

DESCRIPTION (provided by applicant): The acetylation of lysine residues by histone acetyltransferases regulates protein function. Cells contain multiple, distinct families of histone acetyltransferases, including Tip60. Tip60 is implicated in the pathogenesis of several human disease processes, including cancer progression, neurodegenerative diseases and viral replication. The participation of Tip60 in these diverse disease processes reflects Tip60's ability to regulate transcription, viral replication and the cells response to DNA damage. Tip60 regulates the cells response to genotoxic stress through its ability to acetylate and activate the ATM protein kinase. Loss of ATM function is associated with increased incidence of cancer, sensitivity to ionizing radiation and cerebellar neurodegeneration. Tip60 is therefore a key regulator of the cells ability to repair and survive DNA damage. Radiation therapy is a major treatment modality for human cancer. However, many tumor types are relatively resistant to the cytotoxic effects of radiation therapy. Accordingly, selective pharmacological inhibition of Tip60 provides a potentially powerful therapeutic target for enhancing the efficacy of radiotherapy and chemotherapy in the treatment of human tumors. Currently, no selective inhibitors of histone acetyltransferases in general, and particularly of Tip60, have been identified. Thus, in this proposal we propose to develop a high throughput assay to identify novel, specific inhibitors of the Tip60 histone acetyltransferases. 2 specific aims will be undertaken. In specific aim 1, a novel high throughput ELISA assay will be developed which utilizes the ability of recombinant Tip60 to acetylate immobilized peptides. In specific aim 2, the assay will be formatted to a 384-well format, and preliminary screening of active library compounds will be carried out. Tip60 represents a target for which an inadequate array of selective and potent small molecule inhibitors exist; screening and identifying such inhibitors would have a significant impact in studying the role of Tip60 and related HATs in the progression of human disease. This proposal will develop specific inhibitors of the Tip60 HAT which can be employed to disrupt cellular protein acetylation levels with the aim of improving treatment of human tumors. These inhibitors can be used in animal and clinical studies to test if Tip60 In activation sensitizes human tumors to radiation therapy and chemotherapy. In addition, Tip60 inhibitors can be employed to probe the basic signaling mechanisms by which Tip60 exerts its biological effects, and to identify new Tip60 substrates. Finally, Tip60 inhibitors can be used in complex organisms, such as mice, to examine the contribution of Tip60 to disease processes such as cancer progression, neurodegenerative diseases and genotoxic stress.