Chemical Modulators for p53 Functions in Transcriptional Regulation

描述（由申请人提供）：最重要的人类肿瘤抑制蛋白p53在超过50%的人类癌症中突变。p53的肿瘤抑制功能由高密度翻译后修饰（PTM）严格控制，所述PTM位于p53蛋白的N-和C-末端尾部。应激诱导的p53蛋白C末端赖氨酸乙酰化在p53蛋白转录活性中起着重要作用，调节细胞周期阻滞、衰老或凋亡。我们的研究表明，p53的共激活因子CBP（CREB结合蛋白）的募集需要CBP的保守溴结构域（CBP BRD）与p53在乙酰化赖氨酸382（p53 K382 ac）：一个特定的分子相互作用，是必不可少的p53诱导的转录激活的细胞周期蛋白依赖性激酶抑制剂p21，参与G1细胞周期阻滞。本研究计划的长期目标是确定CBP BRD的小分子抑制剂（SMI），以调节癌细胞中的p53功能。虽然已经报道了p53的C-末端尾部中的多个乙酰化位点，但是这些赖氨酸残基的单独或组合乙酰化对p53活性的具体影响仍然是难以捉摸的。此外，尽管p53是序列特异性DNA结合转录因子，但染色质上的PTM以及p53在调节下游基因中的作用也是未知的。所呈现的初步数据评估乙酰化诱导的p53激活响应于DNA损伤依赖于共激活因子募集，并且进一步评估了p21启动子指导的荧光素酶报告系统被开发为用于自动化高通量筛选测定（HTS）的稳健的基于细胞的测定的潜力。我们假设，CBP BRD的高度特异性和选择性SMI可以改变癌细胞的命运，从生长停滞到凋亡。提出了一种多方面的方法来解决p53转录激活的机制基础，重点是C-末端翻译后修饰在p53激活中的作用。为实现这一目标，提出了三个具体目标：（1）开发和验证基于p21荧光素酶报告细胞的检测方法，该方法可用于HTS，以鉴定SMI与CBP BRD的结合;（2）通过二级检测方法评价CBP BRD结合SMI，以排除体外和体内方法的伪影;以及（3）使用新开发的CBP Bromodomain结合SMI在细胞生长停滞和凋亡之间调节p53活性。从拟议的研究中出现的结果，预计将提高我们的理解p53靶基因调控的C-末端修饰的分子基础。鉴于p53在癌症中的核心作用，这些研究将对人类肿瘤的预后和治疗产生重要影响。公共卫生相关性：肿瘤抑制蛋白p53在至少50%的人类癌症中发生突变。开发的基于细胞的测定将鉴定小分子抑制剂（SMI），以通过改变癌细胞的命运来调节p53功能。新开发的CBP BRD SMI也将被用作剖析p53分子相互作用的创新工具，这可能为治疗开发开辟新的途径。

DESCRIPTION (provided by applicant): The most essential human tumor suppressor protein p53 is mutated in more than 50% of the human cancers. The tumor suppressor function of p53 is tightly controlled by high density post-translational modifications (PTMs), which are localized on N- and C-terminal tails of the p53 protein. Stress-induced C-terminal lysine acetylation of p53 plays a central role in the p53 transcriptional activities that regulates cell cycle arrest, senescence or apoptosis. Our study revealed that p53 recruitment of the co-activator CBP (CREB binding protein) requires association of the conserved bromodomain of CBP (CBP BRD) with p53 at acetylated Iysine 382 (p53K382ac): a specific molecular interaction that is essential for p53-induced transcriptional activation of the cyclin dependent kinase inhibitor p21, involved in G1 cell cycle arrest. The long-term goal of this research plan is to identify small molecule inhibitors (SMIs) of CBP BRD to modulate the p53 function in cancer cells. While multiple acetylation sites in the C-terminal tail of p53 have been reported, specific effects of individual or combined acetylation of these lysine residues on p53 activity remain elusive. Further, despite p53 being a sequence-specific DNA binding transcription factor, the role of PTMs on chromatin as well as p53 in regulating downstream genes is also unknown. The presented preliminary data evaluates acetylation-induced p53 activation in response to DNA damage is dependent on co-activator recruitment, and further, potential of the p21 promoter-directed luciferase reporter system to be developed as a robust cell based assay for automated high throughput screening assay (HTS). We hypothesize that highly specific and selective SMIs for CBP BRD could alter the fate of the cancer cells from growth arrest to apoptosis. A multifaceted approach is proposed to address mechanistic underpinnings of p53 transcriptional activation with emphasis on the role of C-terminal post-translational modifications in p53 activation. Three specific aims are proposed to achieve this goal: (1) to develop and validate p21 luciferase reporter cell based assay that could be used for HTS to identify SMI binding to CBP BRD; (2) to evaluate CBP BRD binding SMIs by secondary assays to rule out artifacts by in vitro and in vivo approaches; and (3) to modulate p53 activity between cell growth arrest and apoptosis using the newly developed CBP Bromodomain-binding SMIs. The emerging results from the proposed studies are expected to enhance our understanding of the molecular basis of p53 target gene regulation by its C-terminal modifications. Given the central role of p53 in cancer, these studies will have important implications for the prognosis and treatment of human tumors. PUBLIC HEALTH RELEVANCE: The tumor suppressor protein p53 is mutated in atleast 50% of human cancers. The developed cell based assay will identify small molecule inhibitors (SMIs) to modulate p53 function by changing the fate of cancer cells to cell death. The newly developed CBP BRD SMIs will be also used as innovative tools to dissect molecular interactions of p53, which could open new avenues for therapeutic development.