Chemical Modulators for p53 Functions in Transcriptional Regulation

p53 转录调控功能的化学调节剂

• 批准号: 8196764
• 负责人: SYED Shiraz MUJTABA
• 金额: $ 30.7万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-11 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8196764
• 关键词: Acetylation; Address; Apoptosis; Automation; BRD2 gene; Binding; Biological; Biological Assay; Biological Models; Bromodomain; C-terminal; CDKN1A gene; CREB-binding protein; Cancer Cell Growth; Cancer Etiology; Cancer cell line; Cell Cycle Arrest; Cell Death; Cells; Chemicals; Chromatin; DNA Binding; DNA Damage; DNA Methyltransferase; DNA Modification Methylases; Data; Deacetylation; Disease; Dose; EP300 gene; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Evaluation; Event; Fluorescence; Gene Expression Regulation; Gene Targeting; Genes; Goals; Growth; HIV; Health; Histone Code; Histone Deacetylase; Histone Deacetylase Inhibitor; Histones; Human; In Vitro; Individual; Inhibitory Concentration 50; Investigation; Libraries; Ligands; Luciferases; Lysine; Malignant Neoplasms; Measures; Mediating; Methylation; Modification; Molecular; Morphologic artifacts; Mutate; Normal Cell; Outcome; Outcome Study; PCAF gene; Pattern; Play; Post-Translational Protein Processing; Protein p53; Proteins; Quantitative Reverse Transcriptase PCR; Recruitment Activity; Regulation; Reporter; Reporter Genes; Reporting; Reproducibility; Research; Response Elements; Role; Screening procedure; Site; Specificity; Stress; Structure-Activity Relationship; System; Tail; Testing; Therapeutic; Toxic effect; Transcriptional Activation; Transcriptional Regulation; Tumor Cell Line; Tumor Suppressor Proteins; Ubiquitination; analog; base; cancer cell; cell growth; density; developmental disease; high throughput screening; histone acetyltransferase; human CREBBP protein; in vivo; inhibitor/antagonist; innovation; knock-down; miniaturize; nervous system disorder; oncoprotein p21; outcome forecast; promoter; response; senescence; small molecule; therapeutic development; tool; transcription factor; tumor

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.

描述（由申请人提供）：在超过50％的人类癌症中，最重要的人类肿瘤抑制蛋白p53突变。 p53的肿瘤抑制功能受到高密度翻译后修饰（PTM）的紧密控制，该修饰位于p53蛋白的N-和C末端尾巴上。 p53的压力诱导的C末端赖氨酸乙酰化在调节细胞周期停滞，衰老或凋亡的p53转录活性中起着核心作用。 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 p21,参与G1细胞周期停滞。该研究计划的长期目标是鉴定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，但这些赖氨酸残基对p53活性的个体或乙酰化的特定作用仍然难以捉摸。此外，尽管p53是序列特异性的DNA结合转录因子，但PTM在调节下游基因中的染色质以及p53的作用也未知。提出的初步数据评估乙酰化诱导的p53激活对DNA损伤的响应取决于共激活器的募集，并且进一步的p21启动子指导的荧光素酶报道器系统的潜力是为可自动化的高吞吐量筛选分析（HTS）开发为可靠的细胞测定法（HTS）。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转录激活的机械基础，重点是C末端翻译后修饰在p53激活中的作用。提出了三个特定的目标来实现此目标：（1）开发和验证基于P21荧光素酶报告基细胞的测定法，可用于HTS来识别与CBP BRD的SMI结合； （2）通过次级测定评估CBP BRD结合SMI，以排除体外和体内方法的伪像； （3）使用新开发的CBP溴结构域结合SMI来调节细胞生长停滞和凋亡之间的p53活性。预计拟议研究的新出现的结果将增强我们对p53靶基因调节的分子基础的理解。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具有重要意义。公共卫生相关性：肿瘤抑制蛋白p53在至少50％的人类癌症中突变。开发的基于细胞的测定方法将通过将癌细胞的命运变成细胞死亡来鉴定小分子抑制剂（SMI）来调节p53功能。新开发的CBP BRD SMI还将用作剖析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。

项目成果

The Biological Significance of Targeting Acetylation-Mediated Gene Regulation for Designing New Mechanistic Tools and Potential Therapeutics.

• DOI: 10.3390/biom11030455
• 发表时间: 2021-03-18
• 期刊: Biomolecules
• 影响因子: 5.5
• 作者: O'Garro C;Igbineweka L;Ali Z;Mezei M;Mujtaba S
• 通讯作者: Mujtaba S