Small Molecule Protein Ligands as Modulators of Hypoxia-Inducible Transcription

小分子蛋白配体作为缺氧诱导转录的调节剂

• 批准号: 8865244
• 负责人: Bogdan Olenyuk
• 金额: $ 33.3万
• 依托单位: PROTEOGENOMICS RESEARCH INSTIT/SYS/ MED
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-05 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8865244
• 关键词: Accounting; Affinity; Apoptotic; Arts; Binding; Biological Assay; Biological Factors; Blood Vessels; C-terminal; CREB-binding protein; CXCR4 gene; Cell Culture Techniques; Cell Nucleus; Cells; Characteristics; Chemicals; Chronic; Complex; DNA Sequence; Development; Disease; Down-Regulation; E1A-associated p300 protein; EP300 gene; Energy Metabolism; Enzyme-Linked Immunosorbent Assay; Event; Fluorescence Polarization; Future; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Transcription; Goals; Growth; HIF1A gene; Human; Hydroxylation; Hypoxia; Hypoxia Inducible Factor; In Vitro; Interleukin-2; LOX gene; Laboratories; Lead; Length; Libraries; Ligand Binding; Ligands; Link; Luciferases; Malignant Neoplasms; Mediating; Membrane Proteins; Methodology; Methods; Molecular; Molecular Biology; Mus; Neoplasm Metastasis; Organ; Oxidation-Reduction; Oxygen; Pathway Analysis; Pathway interactions; Patients; Pharmacology; Play; Polyubiquitination; Process; Prognostic Factor; Property; Proteins; Proteomics; Recruitment Activity; Regulation; Research; Response Elements; Selenium; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Solid; Solid Neoplasm; Structure; Surface; Surface Plasmon Resonance; Testing; Therapeutic; Titrations; Transactivation; Transcription Coactivator; Transcriptional Activation; Vascular Endothelial Growth Factors; Work; Xenograft Model; analog; angiogenesis; base; cancer therapy; chaetocin; chemical synthesis; chetomin; design; efficacy testing; genome-wide; human CREBBP protein; human genome sequencing; hypoxia inducible factor 1; in vitro testing; in vivo; inhibitor/antagonist; microcalorimetry; mimetics; neovascularization; novel; nuclear receptor coactivator 1; overexpression; prevent; protein protein interaction; public health relevance; receptor; research study; response; small molecule; therapy development; tool; transcription factor; tumor; tumor growth; tumor progression; tumor xenograft

 DESCRIPTION (provided by applicant): Hypoxia, the state of reduced oxygen levels, triggers a multifaceted adaptive response mediated by oxygen- dependent transcription factors, termed hypoxia-inducible factors (HIFs). Among these, hypoxia-Inducible Factor 1 (HIF1) is the main regulator of oxygen-dependent transcription in a majority of organs and accounts for the increase in expression of hypoxia-inducible genes. HIF controls an expression of over 100 genes involved in angiogenesis, altered energy metabolism, anti-apoptotic, and pro-proliferative mechanisms. Elevated levels of HIF1 are an independent, prognostic factor for a diverse range of malignant neoplasms. We are developing small molecules chemical tools that provide exquisite control over transcriptional activation of hypoxia-inducible genes implicated in cancer progression. Specifically, we will target three key signaling events mediated by three distinct protein-protein interactions with unique synthetic ligands. Small molecules often interact non-specifically with large protein surfaces; and hence inhibition of protein-protein interactions with small molecules remains difficult. Nevertheless, we recently demonstrated that hypoxia-inducible signaling can be effectively modulated in vitro and in vivo by rationally designed dimeric epidithiodiketopiperazines (ETPs) - ligands that bind p300/CBP CH1 region, induce structural change to these domains and prevent it from interacting with HIF1 C-terminal transactivation domain. The specific hypothesis behind the proposed research is that tumor progression could be effectively down-regulated by targeting of HIF1 C-TAD in complex with the CH1 domain of the coactivator protein p300/CBP with these designed molecules. Specific Aim 1 focuses on synthesis of conformational mimetics of the dimeric epidithiodiketopiperazine (ETP) natural products, characterization of the ETPs binding properties via isothermal titration microcalorimetry (ITC), surface plasmon resonance (SPR), and fluorescence polarization competition experiments. In Specific Aim 2 we will evaluate the stability of dimeric ETPs in cell culture and test efficacy in inhibiting HIF-inducible transcription with luciferase assays, qRT-PCR, ELISA. In this Aim we will also investigate structural basis of the inhibition of HIF1-coactivator interactions by employing site- directed mutagenesis and structural NMR. In Specific Aim 3 we will explore mechanistic framework of ETPs with pathway-based RT2 Profiler assays, gene expression profiling, and pathway analysis. We will also conduct in vivo experiments in order to determine efficacy of the ETPs in our mouse tumor xenograft models. Combined the three aims will validate our hypothesis and could lead to novel, unique tools for dissecting the hypoxia-inducible signaling pathway in cancer and facilitate development of future therapeutics.

 描述（由申请人提供）：缺氧，即氧水平降低的状态，触发由称为缺氧诱导因子（HIF）的氧依赖性转录因子介导的多方面适应性反应。其中，缺氧诱导因子1（HIF 1）是大多数器官中氧依赖性转录的主要调节因子，并导致缺氧诱导基因表达增加。HIF控制100多个基因的表达，这些基因参与血管生成、改变能量代谢、抗凋亡和促增殖机制。HIF 1水平升高是多种恶性肿瘤的独立预后因素。我们正在开发小分子化学工具，这些工具可以精确控制与癌症进展有关的缺氧诱导基因的转录激活。具体来说，我们将针对三个关键的信号传导事件介导的三个不同的蛋白质-蛋白质相互作用与独特的合成配体。小分子通常与大蛋白质表面非特异性相互作用;因此抑制蛋白质-蛋白质相互作用， 小分子仍然很难。然而，我们最近证明，缺氧诱导的信号可以有效地调制在体外和体内通过合理设计的二聚epidithiodiketopiperazines（ETP）-配体结合p300/CBP CH 1区，诱导这些结构域的结构变化，并防止它与HIF 1 α C-末端的反式激活结构域相互作用。这项研究背后的具体假设是，通过用这些设计的分子靶向与共激活蛋白p300/CBP的CH 1结构域复合的HIF 1 α C-peptide，可以有效地下调肿瘤的进展。具体目标1侧重于合成的构象模拟物的二聚epidithiodiketopiperazine（ETP）天然产物，表征的ETP结合性能通过等温滴定微量热法（ITC），表面等离子体共振（SPR），和荧光偏振竞争实验。在具体目标2中，我们将评估二聚体ETP在细胞培养物中的稳定性，并使用荧光素酶测定、qRT-PCR、ELISA测试抑制HIF诱导型转录的功效。在本研究中，我们还将通过定点突变和结构核磁共振研究HIF 1 α-辅激活因子相互作用抑制的结构基础。在具体目标3中，我们将探索ETP的机制框架与基于路径的RT 2 Profiler分析，基因表达谱分析和路径分析。我们还将进行体内实验以确定ETP在我们的小鼠肿瘤异种移植模型中的功效。结合这三个目标将验证我们的假设，并可能导致新的，独特的工具，解剖缺氧诱导的信号通路在癌症和促进未来的治疗方法的发展。