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(申请人提供)：缺氧，氧气水平降低的状态，触发由氧气依赖的转录因子介导的多方面适应性反应，称为缺氧诱导因子(HIF)。其中，低氧诱导因子1(HIF1)是大多数器官中氧依赖转录的主要调节因子，并解释了低氧诱导基因表达的增加。HIF控制着100多个基因的表达，这些基因涉及血管生成、能量代谢改变、抗凋亡和促增殖机制。HIF1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，提供对与癌症进展有关的低氧诱导基因转录激活的精细控制。具体地说，我们将针对由三种不同的蛋白质-蛋白质与独特的合成配体相互作用所介导的三个关键信号事件。小分子经常与大的蛋白质表面非特异性地相互作用；因此，抑制蛋白质与蛋白质之间的相互作用 小分子仍然是困难的。然而，我们最近证明，在体外和体内，通过合理设计二聚体的表硫二酮基哌嗪(ETP)，可以有效地调节缺氧诱导的信号转导。ETP是一种与p300/CBPCH1区域结合的配体，可以诱导这些区域的结构变化，并阻止其与HIF1C端反式激活结构域的相互作用。这项研究背后的具体假设是，通过将HIF1CBP C-TAD与辅激活蛋白p300/的CH1结构域与这些设计的分子进行靶向，可以有效地下调肿瘤的进展。具体目标1着重于合成二聚体二硫代二酮基哌嗪(ETP)天然产物的构象模拟物，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表征ETP的结合性质。在特定的目标2中，我们将评估二聚体ETP在细胞培养中的稳定性，并通过荧光素酶分析、qRT-PCR、ELISA法测试抑制HIF诱导转录的效果。在这一目标中，我们还将通过定点突变和结构核磁共振来研究抑制HIF1-辅活化子相互作用的结构基础。在具体目标3中，我们将通过基于途径的RT2 Profiler分析、基因表达谱分析和途径分析来探索ETP的机制框架。我们还将进行体内实验，以确定ETPS在我们的小鼠肿瘤异种移植模型中的有效性。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，在癌症中解剖低氧诱导的信号通路，并促进未来的治疗方法的发展。