Inhibition of Protein-Protein Contacts in VEGF Transcription by Synthetic Helices

描述（由申请人提供）：血管生成是程序性生长的血管，受许多特异性促有丝分裂因子的严格控制，其中血管内皮生长因子（VEGF）及其受体起着核心作用。VEGF的水平在广泛的肿瘤中上调，并参与癌症生物学的关键方面。作为许多癌症的标志，慢性缺氧与某些致癌信号传导途径的激活一起导致VEGF水平升高，并与侵袭和能量代谢改变相关。由于严格控制缺氧诱导基因的表达对细胞的存在至关重要，因此首要的目标是开发控制恶性细胞中缺氧诱导基因同时使正常细胞不受影响的方法。为了实现这一目标，我们寻求发现特异性调节缺氧诱导转录的合成分子。我们假设，转录过程可以通过破坏关键的转录因子-共激活因子相互作用来有效地调节，所述相互作用涉及蛋白p300的CH 1结构域或同源CBP和缺氧诱导因子1的C末端反式激活结构域（C-CET）。这种复合物的特点是界面处的短螺旋结构域，并表明这些螺旋的合成模拟物将调节蛋白质-蛋白质相互作用。在本申请中，我们利用一类新的具有二级结构的人工α-螺旋，其模拟HIF-1的生物学相关片段（C-CRYSTAL）。在初步研究中，我们已经表明，破坏Hif-1 β/p300与我们的合成螺旋的相互作用，导致细胞培养物中癌症进展中重要的缺氧诱导基因（包括VEGF）的快速下调。我们的具体目标是：（1）设计和合成人工螺旋作为转录因子-辅激活因子复合物的抑制剂;（2）评估每种抑制剂与其蛋白质靶标的结合热力学，测试它们在体外抑制同源蛋白质-蛋白质相互作用的能力，和（3）测试人工螺旋破坏HIF-1 α转录的能力。癌症中的诱导基因，并在分子水平上探索这种破坏的机制细节。结合这三个目标将验证我们的假设，并为开发一类新的基于结构和机制的癌症治疗方法奠定基础。 人类基因组测序和蛋白质组学的最新进展使人们更好地了解遗传内容与疾病之间的关系。因此，基于控制患病细胞中基因表达的新型疗法的开发已成为越来越重要的目标。通过将化学合成技术与分子生物学和遗传学方法相结合，我们的目标是发现独特的特异性小分子，这些小分子可作为癌症中缺氧诱导转录的抑制因子，长期目标是开发新的治疗方法来治疗有氧糖酵解和血管生成。

DESCRIPTION (provided by applicant): Angiogenesis, the programmed growth blood vessels, is tightly controlled by a number of specific mitogenic factors, among which vascular endothelial growth factor (VEGF) and its receptors play a central role. The levels of VEGF are up-regulated across a broad range of tumors and are involved in key aspects of cancer biology. A hallmark of many cancers, chronic hypoxia, in conjunction with activation of certain oncogenic signaling pathways, is responsible for the elevated levels of VEGF and is associated with invasion and altered energy metabolism. Because tight control of hypoxia-inducible gene expression is critical for cellular existence, the goal of primary importance has been to develop methods of controlling hypoxia-inducible genes in malignant cells while leaving normal cells unaffected. To address this goal, we seek to uncover synthetic molecules that specifically regulate hypoxia-inducible transcription. We hypothesize that the process of transcription could be effectively modulated via disruption of key transcription factor-coactivator interactions involving CH1 domain of protein p300 or the homologous CBP and the C-terminal transactivation domain (C-TAD) of the hypoxia-inducible factor 1(. This complex features short (-helical domains at the interface and suggests that synthetic mimics of these helices would modulate the protein-protein interaction. In this application, we utilize a new class of artificial (-helices with secondary structure that mimics the biologically relevant fragment of HIF-1( C-TAD. In preliminary studies, we have shown that disruption of the Hif-1(/p300 interaction with our synthetic helices results in rapid downregulation of important in cancer progression hypoxia-inducible genes, including VEGF, in cell culture. Our specific aims are to: (1) to design and synthesize artificial helices as inhibitors of the transcription factor-coactivator complex; (2) evaluate binding thermodynamics of each inhibitor toward their protein targets, test their ability to inhibit the cognate protein-protein interaction in vitro, and (3) test the ability artificial helices to disrupt transcription of HIF-inducible genes in cancer and explore the mechanistic details of this disruption at the molecular level. Combined these three aims will validate our hypothesis and create a foundation for the development of a new class of structure and mechanism-based cancer therapeutics. PUBLIC HEALTH RELEVANCE The sequencing of the human genome and recent advances in proteomics have led to a better understanding of the relationship between genetic content and disease. As a result, the development of novel therapies based on controlling gene expression in diseased cells has become an increasingly important goal. By combining the art of chemical synthesis with the methods of molecular biology and genetics, we aim to uncover uniquely specific small molecules that act as suppressors of hypoxia-inducible transcription in cancer with the long-term goal of developing new therapeutics for treatment of aerobic glycolysis and angiogenesis.