Targeting Tumor Angiogenesis with G-Quadruplex Binders

描述（由申请人提供）：血管内皮生长因子（VEGF）和缺氧诱导因子（HIF）-la在肿瘤血管生成中起关键作用。该提议的总体目标是探索一种新的治疗策略，其旨在通过用小分子靶向在这些基因的启动子区域中形成的G-四链体结构来抑制VEGF和HIF-1 α基因的转录，从而预防肿瘤血管生成。（1）研究肿瘤细胞中VEGF和HIF-1 α基因启动子区是否存在G-四链体结构，并能被小分子靶向。(2)确定VEGF和HIF-1 α启动子区域中的G-四链体结构在这些基因的调节中的作用，以及G-四链体相互作用化合物如何调节这些基因的调节。(3)探讨G-四链体相互作用剂作为一类新型抗血管生成剂的潜在应用，其可以抑制VEGF和HIF-1 α基因的转录活性。(4)为了发现新类别的G-四链体化合物或基于已知类别的G-四链体相互作用剂的新先导化合物，其特异性抑制由VEGF和HIF-1 α基因的启动子活性介导的转录激活。将通过使用体内化学和酶探测技术研究VEGF和HIF-1 α基因的启动子区中G-四链体结构的形成。我们将使用突变分析和启动子报告分析，以确定在这些基因的启动子区域中形成的G-四链体结构的作用。利用多重RT-PCR，微阵列分析，ChIP测定，生物发光成像技术，和血管生成测定，我们将评估在体外和体内的目标选择性和抗血管生成活性的G-四链体相互作用剂。最后，将利用无细胞生物化学测定和基于细胞的高通量筛选系统来鉴定在细胞培养系统中选择性下调VEGF或HIF-1 α表达的其他先导化合物。这些研究直接导致了G-四链体相互作用剂在人肾癌的临床前模型中的抗血管生成和抗肿瘤作用的评价，人肾癌是一种肿瘤类型，其特征在于由于von Hippel Lindau（VHL）肿瘤抑制基因的频繁丢失而导致HIF-1 α和VEGF的异常过表达。

DESCRIPTION (provided by applicant): The vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF)-la play a pivotal role in tumor angiogenesis. The overall objective of this proposal is to explore a new therapeutic strategy aimed at preventing tumor angiogenesis by repressing the transcription of VEGF and HIF-la genes through targeting G-quadruplex structures formed in the promoter region of these genes with small molecules. The specific aims of this proposal are as follows: (1) To investigate whether G-quadruplex structures are formed in vivo in the promoter regions of VEGF and HIF-la genes in tumor cells and can be targeted with small molecules. (2) To determine the role of G-quadruplex structures in VEGF and HIF-la promoter regions in the regulation of these genes, and how G-quadruplex interactive compounds modulate the regulation of these genes. (3) To explore the potential application of G-quadruplex-interactive agents as a new class of anti-angiogenic agents, which can repress the transcriptional activity of the VEGF and HIF-la genes. (4) To discover new classes of G-quadruplex compounds or new lead compounds based on known classes of G-quadruplex-interactive agents that specifically inhibit the transcriptional activation mediated by the promoter activity of VEGF and HIF-la genes. The formation of G-quadruplex structures in the promoter region of the VEGF and HIF-la genes will be studied by using in vivo chemical and enzymatic probing techniques. We will use mutation analysis and promoter reporter assays to determine the role of G-quadruplex structures formed in the promoter region of these genes. Utilizing multiple RT-PCR, microarray analysis, ChIP assay, bioluminescent imaging techniques, and angiogenesis assays, we will evaluate in vitro and in vivo target selectivity and antiangiogenic activity of G-quadruplex-interactive agents. Lastly, cell free biochemical assays and cell based high-throughput screening system will be utilized to identify other lead compounds that selectively downregulate either VEGF or HIF-la expression in a cell culture system. These studies lead directly to the evaluation of the anti-angiogenic and antitumor effects of G- quadruplex-interactive agents in a preclinical model of human kidney cancer, a tumor type characterized by the abnormal overexpression of HIF-la and VEGF due to the frequent loss of the von Hippel Lindau (VHL) tumor suppressor gene.