G-quadruplexes formed in human oncogene promoters and their drug complexes

描述(申请人提供)：四个癌基因，c-myc，bcl-2，VEGF，和HIF-1a，都在其转录激活关键的启动子区域含有PolyG/PolyC区域。在这四种癌基因的启动子区域已经证实了DNA G-四链二级结构的存在，并被证明是一种转录调节因子。虽然DNA G-四链体是很有希望的新药靶点，但它们作为癌症治疗靶点的潜力的评估取决于对生物相关的G-四链体结构的理解。我们的初步研究使我们能够确定c-myc、bcl-2、VEGF和HIF-1a启动子区域中G-四联体的主要形式，它们似乎代表了三种不同的基本G-四联体结构，其中VEGF/HIF-1a G-四联体是相同的基本类型。启动子G-四链体的不同分子结构使这些结构成为特定途径药物设计的靶点。在这项提案中，我们打算定义每个G-四链体及其药物复合体的特定分子结构。获得的结构信息将与生物数据相关联，以了解有效的基因调控。深入了解启动子G-四链体及其药物复合体的结构，将为基于结构的合理药物设计提供重要依据。我们将检验我们的假设，即每个启动子G-四链可以被不同的小分子药物化合物特异性地靶向。除了通过与不同的G-四链核心结构相互作用来实现选择性的原理外，我们预计选择性也可以通过产生结合口袋的外部环和封顶结构内的相互作用来实现。在这方面，原则证明将是重要的。我们将结合使用核磁共振、CD、分子模拟和微量热学数据，并结合适当的突变启动子元件。我们的主要方法，高场核磁共振波谱，是确定生物相关的G-四链体结构的主要工具，因为这样的结构很难结晶。我们的最终目标是使用基于结构的方法来合理设计小分子G-四链相互作用化合物，这些化合物专门针对每个启动子特有的G-四链结构并调控基因转录。具体地说，我们计划1)确定c-myc启动子区域形成的生物相关G-四链体的分子结构及其药物相互作用；2)确定bcl2启动子区域形成的生物相关G-四链体的分子结构及其与药物的相互作用；以及3)确定在VEGF/HIF-1a的启动子区域形成的生物相关G-四链体的分子结构及其药物相互作用。

DESCRIPTION (provided by applicant): The four oncogenes, c-MYC, bcl-2, VEGF, and HIF-1a, all contain polyG/polyC tracts in their promoter regions critical for transcriptional activation. The occurrence of DNA G-quadruplex secondary structures has been demonstrated in the promoter regions of these four oncogenes and has been shown to be a transcriptional modulator. While the DNA G-quadruplexes are promising new drug targets, the evaluation of their potential as cancer therapeutic targets depends on the understanding of biologically relevant G-quadruplex structures. Our preliminary studies have allowed us to identify the predominant forms of G-quadruplexes in the promoter regions of c-MYC, bcl-2, VEGF, and HIF-1a, which appear to represent three different basic G-quadruplex structures with VEGF/HIF-1a G-quadruplexes being the same basic type. The different molecular structures of the promoter G-quadruplexes make these structures attractive targets for pathway-specific drug design. In this proposal we intend to define the specific molecular structure of each G-quadruplex and its drug-complex(es). The structural information obtained will be correlated with the biological data to understand the effective gene modulation. Insight into the structures of the promoter G-quadruplexes and their drug complexes will provide an important basis for structure-based rational drug design. We will test our hypothesis that each promoter G-quadruplex can be specifically targeted by different small molecule drug compounds. In addition to the principle that selectivity can be achieved by interactions with different G-quadruplex core structures, we expect that selectivity can also be achieved by interactions within the external loops and capping structures in which binding pockets are generated. Proof of principle will be important in this regard. We will use a combination of NMR, CD, molecular modeling, and microcalorimetry data in concert with appropriate mutant promoter elements. Our primary approach, high field NMR spectroscopy, represents a major tool for structure determination of biologically relevant G-quadruplexes, due to the difficulty of crystallization of such structures. Our ultimate objective is to use a structure-based approach to rationally design small molecule G-quadruplex-interactive compounds that specifically target the G-quadruplex structure unique to each promoter and modulate gene transcription. Specifically, we plan to 1) determine the molecular structure of the biologically relevant G-quadruplex formed in the promoter region of c-MYC and its drug interactions; 2) determine the molecular structure of the biologically relevant G-quadruplex formed in the promoter region of bcl-2 and its drug interactions; and 3) determine the molecular structure of the biologically relevant G-quadruplex formed in the promoter region of VEGF/HIF-1a and its drug interactions.