Targeting DNA Secondary Structures for Bcl-2 Gene Regulation

针对 Bcl-2 基因调控的 DNA 二级结构

基本信息

批准号：
8215900
负责人：
DANZHOU YANG
金额：
$ 29.46万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8215900
关键词：
Address Affect Affinity Apoptosis BCL2 gene Bacteriophage lambda Binding Binding Proteins Binding Sites Biochemical Biological Biological Assay Cardiovascular Diseases Cells Complex Crystallization DNA DNA Sequence Data Disease Drug Delivery Systems Drug Design Drug Interactions EMSA Effectiveness Fluorescence Resonance Energy Transfer G-Quartets GC Rich Sequence Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Goals Guanine Human Lead Malignant Neoplasms Mediating Membrane Proteins Methods Molecular Molecular Probes Molecular Structure NMR Spectroscopy Operator Regions Pharmaceutical Preparations Physiological Plasmids Play Promoter Regions Protein Binding Proteins Regulation Reporter Repressor Proteins Research Role Running Screening procedure Specificity Structure Techniques Testing Transcriptional Regulation Transcriptional Silencer Elements Up-Regulation WT1 Protein Work base c-myc Genes design drug structure human disease in vivo inhibitor/antagonist insight nervous system disorder novel promoter protein function public health relevance small molecule small molecule libraries tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): Bcl-2 is a membrane protein that functions as an inhibitor of cell apoptosis. Aberrant levels of bcl-2 are associated with many human diseases including cancer, neurological disorders, and cardiovascular diseases. Effective modulation of bcl-2 expression offers promise for the treatment of these diseases. We have found that the human bcl-2 gene contains a GC-rich proximal promoter region that can form two stable intramolecular G-quadruplex DNA secondary structures using overlapping guanine-rich DNA sequences. This GC-rich region contains a binding site of the WT1 protein which has been shown to be a negative regulator of the bcl-2 gene expression. We have recently developed a screening assay of small molecule compounds that can selectively bind the bcl-2 promoter G-quadruplex structures. Intriguingly, these compounds have been shown to upregulate the bcl-2 gene transcription. The hypothesis to be tested is that stabilization of the bcl-2 promoter G-quadruplex secondary structure(s) with small molecules upregulates bcl-2 gene transcription by inhibiting the binding of the negative regulator WT1 protein. A G-quadruplex DNA secondary structure has been demonstrated to be a transcriptional silencer element in the proximal promoter region of the human c-Myc gene and is amenable to small molecule drug targeting. The G-quadruplexes formed in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-Myc promoter in that their formation could serve to modulate gene transcription. However, the complexity of the G-quadruplex structures in the bcl-2 promoter is higher than is the case for the c-Myc promoter. The presence of two interchangeable G-quadruplexes overlapping in the region of the G-rich strand is likely to be important for the precise regulation of bcl-2 gene transcription, as each G-quadruplex may bind to different proteins leading to different gene modulation, in a manner analogous to the genetic switch in the bacteriophage lambda controlled by the interactive Cro and Repressor proteins, whose operator regions (ORs) overlap with each other's promoter regions and thereby inhibit each other's transcription. In this proposal we aim to determine the biological roles and molecular structures for the bcl-2 promoter G-quadruplex structures. Our primary approach, high-field NMR spectroscopy, represents a major tool for determination of DNA secondary structures under physiological conditions, due to the difficulty of crystallization of such structures. Our long-term goal is to use structure-based rational design to develop small molecule compounds that specifically target the bcl-2 promoter G-quadruplex structures and effectively modulate bcl-2 gene expression. Specifically, we plan to 1) determine the functional significance of the two interchangeable bcl-2 promoter G-quadruplexes in bcl-2 gene regulation and how binding of G-quadruplex-stabilizing compounds affects the regulation of the bcl-2 gene; and 2) to determine the structures of the two interchangeable bcl-2 promoter G-quadruplexes and their complexes with G-quadruplex-stabilizing compounds. PUBLIC HEALTH RELEVANCE: Aberrant levels of bcl-2 are associated with many human diseases including cancer, neurological disorders, and cardiovascular diseases. Effective modulation of bcl-2 expression offers promise for the treatment of these diseases. The proposed research represents a novel new strategy for modulating bcl-2 gene expression by small molecule drugs.

描述（由申请人提供）：Bcl-2是一种膜蛋白，可作为细胞凋亡的抑制剂。异常水平的Bcl-2与许多人类疾病有关，包括癌症，神经系统疾病和心血管疾病。 BCL-2表达的有效调节为治疗这些疾病提供了希望。我们发现，人Bcl-2基因包含一个富含GC的近端启动子区域，可以使用富含鸟嘌呤的DNA序列形成两种稳定的分子内G-四链体DNA二级结构。该富含GC的区域包含WT1蛋白的结合位点，已证明是Bcl-2基因表达的负调节剂。我们最近开发了针对小分子化合物的筛选测定法，该测定方法可以选择性地结合Bcl-2启动子G Qu-Quadruplex结构。有趣的是，这些化合物已被证明可以上调Bcl-2基因转录。要检验的假设是，用小分子稳定Bcl-2启动子G四链体二级结构，通过抑制负调节剂WT1蛋白的结合来上调Bcl-2基因转录。 G-四链体DNA二级结构已被证明是人类C-MYC基因近端启动子区域中的转录消音器元件，并且适合小分子药物靶向。 Bcl-2基因启动子区域中形成的G四链体可能与C-MYC启动子中的G Qu-四链体的作用相似，因为它们的形成可以调节基因转录。然而，Bcl-2启动子中G-四链体结构的复杂性高于C-MYC启动子的情况。富含G链区域中两种可互换的G四链体的存在对Bcl-2基因转录的精确调节可能很重要，因为每种G Quadruplex可能与不同的基因调节的不同蛋白质结合，以不同的方式与不同的基因模型相似，而与细菌刺激的遗传型在相似的方式相似，并与细菌蛋白质相似，并将其相互作用的蛋白质转移在细菌protister lamb Doties Croper中，并以互动型的影响，并将其相互作用的蛋白质转移到相互作用。（ORS）与彼此的启动子区域重叠，从而抑制彼此的转录。在此提案中，我们旨在确定Bcl-2启动子G季链体结构的生物学作用和分子结构。由于这种结构的结晶难度，我们的主要方法，高场NMR光谱法代表了在生理条件下确定DNA二级结构的主要工具。我们的长期目标是使用基于结构的合理设计来开发小分子化合物，这些化合物专门针对Bcl-2启动子G Qu-quadruplex结构并有效调节Bcl-2基因表达。具体而言，我们计划以1）确定Bcl-2基因调节中两个可互换Bcl-2启动子G四链体的功能意义以及G-四链体稳定化合物的结合如何影响Bcl-2基因的调节； 2）确定两个可互换的Bcl-2启动子G四链体的结构及其与G-四链体稳定化合物的复合物。公共卫生相关性：异常水平的Bcl-2与许多人类疾病有关，包括癌症，神经系统疾病和心血管疾病。 BCL-2表达的有效调节为治疗这些疾病提供了希望。拟议的研究代表了一种新的新策略，用于通过小分子药物调节Bcl-2基因表达。