Site Specific Alkylation of the HER2/neu Promoter

HER2/neu 启动子的位点特异性烷基化

• 批准号: 6729846
• 负责人: SCOT W EBBINGHAUS
• 金额: $ 31.92万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2005-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6729846
• 关键词: Adenoviridae; alkylation; drug design /synthesis /production; epidermal growth factor; gene mutation; genetic promoter element; growth factor receptors; neoplasm /cancer genetics; neoplasm /cancer pharmacology; nitrogen mustard; nonsmall cell lung cancer; oligonucleotides; polymerase chain reaction; protooncogene; southern blotting; tissue /cell culture; transfection /expression vector

DESCRIPTION (PROVIDED BY APPLICANT): The HER-2/neu oncogene appears to play an important role in the initiation and progression of many types of human cancer, including approximately 25 percent of non-small cell lung cancer (NSCLC), the leading cause of cancer death in both men and women in the U.S. The overall goal of this project is to find novel ways to specifically inhibit HER-2/neu expression by developing triplex forming oligonucleotide (TFO)-alkylator conjugates what will bind in a site-specific manner to the HER-2/neu promoter by triplex DNA formation and lead to covalent DNA modification at specific guanine bases by the DNA alkylating agent. The specific aims of this proposal are designed to address the major obstacles to the successful development of a TFO-based DNA binding drug. Site-specific alkylation with TFOs conjugated to nitrogen mustards will be demonstrated in the HER-2/neu promoter. These data will be rationalized by molecular modeling, and these models will be used to further refine the design of the TFO-alkylator conjugates (Specific Aim 1). Much of this work has now been accomplished, and future work will focus on the design of novel conjugates with a minor groove DNA binding agent and the investigation of nuclease resistant oligonucleotide backbone modifications. The ability of these compounds to inhibit transcription initiation will be studied by triple helix formation in a reporter plasmid transiently transfected into NSCLC cell lines, and the ability of NSCLC cells to recognize and repair triplex directed DNA alkylation will be characterized after transfection (Specific Aim 2). Our preliminary data demonstrates that a TFO conjugated to a nitrogen mustard at both the 3' and 5' ends can direct two guanine adducts adjacent to both ends of the triple helix to resist DNA repair and suppress HER-2/neu promoter activity in NSCLC cells. An adenovirus based ODN delivery system will be developed, and the ability of adenoviruses to mediate ODN uptake and nuclear localization will be determined when adenovirus-ODN complexes are formed by a semi-stable chemical linker (Specific Aim 3). The ability of triplex forming ODNs to bind to the endogenous HER-2/neu gene and direct site-specific DNA alkylation will be studied by Southern blot and ligation mediated-PCR after the treatment of NSCLC cells with the TFO-alkylator conjugates (Specific Aim 4). The therapeutic potential of these compounds to inhibit HER-2/neu expression and alter the malignant and invasive phenotype of NSCLC cells will be evaluated in tissue culture (Specific Aim 5). The development of a HER-2/neu specific anti-gene compound will provide a great deal of information about the role of the HER-2/neu gene in the initiation and progression of NSCLC and may lead to novel treatment approaches for HER-2/neu expressing cancers such as NSCLC.

描述（由申请人提供）：HER-2/neu 癌基因似乎发挥作用 在多种人类癌症的发生和进展中发挥重要作用， 包括约 25% 的非小细胞肺癌 (NSCLC) 癌症是美国男性和女性死亡的主要原因 该项目的目标是寻找特异性抑制 HER-2/neu 的新方法 通过开发三链体形成寡核苷酸 (TFO) 烷基化剂进行表达 缀合将以位点特异性方式与 HER-2/neu 启动子结合的物质 通过三链体 DNA 形成并导致特定的共价 DNA 修饰 DNA烷化剂对鸟嘌呤碱基进行修饰。本提案的具体目标 旨在解决成功开发的主要障碍 基于TFO的DNA结合药物。用 TFO 进行位点特异性烷基化 氮芥将在 HER-2/neu 启动子中得到证实。这些数据 将通过分子建模合理化，这些模型将用于 进一步完善 TFO-烷基化剂缀合物的设计（具体目标 1）。 目前大部分工作已经完成，未来的工作重点是 具有小沟 DNA 结合剂的新型缀合物的设计和 核酸酶抗性寡核苷酸主链修饰的研究。这 将研究这些化合物抑制转录起始的能力 通过在瞬时转染的报告质粒中形成三螺旋 NSCLC细胞系以及NSCLC细胞的识别和修复能力 转染后将表征三链体定向 DNA 烷基化 （具体目标 2）。我们的初步数据表明，TFO 与 3' 和 5' 末端的氮芥可以引导两个鸟嘌呤加合物 邻近三螺旋两端，抵抗DNA修复并抑制 NSCLC 细胞中的 HER-2/neu 启动子活性。基于腺病毒的 ODN 递送 系统将被开发，腺病毒介导ODN摄取的能力 当腺病毒-ODN复合物被确定时，核定位将被确定。 由半稳定化学连接体形成（具体目标 3）。的能力 三链体形成 ODN，结合内源 HER-2/neu 基因并指导 将通过 Southern 印迹和连接研究位点特异性 DNA 烷基化 用 TFO 烷化剂处理 NSCLC 细胞后介导的 PCR 缀合物（具体目标 4）。这些化合物的治疗潜力 抑制 HER-2/neu 表达并改变恶性和侵袭性表型 将在组织培养中评估 NSCLC 细胞（具体目标 5）。这 HER-2/neu 特异性抗基因化合物的开发将为 有关 HER-2/neu 基因在启动和 NSCLC 的进展并可能导致 HER-2/neu 的新治疗方法 表达癌症，例如 NSCLC。

项目成果

Targeting and regulation of the HER-2/neu oncogene promoter with bis-peptide nucleic acids.

• DOI: 10.1089/oli.2005.15.36
• 发表时间: 2005-03
• 期刊: Oligonucleotides
• 作者: A. Ziemba;Zhanna V. Zhilina;Yulia Krotova-Khan;L. Staňková;S. Ebbinghaus

A bis-alkylating triplex forming oligonucleotide inhibits intracellular reporter gene expression and prevents triplex unwinding due to helicase activity.

双烷基化三链体形成寡核苷酸抑制细胞内报告基因表达并防止由于解旋酶活性而导致的三链体解旋。

• DOI: 10.1021/bi0273112
• 发表时间: 2003
• 期刊: Biochemistry.
• 作者: Ziemba,AmyJ;Reed,MichaelW;Raney,KevinD;Byrd,AliciaB;Ebbinghaus,ScotW
• 通讯作者: Ebbinghaus,ScotW

Synthesis and evaluation of a triplex-forming oligonucleotide-pyrrolobenzodiazepine conjugate.

三链体形成寡核苷酸-吡咯并苯二氮卓缀合物的合成和评估。

• DOI: 10.1021/bc0498673
• 发表时间: 2004
• 期刊: Bioconjugate chemistry.
• 作者: Zhilina,ZhannaV;Ziemba,AmyJ;Trent,JohnO;Reed,MichaelW;Gorn,Vladimir;Zhou,Qun;Duan,Wenhu;Hurley,Laurence;Ebbinghaus,ScotW
• 通讯作者: Ebbinghaus,ScotW