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 ONCOGENE似乎在玩 在许多类型的人类癌症的启动和发展中的重要作用， 包括大约25％的非小细胞肺癌（NSCLC）， 美国男性和女性的主要原因总体 该项目的目标是寻找新颖的方法来特别抑制HER-2/NEU 通过开发三核苷酸（TFO） - 烷基的表达 结合将以特定地点的方式与HER-2/NEU启动子结合的内容 通过三个形成，并导致特定的共价DNA修饰 DNA烷基化剂鸟嘌呤碱基。该提议的具体目的 旨在解决成功发展的主要障碍 基于TFO的DNA结合药。与TFO共轭的位点特异性烷基化 氮芥末将在HER-2/NEU启动子中展示。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-烷基偶联物的设计（特定目标1）。 这项工作的大部分已经完成，未来的工作将集中在 与次要凹槽DNA结合剂和 研究抗核酸酶抗性寡核苷酸主链修饰。这 将研究这些化合物抑制转录启动的能力 通过瞬时转染的报告基因质粒中的三重螺旋形成 NSCLC细胞系以及NSCLC细胞识别和修复的能力 转染后将表征为三元的DNA烷基化 （特定目标2）。我们的初步数据表明，TFO共轭 在3'和5'末端的氮芥末都可以指导两个鸟嘌呤加合物 毗邻三螺旋的两端，以抵抗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交付 将开发系统，以及腺病毒介导ODN摄取的能力 当腺病毒-ODN复合物为时，将确定核定位 由半稳定的化学连接器形成（特定目标3）。能力 三胞体形成ODN以结合内源性HER-2/NEU基因并直接 位点特异性DNA烷基化将通过Southern印迹和连接研究 用TFO-烷基处理NSCLC细胞后介导的PCR 共轭（特定目标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入性表型 NSCLC细胞将在组织培养中进行评估（特定目标5）。这 开发HER-2/NEU特定的抗基因化合物将为您提供伟大的 有关HER-2/NEU基因在启动中的作用的信息交易和 NSCLC的进展可能会导致HER-2/NEU的新型治疗方法 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