Development of a HTS system:topoisomerase targets (RMI)

HTS系统的开发：拓扑异构酶靶标（RMI）

• 批准号: 6879445
• 负责人: Yuk-Ching Tse-Dinh
• 金额: $ 7.8万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879445
• 关键词: DNA damage; DNA gyrase; DNA topoisomerases; Escherichia coli; Poxviridae disease; active sites; antiviral agents; biotechnology; bioterrorism /chemical warfare; camptothecin; chemical cleavage; chemical registry /resource; circular DNA; drug discovery /isolation; enzyme complex; gene mutation; genetic strain; genetic transcription; high throughput technology; microorganism disease chemotherapy; phenylalanine; recombinant proteins; reporter genes; smallpox virus; technology /technique development; vaccinia virus; virus DNA

DESCRIPTION (provided by applicant): DNA topoisomerases are important targets for therapy against potential viral and bacterial pathogens. The removal of transcription-driven positive and negative supercoils in DNA is a major function of topoisomerases, including poxvirus topoisomerase I. The trapping of covalent intermediates complexed with cleaved DNA formed by topoisomerases during removal of transcription-driven supercoiling can lead to immediate loss of infectivity. However, cell based viral or bacterial count assays cannot distinguish between such topoisomerase-targeting mechanism and other modes of anti-infectivity. In vitro assays with purified enzyme and DNA substrates do not provide twin domains of positive and negative supercoiling that are the sites of action of topoisomerases involved in transcription in vivo. An E. coli based assay would place the target topoisomerase at sites of transcription-driven supercoils, allowing identification of agents that would trap the target topoisomerases during active transcription. The concurrent use of a target topoisomerase active site mutant can immediately confirm if the positive result is due to trapping of covalent topoisomerase complex. Based on our previous work on the effect of vaccinia virus topoisomerase I expression on E. coli DNA supercoiling, we plan to develop a system suitable for high-throughput screening of chemical libraries for potential small pox therapeutic agents that will act by trapping poxvirus topoisomerase complexed with cleaved viral DNA. The screening system should also be applicable for targeting topoisomerases in pathogenic bacteria relevant for biodefense.

描述（由申请人提供）：DNA 拓扑异构酶是治疗潜在病毒和细菌病原体的重要靶点。 去除 DNA 中转录驱动的正超螺旋和负超螺旋是拓扑异构酶（包括痘病毒拓扑异构酶 I）的主要功能。在去除转录驱动的超螺旋过程中，捕获与拓扑异构酶形成的切割 DNA 复合的共价中间体可导致感染性立即丧失。 然而，基于细胞的病毒或细菌计数测定无法区分这种拓扑异构酶靶向机制和其他抗感染模式。 使用纯化的酶和 DNA 底物进行的体外测定不提供正负超螺旋的双结构域，而正负超螺旋是参与体内转录的拓扑异构酶的作用位点。 基于大肠杆菌的检测会将目标拓扑异构酶置于转录驱动的超螺旋位点，从而识别在活性转录过程中捕获目标拓扑异构酶的试剂。 同时使用目标拓扑异构酶活性位点突变体可以立即确认阳性结果是否是由于共价拓扑异构酶复合物的捕获所致。 基于我们之前关于痘苗病毒拓扑异构酶 I 表达对大肠杆菌 DNA 超螺旋影响的研究，我们计划开发一种适合高通量筛选化学文库的系统，以寻找潜在的天花治疗剂，该治疗剂将通过捕获痘病毒拓扑异构酶复合物来发挥作用。带有切割的病毒DNA。 该筛选系统还应适用于针对与生物防御相关的病原菌中的拓扑异构酶。