Development of a HTS system:topoisomerase targets (RMI)

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、负超卷曲是拓扑异构酶的主要功能，包括天花病毒拓扑异构酶i。在去除转录驱动的超卷曲过程中，拓扑异构酶形成的共价中间体与断裂DNA的复合物被捕获，可导致传染性立即丧失。然而，基于细胞的病毒或细菌计数检测不能区分这种拓扑异构酶靶向机制和其他抗感染模式。用纯化酶和DNA底物进行的体外分析没有提供正、负超旋的双结构域，而这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将目标拓扑异构酶放置在转录驱动的超级线圈的位置，允许鉴定在活性转录期间捕获目标拓扑异构酶的药物。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。基于我们之前对牛痘病毒拓扑异构酶I表达对大肠杆菌DNA超卷的影响的研究，我们计划开发一种适合于高通量筛选化学文库的系统，用于潜在的天花治疗药物，该系统将通过捕获痘病毒拓扑异构酶与裂解病毒DNA复合物来起作用。该筛选系统也适用于与生物防御相关的病原菌的拓扑异构酶。

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.