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。去除转录驱动的DNA中的正负超螺旋是包括痘病毒拓扑异构酶I在内的拓扑异构酶的主要功能。在去除转录驱动的超螺旋时，捕获与拓扑异构酶形成的裂解DNA形成的共价中间体结合的共价中间产物可导致立即丧失感染性。然而，基于细胞的病毒或细菌计数分析不能区分这种拓扑异构酶靶向机制和其他抗感染模式。在体外，用纯化的酶和DNA底物进行的检测没有提供正负超卷曲的双结构域，这是体内参与转录的拓扑异构酶的作用部位。以大肠杆菌为基础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，从而能够识别在激活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捕获共价拓扑异构酶复合体。在我们之前关于痘苗病毒拓扑异构酶I表达对大肠杆菌DNA超螺旋的影响的工作的基础上，我们计划开发一种适合于高通量筛选潜在的小痘治疗药物的化学文库的系统，这些药物将通过捕获与切割的病毒DNA复合的痘病毒拓扑异构酶而发挥作用。该筛选系统还应适用于靶向与生物防御相关的病原菌中的拓扑异构酶。

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.