HTS assay development targeting Yersinia pestis topoisomerase I

描述（由申请人提供）：拓扑异构酶是普遍存在的酶，通过克服DNA中的拓扑障碍来实现重要的细胞过程。细菌拓扑异构酶I影响全局和局部DNA超螺旋，影响DNA复制、转录、重组和修复。在细菌拓扑异构酶I的催化循环中特异性靶向细菌拓扑异构酶I的小分子将是研究DNA超螺旋动力学及其与细菌生理学关系的非常有用的工具。还迫切需要确定开发新型抗菌药物的新线索，以对抗多重耐药病原体。缺乏针对革兰氏阴性菌的药物尤其关键。鼠疫耶尔森菌是鼠疫的病原体，耐多药耶尔森菌是鼠疫的主要致病菌。鼠疫有可能被用作未来恐怖袭击的武器。由细菌拓扑异构酶I形成的共价DNA切割中间体的捕获已显示导致细菌细胞快速死亡。新鉴定的可以增强细菌拓扑异构酶I DNA切割产物的积累的小分子应该是有效的杀菌剂，类似于靶向细菌IIA型拓扑异构酶的氟喹诺酮，但不会受到流行的氟喹诺酮耐药性的影响。 在这个拟议的项目中，将开发一种体外高通量检测方法，利用荧光分析来检测可以增强重组Y染色体DNA切割产物形成的小分子。鼠疫拓扑异构酶I在项目的第一年期间，将优化以下测定参数：（i）寡核苷酸底物序列，以及切割产物荧光检测的修饰选择;（ii）与HTS设置兼容的孵育条件，以实现信号检测的最大灵敏度。将根据从NIH获得的384孔板中的一小组化合物测定信号背景比、变异系数、平板间和当天变异。此外，将在与哈佛医学院相关的NERCE筛选机构的HTS机构中进行中试筛选，对5，000至10，000种化合物进行检测。二级拓扑异构酶松弛和切割活性测定已经到位。目前的体内反筛选将进一步优化，以改善选择特异性靶向细菌中拓扑异构酶I酶的先导物。Y.鼠疫拓扑异构酶I与大肠杆菌拓扑异构酶I具有广泛的序列同源性。大肠杆菌和其他革兰氏阴性菌。预期鉴定的Y. pestis拓扑异构酶I可用于靶向大肠杆菌拓扑异构酶I。大肠杆菌和许多革兰氏阴性病原体。检测试剂盒开发和配置的结果应允许在MLPCN下快速提交筛查，以在第二年与国家筛查中心建立合作。 公共卫生相关性：在这个项目中，将开发一种体外高通量检测方法，以首次鉴定特异性靶向细菌拓扑异构酶I的新型化合物。拟议的工作将解决开发新的抗菌疗法的需求，以对抗多重耐药革兰氏阴性细菌病原体的严重公共卫生问题，其中一些可能用于生物恐怖袭击。

DESCRIPTION (provided by applicant): Topoisomerases are ubiquitous enzymes that enable vital cellular processes by overcoming topological barriers in DNA. Bacterial topoisomerase I affects global and local DNA supercoiling influencing DNA replication, transcription, recombination and repair. Small molecules that target bacterial topoisomerase I specifically during its catalytic cycle would be very useful tools for studying the dynamics of DNA supercoiling and its relationship with bacterial physiology. There is also an urgent need to identify new leads for development of novel antibacterial drugs to combat multi-drug resistant pathogens. The lack of drugs in the pipeline for gram-negative bacteria is especially critical. Yersinia pestis is the causative agent for plague, and multi-drug resistant Y. pestis has the potential to be used as a weapon in future terrorist attacks. Trapping of covalent DNA cleavage intermediate formed by bacterial topoisomerase I has been shown lead to rapid bacterial cell death. Newly identified small molecules that can enhance the accumulation of bacterial topoisomerase I DNA cleavage products should be effective bactericidal agents, similar to fluoroquinolones that target bacterial type IIA topoisomerases, but would not be affected by the prevalent fluoroquinolone resistance. In this proposed project, an in vitro high throughput assay will be developed utilizing a fluorescence assay to detect small molecules that can enhance the formation of DNA cleavage products by recombinant Y. pestis topoisomerase I. During the first year of the project, the following assay parameters will be optimized: (i) oligonucleotide substrate sequence, and the choice of modifications for fluorescence detection of cleavage product; (ii) incubation conditions that are compatible with the HTS setting to achieve maximum sensitivity of signal detection. Signal-to background ratio, coefficient of variation, between-plate and day-today variations will be determined against a small set of compounds obtained from NIH in the 384 well format. In addition, the assay will be tested against 5,000 to 10,000 compounds in a pilot screen at the HTS facility at the NERCE screening facility associated with Harvard Medical School. Secondary topoisomerase relaxation and cleavage activity assays are already in place. Current in vivo counterscreens will be further optimized to improve on selecting leads that specifically target topoisomerase I enzyme in bacteria. Y. pestis topoisomerase I shares extensive sequence homology with topoisomerase I from E. coli and other gram negative bacteria. It is expected that the molecules identified for Y. pestis topoisomerase I could be used for targeting topoisomerase I in E. coli and many gram negative pathogens. The results from the assay development and configuration should allow a fast track submission for screening under the MLPCN to establish collaboration with the National Screening Center in the second year. PUBLIC HEALTH RELEVANCE: In this project, an in vitro high throughput assay will be developed to identify for the first time, novel compounds targeting bacterial topoisomerase I specifically. The proposed work would address the need for development of new antibacterial therapeutics to combat the serious public health problem of multi- resistant gram negative bacterial pathogens, some of which could potentially be used in bioterrorist attacks.