HTS assay development targeting Yersinia pestis topoisomerase I

描述(申请人提供)：拓扑异构酶是一种普遍存在的酶，它通过克服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。在催化循环中针对细菌拓扑异构酶I的小分子分子将是研究DNA超螺旋动力学及其与细菌生理关系的非常有用的工具。还迫切需要为开发新的抗菌药物找到新的线索，以对抗耐多药病原体。缺乏治疗革兰氏阴性细菌的药物尤其关键。鼠疫耶尔森氏菌是鼠疫的病原体，而多重耐药的鼠疫杆菌有可能在未来的恐怖袭击中被用作武器。细菌拓扑异构酶I形成的共价DNA裂解中间体被捕获，导致细菌细胞迅速死亡。新发现的能够促进细菌拓扑异构酶I DNA裂解产物积累的小分子应该是有效的杀菌剂，类似于针对细菌IIA拓扑异构酶的氟喹诺酮类药物，但不会受到普遍存在的氟喹诺酮类耐药的影响。在这项拟议的项目中，将开发一种体外高通量检测方法，利用荧光检测方法来检测能够促进重组鼠疫杆菌拓扑异构酶I形成DNA切割产物的小分子。在项目的第一年，将优化以下检测参数：(I)寡核苷酸底物序列，以及用于切割产物荧光检测的修饰的选择；(Ii)与HTS设置相兼容的孵育条件，以实现最高的信号检测灵敏度。信号背景比、变异系数、板间变化和日变化将根据从NIH获得的384井格式的一小组化合物来确定。此外，该分析将在与哈佛医学院相关的NERCE筛查设施的HTS设施的试点筛查中对5,000到10,000种化合物进行测试。次级拓扑异构酶松弛和切割活性分析已经到位。目前在体内的反筛选将进一步优化，以改进选择专门针对细菌拓扑异构酶I酶的先导。鼠疫杆菌拓扑异构酶I与来自大肠杆菌和其他革兰氏阴性细菌的拓扑异构酶I具有广泛的序列同源性。鉴定出的鼠疫杆菌拓扑异构酶I分子有望用于靶向大肠杆菌和许多革兰氏阴性病原菌的拓扑异构酶I。化验开发和配置的结果应允许快速提交MLPCN下的筛查，以便在第二年与国家筛查中心建立合作。 与公众健康相关：在这个项目中，将开发一种体外高通量检测方法，以首次鉴定针对细菌拓扑异构酶I的新化合物。拟议的工作将解决开发新的抗菌疗法的需要，以应对多重耐药革兰氏阴性细菌病原体的严重公共卫生问题，其中一些可能被用于生物恐怖袭击。

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.