HTS assay development targeting Yersinia pestis topoisomerase I

针对鼠疫耶尔森菌拓扑异构酶 I 的 HTS 检测开发

• 批准号: 7991064
• 负责人: Yuk-Ching Tse-Dinh
• 金额: $ 15.9万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7991064
• 关键词: Address; Affect; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial DNA Topoisomerase I; Bacterial Physiology; Bacterial Typing; Biological Assay; Cell Death; Cell physiology; Cells; Collaborations; DNA; DNA biosynthesis; Data; Detection; Development; Enzymes; Equilibrium; Escherichia coli; Fluorescence; Fluoroquinolones; Future; Genetic; Genetic Transcription; Goals; Gram-Negative Bacteria; In Vitro; Lead; Mammalian Cell; Modification; Multi-Drug Resistance; Oligonucleotides; Pharmaceutical Preparations; Plague; Production; Public Health; Reaction; Recombinants; Relaxation; Resistance; Screening procedure; Sequence Homology; Signal Transduction; Superhelical DNA; Testing; Therapeutic; Time; Topoisomerase; Toxic effect; Type I DNA Topoisomerases; United States National Institutes of Health; Variant; Work; Yersinia pestis; assay development; bactericide; base; combat; counterscreen; fluoroquinolone resistance; follow-up; high throughput screening; improved; in vivo; inhibitor/antagonist; medical schools; novel; pathogen; public health relevance; recombinational repair; small molecule; tool; weapons

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.

描述（由申请人提供）：拓扑异构酶是普遍存在的酶，可以通过克服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会影响全局和局部DNA超螺旋，从而影响DNA复制，转录，重组和修复。针对细菌拓扑异构酶I的小分子在其催化循环过程中，将是研究DNA超螺旋的动力学及其与细菌生理学的关系的非常有用的工具。迫切需要确定新的潜在客户来开发新型抗菌药物，以抗击多药的耐药病原体。革兰氏阴性细菌的管道中缺乏药物尤其重要。耶尔西尼亚柴油是鼠疫的病因，多药耐药的Y.佩斯蒂斯有可能在未来的恐怖袭击中用作武器。通过细菌拓扑异构酶I形成的共价DNA裂解中间体的捕获已显示导致细菌细胞死亡快速。新鉴定的小分子可以增强细菌拓扑异构酶I DNA裂解产物的积累，应该是有效的杀菌剂，类似于靶向细菌型IIA型拓扑异构酶的氟喹诺酮类药物，但不会受到普遍的氟氯酚抗抗性的影响。 在该提出的项目中，将利用荧光测定法来开发一个体外高通量测定法，检测小分子可以通过重组鼠疫tpeasomerase I.在项目的第一年中增强DNA裂解产物的形成。切割产品； （ii）与HTS设置兼容以达到信号检测的最大灵敏度的孵化条件。信噪比比率，变化系数，板间和日间 - 末日变化将根据384井格式从NIH获得的一组少量化合物确定。此外，该测定法将在与哈佛医学院相关的Nerce筛查设施的HTS设施的试点屏幕中对5,000至10,000种化合物进行测试。次级拓扑异构酶松弛和切割活性测定已经到位。当前的体内圆锥体将进一步优化，以改进细菌中特异性靶向拓扑异构酶I酶的选择。 Y. PESTIS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共享广泛的序列同源性。可以预期，鉴定出针对Pestis拓扑异构酶I的分子可以用于靶向大肠杆菌中的拓扑异构酶I和许多革兰氏阴性病原体。测定开发和配置的结果应允许在第二年与国家筛查中心建立与国家筛查中心合作的快速提交。 公共卫生相关性：在这个项目中，将开发一种体外高通量测定法，以首次识别针对细菌拓扑异构酶I的新型化合物。拟议的工作将解决开发新的抗菌治疗剂的需求，以解决严重的公共卫生问题，即多耐药的革兰氏阴性细菌病原体，其中一些可能有可能用于生物恐怖主义攻击。