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

基本信息

批准号：
8234706
负责人：
Yuk-Ching Tse-Dinh
金额：
$ 3.98万
依托单位：
NEW YORK MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
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/8234706
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

项目摘要

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，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 影响整体和局部 DNA 超螺旋，从而影响 DNA 复制、转录、重组和修复。在其催化循环期间专门针对细菌拓扑异构酶 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。还迫切需要确定新的线索来开发新型抗菌药物来对抗多重耐药病原体。正在研发的革兰氏阴性菌药物的缺乏尤为关键。鼠疫耶尔森氏菌是鼠疫的病原体，而具有多重耐药性的鼠疫耶尔森氏菌有可能被用作未来恐怖袭击的武器。捕获由细菌拓扑异构酶 I 形成的共价 DNA 裂解中间体已被证明会导致细菌细胞快速死亡。新发现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，类似于针对细菌 IIA 型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药物耐药性的影响。在这个拟议的项目中，将开发一种体外高通量测定，利用荧光测定来检测小分子，这些小分子可以增强重组鼠疫杆菌拓扑异构酶 I 形成 DNA 裂解产物。在该项目的第一年，进行了以下测定将优化参数：（i）寡核苷酸底物序列，以及用于裂解产物荧光检测的修饰的选择； (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。信号背景比、变异系数、板间变异和当天变异将根据从 NIH 获得的 384 孔格式的一小组化合物来确定。此外，该检测还将在哈佛医学院附属 NERCE 筛选设施的 HTS 设施的试点筛选中针对 5,000 至 10,000 种化合物进行测试。二级拓扑异构酶松弛和裂解活性测定已经到位。目前的体内反筛选将进一步优化，以改进对细菌中专门针对拓扑异构酶 I 酶的先导化合物的选择。鼠疫耶尔森氏菌拓扑异构酶 I 与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。预计针对鼠疫耶尔森氏菌拓扑异构酶 I 鉴定的分子可用于靶向大肠杆菌和许多革兰氏阴性病原体中的拓扑异构酶 I。测定开发和配置的结果应允许在 MLPCN 下快速提交筛选，以便在第二年与国家筛选中心建立合作。公共健康相关性：在该项目中，将开发一种体外高通量测定法，以首次鉴定专门针对细菌拓扑异构酶 I 的新型化合物。拟议的工作将满足开发新抗菌疗法的需求，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。