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Targeting transcription-coupled DNA supercoiling for discovering antibiotics against bacterial DNA gyrase

靶向转录偶联 DNA 超螺旋以发现针对细菌 DNA 旋转酶的抗生素

基本信息

批准号：
9316780
负责人：
Fenfei Leng
金额：
$ 23.26万
依托单位：
FLORIDA INTERNATIONAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-21 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9316780
关键词：
ATP Hydrolysis Address Adopted Affect Antibiotic Resistance Antibiotics Antimicrobial Resistance Bacteria Bacterial DNA Bacterial Drug Resistance Bacterial Infections Biochemical Biological Assay Bone Marrow Transplantation Cells Cellular Assay Centers for Disease Control and Prevention (U.S.)Chemicals Chemotherapy-Oncologic Procedure Chromosomes Ciprofloxacin Clinical Collection Complex Coupled DNA DNA Gyrase Development Enzymes Escherichia coli Firefly Luciferases Fluorescence Fluorescence Resonance Energy Transfer Fluoroquinolones Genetic Transcription Goals Gram-Negative Bacterial Infections Health Human In Vitro Infection Isopropyl Thiogalactoside Label LacZ Genes Libraries Luc Gene Methods Operative Surgical Procedures Organ Patients Poison Property Public Health Publishing Reporting Reproducibility Research Resistance Site Superhelical DNA Topoisomerase Topoisomerase II United States National Institutes of Health World Health World Health Organization antimicrobial bacterial resistance bactericide base combat cost density disorder prevention fighting fluorophore high risk high throughput screening in vitro Assay inhibitor/antagonist killings luminescence novel pathogen promoter screening small molecule libraries stem

项目摘要

Prokaryotic DNA gyrase is a type II topoisomerase that can introduce negative supercoils to the DNA substrates with the hydrolysis of ATP. Because DNA gyrase only exists in bacterial cells and is an essential enzyme to bacteria, it is possible to identify inhibitors targeting DNA gyrase without affecting host human enzymes. Additionally, DNA gyrase can form covalent enzyme-DNA complex intermediates. This property makes gyrase an excellent bactericidal target for developing antibiotics. Indeed, fluoroquinolones are among the most successful antibiotics targeted to DNA gyrase. Unfortunately, bacterial resistance to fluoroquinolones has emerged and makes the development of new, more effective antibiotics an urgent issue especially for Gram-negative bacterial infections. The long-term goal of the proposed research is to discover and develop new and effective antibiotics that are capable of treating infections of antibiotic resistance bacteria. The objectives of this application are to develop novel biochemical and cell-based assays to screen antimicrobial compounds targeting bacterial DNA gyrase, and screen the NCATS compound library to identify novel DNA gyrase inhibitors. The biochemical primary assay stems from the synthesis of a type of unique fluorescence- labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). The cellular assay is based on one recently constructed E. coli strain FL#1181 that contains a pair of divergently coupled PgyrA and PT7A1/O4 promoters controlling the luc and lacZ genes at the attTn7 site of the E. coli chromosome (84 min of the chromosome). Since transcription-coupled DNA supercoiling (TCDS) provided by a strong IPTG-inducible promoter, such as the T7A1/O4 promoter (PT7A1/O4), is capable of potently inhibiting the divergently coupled, supercoiling-sensitive gyrA promoter (PgyrA), our hypothesis is that DNA gyrase inhibitors should greatly “enhance” the expression of the firefly luciferase under the control of the divergently coupled, supercoiling-sensitive PgyrA. As a result, the luminescence generated from the firefly luciferase will be significantly increased. This unique property of TCDS can be effectively used to screen and identify antimicrobial compounds targeting bacterial DNA gyrase. Three specific aims are: Aim 1. Develop a novel in vitro biochemical assay to screen inhibitors targeting bacterial DNA gyrase. Aim 2. Screen the NCATS compound collection to identify bacterial DNA gyrase inhibitors. Aim 3. Validate hits and identify DNA gyrase poisons using a newly developed cell-based method targeting TCDS. This truely interdisciplinary and collaborative effort brings two labs together (Leng and Smith labs) and offers a novel solution to address an urgent world health problem, antimicrobial resistance. !

原核 DNA 旋转酶是一种 II 型拓扑异构酶，可以向 DNA 引入负超螺旋 ATP 水解的底物。因为DNA旋转酶只存在于细菌细胞中，并且是人体必需的酶对细菌的影响，有可能识别针对 DNA 旋转酶的抑制剂，而不影响宿主人类酶。此外，DNA旋转酶可以形成共价酶-DNA复合物中间体。此属性使旋转酶成为开发抗生素的绝佳杀菌靶标。事实上，氟喹诺酮类药物属于针对 DNA 旋转酶的最成功的抗生素。不幸的是，细菌对氟喹诺酮类药物产生耐药性的出现使得开发新的、更有效的抗生素成为一个紧迫的问题，特别是对于革兰氏阴性细菌感染。拟议研究的长期目标是发现和开发能够治疗抗生素耐药性细菌感染的新型有效抗生素。这该应用的目标是开发新型生化和细胞检测方法来筛选抗菌药物靶向细菌 DNA 旋转酶的化合物，并筛选 NCATS 化合物库以鉴定新的 DNA 旋转酶抑制剂。生化初级检测源于一种独特荧光的合成标记的 DNA 分子，可用于通过荧光研究 DNA 拓扑结构和拓扑异构酶共振能量转移（FRET）。细胞测定基于一种最近构建的大肠杆菌菌株 FL#1181 包含一对不同耦合的 PgyrA 和 PT7A1/O4 启动子，控制 luc 和 lacZ 大肠杆菌染色体 attTn7 位点的基因（染色体的 84 分钟）。由于转录耦合 DNA 超螺旋 (TCDS) 由强 IPTG 诱导型启动子（例如 T7A1/O4 启动子）提供 (PT7A1/O4)，能够有效抑制发散耦合的超螺旋敏感 gyrA 启动子 (PgyrA)，我们的假设是 DNA 旋转酶抑制剂应该大大“增强”萤火虫荧光素酶的表达在发散耦合、超螺旋敏感的 PgyrA 的控制下。结果，发光由萤火虫产生的荧光素酶会显着增加。 TCDS 的这种独特性质可以有效地用于筛选和鉴定针对细菌 DNA 旋转酶的抗菌化合物。三具体目标是：目标 1. 开发一种新型体外生化测定法来筛选针对细菌 DNA 的抑制剂旋转酶。目标 2. 筛选 NCATS 化合物集合以鉴定细菌 DNA 旋转酶抑制剂。目标3。使用新开发的针对 TCDS 的基于细胞的方法验证命中并识别 DNA 旋转酶毒物。这种真正的跨学科和协作努力将两个实验室（Leng 和 Smith 实验室）结合在一起，并提供了解决紧迫的世界健康问题——抗菌素耐药性的新解决方案。！