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

• 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
• 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

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. !

Proximate DNA促旋酶是一种II型拓扑异构酶，可以将负超螺旋引入DNA 底物与ATP的水解。因为DNA旋转酶只存在于细菌细胞中， 酶的细菌，有可能确定抑制剂靶向DNA促旋酶，而不影响宿主人 内切酶此外，DNA促旋酶可以形成共价酶-DNA复合物中间体。此属性 使促旋酶成为开发抗生素的优良杀菌靶标。事实上，氟喹诺酮类药物 最成功的针对DNA促旋酶的抗生素。不幸的是，细菌对氟喹诺酮类的耐药性 已经出现，使新的，更有效的抗生素的开发成为一个紧迫的问题，特别是对 革兰氏阴性菌感染。这项研究的长期目标是发现和开发 能够治疗抗生素抗性细菌感染的新的和有效的抗生素。的 本申请的目的是开发新的生物化学和基于细胞的测定来筛选抗微生物剂 靶向细菌DNA促旋酶的化合物，并筛选NCATS化合物文库以鉴定新的DNA 促旋酶抑制剂生物化学的初级检测源于一种独特的荧光的合成- 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）。细胞分析是基于一个最近构建的E。杆菌菌株 FL#1181，其含有一对控制luc和lacZ的趋异偶联的PgyrA和PT 7A 1/O 4启动子 在E. coli染色体（染色体的84分钟）。由于转录偶联 由强IPTG诱导型启动子如T7 A1/O 4启动子提供的DNA超螺旋（TCDS） （PT 7A 1/O 4），能够有效抑制趋异偶联的超螺旋敏感性gyrA启动子（PgyrA）， 我们的假设是，DNA促旋酶抑制剂应该大大“增强”萤火虫荧光素酶的表达， 在发散耦合的超螺旋敏感的PgyrA的控制下。结果，发光 从萤火虫产生的荧光素酶将显着增加。TCDS的这种独特性质可以 有效地用于筛选和鉴定靶向细菌DNA促旋酶的抗微生物化合物。三个具体 目标是：目标1。建立一种新的筛选细菌DNA靶向抑制剂的体外生化分析方法 旋转酶目标2.筛选NCATS化合物集合以鉴定细菌DNA促旋酶抑制剂。目标3. 使用一种新开发的靶向TCDS的基于细胞的方法检测和鉴定DNA促旋酶毒素。 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， 新的解决方案，以解决一个紧迫的世界卫生问题，抗菌素耐药性。 !