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. !

原核生物DNA回转酶是一种II型拓扑异构酶，它可以向DNA引入负超螺旋