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

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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. !