Abstract 1875 A Novel High Throughput Screening (HTS) Method to Discover Bacterial DNA Gyrase Poisons

The emerging threat of antimicrobial resistance (AMR) poses a significant crisis to global public health.According to the most recent study, in 2023 1.05 million deaths were associated with AMR bacterial infections, and this number is projected to rise to 10 million deaths annually by 2050.To address this crisis, the discovery of new classes of antibiotics is urgently needed.One promising target is bacterial DNA gyrase, a ubiquitous and essential enzyme found in all bacteria but absent in humans.Fluoroquinolones, the only clinically significant poisons of gyrase, a specific type of inhibitors which operate through a unique mechanism that stabilizes the gyrase-DNA cleavage complex, effectively transforming gyrase into an intracellular DNA-damaging machine.However, the development of new antibiotics targeting bacterial DNA gyrase is crucial, as many bacterial strains have evolved resistance to fluoroquinolones (FQs).Furthermore, all FQs carry FDA black box warnings due to potential severe side effects.A key challenge is the need for rapid and efficient high-throughput screening (HTS) assays to identify gyrase poisons from smallmolecule libraries.In this study, we developed a novel and unique HTS assay to discover bacterial DNA gyrase poisons.In the presence of a poison, high concentrations of gyrase cleave circular plasmid DNA into small linear fragments.Using T5 exonuclease, which selectively digests linear DNA while leaving supercoiled and relaxed plasmid DNA intact, this way poisons can be detected via a simple DNA dye, such as SYBR Green.This novel HTS assay was validated against a small inhouse library of 50 compounds.To facilitate screening of larger compound libraries containing thousands of compounds, our HTS assay was adapted to a miniaturized 1536well plate format.This method provides an innovative approach for the rapid identification of gyrase-poisoning compounds and holds the potential to accelerate the discovery of a new class of antibiotics to combat AMR.