Antibiotic resistance poses a severe public health threat, causing 2.8 million infections and 35,000 deaths annually in the US. The lack of new antibiotics in the past three decades raises urgent concerns about the potential revival of deadly infections, emphasizing the necessity for new antibiotics development. This phase I application aims to establish and develop an innovative high throughput screening (HTS) assay to identify specific bacterial DNA gyrase inhibitors, poisoning inhibitors that stabilize the gyrase-DNA cleavage-complexes and convert gyrase into a DNA damaging machinery. Bacterial DNA gyrase is a highly conserved and essential enzyme found in all bacteria, but not in humans. It is an important and clinically validated target for discovering and developing new antibiotics. Indeed, fluoroquinolones (FQs), a type of DNA gyrase poisoning inhibitors, are among the most successful antibiotics targeting DNA gyrase. Unfortunately, bacterial resistance to FQs has emerged. Under certain circumstances, FQs have serious side effects. Furthermore, since FQs have been explored extensively, the potential limit of what FQs can achieve has likely been reached. Therefore, it is necessary to develop compounds with novel chemical scaffold targeting DNA gyrase. One challenge is to rapidly and efficiently identify gyrase poisoning inhibitors from thousands or millions of compounds in small molecule libraries since HTS assays are not available for such efforts. In this application, we propose to establish a unique HTS assay that can rapidly identify specific bacterial DNA gyrase poisoning inhibitors from small molecule libraries. Specific aims are to 1) develop a unique high throughput screening assay to discover bacterial DNA gyrase poisoning inhibitors; 2) utilize T5 exonuclease AT-hairpin- and/or supercoiling-dependent fluorescence quenching (SDFQ)-based HTS assays to confirm the identification of novel gyrase poisoning inhibitors; and 3) miniaturize and automate the HTS assay. Successful execution of the proposed research could lead to commercializing a suite of three biochemical HTS assays to identify inhibitors for DNA topoisomerases including gyrase and screening large compound libraries for gyrase poisoning inhibitors in future HTS campaigns, which will be pursued in a subsequent phase II application.