Discovering novel bacterial DNA gyrase poisons using unique high throughput screening assay.

Matthew Dias, Fence Leng

Florida International University

Antimicrobial resistance (AMR) poses a significant threat to global public health. The overuse and misuse of antibiotics have led to the emergence of novel and more dangerous AMR strains, which are predicted to cause more than 317,000 deaths per year in the United States by 2050. To combat this, new classes of antibiotics are urgently needed. One promising target is bacterial DNA gyrase, a conserved and essential enzyme found in all bacteria but not in humans. Fluoroquinolones (FQs), e.g., ciprofloxacin, are a specific type of gyrase inhibitors called gyrase poisons. The mechanism of action of FQs is to stabilize the gyrase-DNA cleavage-complex and convert DNA gyrase into a DNA damaging machine. This gyrase poisoning mechanism makes FQs among the most important and prescribed antibiotics. In fact, FQs are the only type of antibiotics targeting bacterial DNA gyrase. New antibiotics targeting bacterial DNA gyrase need to be discovered and developed urgently as new bacterial strains have evolved FQ resistance alongside with the FDA black box warning on all FQs. One challenge is to develop rapid and efficient high-throughput screening assays to identify gyrase poisons from the millions of compounds found in small molecule libraries. In this study, based on the principle that gyrase poisons cause gyrase-mediated double-stranded DNA breaks, we developed a novel and unique high throughput screening (HTS) assay to discover bacterial DNA gyrase poisons. With the help of T5 exonuclease that only digests linear DNA and does not degrade supercoiled and relaxed plasmid DNA, gyrase poisons can be discovered by this HTS assay rapidly. We validated this HTS assay by screening a small library containing 50 compounds. Our next step is to establish an automatic and miniature HTS assay to screen compound libraries containing thousands or millions of compounds and identify/discover gyrase poisons that can be developed into new antibiotics.