PROJECT SUMMARY This R21 project is designed to determine structure activity relationships (SARs) of a particular chemotype of 4-(1-methylimidazo[1,2-a]pyridine-1-ium (aka CPD229 and analogs), new antimycobacterial gyrase inhibitors discovered in our recent HTS campaign targeting bacterial DNA gyrase funded by an NIAID grant 1R21AI125973. We will also improve their Mycobacterium tuberculosis (Mtb) whole cell potency and pharmacological properties. Additionally, we will determine the mechanism of action (MoA) of these new gyrase inhibitors both in vitro using purified Mtb gyrase and in vivo within Mtb. DNA gyrase, an essential enzyme to Mtb, is an excellent and validated target for discovering and developing new antibiotics to treat MDR-TB. Fluoroquinolones (FQs), one of the most important and prescribed antibiotics, target bacterial DNA gyrase. FQs are widely used as anti-TB drugs, usually as the second-line antibiotics for MDR-TB patients. However, FQ-resistant Mtb strains have been discovered threatening the utility of FQ for TB treatment. Certain FQs also have serious side effects. These newly identified antimycobacterial gyrase inhibitors, structurally distinct from FQs, provide promising starting points for development and optimization of novel antibiotics that should yield effective therapeutics capable of overcoming FQ-resistance and effectively treating MDR-TB. Success in these proposed studies would provide potent non-FQ antimycobacterial gyrase inhibitors for the development of new antibiotics/therapy for the global public health problem of multi-drug resistant TB.