Development of LpxC Inhibitors Based on the Mechanism of Action of Colistin in <i>Acinetobacter baumannii</i>

Multidrug-resistant (MDR) <i>Acinetobacter baumannii</i> is a major clinical threat with limited treatment options, as current therapies rely on polymyxins such as colistin. Targeting the lipopolysaccharide (LPS) biosynthetic pathway offers a new target for new antibiotic discovery, yet most efforts have focused on enzyme-based assays that do not reflect cell level physiology. Here, we developed a cell-based screening strategy that links colistin resistance to inhibition of lipooligosaccharide (LOS) biogenesis in <i>A. baumannii</i>. Using colistin, we established a phenotypic platform in which compounds that inhibit LOS synthesis rescue bacterial growth from colistin's mode of action. This approach allows direct identification of inhibitors acting on essential LPS enzymes, including LpxC. Screening a library of about 7000 small molecules discovered non-hydroxamate compounds that restored growth under colistin stress. Hit compounds were validated through LpxC enzyme assays and protein-compound binding assays. Furthermore, our molecular docking study suggests that the hit compounds bind to the LpxC catalytic pocket similarly to CHIR-090. Together, our work introduces a novel phenotypic screening strategy for discovering LPS targeted inhibitors and provides new chemical scaffolds for developing antibiotics against <i>A. baumannii</i> and other Gram-negative pathogens.