X-ray Co-crystal Structure of a Novel Pseudomonas aeruginosa DXPS Inhibitor Reveals an Unusual Allosteric Binding Pocket

The enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) catalyzes the first and rate-limiting step of the methylerythritol 4-phosphate (MEP) pathway, representing a promising target for novel anti-infective agents. Given its essential role in the survival of Gram-negative pathogenic bacteria and its absence in humans, drug-discovery efforts to advance our understanding of this enzyme are urgently needed. Here, we unraveled a novel druggable allosteric pocket in DXPS, unexpectedly revealed through co-crystallization of tool compound 14 with Pseudomonas aeruginosa DXPS. This inhibitor, identified via virtual screening and subsequent synthetic optimization, binds within an allosteric site distinct from the active site, engaging the protein through halogen bonding interactions. Compound 14 exhibits comparable IC₅₀ values against both P. aeruginosa and Klebsiella pneumoniae DXPS, highlighting its potential as a broad-spectrum DXPS inhibitor. This first co-crystal structure of a non-substrate analog inhibitor with a pathogenic DXPS establishes 14 as a valuable tool compound and provides a novel structural template for future antibiotic development.