Synthesis and Characterization of Electron-Rich Inhibitors of the α-Carboxyketose Synthases DAHPS And NeuB

Abstract

Antibiotic resistance is a growing global health crisis, creating an urgent need for new antibiotics. The α-carboxyketose synthase (αCKS) family, which includes 3-deoxy-Darabinoheptulosonate 7-phosphate synthase (DAHPS), N-acetylneuraminate synthase (NeuB), and 3-deoxy-D-mannooctulosonate 8-phosphate synthase (KDO8PS), offers attractive targets for antibiotic development because these enzymes are essential for bacterial virulence but are absent from mammalian pathways. Although several αCKS inhibitors have been described, none have advanced to clinical application. This work applied a fragment-based and inhibitor-in-pieces approach to design and assess electron-rich and oxime-derived molecules as inhibitors of DAHPS and NeuB. Eleven compounds were synthesized or obtained, and inhibition constants (Ki) were determined. Several compounds showed strong activity, including Me-OPD, which inhibited DAHPS with nanomolar potency (Ki = 7.7 × 10⁻¹⁰ M), making it the tightest-binding small molecule reported for this enzyme. Clear structure-activity trends emerged: DAHPS favored compact, strongly electronwithdrawing substituents, whereas NeuB tolerated bulkier or more polar oxime variants. The inhibitor fragment N-hydroxyalanine (NHA) showed DAHP oxime-like binding behaviour, especially in the presence of glycerol 3-phosphate (Gro3P), indicating its potential to future fulllength inhibitor designs. This work expands the range of αCKS inhibitors and defines structural features explaining DAHPS and NeuB selectivity, providing a better basis for developing more potent and potentially cell-permeable antibacterial candidates.