Metronidazole and ether derivatives target Helicobacter pylori via simultaneous stress induction and inhibition

Metronidazole is a front-line drug for the treatment of Helicobacter pylori infections. However, its mode of action and cellular targets are poorly defined, and higher dosing and combination therapies are required to overcome resistance. Here we performed activity-based protein profiling with tailored metronidazole probes and identified chaperonin HpGroEL and thiol peroxidase HpTpx as prominent targets, the latter being essential for H. pylori survival under oxidative stress. Alkynylated ether probes exhibited enhanced antibacterial potency compared with the parent drug in vitro, including activity against resistant strains. Biological assays, chemical proteomics and co-crystallization studies confirmed target engagement, with enhanced binding of ether derivatives to HpTpx. Refined ether analogues exhibited favourable pharmacological profiles without cytotoxicity. The in vivo activity of ether analogues using an H. pylori mouse model demonstrated full bacterial eradication at low dosing of 0.3 mg kg^-1 day^-1. Our findings reveal that stress induction and simultaneous inhibition of the stress response represent a mechanism of this compound class.