This proposal aims to develop novel, more effective, and selective therapies for the treatment of Helicobacter pylori infections. Chronic infection with H. pylori causes peptic ulcers and gastric cancer. H. pylori is an important drug-resistant pathogen, which is categorized as a high-priority pathogen by the WHO. Standard therapy consists of a proton pump inhibitor (PPI) and two broad-spectrum antibiotics, usually clarithromycin with either metronidazole or amoxicillin. These combinations are becoming increasingly ineffective with high resistance rates, and substantially disrupt the gut's healthy microbiome. There is a considerable unmet need for novel, more effective, and selective antibiotics to eradicate H. pylori. In our preliminary studies, we identified the NuoD interface of Complex I as a druggable target for H. pylori. Respiration through Complex I is essential for H. pylori, but not for most other bacteria, offering mechanistic selectivity and minimizing disruption of the microbiome. We developed and validated a virtual screening platform for this target to identify leads with improved pharmacological properties using the initial inhibitors. The virtual screen identifed hits that are synthetically tractable with demonstrated on-target antibacterial activity, and both ex vivo and in vivo efficacy against H. pylori. The ultimate aim of the proposal is to obtain carefully validated, narrow spectrum, orally bioavailable, and efficacious NuoD inhibitors that will form the basis of a subsequent preclinical antibiotic development effort. These studies will also provide a deeper understanding of H. pylori respiration and the development of selective antibiotics against H. pylori.