The unique composition of bacterial electron transport chains (ETCs), distinct from the human mitochondrial ETC, presents opportunities for selective antibacterial targeting. Recently, inhibitors of bacterial ETCs have shown promise as potential antimicrobials, particularly against mycobacteria. Notably, bacterial cytochrome c (cyt c) biogenesis systems-Systems I and II-are absent in mammalian mitochondria, which utilize System III. This divergence suggests that cyt c biosynthesis pathways may serve as novel antibiotic targets. In this study, we screened a library of 1,760 FDA-approved compounds for inhibitors of in vivo bacterial cyt c biogenesis. We identified seven compounds that inhibit either System I and/or System II pathways. Among these, artemisinin and its analogs (ARTs) were found to directly inhibit in vitro cyt c biogenesis using purified CcsBA (System II) and human HCCS (System III). ARTs disrupt heme at the active sites of cyt c synthases, indicating their potential as direct inhibitors. However, therapeutic use of ARTs would likely require co-administration with inhibitors targeting alternative bacterial respiratory chains that do not involve cyt c. Additionally, three members of the 8-hydroxyquinoline (8-HQ) class-clioquinol, chloroxine, and broxyquinoline-inhibited in vivo cyt c biogenesis at low micromolar concentrations, below those needed to suppress bacterial growth. Results suggest that 8-HQs act through mechanisms beyond simple metal chelation (e.g., iron, magnesium), likely targeting multiple bacterial processes. Consistent with this hypothesis, 8-HQs did not directly inhibit in vitro cyt c synthases.IMPORTANCEThis manuscript establishes bacterial cytochrome c biogenesis as a viable and previously underexplored antibacterial target that is fundamentally distinct from human mitochondrial pathways. Through systematic screening of 1,760 FDA-approved compounds, the study identifies two chemically and mechanistically distinct inhibitor classes-artemisinins and 8-hydroxyquinolines-that disrupt cytochrome c maturation via Systems I and II. The work moves beyond phenotypic growth inhibition by directly linking compound activity to heme degradation using complementary in vivo assays and in vitro experiments with purified cytochrome c synthases. Demonstrating that artemisinins directly target heme within these enzymes provides mechanistic insight with broad relevance to bacterial bioenergetics and drug-heme interactions. Importantly, the manuscript highlights the metabolic flexibility of bacterial respiratory chains and shows that inhibition of cytochrome c biogenesis alone is insufficient for robust killing. Together, these findings argue that cytochrome c biogenesis inhibitors will be effective in combination therapies and advance understanding of bacterial respiration.
Mendez et al. (Mon,) studied this question.