-Translation inhibitors that killand pathogenic Non-tuberculous Mycobacteria have a dual mechanism of action.

and pathogenic nontuberculous mycobacteria pose a growing challenge to human health, and new antibiotics that target new pathways with novel mechanisms of action are urgently needed. Acylaminooxadiazole derivatives have previously been shown to inhibit thetranslation ribosome rescue pathway and kill. Here, we show that modifications to the acylaminooxadiazole scaffold can improve potency and tune mycobacterial species specificity, resulting in molecules that kill,, andclinical isolates. Free iron was previously shown to antagonize antibacterial activity and decrease the inhibition of-translation by acylaminooxadiazoles, but we found that biologically relevant iron sources such as hemin and transferrin do not affect activity. Mutants depleted for tmRNA and mutants defective in siderophore-mediated iron utilization are both hypersusceptible to acylaminooxadiazole-basedtranslation inhibitors, indicating a dual mechanism of action involving both direct inhibition oftranslation and metal starvation. These findings establish acylaminooxadiazoles as dual-mechanism antimycobacterial agents that couple inhibition of-translation with disruption of iron homeostasis.