-Translation inhibitors that killand pathogenic non-tuberculous mycobacteria also disrupt metal homeostasis.

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 haemin and transferrin do not affect activity. Depletion of transfer-messenger RNA resulted in potentiation of acylaminooxadiazole-based-translation inhibitors, confirming inhibition of-translation as a mechanism of action. Acylaminooxadiazoles disrupted metal homeostasis in, and mutants defective in siderophore-mediated iron utilization were hypersusceptible to some acylaminooxadiazole derivatives, suggesting that these compounds may have a dual mechanism for killing mycobacteria.