<i>trans</i> -Translation inhibitors that kill <i>M. tuberculosis</i> and pathogenic Non-tuberculous Mycobacteria have a dual mechanism of action

<i>Mycobacterium tuberculosis</i> 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 the <i>trans-</i>translation ribosome rescue pathway and kill <i>M. tuberculosis</i>. Here, we show that modifications to the acylaminooxadiazole scaffold can improve potency and tune mycobacterial species specificity, resulting in molecules that kill <i>M. avium</i>, <i>M. abscessus</i>, and <i>M. tuberculosis</i> clinical isolates. Free iron was previously shown to antagonize antibacterial activity and decrease the inhibition of <i>trans</i>-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-based <i>trans-</i>translation inhibitors, indicating a dual mechanism of action involving both direct inhibition of <i>trans-</i>translation and metal starvation. These findings establish acylaminooxadiazoles as dual-mechanism antimycobacterial agents that couple inhibition of <i>trans</i>-translation with disruption of iron homeostasis.