Benzoxaboroles are a class of boron-containing compounds with a broad range of biological activities. A subset of benzoxaboroles have antimicrobial activity due primarily to their ability to inhibit leucyl-tRNA synthetase (LeuRS) via the oxaborole tRNA-trapping mechanism, which involves the formation of a stable tRNA-benzoxaborole adduct in which the boron atom interacts with the 2'- and 3'-oxygen atoms of the terminal 3' tRNA adenosine. We sought to identify other antibacterial targets for this promising class of compounds by means of mode-of-action studies, and we selected a nitrophenyl sulfonamide based oxaborole () as a probe molecule because it had potent antibacterial activity (MIC of 0.4 μg/mL against methicillin-resistant ) but did not inhibit LeuRS (IC > 100 μM). Analogues of were synthesized to explore the importance of the sulfonamide linker and the impact of altering the functionalization of the phenyl ring. These structure-activity-relationship studies revealed that the nitro substituent was essential for activity. To identify the target for , we raised resistant strains of , and whole-genome sequencing revealed mutations in , suggesting that the target for this compound was indeed LeuRS, despite the lack of enzyme inhibition. Subsequent analysis of metabolism demonstrated that bacterial nitroreductases readily converted this compound into the amino analogue, which inhibited LeuRS with an IC of 3.0 ± 1.2 μM, demonstrating that is thus a prodrug.
