Benzoxaborole-modified azithromycins inhibit translation without inducing <i>ermC</i> expression

The rapid increase in antimicrobial resistance underscores the urgent need for new antibacterial agents. One promising strategy involves designing novel compounds through targeted chemical modifications of existing antibiotics. Azithromycin (AZI), a widely used macrolide, has served as a versatile scaffold for developing numerous antibacterial candidates. However, the mechanistic consequences of such modifications remain largely unexplored. Here, we characterize the activity and mechanism of action of three AZI-benzoxaborole (AZI-BB) conjugates. We show that these compounds inhibit bacterial translation <i>in vitro</i> and remain active against a model <i>Escherichia coli</i> strain carrying an inducible <i>ermCL-ermC</i> operon, which confers resistance to macrolide antibiotics. Unlike erythromycin, these derivatives, along with AZI itself, exhibit minimal induction of ErmC expression. Structural analysis reveals that the benzoxaborole moiety of AZI-BB2 forms additional interactions with nucleotides C2441 and C2586 of 23S rRNA, likely contributing to premature ribosome stalling at the <i>ermCL</i> regulatory sequence and thereby preventing ErmC expression. Furthermore, high-throughput toeprinting analysis combined with deep sequencing (Toe-seq) demonstrates that AZI-BB2 exhibits reduced sequence specificity for canonical macrolide-sensitive stalling motifs. Altogether, these findings demonstrate that targeted chemical modification of AZI can reshape its context-specific interaction with the ribosome and attenuate the induction of macrolide resistance mechanisms.