The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB. [Display omitted] • Structure-based medicinal chemistry leads to an optimized sequanamycin-derived lead molecule, SEQ-9 • Sequanamycins are macrolide antibiotics able to overcome inherent Mtb macrolide resistance • SEQ-9 is able to adjust its binding mode to the methylated (resistant) form of the Mtb ribosome • SEQ-9 kills Mtb in vitro and is efficacious in mouse models of TB Macrolides generally have low efficacy against tuberculosis, but a class of macrolides called "sequanamycins" overcomes the resistance of Mycobacterium tuberculosis to macrolides and can be optimized for potent activity against a variety of Mycobacterium tuberculosis strains, including drug-resistant ones. [ABSTRACT FROM AUTHOR]