Systematic Determination of the Impact of Structural Edits on Accumulation into Mycobacteria

The mycomembrane of mycobacteria has long been regarded as the primary barrier to the accumulation of molecules within these bacteria. Understanding accumulation beyond the mycomembrane of Mycobacterium tuberculosis ( Mtb ) is crucial for developing effective antimycobacterial agents. This study investigates two design principles commonly found in natural products and mammalian cell-permeable peptides – backbone N -methylation and macrocyclization – aimed at enhancing accumulation. To assess the impact of these structural edits on accumulation, we employed our recently described Peptidoglycan Accessibility Click-Mediated Assessment (PAC-MAN) assay for live-cell analysis. Our findings indicate that peptide macrocyclization generally enhances permeability and metabolic stability, while N -methylation modifies accumulation in a context-dependent manner. Furthermore, we applied these design principles to the peptide antibiotic Tridecaptin A1, demonstrating improved permeability and efficacy against mycobacteria, in specific contexts. This work suggests that strategic structural modifications can enhance accumulation past the mycomembrane and provide the first systematic governing rules for the rational redesign of potential antimycobacterial agents. Most importantly, our results broadly challenge the notion that large hydrophilic molecules ( e.g ., peptides) cannot readily bypass the mycomembrane to reach the cytosolic space.