Tobramycin-Peptide Nucleic Acids Synergize with Polymyxin B and Other Bioactive Compounds to Inhibit Growth of Gram-Negative Bacteria.

Antisense oligomers show promise for the treatment of infectious diseases but face challenges as therapeutic agents due to limited uptake in bacteria. Although cell-penetrating peptides can serve as effective delivery vehicles, they may exhibit nonspecific effects and can contribute to resistance development. Aminoglycoside-peptide nucleic acid (PNA) conjugates represent a complementary delivery strategy that leverages the intrinsic cationic and membrane-interacting properties of aminoglycosides to enhance cellular uptake. In this work, we synthesized tobramycin-PNA (TOB-PNA) conjugates targeting the translation initiation region of the acyl carrier protein gene (acpP) in Enterobacterales. Conjugation of TOB to PNA enhanced target-binding stability, improved the inhibition of translation in vitro, and enhanced activity against Gram-negative bacteria. TOB-PNA activity was further enhanced in combination with the outer membrane-permeabilizing antibiotic polymyxin B (PMB), leading to improved antibacterial activity against clinically relevant Gram-negative pathogens, including drug-resistant isolates. Resistance studies demonstrated that the TOB-PNA conjugate, both alone and in combination with PMB, maintains antibacterial activity over prolonged exposure, suggesting a higher barrier to resistance evolution than conventional TOB treatment. Mammalian toxicity studies in HEK293T cells showed IC50 > 100 μM. Morphological analyses indicated that TOB-PNA treatment induces cellular stress and division abnormalities consistent with disruption of essential metabolic pathways, while PMB increased membrane permeability. In addition, TOB-PNA behaved synergistically with other compounds targeting distinct cellular processes, including fatty acid biosynthesis (triclosan), folate metabolism (trimethoprim), and cell morphology (A22).