Aminoglycoside-Peptide Nucleic Acid (PNA) Conjugates against Gram-Negative Bacteria

Aminoglycosides (AGs) are among the earliest known classes of antibiotics. Despite decades of clinical utility, they have become largely ineffective due to the spread of antimicrobial resistance. In an effort to improve the activity of AGs against resistant strains, we conjugated them with antisense oligonucleotides, specifically peptide nucleic acids (PNAs). We report the synthesis and biological evaluation of novel neomycin (NEO) and amikacin (AMK) conjugates with 10-mer PNA oligomers targeting the essential bacterial gene encoding the acyl carrier protein. The conjugates were prepared via copper(I)-catalyzed azide-alkyne cycloaddition between 5″-azido-modified NEO or 6″-azido-modified AMK and alkyne-functionalized PNA. The AG-PNA conjugates exhibited higher antibacterial activity against AG-resistant strains than the parent AGs or mixtures of unconjugated components, with the NEO-PNA conjugate showing activity against NEO-resistant Salmonella Typhimurium LT2 at an 8 μM concentration. Experiments using mismatched-sequence conjugates and conjugates with PNA sequences targeting the gene encoding red fluorescent protein confirmed the antisense mechanism's contribution to antibacterial activity. Membrane permeabilization assays demonstrated that PNA conjugation preserves AG interaction with the bacterial outer membrane, but alterations of the inner membrane potential and dependence on the SbmA transporter indicate the inner membrane as the main obstacle to bacterial uptake. These AG-PNA conjugates represent a promising strategy for overcoming AG resistance via a dual-action mechanism combining membrane interaction and antisense activity.