The search for new, non-standard targets is currently a high priority in the design of new antibacterial compounds. Bacterial toxin–antitoxin systems (TAs) are genetic modules that encode a toxin protein that causes growth arrest by interfering with essential cellular processes, and a cognate antitoxin, which neutralizes the toxin activity. TAs have no human analogs, are highly abundant in bacterial genomes, and therefore represent attractive alternative targets for antimicrobial drugs. This study demonstrates how artificial activation of Escherichia coli mazEF and hipBA toxin–antitoxin systems using sequence-specific antisense peptide nucleic acid oligomers is an innovative antibacterial strategy. The growth arrest observed in E. coli resulted from the inhibition of translation of the antitoxins by the antisense oligomers. Furthermore, two other targets, related to the activities of mazEF and hipBA, were identified as promising sites of action for antibacterials. These results show that TAs are susceptible to sequence-specific antisense agents and provide a proof-of-concept for their further exploitation in antimicrobial strategies.

Vitamin B also called cobalamin (Cbl) is an important enzymatic cofactor taken up by mammalian and also by many bacterial cells. Peptide nucleic acid (PNA) is a synthetic DNA analogue that has the ability to bind in a complementary manner to natural nucleic acids. Provided that PNA is efficiently delivered to cells, it could act as a steric blocker of functional DNA or RNA and regulate gene expression at the level of transcription or translation. Recently, Cbl has been examined as a transporter of various molecules to cells. Also, PNA, if covalently linked with Cbl, can be delivered to bacterial cells, but it is crucial to verify that Cbl does not change the desired PNA biological properties. We have analyzed the structure and conformational dynamics of conjugates of Cbl with a PNA monomer and oligomer. We synthesized a cyanocobalamin derivative with a PNA monomer C connected via the triazole linker and determined its NMR spectra. Using microsecond-long molecular dynamics simulations, we examined the internal dynamics of cyanocobalamin-C, its conjugate with a 14-mer PNA, and free PNA. The results suggest that all compounds acquire rather compact structures but the PNA oligomer conformations vary. For the Cbl-C conjugate the cross-peaks from the ROESY spectrum corroborated with the clusters from molecular dynamics trajectories. Within PNA the dominant interaction is stacking but the stacking bases are not necessarily neighboring in the PNA sequence. More bases stack in free PNA than in PNA of the conjugate, but stacking is less stable in free PNA. PNA in the conjugate is slightly more exposed to solvent. Overall, cyanocobalamin attached to a PNA oligomer increases the flexibility of PNA in a way that could be beneficial for its hybridization with natural nucleic acid oligomers.

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