Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA.
Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B12 to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B12 was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B12 conjugates; namely the vitamin B12 azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B12 and PNA were tested, including a disulfide bond. We found that vitamin B12 transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)3K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B12 as a carrier of PNA oligonucleotides into bacterial cells.
Vitamin B12 has been proposed to be a natural vector for the in vivo delivery of biologically active compounds. Most synthetic methodologies leading to vitamin B12 conjugates involve functionalization at the 5' position via either carbamate-based linkages or using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in stable conjugates that are not cleaved within the cell. We have developed a novel vitamin B12 derivative suitably tailored for disulfide-based conjugation that can undergo cleavage in the presence of glutathione, the most abundant thiol in mammalian cells. This active compound is simple to prepare and allows for the direct disulfide-based attachment of therapeutic cargos.
The widespread emergence of bacterial resistance to existing antibiotics forces the development of new therapeutic agents. The use of short modified oligonucleotides, such as peptide nucleic acids (PNAs), seems a promising strategy. However, the uptake of such oligonucleotides is limited by the bacterial cell wall and is species-dependent. Therefore, new carriers for PNAs should be extensively explored. In this study, we examined the antibacterial activity of vitamin B 12 −PNA conjugates. Vitamin B 12 was covalently linked to a PNA oligomer targeted at the mRNA of an essential acpP gene encoding acyl carrier protein in Escherichia coli. PNA−vitamin B 12 conjugates were synthesized using the Cu(I)-catalyzed 1,3-dipolar cycloaddition. We examined two types of linkers between vitamin B 12 and PNA, including a cleavable disulfide bond. As a positive control for PNA uptake, we used PNA conjugated to the most widely used cell-penetrating peptide (KFF) 3 K. We found that vitamin B 12 −PNA conjugates inhibit E. coli growth at a concentration of 5 μM, similar as (KFF) 3 K−PNA. We also showed that vitamin B 12 −PNA conjugates are stable in the presence of biological media. This study provides the foundation for improving and developing PNAs conjugated to vitamin B 12 as antibacterials.
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