Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?

Abstract

The emergence of bacterial resistance to traditional small-molecule antibiotics is fueling the search for innovative strategies to treat infections. Inhibiting the expression of essential bacterial genes using antisense oligonucleotides (ASOs), particularly composed of nucleic acid mimics (NAMs), has emerged as a promising strategy. However, their efficiency depends on their association with vectors that can translocate the bacterial envelope. Vitamin B12 is among the largest molecules known to be taken up by bacteria and has very recently started to gain interest as a trojan-horse vector. Gapmers and steric blockers were evaluated as ASOs against Escherichia coli (E. coli). Both ASOs were successfully conjugated to B12 by copper-free azide-alkyne click-chemistry. The biological effect of the two conjugates was evaluated together with their intracellular localization in E. coli. Although not only B12 but also both B12-ASO conjugates interacted strongly with E. coli, they were mostly colocalized with the outer membrane. Only 6–9% were detected in the cytosol, which showed to be insufficient for bacterial growth inhibition. These results suggest that the internalization of B12-ASO conjugates is strongly affected by the low uptake rate of the B12 in E. coli and that further studies are needed before considering this strategy against biofilms in vivo.

Emerging Target-Directed Approaches for the Treatment and Diagnosis of Microbial Infections

“…However, when incubated in conditions where the bacteria are forced to uptake 63 (Scarlett−Turner medium), both conjugates 66 and 67 were able to inhibit the growth of E. coli at a concentration of 5 μM. 172 Nevertheless, it is relevant to refer to a recent study by Pereira et al 176 involving the synthesis of conjugates between 63 and different antibacterial oligonucleotides, namely two locked nucleic acids also designed to target the acpP gene, which found that the compounds were not effective against E. coli. 176 Internalization studies with those conjugates suggested that the conjugates have mostly remained bound to the outer membrane of E. coli, therefore not reaching the cytosol where the antimicrobial oligonucleotide could exert its activity.…”

“…176 Internalization studies with those conjugates suggested that the conjugates have mostly remained bound to the outer membrane of E. coli, therefore not reaching the cytosol where the antimicrobial oligonucleotide could exert its activity. 176 Thus, there are still challenges to the application of vitamin B12− oligonucleotide conjugates as antimicrobial agents and of 63 as a generalized vehicle for the delivery of oligonucleotides.…”

“…The conjugate with the PNA anchored to the highlighted R 5′ -OH group (Figure A, pink) was more efficiently uptaken than the ones with the PNA anchored to the highlighted amine groups (Figure A, blue) . As a result, subsequent studies that used 63 as a vehicle for oligonucleotides have chosen to use the R 5′ -OH group as a conjugation site. , Two vitamin B12 conjugates ( 66 and 67 , Figure B) have shown relevant activity against E. coli . Compound 66 comprises a non-cleavable carbamate and triazole linker, while conjugate 67 contains a cleavable disulfide bond connecting 63 to a PNA that targets the gene encoding for the acyl carrier protein in E. coli ( acpP ), involved in the fatty acid synthesis, essential for the bacteria’s survival.…”

Bioinformatics and Genomic Analyses of the Suitability of Eight Riboswitches for Antibacterial Drug Targets

“…ASO targeting is a treatment model involving specifically designed antisense oligonucleotide (ASO) molecules [ 46 ]. They are complementary to specific targeted regions of the messenger RNA (mRNA), which means that it works on the primary structure, i.e., sequence [ 47 ].…”

New Insights on Biofilm Antimicrobial Strategies, 2nd Volume

“…Only 6–9% reached the cytosol, which was not sufficient to inhibit bacterial growth. The authors concluded that this low internalization of conjugates was affected by E. coli ’s low uptake for vitamin B12 and further studies are necessary before applying it to infectious biofilms [ 3 ].…”