Targeting glmS Ribozyme with Chimeric Antisense Oligonucleotides for Antibacterial Drug Development

Abstract

Due to the steady rise of multidrug-resistant pathogenic bacteria worldwide, it is critical to develop novel antibacterial drugs. This article presents chimeric antisense oligonucleotides that inhibit the bacterial growth of Staphylococcus aureus, one of the most frequent causes of hospital-acquired infections. The chimeric antisense oligonucleotides have a combination of first- and second-generation chemical modification. To deliver the antisense oligonucleotides into a cell, we apply a cell-penetrating oligopeptide attached to them. We have performed complete bioinformatics analyses of the glmS ribozyme present in S. aureus and its essential role in the biochemical pathway of glucosamine-6-phosphate synthesis. Besides, we have analyzed the bacteria for alternative metabolic pathways, such as the nagA gene. The first antisense oligonucleotide explicitly targets the glmS riboswitch, while the second explicitly targets the nagA mRNA. We have evaluated that combined, the antisense oligonucleotides block the synthesis of glucosamine-6-phosphate entirely and inhibit the bacterial growth of S. aureus. However, the glmS riboswitch targeting the antisense oligonucleotide is sufficient to inhibit the growth of S. aureus with a MIC80 of 5 μg/mL. The glmS ribozyme is a very suitable target for antibacterial drug development with antisense oligonucleotides.

A Riboswitch-Driven Era of New Antibacterials

“…The use of antisense oligonucleotides (ASOs) that target a specific riboswitch as antimicrobial agents was first applied to target the glmS riboswitch/ribozyme [ 63 ]. The same strategy led to the discovery of the antisense oligonucleotide ASO-1, which hybridizes with its complementary FMN riboswitch aptamer.…”

“…Besides small-molecule compounds, the abovementioned alternative strategy that has recently gained attention is the use of antisense oligonucleotides. A recent study used a chimeric antisense oligonucleotide (ASO1) that was designed against the glmS riboswitch of S. aureus [ 63 ]. ASO1 was modified in the alkyl group of the ribose 2′ position to bear a phosphorothioate group (PS) in the phosphodiester bond.…”

“…To enter the cell wall, ASO1 5′-end was attached to the pVEC, similar to the ASO-1 that was designed for the FMN riboswitch. The outcome of this study showed that ASO1 specifically targeted the glmS riboswitch of S. aureus and inhibited its growth with a minimum inhibitory concentration for 80% inhibition (MIC 80 ) of 5 μg/mL, while it did not have any effect on the growth of E. coli and B. subtilis [ 63 ]. Taken together, developing antibacterial agents against the glmS riboswitch/ribozyme is of great importance due to the essential role of this riboswitch to regulate the synthesis of a crucial precursor of the bacterial cell wall, GlcN6P.…”

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

“…In vitro experiments in Penchovsky’s laboratory with antisense oligonucleotides, for the first time designed with cell-penetrating peptides and targeted to GlmS [ 44 ] and FMN riboswitches [ 45 ] in three different bacteria, demonstrated that our previous genome-wide bioinformatics results [ 8 ] were proved true and could be used as a milestone for the antisense technology for antibacterial drug development.…”

“…Recently, new chimeric antisense oligonucleotides targeted to glmS riboswitches have been shown to block the synthesis of glucosamine-6-phosphate to inhibit the growth of Staphylococcus aureus and bacterial growth [ 44 ]. Other ASOs have been engineered to target flavin mononucleotide riboswitches in Escherichia coli , Listeria monocytogenes , and Staphylococcus aureus [ 45 ].…”