Inhibition of AAC(6′)-Ib-Mediated Resistance to Amikacin in Acinetobacter baumannii by an Antisense Peptide-Conjugated 2′,4′-Bridged Nucleic Acid-NC-DNA Hybrid Oligomer

Abstract

bMultiresistant Acinetobacter baumannii, a common etiologic agent of severe nosocomial infections in compromised hosts, usually harbors aac(6=)-Ib. This gene specifies resistance to amikacin and other aminoglycosides, seriously limiting the effectiveness of these antibiotics. An antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6=)-Ib mRNA, one of the copies overlapping the initiation codon, efficiently inhibited translation in vitro. An isosequential nuclease-resistant hybrid oligomer composed of 2=,4=-bridged nucleic acid-NC (BNA NC ) residues and deoxynucleotides (BNA NC -DNA) conjugated to the permeabilizing peptide (RXR) 4 XB ("X" and "B" stand for 6-aminohexanoic acid and ␤-alanine, respectively) (CPPBD4) inhibited translation in vitro at the same levels observed in testing ODN4. Furthermore, CPPBD4 in combination with amikacin inhibited growth of a clinical A. baumannii strain harboring aac(6=)-Ib in liquid cultures, and when both compounds were used as combination therapy to treat infected Galleria mellonella organisms, survival was comparable to that seen with uninfected controls.A cinetobacter baumannii is an opportunistic human pathogen, mainly nosocomial, that causes bacteremia, meningitis, urinary tract infections, pneumonia, and necrotizing fasciitis among other infections (1-4). Multidrug-resistant A. baumannii strains are increasingly found in hospitals, complicating treatment of the infections they cause (4). Antisense technologies could be a path for designing new therapeutic strategies to overcome this problem. Options include the silencing of one or more essential genes (5-12) or the silencing of one or more resistance genes to induce phenotypic conversion to susceptibility (13-16). In the latter case, the antisense compound would be administered in combination with the appropriate antibiotic. However, in spite of important advances, silencing of bacterial genes by antisense oligomers is far from reaching its full potential (10). The main antisense mechanisms of gene silencing include degradation of the target mRNA by double-stranded RNA (dsRNA)-specific RNase, RNase H, or RNase P and steric hindrance of translation (interference with assembly of the ribosome or translation arrest) (10, 17). Practical application of any of these strategies requires that the antisense compounds resist the action of the ubiquitous nucleases and reach the cytosol to exert their action.There are numerous nuclease-resistant nucleotide analogs available that are adequate for different antisense strategies (10,18,19). For example, hybrid molecules containing locked nucleic acid and deoxyribonucleotide residues (LNA-DNA) in different configurations have been successfully utilized in bacteria and eukaryotes (16,(20)(21)(22)(23). New analogs related to LNAs, the 2=,4=-bridged nucleic acid-NC (BNA NC ) analogs (Fig. 1), that exhibit advantages such as higher binding affinity to a cRNA and excellent single-mismatch discriminating ability, have been recently introduced (24). Furthermore,...

Assessment of configurations and chemistries of bridged nucleic acids-containing oligomers as external guide sequences: a methodology for inhibition of expression of antibiotic resistance genes

“…1B), is now available, we also generated chimeric oligomers containing these analogs and tested their efficiency as EGSs. We recently showed that antisense oligonucleotide analogs composed of BNA NC and DNA residues efficiently interfered with gene expression by steric hindrance when used as antisense compounds targeting the translation initiation region of aac(6ʹ)-Ib [37]. Therefore, we decided to compare the activity of LNA/DNA with that of BNA NC /DNA oligomers as EGSs.…”

“…Although LNA/DNAs show a degree of gymnotic uptake by some bacterial species, the levels of internalization seem not enough for effective EGS activity [62]. A strategy to increase the internalization efficiency is to conjugate them to CPPs [37, 63–66]. Chemical conjugation and purification of negatively charged oligonucleotide analogs with CPPs, which are usually cationic, has been quite difficult [67].…”

“…Chemical conjugation and purification of negatively charged oligonucleotide analogs with CPPs, which are usually cationic, has been quite difficult [67]. However, LNA/DNAs or BNA NC /DNAs have now been successfully conjugated to CPPs and the conjugates were able to inhibit gene expression by steric hindrance [37, 68]. Therefore, we conjugated one of the two most potent EGSs, LDA2, to the (RXR) 4 XB peptide.…”

“…It is a multidrug-resistant clinical strain that includes the aac(6ʹ)-Ib gene [37, 47]. Escherichia coli TOP10(pNW1) is a strain that contains the recombinant plasmid pNW1, an F′ derivative that includes a copy of aac(6′)-Ib [48].…”

Identification of a Small Molecule Inhibitor of the Aminoglycoside 6’-N-Acetyltransferase Type Ib [AAC(6’)-Ib] Using Mixture-Based Combinatorial Libraries

“…Although considerable efforts are dedicated to designing new aminoglycosides that can overcome existing mechanisms of resistance [4, 9], other alternatives to counter the action of aminoglycoside modifying enzymes are being researched. They include the utilization of inhibitors of gene expression such as antisense oligonucleotide analogs [10-12], inhibitors of the enzymatic activity like those developed to overcome β-lactamase-mediated resistance to β-lactams [5, 13-18], or metal ion inhibitors of the acetylation reaction, which most probably act by protective chelation of the substrate antibiotic [19-22]. However, in spite of the efforts directed at designing inhibitors of aminoglycoside modifying enzymes or their expression, none are in use in the clinics yet [reviewed in 4, 5].…”