Strategies to Overcome the Action of Aminoglycoside-Modifying Enzymes for Treating Resistant Bacterial Infections

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In this study, we investigated the in vitro antifungal activities, cytotoxicities, and membrane-disruptive actions of amphiphilic tobramycin (TOB) analogues. The antifungal activities were established by determination of MIC values and in time-kill studies. Cytotoxicity was evaluated in mammalian cell lines. The fungal membrane-disruptive action of these analogues was studied by using the membrane-impermeable dye propidium iodide. TOB analogues bearing a linear alkyl chain at their 6؆-position in a thioether linkage exhibited chain length-dependent antifungal activities. Analogues with C 12 and C 14 chains showed promising antifungal activities against tested fungal strains, with MIC values ranging from 1.95 to 62.5 mg/liter and 1.95 to 7.8 mg/liter, respectively. However, C 4 , C 6 , and C 8 TOB analogues and TOB itself exhibited little to no antifungal activity. Fifty percent inhibitory concentrations (IC 50 s) for the most potent TOB analogues (C 12 and C 14 ) against A549 and Beas 2B cells were 4-to 64-fold and 32-to 64-fold higher, respectively, than their antifungal MIC values against various fungi. Unlike conventional aminoglycoside antibiotics, TOB analogues with alkyl chain lengths of C 12 and C 14 appear to inhibit fungi by inducing apoptosis and disrupting the fungal membrane as a novel mechanism of action. Amphiphilic TOB analogues showed broad-spectrum antifungal activities with minimal mammalian cell cytotoxicity. This study provides novel lead compounds for the development of antifungal drugs.

Section: Resultsmentioning

confidence: 99%

Amphiphilic Tobramycin Analogues as Antibacterial and Antifungal Agents

Shrestha

Fosso

Green

et al. 2015

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“…This has accelerated the development of resistance against AGs and reduced the effective agents available for combating ever-evolving pathogens. The most common mechanism of bacterial resistance to AGs is the acquisition of AG-modifying enzymes (AMEs) (6,7). Based on the reactions that they catalyze, AMEs can be classified as AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), or AG O-nucleotidyltransferases (ANTs), among which AACs are responsible for the majority of resistant infections.…”

mentioning

confidence: 99%

Inhibition of Aminoglycoside Acetyltransferase Resistance Enzymes by Metal Salts

Green

Johnson

2015

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Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn 2؉ metal ions displayed an inhibitory effect on the resistance enzyme AAC(6=)-Ib in Acinetobacter baumannii and Escherichia coli. A minoglycosides (AGs) are broad-spectrum bactericidal antibiotics that are used clinically for the treatment of serious bacterial infections (1, 2). These antibiotics were originally isolated from Streptomyces and Micromonospora (3) and display activity against Gram-positive bacteria, aerobic Gram-negative pathogenic bacteria, and Mycobacteria. Since the discovery of streptomycin, the first-in-class AG isolated, many AGs have been discovered and developed with improved efficacy. This property has kept them clinically relevant despite their inherent oto-and nephrotoxicity. Currently, amikacin (AMK), gentamicin (GEN), and tobramycin (TOB) are the most commonly prescribed AGs for systemic administration in the United States against bacterial infections (see Fig. S1 in the supplemental material) (4). Another AG, kanamycin A (KAN), is also used systemically but only to treat resistant strains of Mycobacterium tuberculosis in patients who show no response to first-line antituberculosis treatments. Other AGs, such as neomycin B (NEO), are found in antibiotic ointment formulations for topical use (5).The increased importance and popularity of AGs have, unfortunately, led to lapses in antimicrobial stewardship. This has accelerated the development of resistance against AGs and reduced the effective agents available for combating ever-evolving pathogens. The most common mechanism of bacterial resistance to AGs is the acquisition of AG-modifying enzymes (AMEs) (6, 7). Based on the reactions that they catalyze, AMEs can be classified as AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), or AG O-nucleotidyltransferases (ANTs), among which AACs are responsible for the majority of resistant infections. AACs modify AG substrates and disrupt their binding to the ribosome by transferring the acetyl group from acetyl coenzyme A (AcCoA) onto amine moieties of the AG scaffolds. Although most AACs are regiospecific and modify only a single amino group, the unique enhanced intracellular survival (Eis) protein upregulated in resistant M. tuberculosis strains is capable of acetylating AG substrates at several different positions (8)(9)(10).In an effort to overcome bacterial resistance, a large amount of time and money has been invested toward the development of inhibitors of these resistance enzymes. Most commonly, small organic molecules have been explored for this purpose. For example, in silico and high-throughput screening was used to ide...

“…Part of the management of this problem could be the extension of the useful life of existing antibiotics by finding inhibitors of the resistance mechanisms or their expression (30,46,47). Here we tested one of the latest oligonucleotide analogs, a hybrid oligomer composed of 2=,4=-bridged nucleic acid-NC residues and deoxynucleotides conjugated to the permeabilizing peptide (RXR) 4 XB, as an antisense inhibitor of resistance to AMK mediated by AAC(6=)-Ib, one of the most widespread aminoglycoside-modifying enzymes (30,39).…”

Section: Discussionmentioning

confidence: 99%

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

Lopez

Arivett

Actis

et al. 2015

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,...