Chemical and Structural Insights into the Regioversatility of the Aminoglycoside Acetyltransferase Eis

et al. 2015

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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

et al. 2015

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“…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).…”

mentioning

confidence: 99%

Inhibition of Aminoglycoside Acetyltransferase Resistance Enzymes by Metal Salts

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...

“…This is significant due to the multiacetylating ability this AME has. We have documented this enzyme to modify the 3Љ-amino, 2=-amino, 6=-amino, and 1-amino groups of certain AGs (18,30); while the modified positions of NEO are as of yet unresolved, the fact that these compounds hinder Eis ability to transfer an acetyl group is significant. Further work to optimize the selectivity in ribosomal binding and AME activity within a modified AG will be necessary for the development of more potent antibacterials.…”

Section: Effect Of Linker Of Neomycin Dimers On Activitymentioning

confidence: 99%

Influence of Linker Length and Composition on Enzymatic Activity and Ribosomal Binding of Neomycin Dimers

Watkins

Kumar

et al. 2015

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The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.