A series of new hybrid structures containing fluoroquinolone (ciprofloxacin) and aminoglycoside (neomycin) antibiotics linked via 1,2,3-triazole moiety were designed and synthesized, and their ...antibacterial activities were determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The nature of spacers in both the ciprofloxacin and neomycin parts greatly influenced the antibacterial activity. The majority of hybrids was significantly more potent than the parent neomycin and overcame most prevalent types of resistance associated with aminoglycosides. Selected hybrids inhibited bacterial protein synthesis with the potencies similar to or better than that of neomycin and were up to 32-fold more potent inhibitors than ciprofloxacin for the fluoroquinolone targets, DNA gyrase and toposiomerase IV, indicating a balanced dual mode of action. Significant delay of resistance formation was observed in both E. coli and B. subtilis to the treatment with ciprofloxacin−neomycin hybrid in comparison to that of each drug separately or their 1:1 mixture.
A polymer of partially de-N-acetylated β-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm ...matrices. In Staphyloccocus epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrates metal-dependent de-N-acetylase activity on β-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic analysis reveals the low catalytic efficiency (pentasaccharide k cat/K M 0.03 M–1 s–1) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position specific de-N-acetylation was only observed with penta- and hexasaccharides. The enzyme preferentially de-N-acetylates the second residue from the reducing terminus in the pentasaccharide and second and third residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-positive bacteria. The low catalytic activity of IcaB is consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide processing enzymes.
De‐N‐acetylases of β‐(1→6)‐D‐N‐acetylglucosamine polymers (PNAG) and β‐(1→4)‐D‐N‐acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de‐N‐acetylases are ...necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de‐N‐acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition‐state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger–phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de‐N‐acetylase from Escherichia coli, and PgdA, a peptidoglycan de‐N‐acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de‐N‐acetylases reported to date (Ki=80 μM). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors.
Going through a transition: Methylphosphonamidate‐based transition‐state analogue inhibitors of carbohydrate de‐N‐acetylases were developed by coupling phosphonite and azido sugars. These compounds displayed striking differences in inhibition capabilities against various carbohydrate esterase family 4 (CE4) members, thus key variations within this enzyme class were elucidated.
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To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics ...were synthesized, and their activity was determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The hybrids were antagonistic relative to the ciprofloxacin, but were substantially more potent than the parent kanamycin against Gram-negative bacteria, and overcame most dominant resistance mechanisms to aminoglycosides. Selected hybrids were 42–640 fold poorer inhibitors of bacterial protein synthesis than the parent kanamycin, while they displayed similar inhibitory activity to that of ciprofloxacin against DNA gyrase and topoisomerase IV enzymes. The hybrids showed significant delay of resistance development in both E. coli and B. subtilis in comparison to that of component drugs alone or their 1:1 mixture. More generally, the data suggest that an antagonistic combination of aminoglycoside-fluoroquinolone hybrids can lead to new compounds that slowdown/prevent the emergence of resistance.
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The C5″-OH group in neomycin B was glycosylated with a variety of mono- and di-saccharides to probe the effect of introduction of additional binding elements on antibacterial activity ...and interaction with the aminoglycosides modifying enzyme APH(3′)-IIIa. The designed structures show antibacterial activity superior to that of neomycin B against pathogenic and resistant strains, while in parallel they demonstrate poor substrate activity with APH(3′)-IIIa.
Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide ...poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaBAd) has been determined to 1.7 Å resolution. The structure of IcaBAd reveals a (β/α)7 barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaBAd is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni2+, Co2+, and Zn2+. From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci.IcaB is a poly-β-1,6-N-acetyl-d-glucosamine (PNAG) deacetylase required for polysaccharide intercellular adhesion-dependent biofilm formation by staphylococci.
The structure of Ammonifex degensii IcaB has been determined and its catalytic mechanism and localization characterized.
IcaB is a membrane-associated PNAG deacetylase that uses an altered catalytic mechanism relative to other family 4 carbohydrate esterases.
First structural characterization of a Gram-positive PNAG deacetylase.
Aminoglycosides are highly potent, broad-spectrum antibiotics that kill bacteria by binding to the ribosomal decoding site and reducing the fidelity of protein synthesis. The emergence of bacterial ...strains resistant to these drugs, as well as their relative toxicity, have inspired extensive searches toward the goal of obtaining novel molecular designs with improved antibacterial activity and reduced toxicity. In recent years, a new therapeutic approach that employs the ability of certain aminoglycosides to induce mammalian ribosomes to readthrough premature stop codon mutations has emerged. This new and challenging task has introduced fresh research avenues in the field of aminoglycosides research. In this chapter, our recent observations and current challenges in the design of aminoglycosides with improved antibacterial activity and the treatment of human genetic diseases are discussed.
Functional Characterization of Staphylococcus epidermidis IcaB, a De-N-acetylase Important for Biofilm Formation Pokrovskaya, Varvara; Poloczek JoannaauthorDepartment of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada; Little Dustin J.authorProgram in Molecular Structure & Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, CanadaDepartment of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada ...
2015
Journal Article
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