In Vitro Characterization of the Antibacterial Spectrum of Novel Bacterial Type II Topoisomerase Inhibitors of the Aminobenzimidazole Class

ABSTRACT Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.

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Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Heteroaryl Isothiazolones in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00158-07 ◽

2007 ◽

Vol 51 (7) ◽

pp. 2445-2453 ◽

Cited By ~ 16

Author(s):

Jijun Cheng ◽

Jane A. Thanassi ◽

Christy L. Thoma ◽

Barton J. Bradbury ◽

Milind Deshpande ◽

...

Keyword(s):

Amino Acids ◽

Staphylococcus Aureus ◽

Dna Gyrase ◽

Antibacterial Activities ◽

Multidrug Resistant ◽

Activity Assay ◽

Topoisomerase Iv ◽

Low Frequencies ◽

Dual Targeting

ABSTRACT Heteroaryl isothiazolones (HITZs) are antibacterial agents that display excellent in vitro activity against Staphylococcus aureus. We recently identified a series of these compounds that show potent bactericidal activities against methicillin-resistant Staphylococcus aureus (MRSA). We report here the results of in vitro resistance studies that reveal potential underlying mechanisms of action. HITZs selected gyrA mutations exclusively in first-step mutants of wild-type S. aureus, indicating that in contrast to the case with most quinolones, DNA gyrase is the primary target. The compounds displayed low mutation frequencies (10−9 to 10−10) at concentrations close to the MICs and maintained low MICs (≤0.016 μg/ml) against mutants with single mutations in either gyrA or grlA (parC). These data suggested that HITZs possess significant inhibitory activities against target enzymes, DNA gyrase and topoisomerase IV. This dual-target inhibition was supported by low 50% inhibitory concentrations against topoisomerase IV as measured in a decatenation activity assay and against DNA gyrase as measured in a supercoiling activity assay. Good antibacterial activities (≤1 μg/ml) against staphylococcal gyrA grlA double mutants, as well as low frequencies (10−9 to 10−10) of selection of still higher-level mutants, also suggested that HITZs remained active against mutant enzymes. We further demonstrated that HITZs exhibit good inhibition of both S. aureus mutant enzymes and thus continue to possess a novel dual-targeting mode of action against these mutant strains. In stepwise acquisition of mutations, HITZs selected quinolone resistance determining region mutations gyrA(Ser84Leu), grlA(Ser80Phe), grlA(Ala116Val), and gyrA(Glu88Lys) sequentially, suggesting that the corresponding amino acids are key amino acids involved in the binding of HITZs to topoisomerases. The overall profile of these compounds suggests the potential utility of HITZs in combating infections caused by S. aureus, including multidrug-resistant MRSA.

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Alteration of Escherichia coli Topoisomerase IV to Novobiocin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.941-947.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 941-947 ◽

Cited By ~ 42

Author(s):

Christine D. Hardy ◽

Nicholas R. Cozzarelli

Keyword(s):

Escherichia Coli ◽

Antimicrobial Agents ◽

Wide Spectrum ◽

Dna Gyrase ◽

Primary Role ◽

Topoisomerase Iv ◽

Negative Supercoiling ◽

A Site

ABSTRACT DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.

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Mutations in the gyrA, parC, and parE Genes Associated with Fluoroquinolone Resistance in Clinical Isolates of Mycoplasma hominis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.4.954 ◽

1999 ◽

Vol 43 (4) ◽

pp. 954-956 ◽

Cited By ~ 63

Author(s):

Cécile M. Bebear ◽

Joel Renaudin ◽

Alain Charron ◽

Hélène Renaudin ◽

Bertille de Barbeyrac ◽

...

Keyword(s):

Amino Acid ◽

Mycoplasma Hominis ◽

Dna Gyrase ◽

Quinolone Resistance ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Amino Acid Substitutions ◽

Topoisomerase Iv

ABSTRACT Five clinical isolates of Mycoplasma hominis from three different patients were examined for resistance to fluoroquinolones; some of these isolates were probably identical. All five isolates harbored amino acid substitutions in the quinolone resistance-determining regions of both DNA gyrase (GyrA) and topoisomerase IV (ParC or ParE). Furthermore, the novobiocin MIC for three isolates showed a significant increase. This is the first characterization of fluoroquinolone-resistant clinical mycoplasma isolates from humans.

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Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.5.1166 ◽

1997 ◽

Vol 41 (5) ◽

pp. 1166-1167 ◽

Cited By ~ 100

Author(s):

B Perichon ◽

J Tankovic ◽

P Courvalin

Keyword(s):

Streptococcus Pneumoniae ◽

Fluoroquinolone Resistance ◽

Primary Target ◽

Topoisomerase Iv ◽

Low Level ◽

Sequential Acquisition ◽

Level Resistance

We report a mutation in the parE genes of two in vitro mutants of Streptococcus pneumoniae responsible for low-level resistance to fluoroquinolones. Sequential acquisition of mutations in parE and gyrA leads to higher levels of resistance. This confirms that topoisomerase IV is the primary target of fluoroquinolones in S. pneumoniae.

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Characterization of Mutations in DNA Gyrase and Topoisomerase IV Involved in Quinolone Resistance of Mycoplasma gallisepticum Mutants Obtained In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.2.590-593.2002 ◽

2002 ◽

Vol 46 (2) ◽

pp. 590-593 ◽

Cited By ~ 24

Author(s):

A. K. Reinhardt ◽

C. M. Bébéar ◽

M. Kobisch ◽

I. Kempf ◽

A. V. Gautier-Bouchardon

Keyword(s):

Target Genes ◽

Dna Gyrase ◽

Mycoplasma Gallisepticum ◽

Quinolone Resistance ◽

Mycoplasma Species ◽

Topoisomerase Iv ◽

Genes Encoding ◽

Resistant Mutants

ABSTRACT Mycoplasma gallisepticum enrofloxacin-resistant mutants were generated by stepwise selection in increasing concentrations of enrofloxacin. Alterations were found in the quinolone resistance-determining regions of the four target genes encoding DNA gyrase and topoisomerase IV from these mutants. This is the first description of such mutations in an animal mycoplasma species.

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Characterization of the Novel DNA Gyrase Inhibitor AZD0914: Low Resistance Potential and Lack of Cross-Resistance in Neisseria gonorrhoeae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04456-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1478-1486 ◽

Cited By ~ 37

Author(s):

Richard A. Alm ◽

Sushmita D. Lahiri ◽

Amy Kutschke ◽

Linda G. Otterson ◽

Robert E. McLaughlin ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Oral Treatment ◽

Bacterial Species ◽

Treatment Options ◽

Dna Gyrase ◽

Cross Resistance ◽

Topoisomerase Iv ◽

Content Type

ABSTRACTThe unmet medical need for novel intervention strategies to treatNeisseria gonorrhoeaeinfections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potentin vitroactivities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment ofN. gonorrhoeaeinfections. The propensity to develop resistance against AZD0914 was examined inN. gonorrhoeaeand found to be extremely low, a finding supported by similar studies withStaphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different betweenN. gonorrhoeaeand other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment ofN. gonorrhoeaeinfections.

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In VitroandIn VivoCharacterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00619-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4830-4839 ◽

Cited By ~ 12

Author(s):

Christopher M. Tan ◽

Charles J. Gill ◽

Jin Wu ◽

Nathalie Toussaint ◽

Jingjun Yin ◽

...

Keyword(s):

Broad Spectrum ◽

Antibacterial Agents ◽

Cell Activity ◽

Dna Gyrase ◽

Topoisomerase Iv ◽

Bacterial Dna ◽

Whole Cell ◽

Content Type

ABSTRACTOxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, andin vivocharacterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV fromStaphylococcus aureusandEscherichia coliand displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergencein vitroat concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activityin vitroandin vivo.

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Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.12.2714 ◽

1996 ◽

Vol 40 (12) ◽

pp. 2714-2720 ◽

Cited By ~ 106

Author(s):

F Blanche ◽

B Cameron ◽

F X Bernard ◽

L Maton ◽

B Manse ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Strong Support ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Type Ii ◽

Topoisomerase Iv ◽

E Coli ◽

Dna Relaxation

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

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Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1129 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1129-1136 ◽

Cited By ~ 85

Author(s):

Xiao-Su Pan ◽

L. Mark Fisher

Keyword(s):

Streptococcus Pneumoniae ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Topoisomerase Iv ◽

T7 Promoter ◽

Bacterial Killing ◽

E Coli ◽

Apparent Homogeneity ◽

Comparable Activity

ABSTRACT Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

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