scholarly journals Inhibitory Activities of Gatifloxacin (AM-1155), a Newly Developed Fluoroquinolone, against Bacterial and Mammalian Type II Topoisomerases

1998 ◽  
Vol 42 (10) ◽  
pp. 2678-2681 ◽  
Author(s):  
Masaya Takei ◽  
Hideyuki Fukuda ◽  
Tokutaro Yasue ◽  
Masaki Hosaka ◽  
Yasuo Oomori
Keyword(s):  
Hela Cell ◽  
Topoisomerase Ii ◽  
Dna Gyrase ◽  
Antibacterial Activities ◽  
Type Ii ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Inhibitory Activities ◽  
Bacterial Type

ABSTRACT We determined the inhibitory activities of gatifloxacin againstStaphylococcus aureus topoisomerase IV,Escherichia coli DNA gyrase, and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 forS. aureus, r = 0.972 for E. coli, and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 μg/ml for S. aureustopoisomerase IV; IC50 = 0.109 μg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 μg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureustopoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However, the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones, indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases.

scholarly journals Antibacterial Activities and Inhibitory Effects of Sitafloxacin (DU-6859a) and Its Optical Isomers against Type II Topoisomerases

1998 ◽  
Vol 42 (5) ◽  
pp. 1284-1287 ◽  
Author(s):  
Takaaki Akasaka ◽  
Seiko Kurosaka ◽  
Yoko Uchida ◽  
Mayumi Tanaka ◽  
Kenichi Sato ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Antibacterial Activities ◽  
Optical Isomers ◽  
Type Ii ◽  
Inhibitory Effects ◽  
Topoisomerase Iv ◽  
Bacterial Dna ◽  
Inhibitory Activities ◽  
Bacterial Type

ABSTRACT The in vitro inhibitory effects of sitafloxacin (DU-6859a) and its three stereoisomers on bacterial DNA gyrase from Escherichia coli, topoisomerase IV from Staphylococcus aureus, and topoisomerase II from human placenta were compared. No correlation was observed between the inhibitory activities of quinolones against bacterial type II topoisomerases and those against human topoisomerase II. Sitafloxacin showed the most potent inhibitory activities against bacterial type II topoisomerases and the lowest activity against human type II topoisomerase.

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

1996 ◽  
Vol 40 (12) ◽  
pp. 2714-2720 ◽  
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.

scholarly journals Contribution of the 8-Methoxy Group to the Activity of Gatifloxacin against Type II Topoisomerases of Streptococcus pneumoniae

2003 ◽  
Vol 47 (1) ◽  
pp. 77-81 ◽  
Author(s):  
Ryuta Kishii ◽  
Masaya Takei ◽  
Hideyuki Fukuda ◽  
Katsuhiko Hayashi ◽  
Masaki Hosaka
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pneumoniae ◽  
Methoxy Group ◽  
Dna Gyrase ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Methoxy Groups ◽  
Dual Inhibition ◽  
Inhibitory Activities

ABSTRACT The inhibitory activities (50% inhibitory concentrations [IC50s]) of gatifloxacin and other quinolones against both DNA gyrase and topoisomerase IV of the wild-type Streptococcus pneumoniae IID553 were determined. The IC50s of 10 compounds ranged from 4.28 to 582 μg/ml against DNA gyrase and from 1.90 to 35.2 μg/ml against topoisomerase IV. The inhibitory activity against DNA gyrase was more varied than that against topoisomerase IV among fluoroquinolones. The IC50s for DNA gyrase of the 8-methoxy quinolones gatifloxacin and AM-1147 were approximately seven times lower than those of their 8-H counterparts AM-1121 and ciprofloxacin, whereas the IC50s for topoisomerase IV were 1.5 times lower. Moreover, the IC50 ratios (IC50 for DNA gyrase/IC50 for topoisomerase IV) of gatifloxacin, AM-1147, and moxifloxacin, which possess 8-methoxy groups, were almost the same. The 8-methoxy quinolones showed higher antibacterial activity and less mutant selectivity against IID553 than their 8-H counterparts. These results suggest that the 8-methoxy group enhances both target inhibition, especially for DNA gyrase, leading to potent antipneumococcal activity and dual inhibition against both DNA gyrase and topoisomerase IV in the bacterial cell.

scholarly journals A Mutation in Escherichia coli DNA Gyrase Conferring Quinolone Resistance Results in Sensitivity to Drugs Targeting Eukaryotic Topoisomerase II

2004 ◽  
Vol 48 (12) ◽  
pp. 4495-4504 ◽  
Author(s):  
Thomas Gruger ◽  
John L. Nitiss ◽  
Anthony Maxwell ◽  
E. Lynn Zechiedrich ◽  
Peter Heisig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Antimicrobial Agents ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Quinolone Resistance ◽  
Site Directed Mutagenesis ◽  
Type Ii ◽  
Topoisomerase Iv

ABSTRACT Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (GyrS83W) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of GyrS83W, and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for GyrS83W-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the GyrS83W mutation in E. coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.

scholarly journals Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor

2014 ◽  
Vol 59 (1) ◽  
pp. 331-337 ◽  
Author(s):  
Asha S. Nayar ◽  
Thomas J. Dougherty ◽  
Folkert Reck ◽  
Jason Thresher ◽  
Ning Gao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Second Step ◽  
Type Ii ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
Content Type ◽  
E Coli ◽  
Topoisomerase Inhibitor ◽  
Bacterial Type

ABSTRACTIn a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657–2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations inPseudomonas aeruginosaarose exclusively in thenfxBgene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in bothPseudomonas aeruginosaandEscherichia coli. Second-step mutations inP. aeruginosaarose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-stepnfxBefflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected inE. coli. Genetic introduction of the Asp82 mutations observed inP. aeruginosadid not significantly increase the NBTI MIC inE. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC inE. coli. Only the presence of mutations in both targets ofE. coliled to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

scholarly journals Target Preference of 15 Quinolones against Staphylococcus aureus, Based on Antibacterial Activities and Target Inhibition

2001 ◽  
Vol 45 (12) ◽  
pp. 3544-3547 ◽  
Author(s):  
Masaya Takei ◽  
Hideyuki Fukuda ◽  
Ryuta Kishii ◽  
Masaki Hosaka
Keyword(s):  
Staphylococcus Aureus ◽  
Type Strain ◽  
Wild Type Strain ◽  
Dna Gyrase ◽  
Antibacterial Activities ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Mutant Strains

ABSTRACT The antibacterial activities and target inhibition of 15 quinolones against grlA and gyrA mutant strains were studied. The strains were obtained from wild-type Staphylococcus aureus MS5935 by selection with norfloxacin and nadifloxacin, respectively. The antibacterial activities of most quinolones against both mutant strains were lower than those against the wild-type strain. The ratios of MICs for the gyrA mutant strain to those for the grlA mutant strain (MIC ratio) varied from 0.125 to 4. The ratios of 50% inhibitory concentrations (IC50s) of quinolones against topoisomerase IV to those against DNA gyrase (IC50 ratios) also varied, from 0.177 to 5.52. A significant correlation between the MIC ratios and the IC50ratios was observed (r = 0.919; P < 0.001). These results suggest that the antibacterial activities of quinolones against the wild-type strain are involved not only in topoisomerase IV inhibition but also in DNA gyrase inhibition and that the target preference in the wild-type strain can be anticipated by the MIC ratios. Based on the MIC ratios, the quinolones were classified into three categories. Type I quinolones (norfloxacin, enoxacin, fleroxacin, ciprofloxacin, lomefloxacin, trovafloxacin, grepafloxacin, ofloxacin, and levofloxacin) had MIC ratios of <1, type II quinolones (sparfloxacin and nadifloxacin) had MIC ratios of >1, and type III quinolones (gatifloxacin, pazufloxacin, moxifloxacin, and clinafloxacin) had MIC ratios of 1. Type I and type II quinolones seem to prefer topoisomerase IV and DNA gyrase, respectively. Type III quinolones seem to target both enzymes at nearly the same level in bacterial cells (a phenomenon known as the dual-targeting property), and their IC50 ratios were approximately 2.

scholarly journals Type II Topoisomerase Mutations in Fluoroquinolone-Resistant Clinical Strains of Pseudomonas aeruginosa Isolated in 1998 and 1999: Role of Target Enzyme in Mechanism of Fluoroquinolone Resistance

2001 ◽  
Vol 45 (8) ◽  
pp. 2263-2268 ◽  
Author(s):  
Takaaki Akasaka ◽  
Mayumi Tanaka ◽  
Akihito Yamaguchi ◽  
Kenichi Sato
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Dna Gyrase ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Type Ii ◽  
Topoisomerase Iv ◽  
Major Mechanism ◽  
Inhibitory Activities

ABSTRACT The major mechanism of resistance to fluoroquinolones forPseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) ofgyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC orparE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations ingyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.

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

2006 ◽  
Vol 50 (4) ◽  
pp. 1228-1237 ◽  
Author(s):  
Nagraj Mani ◽  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Brian Hanzelka ◽  
Ute Müh ◽  
...  
Keyword(s):  
Bacterial Resistance ◽  
Wide Spectrum ◽  
Dna Gyrase ◽  
Antibacterial Activities ◽  
Mechanisms Of Action ◽  
Fluoroquinolone Resistance ◽  
Topoisomerase Iv ◽  
Low Frequencies

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.

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

1999 ◽  
Vol 43 (5) ◽  
pp. 1129-1136 ◽  
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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