Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Topoisomerase IV is the primary cellular target for most quinolones in Gram-positive bacteria; however, its interaction with these agents is poorly understood. Therefore, the effects of four clinically relevant antibacterial quinolones (ciprofloxacin, and three new generation quinolones: trovafloxacin, levofloxacin, and sparfloxacin) on the DNA cleavage/religation reaction of Staphylococcus aureus topoisomerase IV were characterized. These quinolones stimulated enzyme-mediated DNA scission to a similar extent, but their potencies varied significantly. Drug order in the absence of ATP was trovafloxacin > ciprofloxacin > levofloxacin > sparfloxacin. Potency was enhanced by ATP, but to a different extent for each drug. Under all conditions examined, trovafloxacin was the most potent quinolone and sparfloxacin was the least. The enhanced potency of trovafloxacin correlated with several properties. Trovafloxacin induced topoisomerase IV-mediated DNA scission more rapidly than other quinolones and generated more cleavage at some sites. The most striking correlation, however, was between quinolone potency and inhibition of enzyme-mediated DNA religation: the greater the potency, the stronger the inhibition. Dose-response experiments with two topoisomerase IV mutants that confer clinical resistance to quinolones (GrlA(Ser80Phe) and GrlA(Glu84Lys)) indicate that resistance is caused by a decrease in both drug affinity and efficacy. Trovafloxacin is more active against these enzymes than ciprofloxacin because it partially overcomes the effect on affinity. Finally, comparative studies on DNA cleavage and decatenation suggest that the antibacterial properties of trovafloxacin result from increased S. aureus topoisomerase IV-mediated DNA cleavage rather than inhibition of enzyme catalysis.
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PMID:Action of quinolones against Staphylococcus aureus topoisomerase IV: basis for DNA cleavage enhancement. 1070 24

The resistance mechanisms to fluoroquinolones in Staphylococcus aureus were clarified by analyzing mutations in the genes encoding target enzymes, and examining the expression of the efflux pump, and determining the inhibitory activities of fluoroquinolones against the altered enzymes. Mutations in the grlA and gyrA genes of 344 clinical strains of S. aureus isolated in 1994 in Japan were identified by combinations of methods - single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing - to identify possible relationships with fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA gene in 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA gene in 188 strains (54.7%). Among these mutations, the grlA mutation of TCC --> TTC or TAC (Ser-80 --> Phe or Tyr) and the gyrA mutation of TCA --> TTA (Ser-84 --> Leu) were the principal ones, being detected in 137 (39.8%) and 121 (35.2%) isolates, respectively. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC greater than or equal 3.13 microg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC50 =50 microg/ml). Those strains with the Ser-80 --> Phe or Tyr alteration in grlA, but wild type in gyrA showed a lower level of ciprofloxacin resistance (MIC50 less than or equal 12.5 microg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of concomitant mutations at codons 73, 84, or 88 in gyrA (MIC less than or equal 6.25 microg/ml). Sitafloxacin (DU-6859a) showed good activity in 186 of 193 isolates (96.4%), with an MIC of less than or equal 6.25 microg/ml. The contribution of membrane-associated multidrug efflux protein (NorA) expression to fluoroquinolone resistance was clarified by the checker-board titration method for determining the MIC of norfloxacin alone and in combination with carbonyl cyanide m-chlorophenylhydrazone. Among 344 clinical isolates, 139 strains (40.4%), in which the MIC of norfloxacin varied from 1.56 to >800 microg/ml, overexpressed the NorA protein. GrlA and GrlB proteins of topoisomerase IV, and GyrA and GyrB proteins of DNA gyrase encoded by genes with or without mutations were purified separately. The inhibitory activities of fluoroquinolones against the topoisomerase IV which contained a single amino acid change (Ser --> Phe at codon 80, Glu --> Lys at codon 84 of grlA, and Asp --> Asn at codon 432 of grlB) were from 5 to 95 times weaker than the inhibitory activities against the non-altered enzyme. These results suggest that the mutations in the corresponding genes may confer quinolone resistance; the active efflux pump, NorA, was considered to be the third quinolone-resistance mechanism. The numerous and complicated mutations seen may explain the rapid and widespread development of quinolone resistance described in S. aureus. Sitafloxacin showed good antibacterial activity against ciprofloxacin- or levofloxacin-resistant mutants because of its high inhibitory activity against both topoisomerase IV and DNA gyrase.
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PMID:Mechanism of quinolone resistance in Staphylococcus aureus. 1181 May 52

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlA(S81F) and GrlA(S81Y) quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg(2+) bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg(2+) concentrations to support DNA cleavage by GrlA(S81F) topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the enzyme-quinolone-DNA ternary complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg(2+) bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically relevant quinolone resistance mutations in bacterial type II topoisomerases.
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PMID:Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance. 2212 53

Although quinolones are the most commonly prescribed antibacterials, their use is threatened by an increasing prevalence of resistance. The most common causes of quinolone resistance are mutations of a specific serine or acidic residue in the A subunit of gyrase or topoisomerase IV. These amino acids are proposed to serve as a critical enzyme-quinolone interaction site by anchoring a water-metal ion bridge that coordinates drug binding. To probe the role of the proposed water-metal ion bridge, we characterized wild-type, GrlA(E85K), GrlA(S81F/E85K), GrlA(E85A), GrlA(S81F/E85A) and GrlA(S81F) Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg(2+) concentration required to produce maximal quinolone-induced DNA cleavage and restricted the divalent metal ions that could support quinolone activity. Individual mutation of Ser81 or Glu85 partially disrupted bridge function, whereas simultaneous mutation of both residues abrogated protein-quinolone interactions. Results provide functional evidence for the existence of the water-metal ion bridge, confirm that the serine and glutamic acid residues anchor the bridge, demonstrate that the bridge is the primary conduit for interactions between clinically relevant quinolones and topoisomerase IV and provide a likely mechanism for the most common causes of quinolone resistance.
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PMID:Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance. 2346 Feb 3

We investigated in vitro activity of a novel veterinary fluoroquinolone, pradofloxacin, against methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates and compared with other fluoroquinolones. A total of 38 MRSP isolates were subjected to agar disk diffusion tests for sensitivity to pradofloxacin, orbifloxacin, marbofloxacin, enrofloxacin, and ciprofloxacin. The minimal inhibitory concentration (MIC) values of pradofloxacin, ciprofloxacin, and enrofloxacin were determined. Mutations in the genes encoding DNA gyrase subunit A (GyrA) and topoisomerase IV (GrlA) proteins associated with fluoroquinolone resistance were studied by an analysis of partial sequences of the genes encoding these proteins. Two MRSP isolates were susceptible in disk diffusion and microdilution test to all fluoroquinolones tested, including pradofloxacin. Based on the results of the disk diffusion testing, 33 of 38 isolates showed resistance to pradofloxacin and 3 were intermediate, whereas, by pradofloxacin MIC testing, 35 isolates were classified as resistant and 1 as intermediate. Single alterations in GyrA and GrlA proteins were observed in the 35 resistant isolates and the 1 intermediate isolate (MIC results). These same 36 isolates were also resistant to the other tested fluoroquinolones. The results of the current study showed that MRSP isolates are usually resistant to all fluoroquinolones, including pradofloxacin. Therefore, in routine susceptibility testing to pradofloxacin by disk diffusion, the results should be carefully interpreted for MRSP isolates, especially those resistant to other fluoroquinolones and, in questionable cases, the pradofloxacin MIC should be determined to confirm the susceptibility testing results.
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PMID:Resistance of canine methicillin-resistant Staphylococcus pseudintermedius strains to pradofloxacin. 2744 31


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