Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

In order to clarify the mechanism of action of quinolones against Staphylococcus aureus, GrlA and GrlB proteins of topoisomerase IV encoded by genes with or without mutations were purified separately as fusion proteins with maltose-binding protein in Escherichia coli. The reconstituted enzymes showed ATP-dependent decatenation and relaxing activities but had no supercoiling activity. The inhibitory effects of quinolones on the decatenation activity of topoisomerase IV were determined by quantitative electrophoresis with kinetoplast DNA as a substrate. The 50% inhibitory concentrations (IC50s) of levofloxacin, DR-3354, DU-6859a, DV-7751a, ciprofloxacin, sparfloxacin, and tosufloxacin against topoisomerase IV of S. aureus FDA 209-P were 2.3, 97, 0.45, 1.5, 2.5, 7.4, and 1.8 microg/ml, respectively, and were correlated well with their MICs. The IC50s of these drugs were from 2 to 20 times lower than those for the DNA gyrase. These results support genetic evidence that the primary target of new quinolones is topoisomerase IV in quinolone-susceptible strains of S. aureus. Three altered proteins of topoisomerase IV containing Ser-->Phe changes at codon 80 or Glu-->Lys changes at codon 84 of grlA, or both, were also purified. The inhibitory activities of quinolones against the topoisomerase IV which contained a single amino acid change were from 8 to 95 times weaker than those against the nonaltered enzyme. These results suggest that the mutations in the corresponding genes confer quinolone resistance.
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PMID:Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV purified from Staphylococcus aureus. 937 34

Previous studies have shown that topoisomerase IV and DNA gyrase interact with quinolones and coumarins in different ways. The MICs of coumarins (novobiocin and coumermycin) for MT5, a Staphylococcus aureus nov mutant, are higher than those for wild-type strains. Sequencing the gyrB gene encoding one subunit of the DNA gyrase revealed the presence of a double mutation likely to be responsible for this resistance: at codon 102 (Ile to Ser) and at codon 144 (Arg to Ile). For single-step flqA mutant MT5224c9, previously selected on ciprofloxacin, the fluoroquinolone MIC was higher and the coumarin MIC was lower than those for its parent, MT5. Sequencing the grlB and grlA genes of topoisomerase IV of MT5224c9 showed a single Asn-470-to-Asp mutation in GrlB. Genetic outcrosses by transformation with chromosomal DNA and introduction of plasmids carrying either the wild-type or the mutated grlB gene indicated that this mutation causes both increased MICs of fluoroquinolones and decreased MICs of coumarins and that the mutant grlB allele is codominant for both phenotypes with multicopy alleles. Integration of these plasmids into the chromosome confirmed the codominance of fluoroquinolone resistance, but grlB+ appeared dominant over grlB (Asp-470) for coumarin resistance. Finally, the gyrA (Leu-84) mutation previously described as silent for fluoroquinolone resistance increased the MIC of nalidixic acid, a nonfluorinated quinolone. Combining the grlA (Phe-80) and grlB (Asp-470) mutations with this gyrA mutation also had differing effects. The findings indicate that alterations in topoisomerases may have pleiotropic effects on different classes of inhibitors as well as on inhibitors within the same class. A full understanding of drug action and resistance at the molecular level must take into account both inhibitor structure-activity relationships and the effects of different classes of topoisomerase mutants.
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PMID:Mutations in topoisomerase IV and DNA gyrase of Staphylococcus aureus: novel pleiotropic effects on quinolone and coumarin activity. 944 71

Proteasomes and mitochondrial membrane changes are involved in thymocyte apoptosis. The hierarchical relationship between protease activation and mitochondrial alterations has been elusive. Here we show that inhibition of proteasomes by two specific agents, lactacystin or MG132, prevents all manifestations of thymocyte apoptosis induced by the glucocorticoid receptor agonist dexamethasone or by the topoisomerase II inhibitor etoposide. Lactacystin and MG132 prevent the early disruption of the mitochondrial transmembrane potential (delta psi(m)), which precedes caspase activation, exposure of phosphatidylserine, and nuclear DNA fragmentation. In contrast, stabilization of the delta psi(m) using the permeability transition pore inhibitor bongkrekic acid or inhibition of caspases by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone does not prevent the activation of proteasomes, as determined with the fluorogenic substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin . Thus, proteasome activation occurs upstream from mitochondrial changes and caspase activation. Whereas the proteasome-specific agents lactacystin and MG132 truly maintain thymocyte viability, a number of protease inhibitors that inhibit nuclear DNA fragmentation (acetyl-Asp-Glu-Val-Asp-fluoromethylketone; N-Boc-Asp(OMe)-fluoromethylketone; N-tosyl-L-Phe-chloromethylketone) do not prevent the cytolysis induced by DEX or etoposide. These latter agents fail to interfere with the preapoptotic delta psi(m) disruption. Altogether, our data indicate that different proteases may be involved in the pre- or postmitochondrial phase of apoptosis. Only those protease inhibitors that interrupt the apoptotic process at the premitochondrial stage can actually preserve cell viability.
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PMID:Proteasome activation occurs at an early, premitochondrial step of thymocyte apoptosis. 964 4

The nucleotide sequences of the quinolone resistance-determining regions (QRDRs) of the parC and gyrA genes from seven ciprofloxacin-resistant (Cpr) isolates of viridans group streptococci (two high-level Cpr Streptococcus oralis and five low-level Cpr Streptococcus mitis isolates) were determined and compared with those obtained from susceptible isolates. The nucleotide sequences of the QRDRs of the parE and gyrB genes from the five low-level Cpr S. mitis isolates and from the NCTC 12261 type strain were also analyzed. Four of these low-level Cpr isolates had changes affecting the subunits of DNA topoisomerase IV: three in Ser-79 (to Phe or Ile) of ParC and one in ParE at a position not previously described to be involved in quinolone resistance (Pro-424). One isolate did not show any mutation. The two high-level Cpr S. oralis isolates showed mutations affecting equivalent residue positions of ParC and GyrA, namely, Ser-79 to Phe and Ser-81 to Phe or Tyr, respectively. The parC mutations were able to transform Streptococcus pneumoniae to ciprofloxacin resistance, while the gyrA mutations transformed S. pneumoniae only when mutations in parC were present. These results suggest that DNA topoisomerase IV is a primary target of ciprofloxacin in viridans group streptococci, DNA gyrase being a secondary target.
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PMID:Fluoroquinolone resistance mutations in the parC, parE, and gyrA genes of clinical isolates of viridans group streptococci. 979 5

We examined the response of Streptococcus pneumoniae 7785 to clinafloxacin, a novel C-8-substituted fluoroquinolone which is being developed as an antipneumococcal agent. Clinafloxacin was highly active against S. pneumoniae 7785 (MIC, 0.125 microg/ml), and neither gyrA nor parC quinolone resistance mutations alone had much effect on this activity. A combination of both mutations was needed to register resistance, suggesting that both gyrase and topoisomerase IV are clinafloxacin targets in vivo. The sparfloxacin and ciprofloxacin MICs for the parC-gyrA mutants were 16 to 32 and 32 to 64 microg/ml, respectively, but the clinafloxacin MIC was 1 microg/ml, i.e., within clinafloxacin levels achievable in human serum. S. pneumoniae 7785 mutants could be selected stepwise with clinafloxacin at a low frequency, yielding first-, second-, third-, and fourth-step mutants for which clinafloxacin MICs were 0.25, 1, 6, and 32 to 64 microg/ml, respectively. Thus, high-level resistance to clinafloxacin required four steps. Characterization of the quinolone resistance-determining regions of the gyrA, parC, gyrB, and parE genes by PCR, HinfI restriction fragment length polymorphism, and DNA sequence analysis revealed an invariant resistance pathway involving sequential mutations in gyrA or gyrB, in parC, in gyrA, and finally in parC or parE. No evidence was found for other resistance mechanisms. The gyrA mutations in first- and third-step mutants altered GyrA hot spots Ser-83 to Phe or Tyr (Escherichia coli coordinates) and Glu-87 to Gln or Lys; second- and fourth-step parC mutations changed equivalent hot spots Ser-79 to Phe or Tyr and Asp-83 to Ala. gyrB and parE changes produced novel alterations of GyrB Glu-474 to Lys and of Pro-454 to Ser in the ParE PLRGK motif. Difficulty in selecting first-step gyrase mutants (isolated with 0.125 [but not 0.25] microg of clinafloxacin per ml at a frequency of 5.0 x 10(-10) to 8.5 x 10(-10)) accompanied by the small (twofold) MIC increase suggested only a modest drug preference for gyrase. Given the susceptibility of defined gyrA or parC mutants, the results suggested that clinafloxacin displays comparable if unequal targeting of gyrase and topoisomerase IV. Dual targeting and the intrinsic potency of clinafloxacin against S. pneumoniae and its first- and second-step mutants are desirable features in limiting the emergence of bacterial resistance.
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PMID:DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae. 979 8

We determined the sequences of the quinolone resistance-determining regions of gyrA, gyrB, and parC genes for 30 clinical strains of Pseudomonas aeruginosa resistant to ciprofloxacin that were previously complemented by wild-type gyrA and gyrB plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248-2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in gyrA (codon 83 or 87). Ten of them had an additional mutation in parC (codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type gyrA allele. The remaining two strains harbored the first gyrB mutation described in P. aeruginosa, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e., gyrA and parC) were significantly more resistant to fluoroquinolones than those with a single mutation in gyrA or gyrB (geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 microg/ml, P < 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6, P < 0. 01). No mutant with a parC mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that gyrA mutations are the major mechanism of resistance to fluoroquinolones for clinical strains of P. aeruginosa and that additional mutations in parC lead to a higher level of quinolone resistance.
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PMID:Type II topoisomerase mutations in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. 986 66

Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, > or = 8 micrograms/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 micrograms/ml) and trovafloxacin (MICs, 0.5 to 1 microgram/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA, parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.
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PMID:Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC, and parE loci. 992 27

Human leukemic HL60 cells were selected for resistance to alkylating agents by stepwise exposure to increasing concentrations of L-phenylalanine mustard (melphalan). The resulting resistant cell line (R-HL60) was 4-fold resistant (melphalan IC50 value, 27.84 +/- 4.2 microM) to melphalan compared with parental HL60 cells (melphalan IC50 value, 6.9 +/- 1.78 microM). Nuclear extracts from R-HL60 cells possess a approximately 4-fold increase in DNA topoisomerase II activity compared with parental HL60 cells. As determined using Western blot analysis, the level of topoisomerase IIalpha protein expressed in R-HL60 cells was approximately 3-fold that of parental HL60 cells. However, there were no differences observed in the level of topoisomerase IIbeta protein, in the topoisomerase I activity, or in the level of topoisomerase I protein expression comparing the two cell lines. R-HL60 cells were 5-fold more sensitive than parental HL60 cells to the cytotoxic effect of the topoisomerase II inhibitor doxorubicin. The sensitivity to the cytotoxic effects of the topoisomerase I inhibitor camptothecin did not differ in R-HL60 and parental HL60 cell lines. Preincubation with doxorubicin significantly increased melphalan-induced interstrand DNA cross-link formation and cytotoxicity in R-HL60 cells compared with the parental HL60 cells. The affinity of topoisomerase II for UV-irradiated cross-linked HL60 DNA was increased by approximately 2.5-fold compared with that of HL60 native DNA. The affinity of topoisomerase II for both UV-irradiated (cross-linked) and native DNA was significantly decreased after doxorubicin pretreatment. Elevated topoisomerase II activity and the increased affinity of topoisomerase II for cross-linked DNA in melphalan-resistant cells seems to contribute to alkylator resistance by changing DNA topology, thereby facilitating DNA repair.
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PMID:Induction of alkylator (melphalan) resistance in HL60 cells is accompanied by increased levels of topoisomerase II expression and function. 1038 95

Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptible Staphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 x 10(-10) at 2 to 4x the MIC) than with levofloxacin or ciprofloxacin (3.0 x 10(-7) to 3.0 x 10(-8) at 2 to 4x the MIC). Mutants with a change in GrlA (Ser80-->Phe or Tyr) were most commonly selected with trovafloxacin, ciprofloxacin, levofloxacin, or pefloxacin. First-step mutants were difficult to select with sparfloxacin; however, second-step mutants with mutations in gyrA were easily selected when a preexisting mutation in grlA was present. Against 29 S. aureus clinical isolates with known mutations in gyrA and/or grlA, trovafloxacin was the most active quinolone tested (MIC at which 50% of isolates are inhibited [MIC(50)] and MIC(90), 1 and 4 microg/ml, respectively); in comparison, MIC(50)s and MIC(90)s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 microg/ml for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin, respectively. Strains with a mutation in grlA only were generally susceptible to all of the quinolones tested. For mutants with changes in both grlA and gyrA MICs were higher and were generally above the susceptibility breakpoint for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin. Addition of reserpine (20 microg/ml) lowered the MICs only of ciprofloxacin fourfold or more for 18 of 29 clinical strains. Topoisomerase IV and DNA gyrase genes were cloned from S. aureus RN4220 and from two mutants with changes in GrlA (Ser80-->Phe and Glu84-->Lys). The enzymes were overexpressed in Escherichia coli GI724, purified, and used in DNA catalytic and cleavage assays that measured the relative potency of each quinolone. Trovafloxacin was at least five times more potent than ciprofloxacin, sparfloxacin, levofloxacin, or pefloxacin in stimulating topoisomerase IV-mediated DNA cleavage. While all of the quinolones were less potent in cleavage assays with the altered topoisomerase IV, trovafloxacin retained its greater potency relative to those of the other quinolones tested. The greater intrinsic potency of trovafloxacin against the lethal topoisomerase IV target in S. aureus contributes to its improved potency against clinical strains of S. aureus that are resistant to other quinolones.
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PMID:Activities of trovafloxacin compared with those of other fluoroquinolones against purified topoisomerases and gyrA and grlA mutants of Staphylococcus aureus. 1042 1

In this study, we assessed the activity of ciprofloxacin, levofloxacin, sparfloxacin, and trovafloxacin against clinical isolates of Streptococcus pneumoniae that were resistant to the less-recently developed fluoroquinolones by using defined amino acid substitutions in DNA gyrase and topoisomerase IV. The molecular basis for resistance was assessed by using mutants selected with trovafloxacin, ciprofloxacin, and levofloxacin in vitro. This demonstrated that the primary target of trovafloxacin in S. pneumoniae is the ParC subunit of DNA topoisomerase IV, similar to most other fluoroquinolones. However, first-step mutants bearing the Ser79-->Phe/Tyr substitution in topoisomerase IV subunit ParC were susceptible to trovafloxacin with a minimum inhibitory concentration of 0.25 microg/ml, and mutations in the structural genes for both topoisomerase IV subunit ParC (parC) and the DNA gyrase subunit (gyrA) were required to achieve levels of resistance above the breakpoint. The data also suggest that enhanced activity of trovafloxacin against pneumococci is due to a combination of factors that may include reduced efflux of this agent and an enhanced activity against both DNA gyrase and topoisomerase IV.
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PMID:Contribution of topoisomerase IV and DNA gyrase mutations in Streptococcus pneumoniae to resistance to novel fluoroquinolones. 1042 26


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