New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd, 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan
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Abstract |
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Introduction |
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Sitafloxacin (also known as DU-6859a;()-7-[(7S)-7-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6-fluoro-1-[(1R,S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid sesquihydrate) is a novel quinolone antibacterial agent. It has activity against a wide range of bacteria and is particularly effective against Gram-positive bacteria.6,7 In order to clarify the mechanism of action of sitafloxacin, we purified S. pneumoniae DNA gyrase and topoisomerase IV and determined the inhibitory activities of sitafloxacin against the purified enzymes.
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Materials and methods |
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All quinolones used in this study were synthesized at New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan. Benzylpenicillin was purchased from Sigma-Aldrich Co., Ltd, St. Louis, MD, USA. The bacterial strain used in this study was quinolone- and penicillin-sensitive S. pneumoniae J24.
Determination of MIC
The MICs were determined by the standard agar dilution method8 with MuellerHinton agar (Difco Laboratories, Detroit, MI, USA) containing 5% horse blood. Drug-containing agar plates were inoculated with one loopful (5 µL) of an inoculum corresponding to approximately 104 cfu/spot and were incubated for 18 h at 37°C. The MIC was defined as the lowest drug concentration that prevented visible growth of bacteria.
Construction of expression vectors
Four sets of oligonucleotide primers were designed for amplification of gyrA, gyrB, parC and parE genes and subsequent insertion into the pMAL-c2
fusion protein expression vector (New England Biolabs, Beverly, MA, USA). In each case, the
sequence of the forward primer was chosen at the initiation codon. For reverse primers, an XbaI site was introduced for cloning purposes. For gyrA, the forward primer was
5'-ATGCAGGATAAAAATTTAGTG-3' (containing the
121 bp region of gyrA) and the reverse primer was
5'-AAATCCTATATTTGTCAGC-3'
(251236).
Primers for the gyrB gene were
5'-ATGACAGAAGAAATCAAAAATCTG-3' (124) and
5'-CATATTT
CCAAGGGAAC-3'
(196486),
5'-ATGTCTAACATTCAAAACATGTCCCTGG-3' (128)
and 5'-CCACTCCTTAT
AACCTATTTC-3'
(257298) for parC, 5'-GTGTCA-
AAAAAGGAAATCAATATTAAC-3' (127) and
5'-GCGCCTCT
TAAGCACTC-3' for parE.
PCR was carried out on genomic DNA from strain J24 using the Expand High-Fidelity PCR
System (Boehringer Mannheim, Mannheim, Germany). Each gene was amplified for 25 cycles,
in which the conditions were 0.5 min at 94°C for denaturation, 0.5 min at 60°C for
annealing and 2 min at 72°C for polymerization. The DNA fragments were digested with XbaI, ligated into the XmnI and XbaI sites of the pMAL-c2
expression vector and transformed into Escherichia coli MC1061.
Purification of the enzymes
The GyrA and GyrB proteins of DNA gyrase, and ParC and ParE of topoisomerase IV, were purified separately as maltose-binding protein (MBP) fusion products from overproducing strains of E. coli. The E. coli MC1061/pMAL-c2 cells containing one of the above genes were incubated in LuriaBertani broth until log phase growth and isopropyl-ß-D-thiogalactopyranoside was added to the culture at a final concentration of 0.3 mM to induce protein synthesis. After a 2 h incubation, the cells were harvested and resuspended in TGED buffer (50 mM TrisHCl pH 8.0, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT)) supplemented with 0.5 mg/mL lysozyme and then incubated at 4°C for 30 min. The suspension was centrifuged at 100,000g for 40 min and the supernatant was loaded on to an amylose resin column previously equilibrated with TGED buffer. The column was washed with 10 volumes of TGED buffer, and the fusion proteins were eluted with 10 mM maltose. The eluted fractions were dialysed twice against TGED buffer for 6 h at 4°C and concentrated by dialysis against 50 mM TrisHCl pH 8.0/50% glycerol/1 mM EDTA/1 mM DTT.
Determination of inhibitory activity of drugs
The supercoiling activity of DNA gyrase and the decatenation activity of topoisomerase IV were measured by methods described previously.9 One unit of supercoiling activity was defined as the amount of GyrA and GyrB proteins required to supercoil 50% of 0.2µg of relaxed pBR322 plasmid DNA. One unit of decatenation activity was defined as the amount of ParC and ParE proteins required to fully decatenate 0.4µg of kDNA. The IC50 was defined as the drug concentration that reduced the enzymatic activity observed with drug-free controls by 50%.
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Results |
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The ParC and ParE proteins of topoisomerase IV of S. pneumoniae J24 were purified separately as MBP fusion proteins and single bands for each protein were observed by SDSPAGE at about 130 kDa and 110 kDa for MBPParC and MBPParE, respectively (Figure, a). A factor Xa recognition site was introduced into the fusion proteins and, after factor Xa digestion, the ParC and ParE proteins migrated at 93 and 72 kDa, respectively (Figure, a). Although no single protein had enzymatic activity, ParC and ParE together showed decatenation activity (Figure, b). Because these activities were not detected in the absence of ATP and Mg2+, these enzymes were ATP- and Mg2+-dependent (Figure, b). The optimum concentration range for the potassium cation was 1040 mM, and that for the magnesium cation was>5 mM (data not shown). From these results, the conditions for the decatenation assay were determined as described in Materials and methods. The GyrA and GyrB proteins of DNA gyrase were purified in a similar fashion, and reconstituted enzyme showed ATP-dependent supercoiling activity (Figure, c).
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The quinolones inhibited the activities of the enzymes in a concentration-dependent manner (Figure, d). In contrast, benzylpenicillin, which does not inhibit either enzyme, had no effect on their activity (data not shown). The IC50s of the quinolones were calculated from the quantified bands, which corresponded to fully decatenated substrate or supercoiled DNA (Table). The IC50 values of the quinolones against type II topoisomerases compared with their MICs had correlation coefficients of 0.88 for topoisomerase IV and 0.87 for DNA gyrase. Of the quinolones tested, sitafloxacin showed the highest inhibitory activity against the enzymes. Moreover, unlike the other quinolones, the IC50s of sitafloxacin against DNA gyrase and topoisomerase IV were almost equal.
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Discussion |
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Against S. pneumoniae, the first target of quinolones differs between the drugs. The target of levofloxacin and ciprofloxacin is thought to be topoisomerase IV,4,5 whereas that of sparfloxacin is reported to be DNA gyrase.10 When resistant mutants were selected stepwise with increasing ciprofloxacin concentrations, a parC mutation was found in low-level resistant mutants, and parC and gyrA double mutations were detected in high-level resistant strains.5 S. pneumoniae acquires greater resistance to quinolones step-by-step. However, as sitafloxacin showed similar and the lowest IC50 values against the two target enzymes, the incidence of sitafloxacin-resistant strains should be very low, for the acquisition of sitafloxacin resistance would necessitate mutation of both enzymes at the same time. Furthermore, this drug would be effective against parC mutants and gyrA mutants, since it has the ability to inhibit the wild-type enzymes. From these results, sitafloxacin should be an effective agent against pneumococcal infections. The activity of sitafloxacin against the mutated enzymes and the role of other quinolone-resistant factors, such as the efflux pump, will be clarified by further study.
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Notes |
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References |
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2 . 2.Yoshida, H., Bogaki, M., Nakamura, M. & Nakamura, S. (1990). Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrobial Agents and Chemotherapy 34, 12712.[ISI][Medline]
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7 . 7.Tanaka, M., Hoshino, K., Hohmura, M., Ishida, H., Kitamura, A., Sato, K. et al. (1996). Effect of growth conditions on antimicrobial activity of DU-6859a and its bactericidal activity determined by the killing curve method. Journal of Antimicrobial Chemotherapy 37, 1091102.[Abstract]
8 . 8.National Committee for Clinical Laboratory Standards. (1990). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallySecond edition: Approved Standard M7-A2.NCCLS, Villanova, PA.
9 . 9.Tanaka, M., Onodera, Y., Uchida, Y., Sato, K. & Hayakawa, I. (1997). Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV purified from Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 41, 23626.[Abstract]
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Received 12 January 1999; returned 30 March 1999; revised 14 May 1999; accepted 21 May 1999