Dual inhibitory activity of sitafloxacin (DU-6859a) against DNA gyrase and topoisomerase IV of Streptococcus pneumoniae

Yoshikuni Onodera*, Yoko Uchida, Mayumi Tanaka and Kenichi Sato

New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd, 16-13, Kitakasai 1-Chome, Edogawa-ku, Tokyo 134-8630, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in-vitro inhibitory activities of sitafloxacin (DU-6859a) and other quinolones against Streptococcus pneumoniae DNA gyrase and topoisomerase IV were measured. IC50s of levofloxacin, ciprofloxacin, sparfloxacin and tosufloxacin against DNA gyrase were almost three to 12 times higher than those against topoisomerase IV. On the other hand, sitafloxacin showed dual inhibitory activity against both enzymes and its IC50s were the lowest among those of the quinolones tested. These results suggest that sitafloxacin is an effective agent against pneumococcal infections and that the incidence of drug-resistant mutants is low.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is the major cause of community acquired pneumonia. In recent years, fluoroquinolones have been used to treat pneumococcal infection, resulting in the emergence of quinolone-resistant pneumococci. The main mechanism of quinolone resistance is due to mutations in the target enzymes, DNA gyrase and topoisomerase IV. 1 ,2 ,3 Topoisomerase IV of S. pneumoniae is believed to be the primary target for most quinolones. Strains with low-level resistance contain parC mutations, whereas those with a high level of resistance have mutations in both gyrA and parC, which occurs by a two-step mutation.4,5 The increasing resistance observed in this species worldwide has led to the continued search for more active compounds.

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibacterial agents and bacterial strains

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 Mueller–Hinton 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 1–21 bp region of gyrA) and the reverse primer was 5'-AAATCCTATATTTGTCAGC-3' (2512–36). Primers for the gyrB gene were 5'-ATGACAGAAGAAATCAAAAATCTG-3' (1–24) and 5'-CATATTTCCAAGGGAAC-3' (1964–86), 5'-ATGTCTAACATTCAAAACATGTCCCTGG-3' (1–28) and 5'-CCACTCCTTATAACCTATTTC-3' (2572–98) for parC, 5'-GTGTCA- AAAAAGGAAATCAATATTAAC-3' (1–27) and 5'-GCGCCTCTTAAGCACTC-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 Luria–Bertani 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 Tris–HCl 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 Tris–HCl 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%.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Purification and characterization of topoisomerase IV and DNA gyrase subunit proteins

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 SDS–PAGE at about 130 kDa and 110 kDa for MBP–ParC and MBP–ParE, 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 10–40 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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Figure. (a) SDS–PAGE of purified proteins stained with Coomassie brilliant blue. Lane 1, purified MBP–ParC fusion protein; lane 2, purified MBP–ParE fusion protein; lane 3, MBP–ParC fusion protein after factor Xa cleavage; lane 4, MBP–ParE fusion protein after factor Xa cleavage. Numbers on the left are in Daltons. (b) Enzymatic activities of purified topoisomerase IV of S. pneumoniae J24. Lane 1, ParC; lane 2, ParE; lane 3, ParC and ParE; lane 4, ParC and ParE without ATP; lane 5, ParC and ParE without Mg2+; lane 6, without ParC and ParE. (c) Enzymatic activities of the purified DNA gyrase of S. pneumoniae J24. Lane 1, GyrA; lane 2, GyrB; lane 3, GyrA and GyrB; lane 4, GyrA and GyrB without ATP; lane 5, GyrA and GyrB without Mg2+; lane 6, without GyrA and GyrB. (d) Inhibitory activity of sitafloxacin against the decatenation activity of topoisomerase IV from S. pneumoniae J24. Lanes 1–7, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20 and 0 mg/L of sitafloxacin, respectively.

 
Comparison of inhibitory activities of antibacterial agents against topoisomerase IV and DNA gyrase

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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Table. Inhibition of S. pneumoniae J24 topoisomerase IV and DNA gyrase by quinolones and benzylpenicillin
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We expressed and purified the S. pneumoniae DNA gyrase and topoisomerase IV subunit proteins. The GyrA and GyrB proteins together, and the ParC and ParE proteins together, reconstituted ATP-dependent supercoiling activity characteristic of a DNA gyrase and ATP-dependent decatenation activity characteristic of a topoisomerase IV. The quinolones inhibited both enzymes, and the inhibitory activity of sitafloxacin was especially high. In addition, the ratios of the IC50s of the new quinolones tested for type II topoisomerase (IC50 for DNA gyrase/IC50 for topoisomerase IV) were all greater than 2.8, with the exception of sitafloxacin with a ratio of almost 1. This indicates that sitafloxacin has relatively equivalent inhibitory activity against both enzymes.

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.


    Notes
 
* Tel: +81-3-36890-0151; Fax: +81-3-5696-8344; E-mail: onode90j{at}daiichipharm.co.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . 1.Ferrero, L., Cameron, B., Manse, B., Lagneux, D., Crouzet, J., Famechon, A. & Blanche, F. (1994). Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones. Molecular Microbiology 13, 641–53.[ISI][Medline]

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, 1271–2.[ISI][Medline]

3 . 3.Hoshino, K., Kitamura, A., Morrissey, I., Sato, K., Kato, J. & Ikeda, H. (1994). Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition. Antimicrobial Agents and Chemotherapy 38, 2623–7.[Abstract]

4 . 4.Janior, C., Zeller, V., Kitzis, M.-D., Moreau, N. J. & Gutmann, L. (1996). High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA. Antimicrobial Agents and Chemotherapy 40, 2760–4.[Abstract]

5 . 5.Pan, X. S., Ambler, J., Mehtar, S. & Fisher, L. M. (1996). Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 2321–6.[Abstract]

6 . 6.Sato, K., Hoshino, K., Tanaka, M., Hayakawa, I. & Osada, Y. (1992). Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates. Antimicrobial Agents and Chemotherapy 36, 1491–8.[Abstract]

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, 1091–102.[Abstract]

8 . 8.National Committee for Clinical Laboratory Standards. (1990). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Second 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, 2362–6.[Abstract]

10 . 10.Pan, X. S. & Fisher, L. M. (1997). Targeting of DNA gyrase in Streptococcus pneumoniae by sparfloxacin: selective targeting of gyrase or topoisomerase IV by quinolones. Antimicrobial Agents and Chemotherapy 41, 471–4.[Abstract]

Received 12 January 1999; returned 30 March 1999; revised 14 May 1999; accepted 21 May 1999