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Antimicrobial Agents and Chemotherapy, April 2004, p. 1281-1288, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1281-1288.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis DNA Gyrase: Interaction with Quinolones and Correlation with Antimycobacterial Drug Activity

Alexandra Aubry,1 Xiao-Su Pan,2 L. Mark Fisher,2 Vincent Jarlier,1 and Emmanuelle Cambau1*

Laboratoire de Bactériologie, Faculté de Médecine Pitié-Salpêtrière, and Laboratoire de Recherche Moléculaire sur les Antibiotiques, INSERM 0004, Université Pierre et Marie Curie, Paris, France,1 Molecular Genetics Group, Department of Biochemistry and Immunology, St. George's Hospital Medical School, University of London, London SW17 ORE, United Kingdom2

Received 21 July 2003/ Returned for modification 4 November 2003/ Accepted 9 December 2003

Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in Mycobacterium tuberculosis. Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of M. tuberculosis have precluded direct purification of its gyrase and detailed analysis of quinolone action. To address these issues, we separately overexpressed the M. tuberculosis DNA gyrase GyrA and GyrB subunits as His-tagged proteins in Escherichia coli from pET plasmids carrying gyrA and gyrB genes. The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity. The drug concentration that inhibited DNA supercoiling by 50% (IC50) was measured for 22 different quinolones, and values ranged from 2 to 3 µg/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 µg/ml (pipemidic acid and nalidixic acid). By comparison, MICs measured against M. tuberculosis ranged from 0.12 µg/ml (for gatifloxacin) to 128 µg/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC50s (R2 = 0.9). Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion. Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (R2 = 0.7). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against M. tuberculosis. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting M. tuberculosis gyrase.

* Corresponding author. Mailing address: Faculté de Médecine Pitié-Salpêtrière, 91, Boulevard de l'Hôpital, 75634 Paris Cedex 13, France. Phone: 33 1 40 77 97 46. Fax: 33 1 45 82 75 77. E-mail: cambau{at}chups.jussieu.fr.

Antimicrobial Agents and Chemotherapy, April 2004, p. 1281-1288, Vol. 48, No. 4
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.4.1281-1288.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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