title: Expression and purification of an active form of the Mycobacterium leprae DNA gyrase and its inhibition by quinolones.
abstract: Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the mouse footpad system, which requires at least 12 months before the results become available. We have developed an in vitro assay for studying the activities of quinolones against the DNA gyrase of M. leprae. We overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits separately as His-tagged proteins by using a pET plasmid carrying the gyrA and gyrB genes. The soluble 97.5-kDa GyrA and 74.5-kDa GyrB subunits were purified by nickel chelate chromatography and were reconstituted as an enzyme with DNA supercoiling activity. Based on the drug concentrations that inhibited DNA supercoiling by 50% or that induced DNA cleavage by 25%, the 13 quinolones tested clustered into three groups. Analysis of the quinolone structure-activity relationship demonstrates that the most active quinolones against M. leprae DNA gyrase share the following structural features: a substituted carbon at position 8, a cyclopropyl substituent at N-1, a fluorine at C-6, and a substituent ring at C-7. We conclude that the assays based on DNA supercoiling inhibition and drug-induced DNA cleavage on purified M. leprae DNA gyrase are rapid, efficient, and safe methods for the screening of quinolone derivatives with potential in vivo activities against M. leprae.
authors.affiliation: Faculté de Médecine Pierre et Marie Curie, Site Pitié-Salpêtrière, 91, Boulevard de l'Hôpital, Paris cedex 13, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Matrat
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Petrella
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Cambau
authors.forename: Wladimir
authors.initials: W
authors.lastname: Sougakoff
authors.forename: Vincent
authors.initials: V
authors.lastname: Jarlier
authors.forename: Alexandra
authors.initials: A
authors.lastname: Aubry
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D027081:DNA Gyrase / Q000302:isolation & purification; D004278:DNA, Superhelical / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009166:Mycobacterium leprae / Q000201:enzymology*; D015363:Quinolones / Q000494:pharmacology*; D059005:Topoisomerase II Inhibitors
chemical_list: D000890:Anti-Infective Agents; D004278:DNA, Superhelical; D004791:Enzyme Inhibitors; D015363:Quinolones; D059005:Topoisomerase II Inhibitors; D004247:DNA; D027081:DNA Gyrase