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Antimicrobial Agents and Chemotherapy, September 2009, p. 3822-3831, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00113-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV{triangledown} ,{dagger}

Xiao-Su Pan, Katherine A. Gould, and L. Mark Fisher*

Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St. George's, University of London, London SW17 0RE, United Kingdom

Received 26 January 2009/ Returned for modification 4 April 2009/ Accepted 22 June 2009

Quinazoline-2,4-diones, such as PD 0305970, are new DNA gyrase and topoisomerase IV (topo IV) inhibitors with potent activity against gram-positive pathogens, including quinolone-resistant isolates. The mechanistic basis of dione activity vis-à-vis quinolones is not understood. We present evidence for Streptococcus pneumoniae gyrase and topo IV that PD 0305970 and quinolones interact differently with the enzyme breakage-reunion and Toprim domains, DNA, and Mg2+-four components that are juxtaposed in the topoisomerase cleavage complex to effect DNA scission. First, PD 0305970 targets primarily gyrase in Streptococcus pneumoniae. However, unlike quinolones, which select predominantly for gyrA (or topo IV parC) mutations in the breakage-reunion domain, unusually the dione selected for novel mutants with alterations that map to a region of the Toprim domain of GyrB (R456H and E474A or E474D) or ParE (D435H and E475A). This "dione resistance-determining region" overlaps the GyrB quinolone resistance-determining region and the region that binds essential Mg2+ ions, each function involving conserved EGDSA and PLRGK motifs. Second, dione-resistant gyrase and topo IV were inhibited by ciprofloxacin, whereas quinolone-resistant enzymes (GyrA S81F and ParC S79F) remained susceptible to PD 0305970. Third, dione-promoted DNA cleavage by gyrase occurred at a distinct repertoire of sites, implying that structural differences with quinolones are sensed at the DNA level. Fourth, unlike the situation with quinolones, the Mg2+ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg2+-dependent DNA unwinding. It appears that PD 0305970 interacts uniquely to stabilize the cleavage complex of gyrase/topo IV perhaps via an altered orientation directed by the bidentate 3-amino-2,4-dione moiety.

* Corresponding author. Mailing address: Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St. George's, University of London, Cranmer Terrace, London SW17 0RE, United Kingdom. Phone: 44 208 725 5782. Fax: 44 208 725 2992. E-mail: lfisher{at}sgul.ac.uk

{triangledown} Published ahead of print on 29 June 2009.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

Antimicrobial Agents and Chemotherapy, September 2009, p. 3822-3831, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00113-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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