Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

Abstract

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 Mg 2؉ -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 Mg 2؉ 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 Mg 2؉ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg 2؉ -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.The established and emerging resistance of gram-positive pathogens to fluoroquinolones, macrolides, and beta-lactams has underlined the need for new antimicrobial agents (2,6,28,32,35,46,52). Recent work has shown that quinazolinediones constitute a promising new class of antibacterial inhibitors of DNA gyrase and topoisomerase IV (topo IV) that share structural similarity with fluoroquinolones (14,50,51). Among this group of inhibitors, the 3-aminoquinazolinedione PD 0305790 (3-amino-7-[(R)-3-[(S)-1-aminoethyl]-pyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-8-methyl-1H-quinazoline-2,4-dione) (Fig. 1) has particularly potent activity against susceptible and drugresistant gram-positive species, especially Streptococcus pneumoniae (23). The drug differs most notably from quinolones, e.g., ciprofloxacin, in having the 3-aminoquinazoline 2,4-dione system in place of the 3-carboxy-substituted 4-quinolone ring (Fig. 1). Surprisingly, these subtle differences in drug structure confer activity against quinolone-resistant strains (23).Initial work with Escherichia coli gyrase has shown that, like quinolones, 3-aminodiones, such as PD 0305790, stabilize an enzyme "cleavage complex" on DNA (50). This complex is thought to ref...

Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase

“…In several other species, quinazolinediones containing a 3′-(aminomethyl) pyrrolidinyl [3′-(AM)P] (or a related) group at C7 maintain activity against enzymes containing mutations that disrupt the water-metal ion bridge by forming binding interactions primarily through the C7 substituent (20,21,32,(38)(39)(40)(41)(42). As seen in Fig.…”

Challenges of Antibacterial Discovery

“…Recent work from Fisher's laboratory, using selection of stepwise mutations, confirmed that the primary target of diones is GyrB, with secondary mutations in ParE. Furthermore, it was shown that dione inhibition leads to double-stranded DNA breakage (288). Thus, it appears that while mutations arise in the ATPase subunit, it is the catalytic activity of the topoisomerases that is inhibited.…”

“…As might be expected from the site of resistance mutations, diones are not cross resistant with FQs. In fact, while FQ resistance does not alter susceptibility to diones, resistance to the diones may cause hypersusceptibility to FQs (288). No characterization of toxicity (including selectivity for bacterial over mammalian topoisomerases) of the diones has been reported.…”

Comparison of In Vitro Activities of Fluoroquinolone-Like 2,4- and 1,3-Diones

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