Mutagenesis of E477 or K505 in the B‘ Domain of Human Topoisomerase IIβ Increases the Requirement for Magnesium Ions during Strand Passage

125

cThe unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Characterization of the Novel DNA Gyrase Inhibitor AZD0914: Low Resistance Potential and Lack of Cross-Resistance in Neisseria gonorrhoeae

Alm

Lahiri

Kutschke

et al. 2015

125

“…We identified a novel mutation in the parE gene of P. aeruginosa, which changed Asp-419 to Asn, located in the EGDSA motif, which is highly conserved in type II topoisomerase B subunits (28). An alteration in this motif has been implicated in a fluoroquinolone-resistant E. coli GyrB (33).…”

Section: Resultsmentioning

confidence: 99%

Type II Topoisomerase Mutations in Fluoroquinolone-Resistant Clinical Strains of Pseudomonas aeruginosa Isolated in 1998 and 1999: Role of Target Enzyme in Mechanism of Fluoroquinolone Resistance

Akasaka¹,

Tanaka²,

Yamaguchi

et al. 2001

164

132

The major mechanism of resistance to fluoroquinolones for Pseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinoloneresistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC or parE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in gyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.

“…Moreover, we have solved the crystal structures of these complexes, revealing two quinolone drugs intercalated at the highly bent DNA gate (25). It is known that different quinolones can preferentially target gyrase or topo IV in Streptococcus pneumoniae (1,17,20,33,38,41), and these structures provide new insight into the nature of the cleavage complex.Biochemical evidence points to the participation of two Mg 2ϩ ions in DNA strand scission (11,34,49,54). One Mg 2ϩ ion is suggested to bind the 3Ј bridging oxygen of the scissile DNA phosphodiester bond, thereby promoting DNA cleavage by stabilizing the leaving 3Ј-OH group (11,34).…”

mentioning

confidence: 99%

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

Pan

Gould

Fisher

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 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...