Drug Interactions with Bacillus anthracis Topoisomerase IV: Biochemical Basis for Quinolone Action and Resistance

Abstract

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlAS81F and GrlAS81Y quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg2+ bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg2+ concentrations to support DNA cleavage by GrlAS81F topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the ternary enzyme-quinolone-DNA complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg2+ bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically-relevant quinolone resistance mutations in bacterial type II topoisomerases.

Fluoroquinolone-Gyrase-DNA Complexes

“…In the binding mode revealed by crystal structures (Fig. 1B), the carboxyl end of fluoroquinolones forms a stabilizing magnesium-water bridge with GyrA residues 83 and 87 (17,18). This magnesium-mediated binding contributes to drug activity, and the loss of the interaction correlates with GyrA-mediated quinolone resistance (17,18).…”

“…1B). The quinolone 3-carboxyl, along with an aspartic/glutamic acid and a serine in helix-IV of GyrA (ParC), participates in a magnesium-water bridge that stabilizes the drug-enzyme-DNA complex (17,18). Although existing crystal structures explain many aspects of quinolone action, it is not clear why a spontaneous GyrA resis-tance substitution in Mycobacterium smegmatis (GyrA-Cys 89 ) preferentially restricts the bacteriostatic action of fluoroquinolones that have bulky substitutions at the distal end of the fluoroquinolone C-7 piperazinyl ring (19): this region of the drug should be far from the GyrA-Cys 89 residue in the cleaved complex.…”

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

“…Recent structural (19) and functional (20,21) studies with topoisomerase IV indicate that quinolones interact with bacterial type II enzymes primarily through a water-metal ion bridge. This bridge is formed by a divalent metal ion that is chelated by the C3/C4 keto acid of the drug and stabilized by four water molecules (19).…”

“…Amino acid residues A90 and D94 occur at the positions that, by sequence homology, are predicted to anchor the water-metal ion bridge if it is used to mediate quinolone-enzyme interactions in this species (19)(20)(21). The GyrA A90V , GyrA…”

“…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.…”