Quinolone-DNA interaction: sequence-dependent binding to single-stranded DNA reflects the interaction within the gyrase-DNA complex

Abstract

We have investigated the interaction of quinolones with DNA by a number of methods to establish whether a particular binding mode correlates with quinolone potency. The specificities of the quinolone-mediated DNA cleavage reaction of DNA gyrase were compared for a number of quinolones. Two patterns that depended on the potency of the quinolone were identified. Binding to plasmid DNA was examined by measuring the unwinding of pBR322 by quinolones; no correlation with quinolone potency was observed. Quinolone binding to short DNA oligonucleotides was measured by surface plasmon resonance. The quinolones bound to both single- and double-stranded oligonucleotides in an Mg(2+)-dependent manner. Quinolones bound to single-stranded DNA with a higher affinity, and the binding exhibited sequence dependence; binding to double-stranded DNA was sequence independent. The variations in binding in the presence of metal ions showed that Mg(2+) promoted tighter, more specific binding to single-stranded DNA than softer metal ions (Mn(2+) and Cd(2+)). Single-stranded DNA binding by quinolones correlated with the in vitro quinolone potency, indicating that this mode of interaction may reflect the interaction of the quinolone with DNA in the context of the gyrase-DNA complex.

FIG. 1.

Structures of compounds used in this study.

FIG. 2.

Cleavage specificity assay. A 1.35% agarose gel shows the cleavage of relaxed pBR322 DNA by gyrase in the presence of quinolone drugs. Lanes: M, hyperladder DNA marker (Bioline); Rel, relaxed pBR322 DNA substrate alone; L, EcoRI-linearized substrate. The remaining lanes show the cleavage patterns for ciprofloxacin (CFX; 5 μM), oxolinic acid (OXO; 50 μM), enoxacin (ENX; 25 μM), and pipemidic acid (PPA; 150 μM), classified as set I or set II. The arrows show the approximate positions of the products generated by cleavage at the 990 site (12).

FIG. 3.

SPR sensograms and drug-DNA binding isotherms. (A and C) Examples of the SPR sensograms produced for ciprofloxacin (10, 20, 50, 100, 200, and 500 μM) binding to TGGCCT and TAACCT. Experiments were performed with 1 mM MgCl₂. The increase in RUs from the baseline was measured and used to calculate K_d^apps. (B and D) Examples of drug-DNA binding isotherms for TGGCCT and TAACCT. The amplitudes from the SPR sensogram experiments were fitted to a single-site binding equation. The stoichiometries (Table 3) were calculated from the ratio of the increase in the numbers of RUs for quinolone binding to the mass (in RUs) of DNA attached to the chip surface.

FIG. 4.

Correlations between IC₅₀s and K_d^apps for single-stranded and double-stranded DNAs. Correlations were plotted for the single-stranded 990 site sequence of pBR322 (TGGCCT) (A), the single-stranded substituted sequence (TAACCT) (B), the hairpin that formed the 990 site sequence (C), and the hairpin that formed the substituted sequence (D). The plots include all the datum points (insets) or the datum points for just the compounds with cleavage pattern I (main plots). The correlation coefficients for the main plots were as follows: 0.80 (A), 0.54 (B), −0.50 (C), and −0.18 (D).

FIG. 5.

Ciprofloxacin binding to the 6-mer oligonucleotide (TGGCCT). Experiments were performed with 1 mM MgCl₂, CaCl₂, MnCl₂, or CdCl₂; and the data were fitted to a single-site binding equation.