Ciprofloxacin dimers target gyrase in Streptococcus pneumoniae

Abstract

We have examined the antipneumococcal activities of novel quinolone dimers in which ciprofloxacin was tethered to itself or to pipemidic acid by linkage of C-7 piperazinyl rings. Symmetric 2,6-lutidinyl- and trans-butenyl-linked ciprofloxacin dimers (dimers 1 and 2, respectively) and a pipemidic acid-ciprofloxacin dimer (dimer 3) had activities against Streptococcus pneumoniae strain 7785 that were comparable to that of ciprofloxacin, i.e., MICs of 2, 1, and 4 to 8 microg/ml versus an MIC of 1 to 2 microg/ml, respectively. Surprisingly, unlike ciprofloxacin (which targets topoisomerase IV), several lines of evidence revealed that the dimers act through gyrase in S. pneumoniae. First, ciprofloxacin-resistant parC mutants of strain 7785 remained susceptible to dimers 1 to 3, whereas a gyrA mutation conferred a four- to eightfold increase in the dimer MIC but had little effect on ciprofloxacin activity. Second, dimer 1 selected first-step gyrA (S81Y or S81F) mutants (MICs, 8 to 16 microg/ml) that carried wild-type topoisomerase IV parE-parC genes. Third, dimers 1 and 2 promoted comparable DNA cleavage by S. pneumoniae gyrase and topoisomerase IV, whereas ciprofloxacin-mediated cleavage was 10-fold more efficient with topoisomerase IV than with gyrase. Fourth, the GyrA S81F and ParC S79F enzymes were resistant to dimers, confirming that the resistance phenotype is largely silent in parC mutants. Although a dimer molecule could bind very tightly by bridging quinolone binding sites in the enzyme-DNA complex, the greater potency of ciprofloxacin against gyrase and topoisomerase IV suggests that dimers 1 to 3 bind in a monomeric fashion. The bulky C-7 side chain may explain dimer targeting of gyrase and activity against efflux mutants. Tethered quinolones have potential as mechanistic tools and as novel antimicrobial agents.

FIG. 1.

Structures of quinolone dimers used in this study.

FIG. 2.

Ciprofloxacin dimers inhibit DNA supercoiling by S. pneumoniae DNA gyrase. Relaxed pBR322 DNA (0.4 μg) was incubated with S. pneumoniae GyrA (1 U), GyrB (1 U), and 1.4 mM ATP in the absence or presence of dimer 1, dimer 2, or ciprofloxacin (CIP) at the indicated concentrations. The DNA reaction products were analyzed by electrophoresis in a 1% agarose gel. The concentrations of dimers and ciprofloxacin present in the reaction mixtures are indicated above the wells. Lane a, relaxed pBR322. All reaction mixtures contained 5% dimethyl sulfoxide. R and S, relaxed and supercoiled pBR322 DNA, respectively.

FIG. 3.

Ciprofloxacin is more potent than its dimers as an inhibitor of DNA decatenation by S. pneumoniae topoisomerase IV. kDNA (0.4 μg) was incubated with recombinant ParC and ParE (1 U each) in the absence or presence of dimers and ciprofloxacin (CIP) at the indicated concentrations. DNA products were separated by agarose gel electrophoresis. Lane a, kDNA; lane b, kDNA plus ParC, ParE, and ATP in the absence of drug and dimethyl sulfoxide. All other reaction mixtures contained 5% dimethyl sulfoxide. Monomer, released minicircles.

FIG. 4.

Quinolone-mediated DNA cleavage by S. pneumoniae gyrase (A) and topoisomerase IV (Topo IV) (B). Supercoiled pBR322 (0.4 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in the absence and presence of dimers 1, 2, and 3, ciprofloxacin (CIP), and pipemidic acid (PIP) at the indicated concentrations. After treatment with SDS and proteinase K, the DNA products were examined by electrophoresis in a 1% agarose gel. Lanes M, linear markers; lanes a and b, supercoiled and linear pBR322 DNA, respectively; lanes c, supercoiled pBR322 plus enzyme in the absence of drug but with 5 mM NaOH added. All the dimer reaction mixtures contained 5% dimethyl sulfoxide, and all ciprofloxacin reaction mixtures contained 5 mM NaOH. N, L, and S, nicked, linear, and supercoiled plasmid pBR322 DNA, respectively.

FIG. 5.

Comparison of dimer-promoted DNA breakage by wild-type and quinolone-resistant S. pneumoniae gyrase (A and B) and topoisomerase IV (Topo IV) (C and D). Supercoiled plasmid pBR322 (0.4 μg) was incubated with recombinant S. pneumoniae GyrB (1.7 μg) and either wild-type GyrA or GyrA S81F (in each case 0.4 μg) (A and B) or ParE (1.7 μg) and either wild-type ParC or ParC S79F (0.45 μg) (C and D). Samples were processed and analyzed as described in the legend to Fig. 4; and lanes a, b, and c are as described in the legend to Fig. 4.

FIG. 6.

Site-specific DNA cleavage by S. pneumoniae topoisomerase IV mediated by ciprofloxacin and its dimers. Plasmid pBR322 DNA linearized at its EcoRI site (0.4 μg) was incubated with ParC (0.45 μg) and GyrB (1.7 μg) in the absence or presence of dimer 1 or 2 or ciprofloxacin (CIP) at the indicated concentrations. After addition of SDS and proteinase K treatment, the DNA samples were analyzed by electrophoresis in a 1% agarose gel. Lanes M, a, and b, DNA size markers, linear pBR322, and linear pBR322 incubated with ParC and ParE in the absence of drug, respectively.

FIG. 7.

Ciprofloxacin and its symmetric dimer (dimer 2) induce unwinding of closed circular DNA. Relaxed plasmid pBR322 (0.8 μg) was incubated with calf thymus DNA topoisomerase I (12 U) in the presence of 10 mM MgCl₂ at room temperature for 10 min before addition of ciprofloxacin (CIP) and dimer 2 at the indicated concentrations. After incubation for 1 h at 37°C, the topoisomerase I was inactivated and the DNA was extracted with phenol to remove any bound ligand prior to agarose gel electrophoresis in Tris-phosphate-EDTA buffer. Half of each DNA sample was run in the absence of chloroquine (−CQ); the other half was electrophoresed in the presence of chloroquine (+CQ). Under the conditions of the gel without chloroquine, all the DNA samples are positively supercoiled and are made more positively coiled by the inclusion of chloroquine. Lanes a, pBR322 DNA (previously relaxed in the absence of Mg²⁺ ions), used as the substrate.