Engineering the specificity of antibacterial fluoroquinolones: benzenesulfonamide modifications at C-7 of ciprofloxacin change its primary target in Streptococcus pneumoniae from topoisomerase IV to gyrase

Abstract

We have examined the antipneumococcal mechanisms of a series of novel fluoroquinolones that are identical to ciprofloxacin except for the addition of a benzenesulfonylamido group to the C-7 piperazinyl ring. A number of these derivatives displayed enhanced activity against Streptococcus pneumoniae strain 7785, including compound NSFQ-105, bearing a 4-(4-aminophenylsulfonyl)-1-piperazinyl group at C-7, which exhibited an MIC of 0.06 to 0.125 microg/ml compared with a ciprofloxacin MIC of 1 microg/ml. Several complementary approaches established that unlike the case for ciprofloxacin (which targets topoisomerase IV), the increased potency of NSFQ-105 was associated with a target preference for gyrase: (i) parC mutants of strain 7785 that were resistant to ciprofloxacin remained susceptible to NSFQ-105, whereas by contrast, mutants bearing a quinolone resistance mutation in gyrA were four- to eightfold more resistant to NSFQ-105 (MIC of 0.5 microg/ml) but susceptible to ciprofloxacin; (ii) NSFQ-105 selected first-step gyrA mutants (MICs of 0.5 microg/ml) encoding Ser-81-to-Phe or -Tyr mutations, whereas ciprofloxacin selects parC mutants; and (iii) NSFQ-105 was at least eightfold more effective than ciprofloxacin at inhibiting DNA supercoiling by S. pneumoniae gyrase in vitro but was fourfold less active against topoisomerase IV. These data show unequivocally that the C-7 substituent determines not only the potency but also the target preference of fluoroquinolones. The importance of the C-7 substituent in drug-enzyme contacts demonstrated here supports one key postulate of the Shen model of quinolone action.

FIG. 1

Structures of fluoroquinolones used in this study.

FIG. 2

Inhibitory effects of NSFQ-105 and ciprofloxacin on the DNA-supercoiling activity of 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 NSFQ-105 or ciprofloxacin (CIP). DNA was analyzed by electrophoresis in 1% agarose. The concentrations of NSFQ-105 and ciprofloxacin included in the reaction mixtures are indicated. Lanes a and b, supercoiled and relaxed pBR322 DNA controls, respectively; lane c, relaxed DNA plus DNA gyrase in the absence of added drug or dimethyl sulfoxide (DMSO); lane d, as lane c but with 1% DMSO. All other gyrase reaction mixtures contained 1% DMSO. N, R, and S, nicked, relaxed, and supercoiled pBR322 DNA, respectively.

FIG. 3

Ciprofloxacin is more potent than NSQF-105 as an inhibitor of DNA decatenation by S. pneumoniae topoisomerase IV. kDNA (0.4 μg) was incubated with ParC (1 U), ParE (1 U), and 1.4 mM ATP in the absence or presence of the indicated concentrations of NSFQ-105 and ciprofloxacin (CIP). DNA samples were analyzed by agarose gel electrophoresis. Lane a, kDNA; lane b, kDNA plus ParC, ParE, and ATP in the absence of drug. Monomers, released minicircles; dimers, catenated dimeric minicircles, which are intermediates in the enzyme reaction.