The binding of plasmid DNA to norfloxacin, a quinolone antibacterial agent, was investigated by fluorescence, electrophoretic DNA unwinding, and affinity chromatography techniques. The amount of quinolone bound to DNA was modulated by the concentration of Mg2+. No interaction was evident in the absence of Mg2+ or in the presence of an excess of Mg2+, whereas maximum binding was observed at a Mg2+ concentration of 1-2 mM. The experimental data can be fitted to the formation of three types of Mg adducts: a binary adduct with norfloxacin and Mg2+, a binary adduct with DNA and Mg2+, and a ternary adduct with quinolone, plasmid, and Mg2+. We propose a model for the ternary complex, in which Mg acts as a bridge between the phosphate groups of the nucleic acid and the carbonyl and carboxyl moieties of norfloxacin. Additional stabilization may arise from stacking interactions between the condensed rings of the drug and DNA bases (especially guanine and adenine), which may account for the preference exhibited by quinolones for single-stranded and purine-rich regions ofnucleic acids. Other possible biochemical pathways of drug action are suggested by the observation that norfloxacin binds Mg2+ under conditions that are close to physiological.Conflicting literature reports have been accumulating on the role played by DNA in the mechanism of action of quinolone compounds. Although a large amount of biological data has indicated that DNA gyrase was the target for quinolone compounds (1-4), recent reports dismissed DNA gyrase as the target and pointed to DNA as the direct binding species (5). In fact, a cooperative interaction was proposed to occur between quinolones and supercoiled DNA. Subsequent publications by the same laboratory have modified this view extensively (6-8). In particular, Shen et al. (7) have proposed that in the presence of ATP bound gyrase induces a specific quinolone binding site in the relaxed DNA substrate. Gelelectrophoresis experiments by Tornaletti and Pedrini (9) showed that norfloxacin (Nor) is able to unwind the DNA double helix in the presence of Mg2+. On the other hand 19F NMR measurements failed to show any direct DNAquinolone interaction (10). We were also unable to detect binding using fluorescence spectroscopy techniques (11).Even if reconsidered in terms of affinity, the interaction with DNA is still of great concern because of the possible long-term genotoxicity of quinolone compounds, which are increasingly adopted as first-choice antibiotics for the treatment of many infections, and because it addresses the real mechanism of action of this class of molecules. To shed some light on this cumbersome problem, we have focused our attention on the role of Mg2+ in the binding of the model quinolone drug Nor to plasmid DNA. Our approach includes fluorescence and affinity chromatography measurements and electrophoretic DNA-unwinding assays. MATERIALS AND METHODSChemicals. Nor and [14C]Nor (specific activity, 46.5 ,uCi/ mg; 1 Ci = 37 GBq) were a kind gift of Merck Sharp & Dohme. Magnesium...

The relationship between cell-killing kinetics and penicillin-binding protein (PBP) saturation has been evaluated in the permeability mutant Escherichia coli DC2 in which the antimicrobial activity of beta-lactams has been described as being directly related to the extent of saturation of the PBP target(s). Saturation of a single PBP by cefsulodin (PBP 1s), mecillinam (PBP 2), and aztreonam (PBP 3) resulted in a slow rate of killing (2.5-, 1.5-, and 0.8-log-unit decreases in the number of CFU per milliliter, respectively, in 6 h). Saturation of two of the three essential PBPs resulted in a marked increase in the rate of killing, which reached the maximum value when PBPs 1s and 2 were simultaneously saturated by a combination of cefsulodin and mecillinam (4.7-log-unit decrease in the number of CFU per milliliter in 6 h). Inactivation of all three essential PBPs by the combination of cefsulodin, mecillinam, and aztreonam further increased the killing kinetics (5.5-log-unit decrease in the number of CFU per milliliter), and this was not significantly changed upon additional saturation of the nonessential PBPs 5 and 6 by cefoxitin. Similar relationships between PBP saturation and killing kinetics were obtained with imipenem and meropenem at concentrations which inhibited only one PBP (PBP 2), only two PBPs (PBP 1s and 2), or all three essential PBPs. Saturation of one or more PBPs also resulted in a different rate of bacteriolysis, the highest rate being obtained by the cefsulodin-mecillinam combination and by 5 micrograms of either imipenem or meropenem per ml. All of these conditions caused saturation of PBP 2 and saturation or extensive binding of PBP 1s. However, none of these conditions caused determined the fastest possible rate of killing, which occurred only when all three essential PBPs were saturated. It was concluded that the actual killing effect of beta-lactams is reflected by killing rates that approach the fastest possible rate for the given microorganism and that the targets for the bactericidal activity are precisely those PBPs whose saturation or binding occurs under conditions.

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