Ofloxacin, a potent quinolone antibacterial agent, has a tricyclic ring structure with a methyl group attached to the asymmetric carbon at the C-3 position on the oxazine ring. The S isomer (DR-3355) of ofloxacin has antibacterial activity up to 2 orders of magnitude greater than that of the R isomer (DR-3354). This differential antibacterial activity was not due to different drug transport mechanisms of the two isomers but was found to be derived from the inhibitory activity against the target enzyme, DNA gyrase. Previous mechanistic studies have suggested that the bactericidal effect of the drug is mediated through the stabilization of a cleavable complex via a cooperative drug binding process to a partially denatured DNA pocket created by DNA gyrase. The drug binds to supercoiled DNA in a manner similar to that to which it binds to the enzyme-DNA complex. In the present studies, we first examined the binding of the two radiolabeled ofloxacin enantiomers to supercoiled pUC9 plasmid DNA. Surprisingly, the two enantiomers possessed similar apparent binding affinities and binding cooperatives. The major difference in binding between the two stereoisomers was the molar binding ratio: 4 for the more active S isomer versus 2 for the less active R isomer. We next examined the relative binding potencies of the stereoisomers to the DNA-DNA gyrase complex. The results of a competition assay showed that (S)-ofloxacin binds 12-fold better to the complex than (R)-ofloxacin. The binding potencies of the two enantiomers and two other quinolones correlated well with their respective concentrations causing 50% inhibition against DNA gyrase. The results are interpreted by a stacking model by using the concept of the cooperative drug-DNA binding mechanism, indicating that the potencies of quinolones cannot be determined solely by the DNA binding affinity and cooperativity but can also be determined by their capability in maximally saturating the binding site. The capability of the drug in saturating the binding pocket manifests itself in an increased efficacy at inhibiting the enzyme through a direct interaction between the drug and the enzyme. The results augment the previous suggestion that the binding pocket in the enzyme-DNA complex involves multiple receptor groups including not only DNA bases but also a gyrase subunit. The higher level of potency of (S)-ofloxacin is proposed to derive from the fact that a greater number of molecules are assembled in the pocket. This greater number of molecules optimizes the interaction between the drug and the enzyme, possibly through a contact between the C-7 substituent and the quinolone pocket on the B subunit of DNA gyrase.

A study was made of the correlation between the in vitro inhibitory effects of several quinolones, including four ofloxacin derivatives, on bacterial DNA gyrase from Escherichia coli KL-16 and on topoisomerase II from fetal calf thymus. No correlation was observed between the inhibitions of DNA gyrase activity and topoisomerase II activity. On the other hand, the inhibitory effects of these quinolones against topoisomerase II were closely correlated with their inhibition of cell growth. Furthermore, among the oxazine derivatives tested, the derilvative with a methyl group at position 3 in an S configuration showed the highest activity against DNA gyrase and derivatives without a methyl group on the oxazine ring were more potent against topoisomerase H than those with a methyl group. Among these derivatives, DR-3355, the S isomer o1 ofloxacin, showed the highest activity against DNA gyrase and low activity against topoisomerase II. These results indicate that the methyl group on the oxazine ring plays an important role in the inhibitory activities of ofloxacin derivatives for these enzymes.Quinolone antibacterial agents are currently used for the therapy of various bacterial infections. These agents interfere with the activity of DNA gyrase (3-5), and their inhibitory activities against DNA gyrase have been shown to correlate well with their antimicrobial activities (2, 10, 11). Regarding the relationships among the quinolone molecule, DNA gyrase~,.and DNA, proposed a ternary complex model for gyrase inhibition by quinolones. Quinolone molecules bind to the staggered 4 bp at the DNA gyrase cleavage site. It is thought that inhibitory mechanisms should also be active against mammalia jtop6isomer-ase II, but the degree of inhibitory activity shoWn against mammalian topoisoilerase II is less than that seen against DNA gyrase (11, 12, i4). On the basis of these observations, quinolones should have an inhibitory potency against the proliferation of mammal'iah cells. Actually, some quinolones have inhibitory potencies against HeLa cells, leukemic cell lines, and bone marrow cells (1,9,15,16,21,22). Oomori et al. (15) reported that the inhibitory activities of quinolones against topoisomerase II from HeLa cells correlated with their inhibitory potencies against growth of those cells. To select compounds that are more potent against bacteria and less active against mammalian cells, we examined the selective toxicities of many quinolone derivatives to DNA gyrase and topoisomerase II (11). In this study we focused on the inhibitory activities of quinolones, especially ofloxacin (OFLX) derivatives, against DNA gyrase and topoisomerase II from fetal calf thymus and against the proliferation of normal murine CFU-GM (CFU of granulocyte-macrophage progenitor cells), and we determined the structure-activity relationship of the OFLX derivatives. MATERIALS AND METHODSChemicals. OFLX (17) and OFLX derivatives DR-3355 (8), DR-3354 (8), DN-9494, and DL-8165 were synthesized in * Corresponding author. our laboratories, and the structu...

A series of 1-(2-fluorocyclopropyl)-3-pyridonecarboxylic acids has been prepared. These derivatives are characterized by having a fluorine atom at the 2-position on the cyclopropane ring as the N1 substituent and consist of both cis and trans stereoisomers. Structure-activity relationship studies indicate that the cis derivatives are more potent against Gram-positive bacteria than the corresponding trans counterparts, but the difference in potency against most Gram-negative bacteria is much smaller. The inhibitory effect of compounds 4, 5, 26, 27, 38, and 39 on supercoiling activity of DNA gyrase obtained from E. coli KL-16 correlated with their MICs against the same strain and also depend on their (26, 27, 38, 39) stereochemistry. Introduction of a fluorine atom on the cyclopropyl group resulted in the reduction of lipophilicity compared with the corresponding nonfluorinated quinolones.

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