Abstract
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.
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Selected References
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