Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: the target of the drugs is DNA.

Norfloxacin is a nalidixic acid analogue and one of the most potent DNA gyrase inhibitors. To study the mechanism of this important class of inhibitors, the binding of [3H]norfloxacin to gyrase and substrate DNA was measured. We found that, contrary to prior belief, norfloxacin does not bind to gyrase but instead binds to DNA. This was demonstrated by both equilibrium dialysis and membrane filtration techniques. Binding to ColE1 and pBR322 plasmids showed a primary process that is saturated at a norfloxacin concentration about equal to its supercoiling Ki (1.8 X 10(-6) M) and is followed by weaker secondary binding. The apparent Kd values are 1 X 10(-6) M for both plasmids. The molar binding ratio at this initial saturation point is extremely low: only 4 X 10(-4) norfloxacin per nucleotide for both plasmids. The binding of norfloxacin to DNA plasmids is nonintercalative, as shown by the fact that the drug binds preferentially to single-stranded DNA rather than to double-stranded DNA. The binding is reduced at high salt concentration, has a pH optimum between 4.5 and 6.5, and does not require divalent ions. The binding affinities of other nalidixic acid analogues were estimated by an indirect competition method. The calculated apparent Kd values of these analogues correlate well with their Ki values, providing strong evidence that the binding affinity of the drug to DNA determines biological potency.