Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: the target of the drugs is DNA.
Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: the target of the drugs is DNA.
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DOI：
10.1073/pnas.82.2.307
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发表时间：
1985
影响因子：
11.1
通讯作者：
L. Shen;A. Pernet
中科院分区：
文献类型：
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作者：
L. Shen;A. Pernet
诺氟沙星是萘啶酸类似物，是最有效的DNA促旋酶抑制剂之一。为了研究这类重要抑制剂的作用机制，测定了[3 H]诺氟沙星与促旋酶和底物DNA的结合。我们发现，与先前的看法相反，诺氟沙星不结合促旋酶，而是结合DNA。平衡透析和膜过滤技术均证明了这一点。与ColE 1和pBR 322质粒的结合显示出一个初级过程，该过程在约等于其超螺旋Ki（1.8 × 10（-6）M）的诺氟沙星浓度下饱和，随后是较弱的次级结合。两种质粒的表观Kd值均为1 × 10 ~（-6）M。在这个初始饱和点的摩尔结合比非常低：对于两种质粒，每个核苷酸只有4 × 10（-4）诺氟沙星。诺氟沙星与DNA质粒的结合是非嵌入性的，如药物优先与单链DNA而不是双链DNA结合的事实所示。结合在高盐浓度下降低，具有4.5和6.5之间的最佳pH，并且不需要二价离子。通过间接竞争法估计其他萘啶酸类似物的结合亲和力。计算的这些类似物的表观Kd值与它们的Ki值很好地相关，提供了药物与DNA的结合亲和力决定生物效力的强有力证据。
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.