Colmenarejo G, Montero F, Orellana G
Departamento de Bioquímica y Biologia Molecular I, Universidad Complutense de Madrid, Spain.
Anticancer Drug Des. 1997 Jun;12(4):239-60.
In order to establish the basis for the rational design of a novel family of intercalating chiral photonuclease drugs aimed at photochemotherapy, namely N, N'-dialkylated 6-(2-pyridinium)phenanthridinium (pyp) dications, a detailed investigation of the DNA binding of the dq2pyp member (where dq2 stands for -CH2CH2-), was conducted. The study addresses the sequence- and enantiospecificity, as well as polyelectrolyte effects in the drug-DNA interaction. Binding isotherms with synthetic polynucleotides, forcefield calculations, affinity chromatography in a DNA-cellulose stationary phase and salt-dependent equilibrium and kinetic studies with DNA were used. dq2pyp shows a strong preference for alternating GC over AT base pairs; binding to homopolymeric DNA is weak (< 3 x 10(4) M-1). Affinity chromatography shows enantiospecific binding of dq2pyp to DNA. The polyelectrolyte contribution to the binding free energy are shown to be relatively important (-4.8 kcal/nmol out of an overall value of -7.2 kcal/mmol at 10.2 mM Na+). The slope of the logkd (dissociation rate constant) vs. log[Na+] plot (0.7) agrees with the values predicted from counterion condensation theory for a dicationic intercalator, giving further support to such a DNA binding mode for dq2pyp. The relatively high kinetic dissociation constants (logkd = 0.70log[Na+] + 3.79) in comparison with those of propidium (two orders of magnitude larger at any Na+ concentration) seems to originate from the absence of amino groups in dq2pyp. The kinetic association constants (logka = -1.06log[Na+] + 5.53) are twice these of propidium, probably due to the less restrictive positioning of dq2pyp at the intercalation site. The kinetic studies support a mechanism of intercalation in which the drug forms a pre-equilibrium outside the complex followed by the intercalation of the drug. Molecular modelling is used throughout to rationalize all the experimental data, as well as to propose new candidates with improved DNA affinity and residence time.
为了建立用于光化学疗法的新型嵌入型手性光核酸酶药物家族（即N，N'-二烷基化6-(2-吡啶基)菲啶鎓（pyp）双阳离子）合理设计的基础，对dq2pyp成员（其中dq2代表-CH2CH2-）与DNA的结合进行了详细研究。该研究探讨了序列和对映体特异性，以及药物与DNA相互作用中的聚电解质效应。使用了与合成多核苷酸的结合等温线、力场计算、DNA-纤维素固定相中的亲和色谱以及与DNA的盐依赖性平衡和动力学研究。dq2pyp对交替的GC碱基对比AT碱基对表现出强烈偏好；与同聚DNA的结合较弱（<3×10⁴ M⁻¹）。亲和色谱显示dq2pyp与DNA的对映体特异性结合。聚电解质对结合自由能的贡献相对重要（在10.2 mM Na⁺时，总结合自由能为-7.2 kcal/mmol，其中-4.8 kcal/nmol来自聚电解质贡献）。logkd（解离速率常数）对log[Na⁺]图的斜率（0.7）与双阳离子嵌入剂的反离子凝聚理论预测值一致，进一步支持了dq2pyp的这种DNA结合模式。与碘化丙啶相比，dq2pyp相对较高的动力学解离常数（logkd = 0.70log[Na⁺] + 3.79）（在任何Na⁺浓度下都大两个数量级）似乎源于dq2pyp中不存在氨基。动力学缔合常数（logka = -1.06log[Na⁺] + 5.53）是碘化丙啶的两倍，可能是由于dq2pyp在嵌入位点的定位限制较小。动力学研究支持一种嵌入机制，即药物在复合物外部形成预平衡，随后药物进行嵌入。整个过程中使用分子建模来合理化所有实验数据，并提出具有改善的DNA亲和力和停留时间的新候选物。
