Fleury F, Sukhanova A, Ianoul A, Devy J, Kudelina I, Duval O, Alix A J, Jardillier J C, Nabiev I
EA2063, Institut Fédératif de Recherche no. 53 "Biomolécules," UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51100 Reims, France.
J Biol Chem. 2000 Feb 4;275(5):3501-9. doi: 10.1074/jbc.275.5.3501.
DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.
天然生物碱法格龙宁（NSC157995）及其新的合成衍生物乙氧啶（12-乙氧基-苯并[c]菲啶）的DNA拓扑异构酶（top）I抑制活性与其在DNA复合物中的分子相互作用和序列特异性相关。流动线性二色性表明，乙氧啶在浓度低10倍时对DNA松弛的抑制作用与法格龙宁相同。top I介导的DNA切割模式显示，在0.016 - 50 microM浓度的法格龙宁作用下，CPT依赖性位点呈线性增强，而乙氧啶则抑制top I特异性位点和CPT依赖性位点。发现乙氧啶对top I介导的切割的抑制作用对特定位点具有特异性，包括A和T之间的链切割位点。法格龙宁和乙氧啶是DNA大沟嵌入剂。乙氧啶在A - T序列中嵌入DNA，其12 - 乙氧基部分（法格龙宁中不存在）延伸到DNA小沟中。这些发现可能解释了乙氧啶对紧邻链切割位点的A(+1)、T(-1)处强top I切割位点抑制的特异性。法格龙宁在DNA嵌入方面没有显示出任何序列特异性，但其羟基的高电负性氧原子（乙氧啶中不存在）被证明是与DNA的G碱基的NH(2)基团形成氢键的受体。法格龙宁稳定top I介导的三元复合物的能力被认为是由其羟基与DNA切割位点(+1)位置的鸟嘌呤的相互作用以及季铵氮与top I的相互作用决定的。所提出的模型为筛选新的top I靶向药物提供了指导，有助于确定其top I抑制作用的分子决定因素。
