Bailly C, Goossens J F, Laine W, Anizon F, Prudhomme M, Ren J, Chaires J B
INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, 59045 Lille, France.
J Med Chem. 2000 Nov 30;43(24):4711-20. doi: 10.1021/jm0003438.
Rebeccamycin derivatives represent a promising class of antitumor agents. In this series, two glycosylated indolocarbazoles, NB-506 and NSC-655649, are currently undergoing clinical trials. Their anticancer activities are associated with their capacities to interact with DNA and to inhibit DNA topoisomerases. Previous studies revealed that the planar indolocarbazole chromophore can intercalate into DNA, locating the appended carbohydrate residue in one of the two helical grooves, probably the minor groove as is the case with the anthracyclines and other DNA-binding antibiotics. The sugar residue contributes significantly to the DNA binding free energy of NB-506. However, the exact positioning of the glycosyl residue of rebeccamycin derivatives in the drug-DNA complex remains poorly understood. To better understand how glycosylated indolocarbazoles interact with DNA, we investigated the interaction of a rebeccamycin derivative (85) bearing a 2'-amino group on the sugar residue. We show that the presence of the 2'-amino function permits the formation of covalent drug-DNA complexes in the presence of formaldehyde. Complementary biochemical and spectroscopic measurements attest that 85 reacts covalently with the 2-amino group of guanines exposed in the minor groove of the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also forms cross-links with an oligonucleotide containing only A.T base pairs. The covalent binding to A.T base pairs was detected using a gel mobility shift assay and was independently confirmed by thermal denaturation studies and by fluorescence measurements using a series of synthetic polynucleotides. The HCHO-mediated alkylation reaction of the drug with A.T base pairs apparently involves the 6-amino group of adenines exposed in the major groove whereas the covalent attachment to G.C base pairs implicates the 2-amino group of guanines situated in the opposite minor groove. Therefore, the results suggest that either the drug is able to switch grooves in response to sequence or it can simultaneously bind to both the minor and major grooves of the double helix. This study will help to guide the rational design of new DNA-binding antitumor indolocarbazole drugs and also provides a general experimental approach for probing minor versus major groove interactions between small molecules and DNA.
瑞贝卡霉素衍生物是一类很有前景的抗肿瘤药物。在这一系列药物中，两种糖基化吲哚咔唑，即NB - 506和NSC - 655649，目前正在进行临床试验。它们的抗癌活性与其与DNA相互作用以及抑制DNA拓扑异构酶的能力有关。先前的研究表明，平面吲哚咔唑发色团可以嵌入DNA，使连接的碳水化合物残基位于两条螺旋沟之一中，可能像蒽环类药物和其他DNA结合抗生素那样位于小沟中。糖残基对NB - 506的DNA结合自由能有显著贡献。然而，瑞贝卡霉素衍生物的糖基残基在药物 - DNA复合物中的精确定位仍知之甚少。为了更好地理解糖基化吲哚咔唑与DNA的相互作用，我们研究了一种在糖残基上带有2'-氨基的瑞贝卡霉素衍生物（85）的相互作用。我们发现，2'-氨基官能团的存在使得在甲醛存在下能够形成共价药物 - DNA复合物。补充的生化和光谱测量证明，85与双螺旋小沟中暴露的鸟嘌呤的2-氨基发生共价反应，就像柔红霉素一样。与柔红霉素不同的是，85还能与仅含A.T碱基对的寡核苷酸形成交联。使用凝胶迁移率变动分析检测到与A.T碱基对的共价结合，并通过热变性研究以及使用一系列合成多核苷酸的荧光测量独立证实。药物与A.T碱基对的HCHO介导的烷基化反应显然涉及大沟中暴露的腺嘌呤的6-氨基，而与G.C碱基对的共价连接则涉及相对小沟中鸟嘌呤的2-氨基。因此，结果表明，要么药物能够根据序列切换沟，要么它可以同时结合双螺旋的小沟和大沟。这项研究将有助于指导新型DNA结合抗肿瘤吲哚咔唑药物的合理设计，也为探究小分子与DNA之间小沟与大沟相互作用提供了一种通用的实验方法。
