Burckhardt G, Walter A, Triebel H, Störl K, Simon H, Störl J, Opitz A, Roemer E, Zimmer C
Department of Molecular Biology, Institute of Molecular Biology, Friedrich Schiller University Jena, Winzerlaer Strasse 10, D-07745 Jena, Germany.
Biochemistry. 1998 Apr 7;37(14):4703-11. doi: 10.1021/bi9724220.
We have investigated the binding ability to DNA of compounds belonging to the 2-azaanthraquinone-type structure and have examined the effect on the activity of DNA gyrase as well as on mammalian topoisomerases in vitro. Using different biophysical techniques it was found that one of these ligands, 9-((2-dimethylamino)ethyl)amino)-6-hydroxy-7-methoxy-5, 10-dihydroxybenzo[g]isoquinoline-5,10-dione (TPL-I), is an intercalating DNA binding agent, whereas the parent compound tolypocladin (TPL) and a derivative (TPL-II) showed almost no similar affinity to DNA. CD measurements demonstrated a significant and selective binding tendency of TPL-I to alternating purine/pyrimidine sequences with some preference for poly(dA-dT). poly(dA-dT). Tm values were increased of the ligand complex with the alternating AT-containing duplex polymer. The binding to various DNAs was characterized by CD and visible absorption spectral changes. From the latter, different binding constants of 6.2 x 10(5) and 1.5 x 10(5) M-1 were obtained for poly(dA-dT).poly(dA-dT) and poly(dA). poly(dT), respectively. Sedimentation measurements with supercoiled pBR322 plasmid DNA clearly indicated an intercalative binding mechanism associated with an unwinding angle of about 18 degrees. These results suggest that the intercalative binding of TPL-I is promoted by the 2-(dimethylamino)ethylamino group substituted on carbon 9 of the anthraquinone system. The cytotoxic compound TPL-I, but not TPL or TPL-II, effectively inhibited the DNA supercoiling reaction of DNA gyrase and the activity of mammalian topoisomerases I and II as measured by the relaxation assay. TPL-I affects the cleavage reaction of topoisomerases on a single site located in alternating purine-pyrimidine sequence regions. The inhibitory potency of TPL-I can be ascribed to a blocking of cleavage sites on the DNA substrate, which correlates with the sequence preference of the ligand.
我们研究了属于2-氮杂蒽醌型结构的化合物与DNA的结合能力，并在体外检测了其对DNA促旋酶活性以及哺乳动物拓扑异构酶活性的影响。使用不同的生物物理技术发现，这些配体之一，9-((2-二甲基氨基)乙基)氨基)-6-羟基-7-甲氧基-5,10-二羟基苯并[g]异喹啉-5,10-二酮（TPL-I），是一种嵌入型DNA结合剂，而母体化合物托普卡汀（TPL）及其衍生物（TPL-II）对DNA几乎没有类似的亲和力。圆二色性（CD）测量表明，TPL-I对交替嘌呤/嘧啶序列具有显著且选择性的结合倾向，对聚（dA-dT）.聚（dA-dT）有一定偏好。与含交替AT的双链聚合物形成的配体复合物的熔解温度（Tm）值升高。通过CD和可见吸收光谱变化对与各种DNA的结合进行了表征。从后者可知，聚（dA-dT）.聚（dA-dT）和聚（dA）.聚（dT）的不同结合常数分别为6.2×10⁵和1.5×10⁵ M⁻¹。用超螺旋pBR322质粒DNA进行的沉降测量清楚地表明了一种与约18度解旋角相关的嵌入结合机制。这些结果表明，蒽醌系统9位碳上取代的2-（二甲基氨基）乙氨基促进了TPL-I的嵌入结合。细胞毒性化合物TPL-I，而非TPL或TPL-II，通过松弛测定法有效抑制了DNA促旋酶的DNA超螺旋反应以及哺乳动物拓扑异构酶I和II的活性。TPL-I影响拓扑异构酶在位于交替嘌呤-嘧啶序列区域的单个位点上的切割反应。TPL-I的抑制效力可归因于对DNA底物上切割位点的阻断，这与配体的序列偏好相关。
