Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface. IMAGES:

DNA拓扑异构酶I（top1）是一种普遍存在的核酶。它可被喜树碱（camptothecin）特异性抑制，喜树碱是一种从喜树（Camptotheca acuminata）树皮中提取的天然产物。目前喜树碱及其多款衍生物正处于临床试验阶段，展现出优异的抗癌活性。本研究进一步探究了该药物与酶-DNA复合物之间的分子相互作用。我们使用了一款烷基化喜树碱衍生物——7-氯甲基-10,11-亚甲二氧基喜树碱（7-ClMe-MDO-CPT），并将其在含有单个top1切割位点的DNA寡核苷酸中对牛胸腺top1的活性，与其非烷基化类似物7-乙基-10,11-亚甲二氧基喜树碱（7-Et-MDO-CPT）的活性进行了对比。在top1存在的条件下，7-ClMe-MDO-CPT可产生一条DNA片段，其迁移速率慢于使用7-Et-MDO-CPT时观测到的top1切割DNA片段。在高NaCl浓度或50℃下的蛋白酶K（proteinase K）处理条件中，top1无法将该片段重新连接。该片段对哌啶（piperidine）处理具有抗性，且在top1切割DNA（碱基+1位）的5'端含有7-脱氮鸟嘌呤（7-deazaguanine）的寡核苷酸中同样可形成该片段。不过，该片段经甲酸（formic acid）处理后再经哌啶处理可被切割。上述观察结果与top1共价复合物存在下7-ClMe-MDO-CPT对+1位碱基（腺嘌呤或鸟嘌呤）的烷基化作用相符，同时为喜树碱类化合物通过结合于酶-DNA界面抑制top1提供了直接证据。IMAGES: