Maleimide is a potent inhibitor of topoisomerase II in vitro and in vivo: A new mode of catalytic inhibition
Maleimide is a potent inhibitor of topoisomerase II in vitro and in vivo: A new mode of catalytic inhibition
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DOI：
10.1124/mol.61.5.1235
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发表时间：
2002-05-01
影响因子：
3.6
通讯作者：
Jensen, PB
中科院分区：
文献类型：
--
作者：
Jensen, LH;Renodon-Corniere, A;Jensen, PB
马来酰亚胺，N-乙基-马来酰亚胺（NEM）和N-甲基-马来酰亚胺（NMM）被确定为纯化的人拓扑异构酶II α的有效催化抑制剂，而环饱和类似物琥珀酰亚胺是完全无活性的。拓扑异构酶II突变完全消除了双二氧代哌嗪化合物对催化抑制的影响，这表明这些马来酰亚胺的不同的作用模式，催化抑制没有被废除。在DNA断裂实验中，马来酰亚胺和NEM均能拮抗足叶乙甙引起的DNA双链断裂。因此，马来酰亚胺在三种不同的体内试验中可以拮抗拓扑异构酶II毒物的作用：1）在碱性洗脱试验中，马来酰亚胺保护依托泊苷诱导的DNA损伤。2)在条带耗竭试验中，马来酰亚胺减少了DNA上拓扑异构酶II α和β的依托泊苷诱导的捕获。3)在克隆形成试验中，马来酰亚胺拮抗依托泊苷和柔红霉素对人类和小鼠来源的四种不同细胞系的细胞毒性。具有降低的核拓扑异构酶II α含量的at-MDR细胞系对马来酰亚胺完全敏感，表明其在体内不是拓扑异构酶II毒物。我们发现拓扑异构酶II对马来酰亚胺、NMM和NEM敏感，但对琥珀酰亚胺不敏感，这表明活性对不饱和环键有严格的要求。我们建议，所观察到的拮抗作用在体外和体内是由拓扑异构酶II半胱氨酸残基的共价修饰减少的催化活性酶的量敏感的拓扑异构酶II毒药的行动。
Maleimide, N-ethyl-maleimide (NEM), and N-methyl-maleimide (NMM) were identified as potent catalytic inhibitors of purified human topoisomerase IIalpha, whereas the ring-saturated analog succinimide was completely inactive. Catalytic inhibition was not abrogated by topoisomerase II mutations that totally abolish the effect of bisdioxopiperazine compounds on catalytic inhibition, suggesting a different mode of action by these maleimides. Furthermore, in DNA cleavage assay maleimide and NEM could antagonize etoposide-induced DNA double-strand breaks. Consistently, maleimide could antagonize the effect of topoisomerase II poisons in three different in vivo assays: 1) In an alkaline elution assay maleimide protected against etoposide-induced DNA damage. 2) In a band depletion assay maleimide reduced etoposide-induced trapping of topoisomerase IIalpha and beta on DNA. 3) In a clonogenic assay maleimide antagonized the cytotoxicity of etoposide and daunorubicin on four different cell lines of human and murine origin. at-MDR cell lines with reduced nuclear topoisomerase IIalpha content are fully sensitive to maleimide, indicating that it is not a topoisomerase II poison in vivo. Our finding that topoisomerase II is sensitive to maleimide, NMM, and NEM but insensitive to succinimide demonstrates a strict requirement for the unsaturated ring bond for activity. We suggest that the observed antagonism in vitro and in vivo is caused by covalent modification of topoisomerase II cysteine residues reducing the amount of catalytically active enzyme sensitive to the action of topoisomerase II poisons.