ANTI TOPOISOMERASE DRUG ACTION IN YEAST

DNA拓扑异构酶是临床上广泛使用的抗癌药物的靶点。虽然拓扑异构酶的基本生化反应途径已经相当清楚，但抗拓扑异构酶药物在产生细胞杀伤所必需的稳定共价的作用机制方面仍然知之甚少。除了拓扑异构酶毒药外，最近人们对抑制拓扑异构酶II （Top2）但不稳定共价复合物的药物也很感兴趣。这一类中的一种重要药物是dexrazoxane (ICRF-187)，临床上用于预防阿霉素心脏毒性。最近的实验表明，ICRF-187具有一种意想不到的细胞杀伤机制，即使它不能稳定共价复合物，也能将Top2转化为毒素。建议对ICRF-187的生物化学作用机制进行研究。遗传学和生物化学方法的结合将进一步揭示ICRF-187等催化抑制剂和依托opo苷等Top2毒物如何通过正常反应途径阻断酶的进展。酵母被用作模型系统来分析Top2突变体的影响，并过度表达突变蛋白。研究策略包括构建新型拓扑异构酶突变体，包括抗拓扑异构酶药物对高血压的突变体。突变蛋白被纯化，其生化特性被用来推断蛋白质：药物相互作用的变化。一种新的工具已经开发出来，可以理解药物如何捕获共价复合物：人类拓扑异构酶II α的突变体，它模仿Top2毒物的作用。此外，对赋予依托泊苷耐药的Top2突变体的研究已经导致了一种新的系统的发展，用于研究拓扑异构酶突变是否在获得性临床耐药中发挥作用。最后，最近的实验表明，拓扑异构酶I和II可能是其他DNA损伤剂杀死细胞的重要决定因素，这导致了细胞如何在DNA损伤后调节拓扑异构酶的研究。这项工作旨在深入了解抗拓扑异构酶药物作用的生化机制，并可能有助于开发新的和更有效的拓扑异构酶抑制剂。

DNA topoisomerases are the targets for a wide range of clinically useful anti-cancer drugs. Although the basic biochemical reaction pathway for the topoisomerases is fairly well understood, there is still little information available regarding the mechanism of action of anti- topoisomerase drugs in generating the stable covalent that is necessary for cell killing. In addition to topoisomerase poisons, there has been recent interest in drugs that inhibit topoisomerase II (Top2), but do not stabilize a covalent complex. An important drug in this category is dexrazoxane (ICRF-187) which is used clinically to prevent doxorubicin cardiotoxicity. Recent experiments have demonstrated that ICRF-187 has an unexpected mechanism of cell killing, with the ability to convert Top2 into a poison even though it does not stabilize covalent complexes. Studies are proposed to understand the biochemical mechanism of action of ICRF-187. A combination of genetic and biochemical approaches will be used to further unravel how both catalytic inhibitors such as ICRF-187 and Top2 poisons such as etoposide block the enzyme's progression through its normal reaction pathway. Yeast is used as a model system for analyzing the effects of Top2 mutants, and for over-expressing the mutant proteins . The research strategy involves the construction of novel topoisomerase mutants, including mutants that are hypertensive to anti- topoisomerase drugs. The mutant proteins are purified, and their biochemical characteristics are used to infer changes in protein: drug interactions. A new tool has been developed understand how drugs trap covalent complexes: a mutant in human topoisomerase II alpha that mimics the action of Top2 poisons. Additionally, work on Top2 mutants that confer resistance to etoposide has led to the development of a new system for investigating whether mutations in topoisomerases play a role in acquired clinical drug resistance. Finally, recent experiments indicating that topoisomerase I and II may be an important determinant in cell killing by other DNA damaging agents has led to studies of how cells may modulate topoisomerases following DNA damage. This work is designed to provide insight into the biochemical mechanisms of anti- topoisomerase drug action, and may be useful in the development of new and more effective topoisomerase inhibitors.