2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658412摘要：第II 型拓撲異構酶(TOP2)是生物體進行DNA 複製、轉錄、重組、轉置、以及同源染色體分離時不可或缺的重要蛋白。利用其催化可逆性轉酯化反應(reversibletranesterification reaction)的活性，TOP2 可將DNA 雙螺旋結構的兩條單股DNA 同時切斷，引入缺口，使另一段雙股DNA 得以由此暫時性的缺口通過，達成DNA 分子超螺旋態(DNA supercoiling state)或拓撲構形(DNA topology)的改變，之後TOP2 會將缺口重新黏合(religation)，以避免DNA 的損傷。先前的研究結果顯示，etoposide、doxorubicin、mitoxantrone 等抗癌藥物會與TOP2之DNA結合-切割活性區(DNA-binding and cleavagecore; DBCC)作用並阻斷DNA 缺口的黏合，誘發永久性DNA 斷裂的形成，導致細胞的凋亡(apoptosis)，且TOP2 含量越高的細胞越容易受到此類藥物的毒殺。人類具有兩種TOP2 同功酶：hTOP2與hTOP2，其中DNA 複製作用旺盛、生長較快的細胞中hTOP2的含量較高，而hTOP2的功能則與轉錄作用的關聯較為密切。由於許多癌細胞中hTOP2皆有大量表達的現象，因此上述之TOP2 針對性藥物已被成功用於多種癌症之臨床治療。然而、此類藥物伴隨的副作用(如心臟毒性與血液毒性等)仍是治療時無法忽視的因素，分析顯示大部分的副作用可能源於藥物與hTOP2的交互作用，因此開發對hTOP2具高度專一性的藥物是當前的研究重點之一。此外、治療過程中抗藥性腫瘤細胞的出現，亦是開發新一代藥物時必須面對的課題，但由於目前尚無任何藉由實驗判定之藥物作用區(即hTOP2_DBCC 與hTOP2_DBCC)的立體結構，因此對於藥物作用以及抗藥性突變導致藥物失效的分子機轉仍待探討。為了輔助新型抗癌藥物的研發，本研究的主要目標在於以X-射線結晶學解析hTOP2與hTOP2、及其與抗癌藥物和DNA 形成之二重或三重複合體的晶體結構。目前本實驗室成功建立hTOP2_DBCC 與hTOP2_DBCC 之表達、純化、與結晶的標準流程，初步X-射線繞射分析顯示hTOP2_DBCC 晶體之繞射解析度約為2.9 Å，目前正致力於其結構解析以及hTOP2_DBCC 晶體品質的改良，並嘗試以抗癌藥物處理hTOP2_DBCC 與hTOP2_DBCC 的結晶，以研究蛋白與藥物之交互作用和抗藥性的分子機轉。我們亦將鑑定與hTOP2_DBCC 和hTOP2_DBCC 親和力較佳的DNA序列，以從事蛋白-藥物-DNA 三重複合體的結構分析。綜合以上敘述可知：本計畫的方向明確，且已獲得關鍵的初步結果，因此預期本研究應能順利進行，並可望為新一代抗癌藥物的研發、TOP2 催化反應的分子機制、以及hTOP2與hTOP2兩種同功酶間活性差異等重要課題提供重要且豐富的結構訊息，這也將是首次針對多細胞真核生物TOP2_DBCC 的結構分析。<br> Abstract: Type II DNA topoisomerases (TOP2s) are ubiquitous proteins that play essential rolesin cellular DNA transactions including replication, transcription, recombination,chromosome condensation and segregation. By catalyzing a reversible transesterificationreaction, TOP2 introduces a transient and enzyme-mediated double-stranded break on oneDNA duplex to allow the passage of another, causing changes in DNA superhelicity ortopology. The DNA cleavage activity of TOP2 has been referred as a double-edged sword;failure to reseal the TOP2-mediated DNA break can lead to permanent DNA damage andcell death. A number of clinically active antitumor drugs, including etoposide, doxorubicin,and mitoxantrone exploit this unique characteristic to exert cell-killing effect by targetingthe TOP2’s DNA-binding and cleavage core (DBCC) to inhibit the religation step.Human possesses two TOP2 isoforms, termed hTOP2 and hTOP2. Despite theirsimilarity, these two isozymes exhibit differential expression patterns and are functionallydistinct. The  isoform is particularly important for DNA replication and is usually presentat high level in fast growing cells, including many types of tumors, whereas hTOP2ismainly involved in transcription-related processes. Recent studies strongly suggest that thehTOP2-targeting is mainly responsible for antitumor activity, whereas hTOP2-poisoninglikely leads to various undesired side effects (such as cardio and hematological toxicity).Thus, it would be clinically desirable to have hTOP2-specific drugs. In addition, thelong-term effectiveness of the currently used TOP2-targeting drugs suffers from theemergence of drug-resistant tumor cells. Due to the lack of an experimentally determinedthree-dimensional structure of the TOP2 drug targeting domains (hTOP2_DBCC andhTOP2_DBCC), molecular basis for the drug-resistant mutations remains largelyuncharacterized. To overcome these problems and to facilitate the development of betterantitumor drugs, a key mission of this research project is to determine the three-dimensionalstructures of hTOP2 and hTOP2 and their binary or ternary complexes with antitumordrugs and DNA.Toward this goal, we have successfully cloned, expressed, purified, and crystallized thehTOP2_DBCC and hTOP2_DBCC. Preliminary analysis indicated that hTOP2_DBCCcrystals can diffract X-ray to ~2.9 Å resolution, it is thus reasonable to expect that thisstructure will be determined in the near future. These crystals will also be used as platformsto study the protein-drug interactions after being exposed to high concentration of antitumordrugs. The preferred DNA-binding sequences and cleavage hot spots for both human TOP2swill be characterized to facilitate the production and crystallization of TOP2-DNA-drugternary complexes. Given the solid preliminary results, we should be able to revealmolecular codes useful for the development of new generation of hTOP2-specific drugs. Inaddition, we may obtain crucial understandings on the structural basis of TOP2 catalysis aswell as how hTOP2 and hTOP2 differ in function. Moreover, this study will provide forthe first time the structure of TOP2_DBCC of a multicellular organism.蛋白質結晶學抗癌藥物第二型拓撲異構酵素蛋白-DNA交互作用蛋白-藥物交互作用protein crystallographyanticancer drugtype II topoisomeraseprotein-DNA interactionprotein-drug interaction