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標題: | 探討Beta-carboline衍生物對於第二型拓樸異構酶亞型之細胞生理作用與酵素專一性 Identify topoisomerase II isozyme-specific targeting agents from β-carboline derivatives |
作者: | Chia-Hao Yen 嚴家濠 |
指導教授: | 李財坤(Tsai-Kun Li) |
關鍵字: | 第二型DNA拓樸異構?,致癌細胞毒性,藥物酵素專一性,Beta-carboline衍生物, topoisomerase II,cancer cell-killing,β-carboline deriviatives,isozyme-specific targenting agents, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 人類第二型拓樸異構酶(Top2), 是細胞中重要調控蛋白,主要利用其可以解決不同DNA的拓樸構型的酵素能力,幫忙解決在細胞DNA複製和RNA轉錄過程伴隨產生的DNA超螺旋構造,使過程能順利進行。另外,針對其細胞生理的重要性,目前臨床上已發展許多抑制第二型拓樸異構酶的藥物;根據藥物作用機制目前分為兩類: (1) Top2 poisons,例如etoposide(VP-16)及doxorubicin,當Top2利用其活性中心的酪胺酸與DNA形成共價鍵,並產生DNA雙股螺旋的斷裂,此時藥物會穩定這個酵素-DNA共價附合體,稱之為切割複合體(cleavable complex),進而累積染色體DNA的斷裂最終引發癌細胞的死亡。(2) Top2 inhibitor,例如dexrazoxane(ICRF-187),此類藥物不會穩定切割複合體而使DNA產生斷裂,相對的是抑制在酵素催化周期的其他步驟,例如ICRF-193它會在酵素活性周期中抑制ADP的釋出,使DNA無法離開進而阻斷酵素活性與作用,甚至降低切割複合體的形成。在人類的細胞中,已知表現兩種亞型的第二型拓樸異構酶:(1) Top2α -α亞型同功酵素(isozyme)主要調控DNA複製及染色體分離,在快速分裂中的細胞會大量表現,因此針對癌細胞的快速複製特性,為目前抗癌藥物主要針對的標的。(2) Top2β- Human topoisomerase II (hTop2) proteins solve topological problems during DNA replication, transcription, chromosome condensation and segregation, thereby playing essential roles in cells. Owing to the essential functions, Top2s have been identified to be excellent targets for antibiotics and anti-cancer drugs. Based on the mechanisms of action, two groups of hTop2-targeting compounds have been classified, namely Top2 poison and inhibitor: (i) hTop2 poisons, such as etoposide (VP-16) and doxorubicin, stabilize hTop2-DNA-drug covalent ternary complex (cleavable complex, hTop2cc) and induce DNA double-stranded break, (ii) hTop2 inhibitors such as dexrazoxane (ICRF-187) only interfere catalytic activity and antagonize hTop2cc-mediated DNA break without hTop2cc induction. It shall be noted that there are two hTop2 isoforms with distinctive cellular functions and pharmacological properties: (i) hTop2α plays the major role for cancer cell killing of Top2 poison (ii) hTop2β is responsible for its side effects such as the doxorubicin-mediated cardiotoxicity and VP16-induced 2nd malignancies. Here, we proposed two specific aims: (I) To identify hTop2α-specific poisons for maximizing the efficacy of cancer cell killing with reduced side effects; and (II) To identify hTop2β-specific inhibitors to antagonize side effects of clinically useful hTop2 drugs. In this regards, previously studies have shown that β-carboline derivatives could not only inhibit hTop2, but also have potential isozyme selectivity against monoamine oxidases (MAOs). Thus, we had first chosen β-carbolines and used cytotoxicity and hTop2 relaxation assays to screen 27 derivatives to categorize desired poison and inhibitor candidates based on obtained results of above assays. We then performed the comet assay to detect their ability of induce hTop2-mediated DNA damage and others in vitro assays with purified hTop2 isozymes for further validation. For hTop2-poison candidates, we found two potential poisons. One is H2-53, however, despite of it has a higher cytotoxicity and induces DNA damage, H2-53’s target is not hTop2. Another is DH-337, which induced Top2-mediated DNA damages, and it was mainly from hTop2β. For Top2-inhibitor candidates, we identified that H9-1, H9-2 and H9-3 those have lower cytotoxicity and could also inhibit Top2 relaxation ability. Using the comet assay-based antagonizing experiments, we observed that H9-1 could antagonize the VP-16-induced chromosomal DNA breaks most efficiently among the above three agents. Coupled with the isozyme-knockdown experiments, we found that the antagonized ability of H9-1 is similarly against Top2α and Top2β. Therefore, we suggested that H9-1 could antagonize on Top2, but unfortunately without the isozyme preference. Together, studied on these β-carboline agents and their structure-function relationship, we have gained the following notions. (1) If there is a positive charge on the structure of carboline ring, the agents seem to have higher cytotoxicity. (2) If these agents have longer side chains on the N-3 of carboline ring, it may become structural obstacles for agents to target on Top2. Further investigation is necessary to identify the isozyme preference of these candidate hits and corresponding action mechanism(s) of Top2 targeting. Nevertheless, our study shall provide some revenues for the search of hTop2 isozyme-specific targeting intervention. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53770 |
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