2011-05-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/660802摘要：抗癌藥物對於腫瘤的選擇性(tumor selectivity)在藥理學上是一個很重要的研究課題。我們的研究顯示，DNA拓樸異構酶的抗癌藥物在tumor selectivity是一個有潛力的來源；值得注意的是，人類細胞有兩種第二型拓樸酶的同功酵素(hTOP2和hTOP2 isozymes)，雖然具有不同的C’端功能區，但都可被臨床藥物作為作用目標。而最近研究亦發現藥物導致的TOP2之標的作用(targeting)與TOP2的毒性(toxicity)分別是造成抗癌活性及治療副作用的原因，由此可知hTOP2專一標的藥物將會在臨床上會有所助益。經過合作及研究努力，我們亦找出一個MACs藥物相關的新穎TOP2標的機轉(具有好的抗癌效果但副作用少)。因此，此子計劃的目的是為TOP2標的治療上，提供分子、生化與藥理層面的支持，並系統性篩選、鑑定對TOP2 isozymes具專一性的先導型化合物(lead compounds)。與子計劃1合作，建立藥物的物化(高通量)、生化與細胞分析平台；與子計劃3合作，提供hTOP2與hTOP2的生化基礎，以利其在功能與藥物作用差異上的結構分析。另外，我們將檢測hTOP2標的lead compounds引起繼發性惡性腫瘤(2nd malignancies)之可能性，也將對新一代MACs的機轉作更深入的瞭解。研究方向為下：1. 研究以hTOP2 isozymes為治療標的的分子及生化基礎。2. 建立藥物分析平台並系統性篩選對hTOP2 isozymes具專一性的先導型化合物。3. 提供抗hTOP2 isozymes先導型抗癌化合物的生化分子鑑定及藥效藥理評估。<br> Abstract: DNA topoisomerases have been proven to be excellent targets for anti-cancer and antibiotic therapies. Human topoisomerase II (hTOP2)-targeting therapy (using etoposide, doxorubicin or mitoxantrone) is among the most efficacious and widely-used anticancer drugs in the clinic. However, toxic side-effects, whose underlying mechanism(s) remains to be studied, greatly limit their usage. Moreover, the lack of any structural information on the hTOP2-DNA-drug ternary complex (cleavable complex, hTOP2cc) has also significantly hindered the subsequent development of better TOP2-targeting drugs. Furthermore, the clinical application of known TOP2-targeting drugs is also hampered by the emergence of drug-resistance and the lack of tumor selectivity. Interestingly, two isozymes, hTOP2 and hTOP2, with distinctive biological regulations at the levels of gene expression and cellular locations have been identified in human cells. Recent advances from our (including structure determination, isozyme biochemistry, biology and pharmacology) and other labs have suggested potential new isozyme-specific targeting approaches to solve above clinical problems. It should be noted here that the TOP2-targeting killing and TOP2-mediated toxicity has been considered to be responsible for the anticancer efficacy and unwanted therapy-related 2nd malignancies, respectively. Therefore, the overall goal of this sub-project is to provide the molecular, biochemical and cellular bases for hTOP2 isozyme-specific targeting approaches and to systematically identify the hTOP2-specific targeting lead compounds. Through a limited screening, we have already identified a novel class of effective TOP2-targeting agents, mitoxantrone-amino acid conjugates (MACs), potentially with favorable clinical profiles. In addition, through collaboration with sub-project 1, several drug screening platforms will be established. Specifically, the cytotoxic subset of chemical libraries (IC50 < 1 M) will be systematically screen using our biochemical and cellular platforms. The identified isozyme-specific lead compounds will also be validated by TOP2 isozyme-specific assays and evaluated of their efficacy and potential in generating 2nd malignancies using mouse models. Through collaboration with sub-project 3, we will provide molecular, biochemical and structural insights into the isozyme-targeting approach. The specific aims are as the followings:(1) Molecular and biochemical insights into the TOP2 isozyme-targeting therapy: We will develop TOP2 isozyme-specific assays and provide further experimental supports and rational designs for the TOP2-targeting anticancer agents. Specifically, biochemical, molecular and cellular technologies (e.g. RNAi approach, comet assay and hTOP2cc analyses) will be employed to facilitate the discovery of the TOP2-specific agents.(2) To identify lead compounds through systematic screening of known TOP2 drugs and chemical libraries. Biochemical (e.g., in vitro TOP2 cleavage assay) and molecular (i.e., the isozyme-specific RNAi knockdown approach) analyses will be used to systematically screen for the lead compounds from the cytotoxic subset of libraries.(3) To validate the lead compounds for their hTOP2-specific targeting. The identified lead compounds will be validated for hTOP2-specific targeting with isozyme-specific cell- and animal-based assays (e.g. the skin-carcinogenesis mouse model for evaluating their carcinogenic activity). Other pharmacological properties (e.g. as drug resistance) will also be determined.It is highly anticipated that the information and hTOP2 isozyme speicifc approaches established from our study will certainly provide great clinical implications as well as crucial insights for future drug development.第二型拓樸酵素抑制劑抗癌藥物蛋白-藥物-DNA三重複合體DNA topoisomerasetopoisomerase-targeting therapyisozyme-specific agentstopoisomerase cleavable complexmutagenesiscarcinogenesis.