請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/48515
標題: 以肺癌主要驅動分子為標靶的新穎治療及抗藥性解決策略之發展-(總計畫與子計畫一)發展以Src酪胺酸激酶為標靶的先導型藥物並評估其抑制肺癌形成之活性( I )
(100cap026-1) to Develop the Novel Compounds Targeting Src Kinase and Evaluate Their Activity on Suppressing Lung Cancer Progression
作者: 陳健尉
侯明宏
吳雨珊
林季千
劉俊吉
關鍵字: 生物技術
商品化
src kinase
tumor suppressor HLJ1
EGFR
computational virtual screening
lead compound
摘要: 在人類癌症中,Src 激酶的表現量和活化是癌細胞生長和轉移的指標。在許多肺癌細胞中皆可發現 Src 的活性增加,進而啟動其下游訊息傳導路徑並造成肺癌的進展,尤其是表皮生長因子受體(EGFR)家族。我們先前的研究顯示,抑癌基因HLJ1(又稱為DNAJB4),能結合至Src SH3 及kinase 區域,並且抑制Src 的活性與阻斷其下游的訊息傳遞。因此,在這項計畫中,我們將利用HLJ1-Src 的胺基酸結合區域,發展以Src 為標靶的先導化合物,並進一步探討此化合物之分子作用機制。首先,將利用計算虛擬篩選方法尋找可抑制肺癌細胞的Src 抑製劑。為了快速的篩選藥物,亦將發展一套藥物篩選資訊系統,可以針對Src 與 HLJ1 的交互作用區域進行大規模的藥物篩選。我們正在發展一個嶄新的演算法,同時整合大規模的基因表現、蛋白質表現及藥物反應 (IC50)數據資料,以預測可能會作用於Src 的藥物。由於蛋白質與藥物結合的模擬計算需要巨量的電腦計算能力,我們將運用 World Community Grid 來克服計算上的困難。找到可能的Src 抑制劑後,將利用ELISA 來測量Src 磷酸激酶的活性。在體外試驗中,使用MTT、細胞群落形成、細胞侵犯和細胞傷口癒合分析來測量可能的Src 抑制劑對於癌細胞存活率和抑制細胞移動以及侵入的影響;於體內試驗中,使用皮下、尾靜脈與原位移植腫瘤注射動物模式,觀察先導化合物對腫瘤生長與轉移的影響。從已知的化合物庫中,透過虛擬篩選找出與Src 有最佳結合的先導化合物,並透過對先導化合物的合成以及抑制活性的評估,我們將能夠選擇最佳的複合結構作進一步修改。藥物設計與合成將分為兩部分:一個是合成模擬HLJ1 與Src- SH3 結合部位結構的胜肽複合物,另一個則是設計合成抑制Src 活性的非胜肽類分子。藉由合成足夠衍生物,我們可建立Src 與衍生物間的構效關係,而此構效關係能相應地優化先導化合物。此外,為了瞭解藥物結合Src 蛋白的機制和最適化Src 激酶抑製劑,得分最佳的化合物和Src 蛋白的複雜結構將採用 X 射線晶體學來解析。最後,我們推測這個嶄新的Src 抑制劑有抑制肺癌細胞成長和轉移的潛力。此外,也將評估臨床試驗的可行性,尤其是臨床第一期。經由這些努力,我們預計將可對新一代肺癌標靶治療藥物的發展和個人化醫療做出巨大的貢獻°
Up-regulation of Src is a common event in human cancers. In lung cancer, src activity andlevel are significantly increased relative to tumor progression, poor prognosis, and metastasis.The increased Src expression and its activity have been implicated in lung cancer progressionthrough positively regulating numerous important intracellular signaling pathways,especially EGFR family. Our previous study demonstrated that tumor suppressor HLJ1 (alsoknown as DNAJB4) associates with Src and represses its activation as well as downstreamsignaling. In this proposal, we intend to discover novel compounds that would suppress Srcactivity based on the known HLJ1-Src interaction, and investigate their underlying molecularmechanism. We will firstly focus on finding Src inhibitors against lung cancer bycomputational virtual screening. To accelerate the drug screening process, we will develop anin silico drug screening framework targeting the crucial domain (or residues) of Src involvedin Src activation (Src-SH3 and kinase domain including Y418). Furthermore, we aredeveloping a new method that could integrate large-scale gene expression, protein expression,and drug response (IC50) data to predict potential drugs targeting Src. To overcome the hightime complexity of the protein-ligand docking algorithm, we will utilize theWorld Community Grid. After identifying the potential Src inhibitors (lead compounds), theenzymatic activities of Src kinase will be measured by enzyme-linked immunosorbent assayand then subjected to the functional analysis of anti-cancer effect. For in vitro assay, theanti-proliferative, anti-invasion and anti-migration activities will be examined by MTT,colony formation, invasion and wound healing assays to confirm the effect of the potential Srcinhibitors. For in vivo assay, we will evaluate the inhibitory effects of the new Src inhibitor ontumor growth and metastasis using the human lung cancer CL1-5 orthotopic implantedxenograft model in athymic mice. The most active compounds selected from above will bestructurally modified through chemical synthesis and their activity will subsequently bedetermined. With sufficient analogues synthesized, we should be able to establish theirstructure-activity relationship (SAR), and optimize lead compounds accordingly. Moreover, inorder to reveal the mechanism of drug bound to Src kinase and optimize novel inhibitor of Srckinase, the complex structures of the best scoring compounds and Src protein will bedetermined by X-ray crystallography. We believe that these novel Src inhibitors will possesspotent activity against lung cancer growth and metastasis, thereby rendering it the leadingcandidate in treating lung cancers with elevated Src tyrosine kinase activity. The feasibility ofclinical trial will also be assessed, especially in phase I. Through these efforts, we will be ableto make a significant contribution to a new generation of targeted therapies for lung cancerdrug development and personalized medicine.
URI: http://hdl.handle.net/11455/48515
其他識別: NSC100-2325-B005-001
顯示於類別:生醫工程研究所

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