發(fā)布時間：2018-04-03 14:12

  本文選題：細胞周期　切入點：CDK2　出處：《大連醫(yī)科大學》2017年碩士論文

【摘要】：研究背景:隨著分子生物學和結(jié)構(gòu)生物學的發(fā)展,作為受體的生物大分子的三維結(jié)構(gòu)被越來越多的發(fā)現(xiàn)和測定。根據(jù)已知生物大分子的結(jié)構(gòu)信息,利用計算化學和計算生物學方法篩選與其相互作用的小分子,即基于結(jié)構(gòu)的藥物設(shè)計方法(structure-based durg design,SBDD)。這個方法能夠大大降低新藥研發(fā)的成本和周期,因此越來越多的應用在目前的藥物研發(fā)領(lǐng)域。腫瘤作為一種嚴重威脅人類健康的疾病,一直是科學家們致力于攻克的目標。細胞周期蛋白依賴性激酶(cyclin dependent kinase,CDK),是絲/蘇氨酸蛋白激酶家族中的一員,是參與細胞周期調(diào)控的關(guān)鍵酶。CDK2作為其中一個亞型,其與cyclinE和cyclin A結(jié)合后,能夠促進細胞G1到S期的轉(zhuǎn)變,同時參與調(diào)控S期進程。有研究發(fā)現(xiàn),超過80%的癌癥都會出現(xiàn)CDK2過表達,如肝癌,乳腺癌等,因此發(fā)現(xiàn)靶向作用于CDK2的抑制劑有利于發(fā)現(xiàn)新型抗癌藥物;信號轉(zhuǎn)導與轉(zhuǎn)錄激活因子3(signal transducer and activator of transcription 3,STAT3)參與JAK-STAT3信號通路的作用,JAK-STAT3信號通路是由細胞因子刺激而激活的信號通路,參與細胞核基因轉(zhuǎn)錄調(diào)控,對細胞正常生理功能的維持起著不可或缺的作用。然而STAT3的持續(xù)激活會誘導一系列基因高表達,促進細胞增殖,惡性轉(zhuǎn)化,抑制細胞凋亡,從而導致癌癥的發(fā)生,促進癌癥進程。近年來也陸續(xù)發(fā)現(xiàn)STAT3在多種癌癥中高表達,使得STAT3成為發(fā)現(xiàn)抗癌藥物重要靶標。目前CDK2-cyclinA復合物和STAT3的三維結(jié)構(gòu)都已發(fā)現(xiàn)并研究報道,這也為研究針對特異蛋白的小分子抑制劑提供了基礎(chǔ)。研究目的:利用CDK2-cyclinA復合物晶體結(jié)構(gòu)設(shè)計發(fā)現(xiàn)作用于CDK2蛋白ATP口袋的新的母核結(jié)構(gòu)的化合物;利用STAT3晶體結(jié)構(gòu)設(shè)計靶向作用于其SH2結(jié)構(gòu)的抑制劑。研究方法:本文以CDK2-cyclinA和STAT3蛋白三維結(jié)構(gòu)為基礎(chǔ),首先運用計算機輔助藥物設(shè)計方法進行分子對接,篩選商業(yè)化合物庫,對小分子進行打分和排序,購買其中打分靠前的小分子。對購買的CDK2潛在小分子抑制劑進行體外活性初篩,挑選活性最好的進行藥物化學改造,以提高小分子的生物活性。將改造后具有相同母核、不同取代基的小分子進行體外活性檢測,得到最好的小分子在癌細胞中進行CCK8實驗,接著運用米氏方程驗證小分子抑制劑的競爭性。運用FP實驗對購買的STAT3潛在小分子抑制劑進行體外活性初篩,將活性較好的幾個在MDA-MB-468細胞中進行CCK8實驗,最終篩出活性最好的一個在多種癌細胞中進行CCK8驗證。使用Biacore實驗驗證其作用位點。實驗結(jié)果:CDK2抑制劑:初篩得到81號化合物,半數(shù)抑制濃度IC50是30.34μM。經(jīng)過藥物化學改造,得到CDK2抑制劑WZ-26,其IC50是3.81μM,較初篩得到的化合物活性提高了近10倍。在人源肝癌細胞HepG2中進行細胞增殖抑制檢測,其IC50是30.58μM;經(jīng)實驗驗證WZ-026是ATP競爭性抑制劑。STAT3抑制劑:FP實驗初篩后挑選幾個對STAT3具有最佳結(jié)合力的小分子進行細胞增殖抑制檢測,最終得到作用于STAT3的抑制劑B9,在MDA-MB-468細胞中,IC50為5.1μM;在MDA-MB-231和DU145細胞中的IC50為18.87μM和63.77μM,表現(xiàn)出了明顯的細胞增殖抑制活性,同時還利用Biacore的方法驗證了小分子的作用區(qū)域是STAT3的SH2結(jié)構(gòu)域。結(jié)論:運用基于結(jié)構(gòu)的藥物設(shè)計方法得到了:(1)具有抑制CDK2活性的小分子81,對其進行藥物化學改造后,得到了小分子WZ-26,它是一個ATP競爭性抑制劑,在人源肝癌細胞中表現(xiàn)出增殖抑制活性;(2)具有抑制STAT3活性的小分子B9,在乳腺癌和前列腺癌細胞系中都表現(xiàn)出了增值抑制活性。其作用區(qū)域位于STAT3蛋白的SH2結(jié)構(gòu)域。
[Abstract]:Background: with the development of molecular biology and structural biology, as the three-dimensional structure of biological macromolecules and the receptor was determined. According to the discovery of more and more structural information of known biological macromolecules and small molecules using the calculation method of chemistry and computational biology screening and interaction, namely the structure based drug design approach (structure-based durg design, SBDD). This method can greatly reduce the cost and cycle of new drug research and development, so more and more used in current drug research and development. As a serious threat to human health and disease cancer, scientists are working to capture the target. Cyclin dependent kinase (cyclin dependent, kinase, CDK) that is a threonine protein kinase family member / wire, is a key enzyme of.CDK2 in cell cycle regulation as one subtype, with cyclinE and cyc Lin combined with A, can promote cell G1 to S transition period, also involved in the regulation of S progression. Studies have found that more than 80% of all cancers occur over expression of CDK2, such as liver cancer, breast cancer, so that targeted inhibitors on CDK2 for the discovery of new anticancer drugs; signal transduction and transcription the activation of factor 3 (signal transducer and activator of transcription 3, STAT3) in the role of JAK-STAT3 signaling pathway, JAK-STAT3 signaling pathway is activated by cytokines, involved in gene transcription regulation, maintain the normal physiological function of cell plays an integral role. However, STAT3 can induce the high expression of a sustained activation a series of genes that promote cell proliferation, malignant transformation, inhibition of apoptosis, which leads to the occurrence of cancer, promote cancer progression. In recent years have found that STAT3 was highly expressed in many cancers To make STAT3 become the important target of anticancer drugs found. At present the three-dimensional structure of CDK2-cyclinA complex and STAT3 have been found and reported, which also provide a basis for the study of small molecule inhibitors of specific proteins. Objective: the structure design of nulei structure of new compounds found in CDK2 protein using ATP pocket CDK2-cyclinA composite crystal; crystal structure design using STAT3 inhibitors targeting its SH2 structure. Methods: on the basis of CDK2-cyclinA and STAT3 protein three-dimensional structure, molecular docking firstly by using the method of computer aided drug design, commercial screening of compound libraries, marking and sorting of small molecules, which marks on the purchase small molecules. The CDK2 potential of small molecule inhibitors for in vitro activity screening, selection of active transformation of pharmaceutical chemistry best, in primary school The molecular biological activity. After the transformation, with the same parent nucleus, small molecules with different substituents were in vitro, obtained the best CCK8 experiment of small molecules in cancer cells, then using the Michaelis equation verification competition of small molecule inhibitors. The use of FP in vitro experiment on STAT3 potential of small molecule inhibitors to buy at the beginning the screen, some good activity for the CCK8 experiment in MDA-MB-468 cells, finally screened the best activity in a variety of cancer cells. Using Biacore CCK8 validation experiments to verify its site. Results: CDK2 inhibitors: preliminary screening of 81 compounds, half inhibitory concentration IC50 is 30.34 M. after transformation medicinal chemistry, CDK2 inhibitor WZ-26, the IC50 is 3.81 M, a preliminary screening of compound activity increased nearly 10 times. The cell proliferation inhibition test in human hepatocellular carcinoma cell line HepG2, IC The 50 is 30.58 M; the experiment WZ-026 is a competitive inhibitor of ATP.STAT3 inhibitors: FP experiment after the screening of STAT3 to select a few small molecules with the best combination of force detecting inhibition of cell proliferation, resulting in the role of STAT3 inhibitor B9 in MDA-MB-468 cells, IC50 5.1 M in MDA-MB-231 and DU145; cells in the IC50 was 18.87 M and 63.77 M, showed obvious inhibitory activity of cell proliferation, but also the use of Biacore method to verify the interaction region of small molecules is the SH2 domain of STAT3. Conclusion: the use of drug design method based on the structure are: (1) is a small molecule that inhibits CDK2 the activity of 81, after the reform of medicinal chemistry, the small molecule WZ-26, which is a competitive inhibitor of ATP, on human hepatocellular carcinoma cells showed inhibition of proliferation activity; (2) with a small molecular B9 inhibited the activity of STAT3, and the former in breast cancer The adenocarcinoma cell lines of the adenocarcinoma show a value added inhibitory activity. The area of action is located in the SH2 domain of STAT3 protein.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R91

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