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研究生:林煥康
研究生(外文):Huan-Kang Lin
論文名稱:探索人類角鯊烯合成酵素胜肽抑制劑作為治療高膽固醇血症的潛力藥物
論文名稱(外文):Peptide Inhibitors of Human Squalene Synthase as Potential Drug Candidates Against Hypercholesterolemia
指導教授:宣大衛宣大衛引用關係
指導教授(外文):David Shiuan
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:103
論文頁數:76
中文關鍵詞:虛擬篩選
外文關鍵詞:phage displayhuman squalene synthase
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膽固醇在人體中扮演很重要的角色,但在身體內累積過量,會引發高膽固醇血症,因而造成許多慢性疾病。目前降膽固醇藥物,皆為膽固醇生成路徑上游之關鍵酵素,HMG-CoA reductase之抑制劑。但此類藥物因抑制HMG-CoA reductase,進而影響下游許多重要生理分子的生合成,所以造成嚴重的副作用。然而,在膽固醇合成下游路徑中,另一個重要關鍵酵素:角鯊烯合成酵素 (squalene synthase, SQS),因SQS位於farnesyl pyrophosphate (FPP) 的下游,可以避免缺乏FPP所導致的副作用,若以SQS作為降膽固醇藥物的標靶,應更具有潛力。而peptide藥物除藥效力高外,其毒性低,且較少累積於組織中,對人體造成之負擔低。因此,本研究以human SQS (PDB 1EZF) 當目標蛋白,首先以重組蛋白的方式,表達及純化SQS,再以phage displaed peptide library篩選會與之結合的phage。經過三輪篩選及親和性測試後,隨機選取10株親和力較強之phage,完成其ssDNA定序,經過docking 分析後,共合成8條peptides:如RVACLY、VCACLY、VCACNW、FTACNW 、ACNW 、CACL、VACL、VACH,進行SQS活性抑制實驗。結果顯示,FTACNW和VACL有較佳的抑制效果,其IC50約為100 μM。此外,藉由ADMET軟體預測出這8條peptides的肝毒性非常低,未來有機會發展更有效治療高血脂的peptide藥物。
Cholesterol plays an important role in living cells. However, very high level of cholesterol (hypercholesterolemia) may lead to many cardiovascular diseases. To prevent the disease-causing accumulation of cholesterol, many drugs have been developed. The currently available cholesterol-lowering drugs are all based on inhibiting the enzyme HMG-CoA reductase in the cholesterol biosynthesis pathway. However these medicines also indirectly inhibit the synthesis of many other important factors at the lower stream and cause severe side effects such as rhabdomyolysis. Therefore, to discovery better drugs to treat hypercholesterolemia remains necessary. Recent reports have revealed that peptide drugs are gaining more and more visibilities and higher potential in many therapeutic areas. In the present study, we employed phage-display techniques to screen for peptide inhibitors against human squalene synthase (hSQS), one of the key enzymes of the cholesterol biosynthetic pathway.
We had cloned, expressed and purified the hSQS as the lure to screen for the tight binders from the phage displayed random peptide libraries. After three rounds of biopanning, 10 phages were randomly picked and their ssDNA prepared for sequencing. Through DNA sequence analysis and molecular modeling of the peptides with hSQS, we synthesized eight peptides for enzymatic assays: RVACLY, VCACLY, VCACNW, FTACNW, ACNW, CACL, VACL, and VACH. We found that peptide “FTACNW” and “VACL” can inhibit hSQS effectively (IC50 = 100 μM). The two peptides were also estimated to possess good ADMET properties and low hepatoxicity. Therefore, it is anticipated that these peptides can be served as the leads to develop potential drugs to treat hypercholesterolemia.

目錄

致謝………………………………………………………………............I

目錄…………………………………………………………….………..II

摘要……………………………………….………………….………VIII

Abstract……………………………………...……………..……....…IX

一、 緒論………………………………………..………………….…...1
1.1研究目的………………………………..……………….…….1
1.2背景及重要性簡介..…………………...……….………….….1
1.3膽固醇合成及相關疾病治療…………………………………2
1.4 Human Squalene Synthase及其抑制劑發展…………………3
1.5噬菌體呈現技術 (Phage Display) 簡介……………………..5
1.6固相胜肽合成法 (Solid-Phase Peptide Synthesis, SPPS)……7
1.7電腦輔助藥物設計……………………………………………8
1.8蛋白質資料庫 (Protein Data Bank, PDB) …….……………..9
1.9蛋白質及結構分析模擬軟體 (Discovery Studio, DS) ………9
1.10 ADMET預測軟體………………………………………….10

二、 材料與方法………………………………………………………13
實驗材料…………………………………………………………13
實驗儀器設備……………………………………………………14
實驗方法…………………………………………………………14
實驗流程…………………………………………………………15
1. Expression of Human Squalene Synthase (hSQS) …….…..16
2. Purification of Human Squalene Synthase (hSQS) .…….….16
3. 噬菌體胜肽庫的保存Titer測試…………………….…..…...18
4. 生物親和性篩選 (Biopanning).……………………….….19
5. 單一溶菌斑之噬菌體擴增 (Plaque amplifyication) ………..21
6. 酵素免疫分析法 (ELISA) …………………………………..22
7. 抽取噬菌體 ssDNA定序與序列分析………..…………….23
8. 候選peptide進行ADMET prediction………………………..25
9. 合成特定peptides…………..…..……………..………..26
10. 酵素活性抑制實驗 (Enzyme Inhibition Assay) …..……..…27
11. 以DS 3.5軟體進行分子對接……………………………..29
12. Ligand-protein interaction 分析……………..…...………30
13. 細胞毒性測試 (MTT Assay)…………………….…………31

三、 結果與討論………………………………………………………33
1. 重組蛋白hSQS之表達及純化…………….………………..33
2. 噬菌體呈現技術篩選………………………………………...35
3. 以酵素免疫分析法(ELISA)挑選結合能力較高之phage…...36
4. Phage之ssDNA定序及peptide序列分析…………….…….37
5. 利用電腦進行分子模擬分析……………..………………….37
6. Peptide 及BPH701與1EZF之結合作用分析………….…38
7. 進行ADMET預測:8條peptide及BPH701…………..…..40
8. hSQS活性抑制實驗………………………………………….40
9. Peptide FTACNW、VACL及BPH701對細胞毒性測試……42

四、 結論與展望………………………………………………………43

五、 參考文獻…………………………………………………………45

六、 圖表………………………………………………………………49

七、 附錄……………………………………………………………..67

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