具有 ALK 基因變異的非小細胞肺癌對於目前抗ALK酪胺酸激酶抑制劑反應效果良好。最早是於西元2011年被美國食品藥物管理局通過的藥物crizotinib用來治療晚期具ALK陽性的非小細胞肺癌，之後陸續通過的 ceritinib、alectinib、brigatinib。然而，大多數病患會在使用標靶藥物一到兩年內產生藥物抗藥性。在1202位點產生的從甘胺酸glycine被取代為精胺酸arginine G1202R突變對以上四種藥物皆無效，而且還未有藥物核准通過用來治療具此突變之ALK陽性的肺癌。

本研究主要目的是利用結構預測以及分子動態模擬的方式去探討研究對具有ALK變異的肺癌細胞對ALK抑制劑產生抗藥性的機轉，特別著重在G1202R突變。包括計算 RMSD、 RMSF、藥物和ALK之間形成之氫鍵數量及時間頻率、以及計算藥物鍵結自由能等方法去分析。

本研究結果證明，利用結構預測及分子動態模擬等工具所建立的分析流程，藥物和ALK蛋白間結合所產生的氫鍵及自由能變化結果，可以吻合細胞及臨床實驗的數據結果，lorlatinib 和原生型或突變型的ALK蛋白結合後的自由能結果相近 (△G= -14 kcal/mol)，證明lorlatinib在突變後的ALK蛋白結合穩定度高，可以驗證在細胞實驗上的結果。

利用結構預測及分子動態模擬等程式所建立的分析法流程，可以用來研究ALK基因突變的肺癌。期能利用此平台作為後續快速藥物篩選開發之用。

Non-small cell lung cancers (NSCLC) with ALK rearrangements are highly sensitive to ALK tyrosine kinase inhibition (TKI). The multi-targeted TKI crizotinib, ceritinib, alectinib, and brigatinib have been approved by the U.S. food and drug administration (FDA) in 2011, 2014, 2015, and 2017 for treating patients with advanced ALK-positive NSCLC. However, most patients develop resistance within one to two years. The G1202R mutation has been reported to be resistant to the TKI above. The possible treatment strategies are still critically lacking.

The purpose of the current study is to utilizing protein structure prediction and molecular dynamics simulation to investigate the possible resistant mechanisms of ALK inhibitors, focus on the G1202R mutation. We calculated RMSD, RMSF, hydrogen bond frequency and distance, binding free energy between drug and ALK wild type and G1202R mutation proteins.

Through molecular dynamic simulation, our analysis indicates that G1202R mutation alters the conformation of the ALK binding pocket surface residues which results in a marked decrease in hydrogen bond interactions between TKIs and ALK. However, compared with binding with wild type ALK, lorlatinib binding with G1202R ALK could had more hydrogen bonds and expressed similar binding free energy between wild type and mutation type ALK protein (△G = -14 kcal/mol). The result is compatible with cell line experiment result that lorlatinib is effective to inhibit G1202R ALK protein phosphorylation.

We proved that through computational protein structure prediction and molecular dynamic simulation, we can utilize this analysis protocol as a tool for investigation of drug resistant mechanisms and for further drug development.

論文審定書..................................................i
目錄........................................................ii
圖次.......................................................iii
表次........................................................iv
中文摘要.....................................................v
英文摘要....................................................vi
第一章 背景知識介紹..........................................1
第一節 肺癌的流行病學與治療..............................1
第二節ALK 基因變異陽性的肺癌.............................3
第三節ALK酪胺酸激酶抑制劑的發展........................5
第四節ALK酪胺酸激酶抑制劑的抗藥性......................7
第五節臨床困境與研究動機................................. 8
第二章 研究方法............................................10
第一節 以結構預測及分子動態模擬方法探討ALK突變與標靶藥物交互作用之影響.....................................10
第二節 分子動態模擬......................................11
第三章 研究結果.............................................15
第一節 結構預測........................................15
第二節 藥物結合ALK蛋白位置之Root Mean Square Deviation..20
第三節 Root Mean Square Fluctuation........................28
第四節 門戶胺基酸Gly1123及Asp1203之間距離............. 31
第五節 氫鍵產生的個數與頻率.............................33
第六節 結合自由能Binding Free Energy........................38
第四章 討論分析.............................................38
第一節 ALK蛋白質結構預測與RMSD.....................38
第二節 ALK蛋白質結構RMSF 與門戶胺基酸之間的距離....39
第三節 氫鍵產生的個數與頻率............................39
第四節 自由能..........................................40
第五章 結論.................................................41
第六章 未來展望.............................................41
參考文獻....................................................43 

圖次

圖 1-1 致癌基因及其訊息傳導路徑............................4
圖 1-2 Crizotinib 抗藥性機轉................................8
圖 1-3 在使用不同標靶藥物後產生續發性ALK突變基因分布圖...9
圖 2-1 拉伸模擬之示意圖...................................13
圖 2-2 分析藥物和ALK結合之複合物的鍵結自由能............14
圖 3-1 ALK蛋白質結構....................................15
圖 3-2 ALK蛋白質和crizotinib結合之結構預測圖..............16
圖 3-3 ALK蛋白質和ceritinib結合之結構預測圖...............17
圖 3-4 ALK蛋白質和alectinib結合之結構預測圖...............17
圖 3-5 ALK蛋白質和brigatinib結合之結構預測圖..............18
圖 3-6 ALK蛋白質和lorlatinib結合之結構預測圖..............18
圖 3-7 ALK蛋白質和crizotinib結合位置圖與其RMSD..........20
圖 3-8 ALK蛋白質和ceritinib結合位置圖與其RMSD...........21
圖 3-9 ALK蛋白質和alectinib結合位置圖與其RMSD...........23
圖 3-10 ALK蛋白質和brigatinib結合位置圖與其RMSD..........25
圖 3-11 ALK蛋白質和lorlatinib結合位置圖與其RMSD..........27
圖 3-12 ALK蛋白質和crizotinib結合之RMSF圖形..............28
圖 3-13 ALK蛋白質和ceritinib結合之 RMSF圖形..............29
圖 3-14 ALK蛋白質和alectinib結合之RMSF圖形...............29
圖 3-15 ALK蛋白質和brigatinib結合之RMSF圖形..............30
圖 3-16 ALK蛋白質和lorlatinib結合之RMSF圖形..............31
圖 3-17 ALK蛋白和配體鍵結之門戶胺基酸Gly1123及Asp1203之間
距離示意圖......................................31
圖 3-18 ALK蛋白門戶胺基酸Gly1123及Asp1203之間距離變化
圖形............................................32
圖 3-19 Crizotinib 和WT and G1202R ALK 形成之氫鍵...........33
圖 3-20 Ceritinib 和WT and G1202R ALK 形成之氫鍵............34
圖 3-21 Alectinib 和WT and G1202R ALK 形成之氫鍵............35
圖 3-22 Brigatinib 和WT and G1202R ALK 形成之氫鍵...........36
圖 3-23 Lorlatinib和WT and G1202R ALK 形成之氫鍵............37
圖 3-24 Crizotinib (VGH) 與 lorlatinib (KVC) 和原生型或G1202R突變
型 ALK結合過程之自由能變化圖...................38

表次

表 1.1 抑制百分之50的ALK蛋白磷酸化所需藥物濃度表........8
表 2.1 抗ALK酪胺酸激酶抑制劑........................... 14

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