設計及合成嘌呤型複合體作為蛋白水解靶向嵌合體來抑制癌細胞生長

Abstract

每年肺癌造成約180萬人死亡，為全世界第二常見的癌症。之前中央研究院及國立臺灣大學的團隊經由高通量藥物篩選分析，選出了嘌呤類化合物1 (GRC0321)可以讓肺癌細胞凋亡。而該研究也發現投入此化合物可促使微管斷裂，因而推測化合物1是經由活化細胞劍蛋白 (katanin)，分解微管以達到抑制肺癌細胞的效果。 因此，我們考慮使用蛋白降解靶向嵌合體(proteolysis targeting chimeras, PROTAC)的策略，以證實化合物1的作用靶點。蛋白質的泛素化是一種轉譯後修飾的過程，目標受質藉由 E1、E2、E3促使泛蛋白(ubiquitin, Ub)連接上目標蛋白的離胺酸。而PROTAC藉由兩個靶點嵌合的特性，分別去招募E3連接酶及目標蛋白，透過化學的方式達成蛋白質的泛素化降解。本論文中，我們將化合物1與E3配位基連接，設計出一個能誘發標的蛋白水解的嵌合體。首先必須決定最適合的連接位置，最佳化嵌合物連接分子的長度及組成，再進行生物抑制活性測試來驗證。 本研究發現化合物2於C6-醯基上接上六個碳的碳鏈，對H1975肺癌細胞及MDA-MB231乳癌細胞具有抑制活性，其IC50分別為56.3 µM及43.5 µM；而於N9-苯環上連接碳鏈的化合物4則有更好的表現，其IC50分別為9.9 µM及0.7 µM。基於此研究結果，我們合成以五個碳作為連接鏈的PROTAC型化合物22、24，然而經測試發現兩者對細胞株都沒有抑制能力，推測為連接鏈不夠長使E3配位基阻礙到受體和嘌呤類藥物間的作用。為解決此問題，我們選擇使用較容易延長的聚乙烯作為連接鏈，其中以五單元聚乙烯作為連接鏈的PROTAC型化合物33 (JMF4675) 對H1975肺癌細胞及MDA-MB231乳癌細胞具有不錯的抑制活性，其IC50分別為0.83 µM及35.1 µM。後續也參考了近期文獻，得到一些連接鏈的範例，方便往後嘗試使用適當的連接鏈，期望能獲得表現更好的PROTAC型化合物。

Lung cancer is the second most common cancer, making up almost 1.8 million deaths per year. By using high throughput screening, the purine-type compound 1 (GRC0321) was found to be cytotoxic to non-small cell lung cancer (NSCLC) by activating katanin, a microtubule severing protein. In addition to the biochemical evidence, we consider using a proteolysis targeting chimera (PROTAC) strategy to validate the protein target of compound 1. Ubiquitination is a post-translational modification process which makes ubiquitin connect to the lysine of the target protein through E1, E2, and E3. PROTAC uses the chimeric properties to recruit E3 ligase and target proteins, achieving protein degradation by chemical means. In this approach, a chimeric molecule is designed to contain structure 1 and an E3 ligand connected by a linker. We have determined a suitable position to place linkers. In addition, several PROTAC compounds have been synthesized and submitted for the cell-based assays to find the best linker with appropriate length and composition. Compound 2, which has a hexylene linker connected to the C6-acyl group, displays good potency to inhibit the growth of H1975 lung cancer cell and MDA-MB231 breast cancer cell, with IC50 values of 56.3 µM and 43.5 µM, respectively. Compound 4, which has a hexylene linker connected to the N9-phenyl ring, shows IC50 values of 9.9 µM and 0.7 µM against H1975 and MDA-MB231 cells, respectively. Based on the results, we synthesized PROTAC compounds 22 and 24 with pentylene linker. However, we found that neither of them has inhibitory activity against H1975 or MDA-MB231 cell lines. The possible reason is that the linker is not long enough, thus the E3 ligand hinders the interaction between katanin receptor and the purine-type drug. To solve this problem, we chose polyethyleneglycol (PEG) linkers with various lengths. Among them, PROTAC-type compound 33 (JMF4675) with a penta-PEG linker showed good inhibitory activity against H1975 lung cancer and MDA-MB231 breast cancer cells, with IC50 values of 0.83 µM and 35.1 µM, respectively. We also referred to recent literature to construct some typical linkers, hoping to design PROTAC-type compounds with better cancer cell cytotoxicity.