發展分子探針運用於鑑定孕烯醇酮結合蛋白質以及位置選擇烷基化鳥嘌呤四股去氧核醣核酸

Abstract

本論文的研究工作分為兩個部分： 第一部分的內容是根據親合型探針 (affinity-based probes, AfBP) 的設計概念，透過在孕烯醇酮於 C7 位置利用醚基衍伸出具有光活化基團 benzophenone 與標籤分子 biotin，用以研究在斑馬魚胚胎細胞發育中孕烯醇酮的結合蛋白。我們同時合成了不同連接長度與位向的孕烯醇酮光親合型探針，探討不同連接長度與位向對於其促進微管蛋白聚合活性的影響。由實驗結果得知，P5-NBPN (圖1) 保有與孕烯醇酮類似的生物活性，所以接著利用 P5-NBPN 在斑馬魚胚胎細胞液進行光標定實驗，並成功地共價性標定到孕烯醇酮結合蛋白。經由 LC-MS/MS 鑑定後，得知此孕烯醇酮結合蛋白為 cytoplasmic linker protein 170 (CLIP-170)。由缺失型突變體 (depletion mutant) 的實驗果得知孕烯醇酮與 CLIP-170的結合區間在胺基酸 920-970；競爭實驗也證明了 P5-NBPN 具有良好的標定選擇性與特異性。 第二部分的內容是探討一系列以 BMVC 骨架為辨識端，導入不同連結端長度的氮芥子氣衍伸物 (BMVC-CnM; n = 2, 3, 6) 或增加氮芥子氣攻擊端作用距離 (BMVC-SW)，對於不同DNA結構 (鳥嘌呤四股、雙股與單股) 烷化能力的影響 (圖2)。為了研究連接長度或作用距離對於三種鳥嘌呤四股結構 (混和型、逆平行與平行) 反應表現的影響，利用電噴灑離子化質譜 (electrospray ionization mass spectroscopy, ESI-MS)、液相層析-質譜分析 (liquid chromatography-mass spectrometry, LC-MS) 與化學/酵素足跡法 (footprinting) 進行共價產物的分析，以準確的決定作用產物的分布與共價位置的結合環境。由結果得知，連結端長度調控選擇性、特異性與反應性；而對細胞較具有傷害的鏈內交聯 (intrastrand cross-link) 的能力則是由反應端之間的作用距離決定。透過研究反應能力與分子結構的關係，希望能提供設計有效率的 DNA 交聯劑的準則，進而應用在選擇性癌細胞毒殺藥物。

The work presented here consists of two parts: Section I describes that pregnenolone (P5) was equipped with benzophenone photoreactive group and biotin tag at C7 position in ether linkage to explore P5-binding proteins in the stage of embryonic development of the zebrafish. Various spacer lengths and orientations of P5-photoaffinity probes had been employed to investigate the influences on the activity of in vitro tubulin polymerization. With the preservation of the biological functions as P5, P5-NBPN was used to label P5-binding proteins from zebrafish embryo lysates and the P5 binding protein (Figure 1), cytoplasmic linker protein 170 (CLIP-170), had ultimately been found by LC-MS/MS identification. The photolabeling experiments of CLIP-170 and/or its various depletion mutants showed that the binding region of P5 on CLIP-170 located in the region between aa 920-970 with remarkable labeling selectivity and specificity. Section II describes that a series of G-quadruplex (G-4)-directing alkylating agents, BMVC-CnM (n = 2, 3, and 6) and BMVC-SW, integrating BMVC with aniline mustard in spacers of various lengths or with longer bridge length to react with different G-4 structures (hybrid-2 type, antiparallel and parallel) (Figure 2). The intact alkylated adducts were elaborately characterized by electrospray ionization mass spectroscopy (ESI-MS), LC-MS, and chemical/enzymatic footprinting to determine precise alkylation sites and plausible binding profiles. These results indicated that alkylation selectivity, specificity, and reactivity are modulated by adjusting linker lengths, whereas intrastrand cross-link efficiency which showed higher cytotoxicity is determined by the distance between two reactive warheads. Our preliminary findings regarding the different distance effects on G-4-specific alkylation provide a structural foundation for the development of G-4-selective bifunctional alkylating agents.