親和型探針的合成與應用：孕烯醇酮結合蛋白質的鑑定以及端粒導向的去氧核醣核酸烷化劑之研究

Synthesis and Application of Affinity-Based Probes: Identification of Pregnenolone Binding Proteins and Study of Telomere-Directed DNA Alkylating Agents

黃子樵(Tzu-Chiao Huang)

指導教授 ： 陳昭岑

國立臺灣大學/理學院/化學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00108

摘要

近期有許多研究團隊發展親和型探針 (affinity-based probes, AfBPs) 的相關技術，第一個成功運用於蛋白質研究的 AfBPs，是由 Cravatt 以及 Yao 教授團隊各自發表的金屬蛋白酶探針。AfBPs 與蛋白質上的特定位置結合後，再經由共價鍵的生成，以達到對特定蛋白質的標記。AfBPs 的研究工作蓬勃發展，讓我們明瞭設計與合成標記不同生物分子的探針，為勢在必行的工作。
　　胚胎細胞的遷移過程對於生物體的形態發生與體型之建立相當重要。本實驗室先前研究指出，類固醇荷爾蒙－孕烯醇酮 (pregnenolone) 可維持微管的穩定，進而有效地促使細胞的遷徙。因此我們推測存在某種蛋白質，能與孕烯醇酮結合並調控微管的形成，我們稱此為孕烯醇酮結合蛋白質 (pregnenolone binding proteins, PBPs)。為了驗證此推論，我們設計與合成以孕烯醇酮為辨識基團、二苯丙酮為光反應基團以及生物素為序號標籤的光反應型探針 (P5C7b-O-NBPN)，並期待能運用此探針來鑑定與純化 PBPs。
　　除了蛋白質的研究之外，我們也對去氧核醣核酸 (DNA) 特殊的二級結構之相關研究深感興趣。在細胞分裂期間，DNA 尾端的端粒序列，能保護基因組的不穩定以及避免細胞衰老。許多腫瘤細胞中有過度表現的端粒酶，可維持端粒序列的動態平衡，使端粒長度不會因細胞分裂而縮短，此為腫瘤細胞不死的重要因素之一。所幸富含鳥嘌呤的端粒序列，在特定的環境下傾向於形成鳥嘌呤四股結構 (G-quardruplex)，此特殊的結構可抑制端粒酶延長端粒序列。近年來咔唑衍生物 BMVC 在文獻中被報導可作為四方結構 (G-quartet) 的穩定劑以及螢光探針。有鑑於此，我們設計與合成一系列以 BMVC 骨架為辨識端的芥子氣衍生物 (BMVC-CnM; n = 2, 3, 6)，以嘗試發展成為端粒導向的 DNA 烷化劑。先前我們已利用多種光譜學以及凝膠電泳實驗，來探討 BMVC-CnM 與四股結構之間的結合辨識。在本論文中，我們藉由螢光共振能量轉移 (FRET) 以及 DNA 足跡 (footprinting) 等分析方法，對 BMVC-CnM 與四股結構間的結合模式做更深入的研究。此外，端粒酶抑制性的測試以及細胞顯影等實驗結果，顯示出 BMVC- CnM 有潛力發展成為抗癌藥物。

關鍵字

親和型探針 ； 孕烯醇酮 ； 端粒 ； 去氧核醣核酸烷化劑

並列摘要

Affinity-based probes (AfBPs) have been developed recently by several research groups. Both of Cravatt and Yao groups independently reported the first AfBPs for metalloproteases. AfBPs achieves labelling by binding at a specific site of a protein followed by a non-specific covalent bond-forming event. The seminal works of AfBPs research enlighten us to design and synthesize probes for targeting different biomolecules of interest.
　　Embryonic cell movement is essential for morphogenesis and establishment of body shape. In the previous research reported by our group, showed the steroid hormone, pregnenolone, can preserve the abundance of microtubules, and thus effectively promotes the cell movement. In light of this study, we speculate that there might be pregnenolone binding proteins (PBPs), which can bind pregnenolone and further control the formation of microtubules. To verify our assumption, the photoreactive probes (P5C7b-O-NBPN) composed of a pregnenolone as the PBPs binding group, benzophenone as the photo cross-linker and a biotin as the reporter were designed and synthesized. It is hoped that by using this set of probes, PBPs could be identified and further purified prospectively.
　　On the other hand, telomeres are specialized DNA ends providing protection against genomic instability and cell senescence during cell division. The homeostasis of telomere is maintained by telomerase, which is selectively expressed in most tumors, and hence has a crucial role in cellular immortalization.38(?) The G-rich sequence of human telomeric DNA has a strong propensity to form the DNA G-quadruplex secondary structure, which can inhibit the activity of telomerase.53(?) Recently, 3,6-bis(1-methyl-4-vinylpyridium) carbazole diiodide (BMVC) was reported as a G-quartet stabilizer and a fluorescence probe. Preserving the key structural features of BMVC, a series of BMVC-mustard conjugates (BMVC-CnM; n = 2, 3, 6) were designed and synthesized in our group in attempts to develop telomere-directed DNA alkylating agents. The recognition between BMVC-CnM and G-quarduplex were analyzed and confirmed by various spectroscopic tools and DNA-PAGE studies previously. In this thesis, FRET analysis and DNA footprinting were applied to investigate the binding modes of BMVC-CnM with G-quarduplex. Furthermore, the TRAP-assay and cell images experimental results indicate BMVC-CnM have great potentials to be developed as anti-tumor agents.

並列關鍵字

affinity-based probe ； pregnenolone ； telomere ； DNA alkylating agents

參考文獻

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