- 學位論文
發展以阿比特龍為辨識基團之光親和性探針 及其標示 CYP17A1 的效用評估
Development of Abiraterone-based Photoaffinity Probe and Evaluation of their Labeling Efficiency for CYP17A1
摘要
Abiraterone 在2012年底經美國食品暨藥物管理局許可，成為治療閹割抗性前列腺癌的二線藥物。其機轉為抑制類固醇生合成路徑中的 CYP17A1 以減少雄性激素的產生。至今大多數的體外實驗或是臨床試驗，為觀測 Abiraterone 影響生合成相關蛋白表現量和上下游產物的分泌量，然而專門探尋未知結合蛋白，所產生的藥物機轉研究卻相對貧乏。在本論文中，我應用 ABPP (Activity-Based Protein Profiling)原理，發展以 Abiraterone 為主體的光親和性探針。此探針是由辨識端Abiraterone、Diazirine光反應性基團和 Biotin為標籤所組成。當辨識端成功與目標蛋白結合時，可藉由 365 nm 波長的光激發使 Diazirine 對目標蛋白產生共價性鍵結，再由 Biotin 標籤做後續的純化或免疫染色。在結構設計上，依據 Docking 實驗結果和已報導的 X-ray 晶體結構，我們選擇 Abiraterone 之 C7a 位置做探針衍生化，以減少與 CYP17A1 結合的干擾。接著我們以單元模組化的合成策略，成功地合成以 Abiraterone 為導引的光親和性探針 Ab-C7a-PB 和 Ab-C7a-P，並以 NMR 技術確認了 C7a 位置的位向。最後進行光親和性探針的功能性鑑定，以 H295 細胞株作為 CYP17A1 來源，in vitro 光標示結果顯示，利用不同的細胞裂解液成分，皆無法萃取出有結合能力的 CYP17A1，故無法成功進行光標示。但以 in vivo 的方式，在維持原始生化功能的細胞組織下，加入 Ab-C7a-PB 進行培養和照光，經後續西方墨漬法分析顯示可成功標示到 CYP17A1。故確認 Ab-C7a-PB 具辨識和光標示的功能性，可用於剖析細胞層級之蛋白質體學研究。
並列摘要
Abiraterone, a drug approved by US Food and Drug Administration at the end of 2012 for Castration-Resistant Prostate Cancer, is an inhibitor of CYP17A1, which catalyzes the steroid biosynthesis to produce the androgen in adrenal cortex. Up to date, most in vitro tumor cell lines research or in vivo clinic trials focused on the evaluation of the abiraterone effects on relative protein up/down regulation or the level of specific sterols in the hormonal signal pathways. Research works involving the drug mechanism of action as well as ligand-based protein screening are relatively rare. In this study, I applied the Activity-Based Protein Profiling (ABPP) concept to develop abiraterone-based photoaffinity probes to profile abiraterone’s possible working targets. The probes are composed of abiraterone (ligand) as a recognition unit, daizirine moiety as a photoreactive group and a biotin tag as an output. The working hypothesis is that once the ligand recognized by the target protein, the daizirine could be brought to the vicinity of the target protein and subsequently forms a covalent bond with the target protein upon irradiation at 365 nm. After washing off the nonspecific binding proteins, the covalent-linked proteins can be further purified and characterized either by the tag purification or tag immunohistochemistry via biotin. Based on the molecular docking result and the reported X-ray structure of abiraterone-CYP17A1 ternary complex, abiraterone was derivatized at the C7 position with α configuration to minimize the structural perturbation to bind CYP17A1. The probes, denoted by Ab-C7α-PB and Ab-C7α-P, were successfully synthesized by assembling different functional modules via the designed connectivities. The configuration at C7 was confirmed to be α by several different NMR techniques. To exploit the utility of the probes, H295 cell line was used since it contains abiraterone binding protein-CYP17A1. In vitro photolabeling results showed that Ab-C7α-PB failed to label CYP17A1 by using many kinds of lysis buffers. On the other hand, if Ab-C7α-PB was incubated and photoactivated under native cellular environments in vivo, subsequent Western Blot showed that Ab-C7α-PB could successfully label CYP17A implicating that Ab-C7α-PB could function as an effective photoaffinity probe for labeling CYP17A1 and profiling the abiraterone regulating proteins.
參考文獻
延伸閱讀
- Hou, C. L. (2016). 探討泛極光激酶抑制劑tozasertib於Mycn擴增之神經母細胞瘤的舊藥新用潛力 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201601400
- 王智鴻（2007）。鑑定梨形鞭毛蟲的ARID/bright同源蛋白質對於cwp1基因的轉錄調控之影響〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2007.02106
- Lin, S. Y. (2012). 阿拉伯芥轉錄因子 HSFA1a/b/d 及 HSFA2 對一群熱逆境記憶效應基因調控之研究 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2012.00265
- 徐榮翊（2020）。Development of Affinity-Based Fluorescent Labeling Probes for Beta-Amyloid Proteins〔碩士論文，國立清華大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0016-2502202114244190
- 黃詠宣（2010）。Development of activity-based probes with high specificity for protein tyrosine phosphatase subfamilies〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2010.10310