發展以 D4A 為導引基團的光親和性探針並探究其對於類固醇生合成路徑中的蛋白質之標示能力

Abiraterone 能夠抑制類固醇生合成路徑中的關鍵酵素 CYP17A1，從而有效減少雄性激素的產生，並因此於 2012 年獲得美國食品及藥物管理局許可上市，被應用於治療去勢抗性的攝護腺癌。然而， Abiraterone 在進入人體後會經由3β-烴類固醇脱氫酶（3β-HSD）的催化得到代謝產物 Δ4-Abiraterone（D4A），相較於 Abiraterone， D4A 不僅對於 CYP17A1 具有抑制作用，同時還可阻斷多種類固醇生成酶，使其有機會成為更加有效的抗攝護腺癌藥物，以及應用於類固醇生合成路徑中對於配基-蛋白質篩選的研究。因此，本研究利用 AfBPP（Affinity-Based Protein Profiling）原理，設計並合成基於 D4A 的光親和性探針 D4APB 以及可利用生物正交點擊化學進行兩步標示的 D4AP。該探針使用 D4A 作為辨識基團，並與目標蛋白進行非共價結合，隨後，光反應基團二氮環丙烯在 365 nm 波長的光激發下將會與目標蛋白形成共價鍵結，將探針固定在目標蛋白上，以利後續生物素標籤基團進行純化及免疫染色。在對於光親和性探針之功能鑑定中，選擇了可完整表達類固醇生合成路徑的腎上腺皮質癌細胞株H295以及攝護腺癌細胞亞系 CWR22Rv1 與 C4-2B 來進行後續的細胞實驗。最終結果顯示 D4APB 能夠特異性標示分子量為 40 kDa 之目標蛋白，然而在使用質譜對其進行蛋白質組學分析時，由於依舊存在非特異性標示，不易準確鑑定目標蛋白身分，故未來可對目標蛋白的富集進行優化，以減少不必要的影響。

關鍵字

蛋白質體學；光親和性探針；攝護腺癌；阿比特龍； Δ4-阿比特龍

並列摘要

Abiraterone, which is an anti-prostate cancer drug approved by US Food and Drug Administration in 2012, can inhibit the enzyme Cytochrome P450 17A1 (CYP17A1) in the stereodogenesis pathway in order to reduce the production of androgens. But it was reported that its metabolite Δ4-Abiraterone (D4A) arising from the action of 3β-hydroxysteroid dehydrogenase (3β-HSD) was actually responsible for the cytotoxicity. In other words, D4A can also inhibit CYP17A1 in the steroidogenesis pathway. Moreover, it can block a variety of steroid-producing enzymes. Potentially, it can be used as a more effective anti-prostate cancer drug and developed into an effective probe to study ligand-protein screening/interactions in the steroidogenesis pathway. Correspondingly, a D4A-based photoaffinity probe D4APB and a clickable probe D4AP, both of which based on the Affinity-Based Protein Profiling (AfBPP) concept, had been designed and synthesized. The probe consists of D4A as a ligand to recognize the target protein via non-covalent interactions, the diazirine photoreactive group will be activated to form a covalent bond with the interacting proteins upon irradiation at 365nm and the biotin tag-containing fragment will be used for subsequent purification and the immunostaining. H295 cell line was chosen for the cell studies since it can fully express the steroidogenesis pathway. In addition, the labeling efficiency of D4APB was also evaluated in CWR22Rv1 and C4-2B prostate cancer sublines. Collectively, D4APB can specifically label a protein of which molecular weight is estimated as 40kDa, followed by proteome analysis using Mass spectrometry. However, some other non-specific labeling increased the difficulties of identification. Hence, the enrichment of target protein needs to be further enhanced.

並列關鍵字

Proteomics ； Photoaffinity Probe ； Abiraterone ； Δ4-Abiraterone

參考文獻

1 Wilkins, M. R.; Sanchez, J.-C.; Gooley, A. A.; Appel, R. D.; Humphery-Smith, I.; Hochstrasser, D. F.; Williams, K. L. Biotechnol. Genet. Eng. Rev. 1996, 13, 19.