TWI384995B - 第一型去氧核糖核酸拓樸異構酶抑制劑 - Google Patents
第一型去氧核糖核酸拓樸異構酶抑制劑 Download PDFInfo
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- TWI384995B TWI384995B TW097147505A TW97147505A TWI384995B TW I384995 B TWI384995 B TW I384995B TW 097147505 A TW097147505 A TW 097147505A TW 97147505 A TW97147505 A TW 97147505A TW I384995 B TWI384995 B TW I384995B
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Description
本發明係關於一種可抑制第一型去氧核糖核酸拓撲異構酶的化合物、包含此化合物之組合物或製備物，並可應用於抑制核酸複製或轉錄作用完成之化學療法，或應用此化合物與第一型去氧核糖核酸拓撲異構酶結合所開發之新藥設計。核酸複製或轉錄作用包含於病毒、細菌、真核細胞等。
DNA拓樸異構酶具有調節DNA合成時起始與延長的功能。當DNA進行複製時，第一型去氧核糖核酸拓樸異構酶可切單股DNA形成缺口，讓DNA鬆解反應得以進行。隨後DNA再接合形成雙股DNA。此酵素反應機制包括兩個連續酯交換反應(transesterification)(Teicher,2008)。在切割反應中，人類第一型去氧核糖核酸拓樸異構酶(human Topo I)中的酪氨酸(Tyr732)是其活性區做為親核劑。Tyr732上的酚氫基氧會攻擊DNA的3’端的磷酸二酯鍵，形成一Tyr732與DNA磷酸根共價結構的中間物質。在再接合(religation)階段，則是DNA的5’端上的氫氧基攻擊其該中間物質，進行酯交換反應。上述切割反應與再接合反應具有一致性與可逆性。拓樸異構酶的酯交換反應若被抑制將導致DNA複製或轉錄作用無法完成，因此處於斷裂的複合中間物狀態。
一些以第一型與第二型去氧核糖核酸拓樸異構酶為標的的藥物，藉由穩定共價結構的拓樸異構酶與DNA複合物，
而無法進行再接合反應(Liu,1989)。該複合物包含有第一型去氧核糖核酸拓樸異構酶與切斷的DNA分子，稱為切割複合體(cleavable complex)。該複合體不具再接合功能可被純化。拓樸異構酶抑制劑應用來作為抗腫瘤(Feun & Savaraj,2008；Wethington,Wright & Herzog,2008)、抗病毒(Sadaie,Mayner & Doniger,2004)、抗菌(Anderson & Osheroff,2001)、抗癲癇(Song,Parker,Hormozi & Tanouye,2008)、免疫調節(Verdrengh & Tarkowaki,2003)。喜樹鹼(Camptothecin,CPT)為穩定第一型去氧核糖核酸拓樸異構酶與DNA共價結構的代表性藥物，喜樹鹼衍生的抗癌藥物儘管臨床應用相當成功，但仍然有許多問題，包括多重抗藥性(Multidrug resistance,MDR)，會將癌細胞內的有效物質大量降低是因為多重抗藥性蛋白質P-醣蛋白(P-glycoprotein)會過度表現(Chu,Kato & Sugiyama,1997)，其臨床應用因容易誘發化學抗藥性而受限。
吳茱萸鹼，其化學式如下：
一種由芸香科植物吳茱萸中分離出來的物質，根據報導指出具有血管舒張、抗肥胖(Wang,Wang,Kontani,Kobayashi,Sato et al.,2008)、抗癌(Ogasawara,Matsunaga,Takahashi,Saiki & Suzuki,2002)、抗發炎(Ko,Wang,Liou,Chen,Chen et al.,2007)生理功能。證據顯示吳茱萸鹼可以藉著讓細胞循環停滯在G2/M時期，誘導細胞程式凋亡(Kan,Huang,Lin & Wang,2004)。在製備過程中產生一些吳茱萸鹼衍生物，包括：吳茱萸次鹼(rutaecarpine)，其化學式如下：
、吳茱萸醯胺(evodiamide)、脫氫吳茱萸鹼(dehydroevodiamine)(Zhou et al.2006)等，其化學式如下：
在所有的生物活性中，從無任何報告揭露吳茱萸鹼衍生物的直接作用目標是在第一型去氧核糖核酸拓樸異構酶。本案之揭露將確定吳茱萸鹼衍生物的作用模式，其將啟動吳茱萸鹼衍生物更廣泛的應用開發及進一步設計改善既有生物活性功效的新藥。
在本發明中，吳茱萸鹼可穩定第一型拓樸異構酶與DNA共價結構的能力，在治療應用的發展上具有益處。
吳茱萸鹼(Evodiamine,EVO)，一種由芸香科植物吳茱萸(Evodia rutaecarpa,(Juss))中分離出來的物質，會影響生理功能。拓樸異構酶抑制劑(Topoisomerase inhibitor)作為臨床上的應用。根據我們的研究報導指出吳茱萸鹼中有第一型去氧核糖核酸拓樸異構酶
(Topoisomerase I,Top I)抑制劑具有更佳的治療益處。吳茱萸鹼可抑制第一型去氧核糖核酸拓樸異構酶與超螺旋質體DNA的鬆解反應。在乳癌細胞(MCF-7)中給予吳茱萸鹼，遊離態第一型去氧核糖核酸拓樸酶減少具時間依賴性(0~120分鐘)，且具濃度依賴性(0~10μM EVO)，此乃因為Top I被吳茱萸鹼固定在核酸分子上。另外再利用K-SDS沉澱分析來檢測第一型拓樸酶結合染色體DNA的程度。KC1/SDS沉澱法正常作用於沉澱蛋白質-不沉澱DNA，若有DNA被K-SDS沉澱出來，只有在蛋白質-DNA共價鍊合狀態下才發生。實驗數據證明吳茱萸鹼會增加第一型去氧核糖核酸拓樸異構酶與DNA形成複合物，且具有濃度依賴性。當給予細胞30μM吳茱萸鹼，DNA結合增加43.6%。結果顯示吳茱萸鹼靠著穩定第一型去氧核糖核酸拓樸異構酶與DNA的共價複合物來抑制第一型去氧核糖核酸拓樸異構酶的作用。
綜上所述，如圖一所示，當DNA進行複製時，第一型DNA拓樸異構酶可切單股DNA形成缺口，讓DNA得以進行拓樸型之鬆解，隨後再催化接合形成雙股DNA(左邊)。當加入吳茱萸鹼或衍生物時，穩定第一型去氧核糖核酸拓樸異構酶與DNA共價結構的能力，拓樸異構酶的酯交換反應被抑制並導致DNA複製或轉錄作用無法完成處於斷裂的複合中間物狀態(右邊)，進而抑制細胞週期進行。
吳茱萸鹼在照光或受熱會自然產生包括吳茱萸次鹼(rutaecarpine)、脫氫吳茱萸鹼(dehydroevodiamine)等衍生物，因此在一般製備過程
中會獲得吳茱萸鹼衍生物。拓樸異構酶抑制劑在臨床及相關先前報導指出可以被應用來作為抗腫瘤、抗病毒、抗菌、抗癲癇、免疫調節等應用。吳茱萸鹼首度證實具有第一型DNA拓樸異構酶的抑制活性，因此預期可以應用於抗腫瘤、抗病毒、抗菌、抗癲癇、免疫調節等應用。對照先前技藝曾揭露吳茱萸鹼或衍生物的抗腫瘤、免疫調節等功效，其抗病毒、抗菌、抗癲癇功效是未曾先前揭露者。
此外，因本案首度揭露吳茱萸鹼的藥物目標為第一型DNA拓樸異構酶，便提供一抗癌藥物新藥開發的平臺，由第一型DNA拓樸異構酶的立體結合特性，可利用吳茱萸鹼(evodiamine)、吳茱萸次鹼(rutaecarpine)、或脫氫吳茱萸鹼(dehydroevodiamine)為引導化合物，設計與DNA拓樸異構酶結合更緊密的新藥，在醫藥發展上將會有很重要的貢獻。其活性分析可利用電腦分子立體模擬技術(Staker,et al.2002)、鬆解DNA超螺旋活性、或細胞內第一型去氧核糖核酸拓樸異構酶與DNA複合體的結合活性進行。
乳癌細胞(MCF-7)培養在Dulbecco’s modified Eagle medium(DMEM)中，添加10%加熱去活化的胎牛血清(Fetal bovine serum,FBS)、100μg/ml penicillin-streptomycin培養液中。培養在37℃下
，95%空氣加5%二氧化碳環境下。利用MTT來分析細胞存活率藉以瞭解添加試劑對於細胞的毒性。將細胞(每孔5000個細胞)培養在96孔培養皿添加含有1%胎牛血清的DMEM培養液中24小時。接著添加0~30μM的喜樹鹼或吳茱萸鹼，藉由MTT的減少來觀察細胞的存活率。添加MTT溶液(每毫升PBS中添加5 mg的MTT)至培養皿中，使其最後濃度達0.5 mg/ml。在波長560 nm下，利用分光光度劑來測量吸光值。所有數值將扣除背景值。
人類乳癌細胞MCF-7添加吳茱萸鹼或喜樹鹼細胞毒性測試。喜樹鹼作為參考藥物。在添加兩種藥物24小時後都有明顯的細胞毒性。計算其IC50(達到50%致死率之藥物濃度)，喜樹鹼為3.23μM而吳茱萸鹼為6.02μM。由此可知吳茱萸鹼對於人類乳癌細胞MCF-7具有細胞毒性。
牛痘病毒的DNA第一型拓樸異構酶(EPICENTRE Biotechnologies,Madison,WI)屬於真核的去氧核糖核酸拓樸異構酶，具有催化打斷或形成雙股DNA分子中的單股上的磷酸雙酯鍵。該酵素會針對DNA上的特定序列[5’(C/T)CCTT-]上的3’端進行切割接著再接合原本的磷酸雙酯鍵藉以鬆解DNA。此反應可將超螺旋DNA轉換為環狀鬆解DNA。藉由超螺旋DNA被第一型去氧核糖核酸拓樸異構酶切割的情形來評估喜樹鹼與吳茱萸鹼的抑制效果。pCDNA3質體DNA(200 ng)在37℃下培養在反應溶
液中(反應溶液：50 mM Tris-acetate,100mM NaCl,2.5 mM MgCl2，和0.1 mM EDTA，調整pH7.5)添加或不添加0~3.0μM抑制劑，最後反應容積20μl(Sekiguchi,Cheng & Shuman,1997)。藉著超螺旋雙股環狀DNA鬆解轉換情形來評估抑制劑的效果。反應後之樣品加入到1%的洋菜膠中在TAE(40 mM Tris-acetate和1 mM EDTA)緩衝溶液跑電泳，隨後洋菜膠照射紫外線並照相。
第一型去氧核糖核酸拓樸異構酶可以鬆解質體DNA的超螺旋結構形成一環狀DNA。而在試管實驗中，喜樹鹼與吳茱萸鹼皆可抑制超螺旋DNA的鬆解。第2圖顯示牛痘病毒的第一型去氧核糖核酸拓樸異構酶作用下超螺旋質體DNA的鬆解情形。在洋菜膠電泳中超螺旋DNA移動速度較鬆解環狀DNA快(第2圖中的第1~2行)。由不同濃度(1~3μM,第2圖第3~5行)的喜樹鹼加入後，濃度越高，維持超螺旋質體DNA量愈多，具有濃度依賴性。而添加不同濃度(1~3μM，第2圖第6~8行)吳茱萸鹼同樣具有濃度依賴性。結果顯示，吳茱萸鹼確實可以抑制牛痘病毒第一型去氧核糖核酸拓樸異構酶鬆解超螺旋質體DNA的作用。
來自重組DNA所製備的第一型去氧核糖核酸拓樸異構酶的鬆解超螺旋DNA的活性也同樣被吳茱萸鹼抑制。吳茱萸次鹼(rutaecarpine)與喜樹鹼、吳茱萸鹼具有實質上相當的第一型去氧核糖核酸拓樸異構酶的鬆解質體DNA活性。
吳茱萸鹼若能夠將Top I固定在核酸分子上，則遊離態第一型去氧核糖核酸拓樸異構酶量應會減少。50~80%滿的MCF-7乳癌細胞株短時間處理藥物，抽取細胞蛋白並以7.5% SDS-PAGE進行電泳後再轉染至PVDF(polyvinylidene difluorite)膜上。該膜於室溫下與第一抗體(rabbit anti-human topoisomerase I antisera)作用2小時，接著再與第二抗體immunoglobulin G(horseradish peroxidase-conjugated secondary Ig G)作用。再添加化學螢光試劑來顯現免疫反應。利用膠體電腦軟體系統拍照。
遊離態第一型去氧核糖核酸拓樸異構酶耗損分析可以用來反映吳茱萸鹼對於第一型去氧核糖核酸拓樸異構酶催化DNA的影響。這個分析係為根據喜樹鹼會和第一型去氧核糖核酸拓樸異構酶與DNA形成一三體複合物，而吳茱萸鹼是否經由類似機轉抑制第一型去氧核糖核酸拓樸異構酶。MCF-7細胞在正常情況下有足夠的第一型去氧核糖核酸拓樸異構酶可被偵測到。將吳茱萸鹼加入MCF-7細胞中0~120分鐘。利用西方點墨法(immunoblotting)來偵測第一型去氧核糖核酸拓樸異構酶的含量。結果顯示，加入10μM吳茱萸鹼時，第一型去氧核糖核酸拓樸異構酶隨著時間增加而減少，具有時間依賴性。在處理120分鐘後，與對照組比較減少20%的量(第3圖A)。在1小時反應時
間下，添加吳茱萸鹼濃度不同，同樣具有濃度依賴性，即濃度越高，第一型去氧核糖核酸拓樸異構酶越少(第3圖B)。與對照組比較，處以0~10μM吳茱萸鹼，遊離態第一型去氧核糖核酸拓樸異構酶減少至小於40%。
利用Yoshinari et al.(1993)改良之KCl/SDS沉澱分析技術來定量切割複合體形成。運用濃度10μM Ci/ml在細胞DNA中的腺嘧啶(3H-Thymidine)上標定放射線。經過一夜培養讓24孔培養皿每孔細胞數量達1×105個，接著給予不同濃度吳茱萸鹼(0~30μM)培養60分鐘。將培養液移除並利用PBS(phosphate-buffered saline)清洗，再加入65℃下預熱過的裂解液(lysis buffer)(1.25% SDS,5 mM EDTA,0.4 mg/ml鮭魚精子DNA)。利用注射針筒連續來回抽吸(21-gauge-needle)細胞。控制組樣品相同進行以上步驟，在裂解液中加入400μg/ml蛋白激酶K(proteinase K)，在50℃下作用2小時。接著所有樣品都加入250μl KCl(325 mM)混合均勻，冷卻10分鐘後，在4℃下離心2500 rpm 10分鐘。每次利用1 ml清洗液(10 mM Tris-HCl,100 mM KCl,1 mM EDTA和0.1 mg/ml鮭魚精子DNA)清洗兩次後，在65℃下作用10分鐘後在冰上冷卻。接著在2500 rpm下離心10分鐘。再利用400μl預熱65℃的水重新懸浮沉澱物，加入4 ml閃爍計數液，以液體閃爍計數器測量放射線
強度。
利用上述K-SDS可以沉澱出第一型去氧核糖核酸拓樸異構酶與DNA的複合體，因此測量該複合體即可反映出吳茱萸鹼的抑制能力。將具有標定放射線的腺嘧啶(3H-Thymidine)分別處以不同濃度(0,5,10,20和30μM)的吳茱萸鹼60分鐘，再利用K-SDS沉殿來分析。吳茱萸鹼在濃度0μM僅有3.1%標定放射線腺嘧啶之共價複合體。而在吳茱萸鹼在濃度30μM時，則增加至43.6%(第4圖)。此結果顯示，濃度越高的吳茱萸鹼能夠形成越多的共價複合體，具有一濃度依賴性。
分子模擬(molecular modeling)係利用分子模擬軟體，設定3D結構新藥物的結合位置為分子定位(molecular docking)的區域作為篩選範圍，喜樹鹼所穩定之第一型去氧核糖核酸拓樸異構酶與DNA共價結構的三維結構已經解出(Staker,et al.2002)，容易以結合進行虛擬篩選。完成DOCK篩選後運用rigid-body docking電腦軟體依化合物與酵素的鍵結方式如凡得瓦力或靜電作用去檢視高評分(high score)的組合模式。這些較高分數的鍵結方式，即為抑制劑在配位體(ligand)上與酵素活性中心部位可能的結合方式。參考分子模擬的結果，分析新藥物在抑制第一型去氧核糖核酸拓樸異構酶與DNA共價結構的結合活性(Chang,et
al.2007)。
第1圖、說明吳茱萸鹼藉由穩定第一型去氧核糖核酸拓樸異構酶-DNA共價結構來抑制之機制。
第2圖、吳茱萸鹼抑制牛痘病毒第一型去氧核糖核酸拓樸異構酶在質體pCDNA3中鬆解DNA的作用。
第3圖、A乳癌細胞(MCF-7)中，給予吳茱萸鹼在各個時間點(0~120分鐘)，第一型去氧核糖核酸拓樸異構酶減少的情形；B乳癌細胞(MCF-7)中給予不同濃度(0~10μM)吳茱萸鹼，第一型去氧核糖核酸拓樸異構酶減少情形。
第4圖、乳癌細胞(MCF-7)中，給予吳茱萸鹼，分析第一型去氧核糖核酸拓樸異構酶與DNA複合體的結合能力。
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Claims (4)
- 一種用以抑制第一型去氧核糖核酸拓樸異構酶之組合物，其包括：吳茱萸鹼(evodiamine)、吳茱萸次鹼(rutaecarpine)和脫氫吳茱萸鹼(dehydroevodiamine)中的任一種。
- 如申請專利範圍第1項所述之組合物，其中該第一型去氧核糖核酸拓樸異構酶係來自病毒。
- 如申請專利範圍第1項所述之組合物，其中該第一型去氧核糖核酸拓樸異構酶係來自真核細胞。
- 如申請專利範圍第1項所述之組合物，其中該第一型去氧核糖核酸拓樸異構酶係來自細菌。
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| CN101829117A (zh) * | 2010-05-05 | 2010-09-15 | 中国医学科学院实验动物研究所 | 吴茱萸碱在制备治疗阿尔茨海默病的药物中的应用 |
| CN106265704A (zh) * | 2016-07-26 | 2017-01-04 | 河南大学 | 一种靶向肿瘤低氧微环境的中药小分子组合在肿瘤治疗中的应用 |
| CN110917198B (zh) * | 2019-11-15 | 2021-05-11 | 中国人民解放军总医院第五医学中心 | 去氢吴茱萸碱在制备治疗急性胃炎药物中的应用 |
| CN116650494B (zh) * | 2023-05-18 | 2024-03-12 | 中国农业大学 | 一种生物碱制剂及其在制备抗猪流行性腹泻病毒活性的制剂中的应用 |
| CN119306720B (zh) * | 2024-10-12 | 2025-07-08 | 济宁市第一人民医院(济宁市医学科学研究院) | 一种吴茱萸碱衍生物、吴茱萸次碱衍生物及其制备方法和应用 |
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| CN1195269A (zh) * | 1996-06-12 | 1998-10-07 | 协和发酵工业株式会社 | 脂质代谢改善剂 |
| US20030202937A1 (en) * | 2001-06-05 | 2003-10-30 | Sabbadini Roger A. | Minicell-based diagnostics |
| CN101005856A (zh) * | 2004-08-19 | 2007-07-25 | 皮肤应用遗传学股份有限公司 | 吴茱萸果实提取物的仿生物用于缓解炎症 |
| WO2008057253A2 (en) * | 2006-10-27 | 2008-05-15 | Bioresponse, L.L.C. | Anti-parasitic methods and compositions utilizing diindolylmethane-related indoles |
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Non-Patent Citations (2)
| Title |
|---|
| International Journal of Cancer 2004 110: (641-651)已揭示：「Inhibitory effects of evodiamine on the growth of human prostate cancer cell line LNCaP.」而可推知 「evodiamine 及rutaecarpine 具有抑制癌細胞之作用」，而不具「新穎性」，不符專利法第22條第1項第1款之規定 * |
| Phytochem Rev (2007) 6 (65-79)已揭示：「Rutaceous alkaloids as models for the design of novel antitumor drugs 」而可開發藥物方法，得到 「evodiamine之相關衍生物」，而不具「新穎性」，不符專利法第22條第1項第1款之規定 * |
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| TW201021819A (en) | 2010-06-16 |
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