建構登革NS2B/NS3蛋白酶計算模型及應用在舊藥新用

Abstract

登革熱為熱帶與亞熱帶地區普遍流行的疾病，每年影響人口超過五千萬，其成因為黃病毒科 (Flaviviridae) 中黃病毒屬 (Flavivirus) 的單股正向核糖核酸登革病毒 (Dengue virus, DENV) 由斑蚊叮咬而傳播，目前尚無有效治療的藥物或疫苗上市。研究報告指出，抑制登革NS2B/NS3 蛋白酶可降低80%的感染力。因此，發展登革NS2B/NS3 蛋白酶抑制劑將是很好的抗登革策略，尤其是針對已批准上市的藥物進行篩選，利用篩選已核准藥物資料庫的方式來加速提供有潛力舊藥新用的藥物給病人。 本研究我們透過實驗室所開發的空間藥理模板尋找與登革NS2B/NS3蛋白酶具相似藥物作用環境的蛋白群，以探討登革NS2B/NS3蛋白酶抑制劑作用機制與可能造成的副作用。發現人類凝血酶與登革NS2B/NS3蛋白酶具有相似的藥理環境，意味著登革NS2B/NS3蛋白酶抑制劑極可能同時抑制人類凝血酶。然而，抑制人類凝血酶會加重登革熱的出血症狀。因此，發展選擇性登革NS2B/NS3蛋白酶抑制劑將是此類抗登革策略成功的關鍵。為了達成此目標，我們運用實驗室獨立發展且在藥物設計上成果豐碩的蛋白結合位點-化合物官能基輿圖 (SiMMap) ，透過此軟體分別針對登革NS2B/NS3蛋白酶與人類凝血酶進行分析，探索登革NS2B/NS3蛋白酶、人類凝血酶與配體 (ligand) 交互作用介面的官能基偏好以及其物理化學特性之間的關係。最後我們從上市藥物中篩選出抗生素—阿米卡星 (Amikacin)等藥物為選擇性登革NS2B/NS3蛋白酶抑制劑。阿米卡星可與登革NS2B/NS3蛋白酶的H51、F130、G133、S135、G151、N152、G153、Y161形成8個氫鍵；其上的芳香環和其他官能基可與登革NS2B/NS3蛋白酶的H51、K131、P132、Y161形成凡得瓦爾力。涵蓋大部分抑制劑與蛋白酶催化的交互作用；反觀凝血酶處，阿米卡星只能與H57、S195、S214、G216形成4個氫鍵﹔其上的芳香環和其他官能基與凝血酶的H57、Y99、C191、Q192、W215、G216形成凡得瓦爾力，其作用力遠低於登革NS2B/NS3蛋白酶。未來，我們將對篩選出的藥物進行溶菌斑 (Plaque assay) 等實驗，以評估其作為抗登革治療策略的潛力。

Dengue virus (DENV), a RNA viruses of the family Flaviviridae, genus Flavivirus are mosquito-borne and single-stranded, positive-sense viruses, remains to be a hazardous infectious epidemic with no effective vaccine or antiviral drugs. Some studies showed that inhibition of DENV NS2B/NS3 protease decreases DENV infection by 80%. Therefore, development of dengue NS2B / NS3 protease potential inhibitor and may be a good strategy to anti-dengue, especially to accelerate promising repurposed drugs to patients by screening approved drug dataset. In this research, we propose a new concept of " Space-related pharma-motifs", to look for protein groups which exhibited similar drug working environment with dengue NS2B/NS3 protease. In this way, we could further explore the mechanism and possible side effect of dengue NS2B/NS3 protease inhibitors. We find that human thrombin exhibits similar binding environment and physicochemical properties with dengue NS2B/NS3 protease. Therefore, dengue NS2B/NS3 protease inhibitors may inhibit human thrombin activity in the meantime. However, inhibition of human thrombin activity will increase the bleeding of dengue fever. Therefore, the development of selective dengue NS2B / NS3 protease inhibitors will be key factor to success in this anti-dengue virus strategy. To achieve this goal, we use a distinctive method, protein binding sites and compound functional group map (SiMMap), which is developed by our laboratory alone and exhibits brilliant performance in drug design. We use SiMMap software to analyze human thrombin and dengue NS2B/NS3 protease, to explore the functional groups bias of interaction of ligands to human thrombin or dengue NS2B/NS3 protease and the physical and chemical properties. Finally, we identify an antibiotic drug, amikacin, from the market as selective dengue NS2B/NS3 protease inhibitor agent. Amikacin could form hydrogen bond with dengue NS2B/NS3 protease at H51、 F130、G133、 S135、G151、N152、G153、 Y161. Aromatic rings and other functional groups of amikacin could from van der Waals force with dengue NS2B / NS3 protease at H51, K131, P132, Y161. These bindings covering most of the protease inhibitors and catalytic interactions; at the other hand, in human thrombin, amikacin only form four hydrogen bonds at H57, S195, S214, G216; aromatic rings and other functional groups bind with thrombin at H57, Y99, C191, Q192, W215, G216 formation of van der Waals force. It's worth noting that the binding affinity of amikacin to thrombin is far lower than that of dengue NS2B/NS3 protease. Moreover, we will test the proposed drugs through plaque assay to determine the efficacy for inhibiting DENV replication.