Title

以結構為基礎探討冠狀病毒核殼蛋白N端功能區之二聚體交界面做為藥物開發之標的

Translated Titles

Structural basis for identification of the interface of the N-terminal domain of CoV N protein dimer as a target for drug development

Authors

許家寧

Key Words

冠狀病毒 ; 中東呼吸道症候群冠狀病毒 ; 核殼蛋白 ; N端功能區 ; coronavirus ; Middle East respiratory syndrome coronavirus ; nucleoprotein ; N terminal domain

PublicationName

中興大學生命科學系所學位論文

Volume or Term/Year and Month of Publication

2018年

Academic Degree Category

碩士

Advisor

侯明宏

Content Language

繁體中文

Chinese Abstract

西元 1973 年冠狀病毒首次被分離出來,這類病毒會引起人類和脊椎動物的呼吸道或腸胃道疾病,更嚴重者會造成死亡。西元 2012 年,中東呼吸道症候群冠狀病毒 (Middle East respiratory syndrome coronavirus, MERS-CoV) 在沙烏地阿拉伯首次被發現,其致死率高達 35%,之後在南韓也爆發大規模感染,直至今日仍有病例。目前對付冠狀病毒並沒有有效的抗病毒藥物,也沒有疫苗,因此了解冠狀病毒的感染途徑,並開發抗病毒藥物成了各國間的熱門議題。冠狀病毒屬於正向單股的 RNA 病毒,RNA 病毒的變異性相當高,容易產生抗藥性,但在冠狀病毒的生命週期中會表現出特定的結構性蛋白,而這些蛋白序列具有高保留性的,其中核殼蛋白 (nucleocapsid protein, NP) 在病毒複製過程中,會與病毒的 RNA 結合形成長鏈螺旋狀的核糖核蛋白複合體 (ribonucleoprotein complex, RNP),對於病毒的轉錄與複製扮演重要的角色。本篇研究針對 MERS-CoV 核殼蛋白中,會與 RNA 結合的 N 端功能區做為藥物設計之標的,在本實驗室先前解析出的 MERS-CoV 核殼蛋白 N 端功能區 (MERS-CoV N-NTD) 中,發現其二聚體之交接面有一個有個重要位點 (W43),可作為藥物設計之結合位。首先從 ZINC 資料庫中篩選與先前使用的 P3 小分子配體之類似物,稱為 P3 系列小分子配體,並針對此位點進行電腦模擬分子對接 (molecular docking) 實驗,共找出兩個有潛力的小分子化合物,在分析其複合體之結晶結構後發現,P3-1 複合體在二聚體交接面多了一個在先前的結構中未發現之融合肽 (M38),這使得藥物無法深入至配體結合位;在 P3-2 複合體中雖然沒有發現 M38,與 W43 也沒有交互作用力,但在螢光光譜實驗中可以看到 P3-2 和 MERS-CoV 核殼蛋白及 MERS-CoV N-NTD 均有些微的交互作用,經小角度 X 光散射 (Small-angle X-ray scattering, SAXS) 實驗分析後,發現 P3-2 複合體之 Rg 值與對照組相比增加了 5.59 Å,但整體而言使 MERS-CoV 核殼蛋白聚合之效果沒有 P3 理想。之後又以保留 P3 配體中的吲哚啉 (indoline),找了四個有潛力的小分子化合物稱為 P4 系列小分子配體,在解析晶體結構後發現,二聚體交接面均多了融合肽 (M38),但在分析其作用力時發現,P4-2 小分子配體跟 W43 有一個 π-π 作用力,和 T40 有氫鍵作用力,這使的 P4-2 相較其他配體更深入到二聚體之交接面,以穩定 MERS-CoV N-NTD 二聚體的結構,經 SAXS 分析其 Rg 值與對照組相比增加了 7.15 Å,證實了 P4-2 具有促進 MERS-CoV 核殼蛋白聚合的能力,雖然 W43 非 RNA 結合位點,但我們認為 P3-2 及 P4-2 是藉由異位調控的方式與二聚體交界面結合,以影響 MERS-CoV 核殼蛋白的功能,並促進其寡聚化 (oligomerization)。本篇藉由深入了解影響 HCoV 中 N-NTD 二聚體間的交互作用,提供一個新的篩選抗病毒藥物之方法。

English Abstract

In 1973, the coronavirus was isolated for the first time. The coronavirus are known to cause the respiratory and gastrointestinal diseases in humans and vertebrates. The more severe infections could cause the death. In 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) was discovered in Saudi Arabia, with a mortality rate of 35% followed by the number of infections in South Korea too. Currently, there are no effective therapeutic as well as prophylactic treatments available against the coronavirus. So understanding the pathways of infection of coronaviruses and developing antiviral drugs has become a hot topic among countries.   Coronavirus is a positive single-stranded RNA virus with high variability and can easily become drug resistant. However, specific structural proteins are expressed in the life cycle of the coronavirus, and these protein sequences are highly conserved. During replication, the nucleocapsid protein (NP) binds to the RNA genome to form a long chain helical ribonucleoprotein complex (RNP). It has been found that the RNP complex plays an important role in the transcription and reproduction of virus life cycle. In the current study, we focus on utilizing the MERS-CoV N-NTD as a target for anti-viral drug development. Initially, the structure-based molecular docking using the ZINC database was performed to identify two P3 derivative compounds namely P3-1 and P3-2 respectively. To understand the possible mechanism of binding of these compounds to the N-NTD, we solve the crystal structure of these compounds with N-NTD protein. The previous studies has shown that the tryptophan 43 (W43) is an important residue at the ligand binding site. In the P3-1 complex with N-NTD, we found that M38 prior to N39 occupies the ligand binding pocket which makes the drug unable to penetrate deeply into the ligand binding site. Although in the P3-2 complex M38 was not found to interfere at binding site, however, there was no interaction of P3-2 with the W43.   In the fluorescence spectra measurement, the intrinsic fluorescence of N protein and N-NTD showed the light shift in the presence of the compound P3-2. In addition, the P3-2 can also enhance the oligomerization of N protein by small angle X-ray scattering (SAXS) assay. The Rg value was increased by 5.59 Å compared to that of the control, but the effect of MERS-CoV NP polymerization was not ideal to that of the original P3 compounds. Thus to retain the P3 ligand in the indoline, we then identify four new derivatives of P3 called as P4 series of small molecules. The crystal structures of P4 series of compounds with N-NTD showed that the M38 occupied the ligand binding pocket in all structures. However, the P4-2 complex showed the π-π interaction with W43, and hydrogen bond with T40, which made P4-2 deeper than dimer interface in the N-NTD to stabilize the structure of the MERS-CoV N-NTD dimer. In addition, the P4-2 compound can also enhance the oligomerization of N protein by small angle scattering assay. The Rg value was increased by 7.15 Å compared with the control which confirms that the P4-2 has the oligomerization properties of MERS-CoV N protein. Although the W43 non-RNA binding site, we consider that P3-2 and P4-2 by allosteric modulation of the ligand binding site to influence the function of MERS-CoV NP and its oligomerization properties. We provide a new method for screening antiviral drugs by dimer interface of HCoV N-NTD

Topic Category 生命科學院 > 生命科學系所
生物農學 > 生物科學
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