病毒3C及3C-Like蛋白酶的鑑定、抑制和改造

嚴重急性呼吸道症候群(Severe Acute Respiratory Syndrome)是由新型人類冠狀病毒 (SARS-CoV) 感染所造成。此疾病在2002~2003年爆發，造成全球性的感染與恐慌。根據世界衛生組織(WHO)指出，這段期間內各國總共有8,465通報病例，而其中有801死亡病例。SARS病毒在宿主體內之成熟需藉由一主要蛋白酶(3CLpro)，其負責的工作為切剪由病毒基因轉錄而產生的蛋白鏈，進而產生病毒內部各個重要且具功能的蛋白質，讓病毒得以感染與複製。在人類小RNA病毒(picornavirus，簡稱PV)的感染機制中，也需要一個類似的蛋白酶(3Cpro)來專職上述功能。因此，3CL與3C蛋白酶遂成為研究抗CoV與PV藥物的重要標靶。然而，目前已知的鼻病毒(rhinovirus，亦屬PV的成員之一)抑制劑，AG7088，並無法有效的抑制SARS 3CLpro活性，這顯示此兩種蛋白酶在結構上具有某程度的差異性。本篇論文利用大腸桿菌表現SARS 3CL蛋白酶，並設計螢光胜肽受質來偵測酵素活性與催化特性，以及篩選抑制劑。配合上蛋白酶之蛋白質晶體結構，我們推論出3CL蛋白質二聚體之成熟(maturation)模式。在藥物篩選方面，我們成功的篩選出多種形式的抑制劑，並透過蛋白質共結晶技術與電腦分子模擬，深入探討這些抑制劑與酵素之間的結合模式，且進一步的將其修飾與改良。在另一方面,我們也針對抗PV藥物進行研究，包括腸病毒(enterovirus)71型與柯薩奇病毒(coxsackievirus)B3型。我們篩選出數個化合物能有效的抑制酵素活性，並證明其可以有效的保護細胞抵抗病毒的感染。此外，我們從6800個小分子中，利用高效能藥物篩選方法來篩選SARS 3CLpro的抑制劑，在篩選出的五個化合物之中，有一個化合物與其類似物可以同時抑制CoV與PV的蛋白酶。接著，我們利用電腦分子模擬運算，釐清出這些抑制劑與此兩類蛋白酶在結合模式上的差異。另一方面，我們也探討SARS 3CL蛋白酶結構中，調控受質P1’位置專一性之重要胺基酸(Threonine)，將其突變成甘胺酸(Glycine)後，可將原本受質P1’位置上的可供辨識的絲胺酸(Serine)置換成甲硫胺酸(methionine)。這個改造過的SARS T25G 3CL蛋白酶，可將利用大腸桿菌與酵母菌所表現出來的融合蛋白(fusion protein)上的標誌蛋白(tag)，作有效率切除，頗具商業應用價值。

關鍵字

嚴重急性呼吸道症候群；腸病毒；柯薩奇病毒；冠狀病毒；小RNA病毒；抑制劑；蛋白酶

並列摘要

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus (CoV), which infected more than 8000 people during the 2003 outbreak. The viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. Accordingly, in human picornavirus (PV) family which consists of over 200 medically relevant viruses, a chymotrypsin-like protease (3Cpro) is required for viral replication through the processing the polyproteins. As a result, the 3CLpro and 3Cpro are regarded as anti-viral drug targets. However, known inhibitors (AG7088) against PV 3Cpro failed to inhibit SARS-CoV 3CLpro, indicating differences in their active-site structures. In this study, we have prepared the recombinant SARS 3CLpro without redundant residues at both N- and C-termini and characterized its kinetic property using a fluorogenic substrate. Combined with our crystallography data, we proposed a model to illustrate the maturation process of SARS 3CLpro. In addition, we evaluated several types of inhibitors and investigated their inhibitory mechanisms. Additionally, the recombinant 3Cpro from PV (including entervirus, coxsackievirus, and rhinovirus, abbreviated as EV, CV, and RV, respectively) were purified and characterized as well. We have identified several inhibitors which show their potencies against viral replication. Furthermore, we tested 6800 small molecules by high-throughput screening for anti-SARS agents and found one hit and its analogues could serve as the common inhibitors against CoV 3CLpro and PV 3Cpro. By computer modeling, the structural features of these compounds were elucidated to enhance our knowledge for developing anti-viral agents against PV and CoV. In order to determine the amino acid residues essential for the substrate specificity and engineer 3CLpro as a tool for tag removal of the recombinant fusion proteins, we developed a mutant 3CLpro (T25G) which has altered substrate specificity to cleave Gln↓Met. We have also constructed E. coli and yeast vectors to express recombinant fusion proteins with the T25G 3CLpro recognition site (Ala-Val-Leu-Gln↓Met) between the tags and the target proteins for tag removal.

並列關鍵字

SARS ； enterovirus ； coxsackievirus ； coronavirus ； picornavirus ； inhibitor ； protease

參考文獻

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病毒3C及3C-Like蛋白酶的鑑定、抑制和改造

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