嚴重急性呼吸道症候群冠狀病毒之似木瓜素蛋白水解酶之野生種與C271A突變種之結構與功能之對比分析

Comparative Structural and Functional Analysis of Wild-Type and C271A Mutant Forms of Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease

穆則鳴(David Charles Moses)

指導教授 ： 陳曜鴻

淡江大學/理學院/化學學系碩士班/碩士(2018年)

https://doi.org/10.6846/TKU.2018.00748

摘要

嚴重急性呼吸道症候群冠狀病毒（SARS-CoV）和中東呼吸系統症候群冠狀病毒（MERS-CoV）的似木瓜素蛋白水解酶（PLpro），由於其在病毒成熟過程中重要的角色，為抗病毒藥物的理想目標。最近的研究發現，酒精厭惡療法藥物disulfiram以競爭性的方式抑制SARS-CoV PLpro，但以非競爭性的方式抑制MERS-CoV PLpro，而且最近刊出的文章中的docking model顯示disulfiram從4.0Å的距離和SARS-CoV PLpro blocking loop 上的C271胺基酸互動。因為MERS-CoV PLpro同功能的位置不是Cys而是Ala，我們針對SARS-CoV PLpro 野生種與C271A突變種進行了一系列的對比性的測試以研究此抑制方式的差別是否跟此位置的胺基酸的差別有關聯。酵素動力學分析顯示，C271A突變種的disulfiram IC50值是野生種的250%，而且CD和AUC測試沒有顯示任何可以說明此IC50值的差別的二級或四級結構的差別。這些結果顯示，SARS-CoV PLpro有C271，而MERS-CoV PLpro同功能的位置有Ala，可能可以說明disulfiram針對SARS-CoV PLpro和MERS-CoV PLpro不同方式的抑制。期待晶體結構提供更可靠的證據。

關鍵字

嚴重急性呼吸道症候群 ； 似木瓜素蛋白水解酶

並列摘要

The papain-like proteases of the SARS and MERS coronaviruses (SARS-CoV and MERS-CoV PLpros) are vital to viral maturation and therefore are promising antiviral drug targets. A recent study suggests that the alcohol-aversion drug disulfiram inhibits SARS-CoV PLpro by a competitive mechanism but MERS-CoV PLpro by a non-competitive mechanism, and a recently published docking model shows blocking-loop residue C271 of SARS-CoV PLpro interacting with disulfiram at a distance of 4.0 Å. Because MERS-CoV PLpro has an alanine rather than a cysteine residue at the analogous position, we performed a series of comparative assays of wild-type and C271A mutant forms of SARS-CoV PLpro to investigate whether this difference in modes of inhibition might be related to this difference in residues at the relevant position. Steady-state kinetic analysis showed a 2.5-fold increase in IC50 value for disulfiram for the mutant in comparison to the wild-type form of the enzyme, and circular dichroism and analytical ultracentrifugation assays showed no difference in secondary or quaternary structure that would account for the difference in IC50 values. We conclude that the presence of a cysteine residue at position 271 in SARS-CoV PLpro in contrast to its absence at the analogous position in MERS-CoV PLpro may explain disulfiram’s different modes of inhibition of the two enzymes. Further confirmation of these findings awaits crystallographic evidence.

並列關鍵字

SARS ； disulfiram ； protease ； PLpro

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