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。

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.

Table of Contents
1   Introduction	1
1.1  SARS and MERS	1
1.2  Rationale for continued anti-SARS drug discovery efforts	2
1.3  The search for commercially available drugs to use against the SARS and MERS coronaviruses	3
1.4  General characteristics of coronaviruses	4
1.5  Life cycle of SARS-CoV and role of SARS-CoV PLpro	5
1.6  Possible targets of anti-SARS and anti-MERS drugs	6
1.7  Structure and mechanism of SARS-CoV PLpro	7
1.8  Disulfiram as an anti-SARS-CoV drug	9
1.9  Disulfiram and BL2 blocking loop residue C271	10
1.10  Motivation of the present study	11
2   Materials	13
2.1  Chemical reagents	13
2.2  Instruments	13
2.3  Buffers used in production of competent cells	14
2.4  Buffers used in protein purification	14
2.5  Solutions used for SDS-PAGE	16
2.6  Buffer used for agarose gel electrophoresis	16
2.7  Buffer used for protein activity assays	16
3   Methods	17
3.1  Culturing broths and plates	17
3.2  Competent cell preparation	17
3.3  Transformation	18
3.4  Plasmid preparation and purification	19
3.5  Agarose gel electrophoresis	20
3.6  Recombinant protein production and purification	20
3.6.1  Recombinant protein production	20
3.6.2  Lysis and resuspension	21
3.6.3  Nickel ion affinity chromatography	21
3.6.4  Gel-filtration chromatography	22
3.6.5  Bradford assay of protein concentration	23
3.6.6  SDS-PAGE	23
3.7  Steady-state kinetic analysis	24
3.8  Inhibition assays	25
3.9  Circular dichroism (CD) assays of secondary structure	26
3.10  Circular dichroism (CD) assays of thermostability	26
3.11  Analytical ultracentrifugation (AUC) assays of quaternary structure	26
3.12  Crystallization screening	27
4   Results and Discussion	28
4.1  Protein production and purification	28
4.2  Steady-state kinetic analysis	29
4.3  Inhibition assay	32
4.4  Crystallization screening	33
4.5  Circular dichroism (CD) assay of secondary structure	34
4.6  Circular dichroism (CD) assay of thermal stability	36
4.7  Analytical ultracentrifugation (AUC) assay of quaternary structure	38
5   Conclusion	40
6   References	43
7   Appendices	47
7.1  Appendix I: Flow chart summarizing logic of experiments	47
7.2  Appendix II: Steady-state kinetic assays	48
7.3  Appendix III: Inhibition assays	49
7.4  Appendix IV: DICHROWEB results	50

List of Figures
Fig. 1: SDS-PAGE gel (left panel) and 280 nm UV spectrum (right panel) from protein purification process	28
Fig. 2: Saturation curves for SARS-CoV PLpro WT and C271A mutant forms in relation to Dabcyl–FRLKGGAPIKGV–EDANS peptidyl substrate. 	31
Fig. 3: Inhibition by disulfiram of SARS-CoV PLpro WT and C271A. 	33
Fig. 4: CD spectra of SARS-CoV PLpro WT and C271A mutant forms	35
Fig. 5: Thermal stability of SARS-CoV PLpro C271A with and without disulfiram	36
Fig. 6: SEDFIT analysis of AUC data  	38

List of Tables
  Table 1: Enzymatic parameters of SARS-CoV PLpro WT and C271A mutant forms	31
  Table 2: IC50 values for inhibition by disulfiram of SARS-CoV PLpro WT and C271A mutant forms	33
  Table 3: Secondary structure information for SARS-CoV PLpro WT and C271A forms obtained by analysis of circular dichroism data on DICHROWEB website	36
  Table 4: TM (“melting temperature”) values of SARS-CoV PLpro WT and C271A forms obtained by circular dichroism	36
  Table 5: Quaternary structure of SARS-CoV PLpro WT and C271A from SEDFIT analysis of AUC data	39

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