臺灣博碩士論文加值系統

專一性泛素水解酶 (Ubiquitin-specific proteases) 為去泛素化酶 (Deubiquitinase) 分類中最大的家族，它們具有相似的催化domain結構和半胱氨酸蛋白酶的活性，研究發現專一性泛素水解酶在許多癌症中都被高度調控或大量的被表現，衍然為抑制癌症的重要標靶，然而截至目前，尚未有抑制專一性泛素水解酶的藥物能通過臨床檢驗。在人類專一性泛素水解酶的家族中，人類第二型與第二十一型專一性泛素水解酶各自在許多癌症中被高度調控，另一方面，它們在演化上被分類在很小的群集且擁有極度相似的catalytic domain結構，先前的研究指出在難以被診斷且惡性的三陰性乳癌 (Triple negative breast cancer) 細胞中，沉默人類第二型與第二十一型專一性泛素水解酶的基因可以抑制腫瘤細胞的擴散，因此，開發抑制人類第二型與第二十一型專一性泛素水解酶的藥物能得利於它們的結構相似性，並用於治療與兩種水解酶皆相關的癌症。在本篇研究中，我們第一個發現臨床的戒酒用藥，戒酒硫(disulfiram)，對於人類第二型與第二十一型專一性泛素水解酶皆具有競爭型的抑制效果，並標示了戒酒硫在兩個專一性泛素水解酶的catalytic domain上可能的結合位，而在戒酒硫藥物與白血病用藥6-硫代鳥嘌呤(6TG)對於人類第二型與第二十一型專一性泛素水解酶的雙抑制實驗中，兩個藥物對於兩個專一性泛素水解酶皆具有協同抑制作用，顯示出針對與兩個專一性泛素水解酶皆相關的癌症，可以合併使用兩個藥物作為治療方式，另外，戒酒硫與6-硫代鳥嘌呤對於人類第二型與第二十一型專一性泛素水解酶皆具有不可逆的抑制效果。綜合以上，我們第一個發現戒酒硫藥物能作為專一性泛素水解酶的抑制劑，亦提供了證據以利臨床使用戒酒硫與6-硫代鳥嘌呤來治療人類第二型與第二十一型專一性泛素水解酶皆被大量表現的癌症作為參考。

Ubiquitin-specific proteases (USPs) are the largest subfamily of deubiquitinases (DUBs), they share a structurally similar catalytic domain and are mostly with cysteine protease activity. USPs have emerged to be attractive anticancer targets as many USPs were found upregulated or overexpressed in a variety of tumors, however, no inhibitors targeting USPs have entered clinical trial so far. Among the USP family, USP2 and USP21 were respectively found upregulated in various cancers. On the other hand, they were phylogenetically categorized in a small cluster and display extremely high similarity in the catalytic domain protein structure. Previous studies have shown silencing either USP2 or USP21 in triple negative breast cancer (TNBC), an aggressive subtype of breast cancer with poor prognosis, inhibits tumor metastasis. Thus, discovering drugs targeting both USPs gains advantage from their structure similarity and serve as a desirable therapeutic strategy to treat cancers correlated to both USPs. In this study, we first reported disulfiram, a clinically available alcohol-aversive drug, is a competitive inhibitor for both USP2 and USP21, and indicated the potential binding sites within the USP catalytic domain. Synergistic inhibition of USP2 and USP21 by disulfiram and 6-thioguanine (6TG), a clinically available leukemia drug, suggested the potential of using these two clinically available drugs for combination treatment on diseases correlated to both USPs. Additionally, both drugs exhibit slow-binding inhibition and irreversibility of enzyme activity on USP2 and USP21. Taken together, our findings demonstrate disulfiram is an inhibitor for USPs and provide evidence for clinical evaluation of using disulfiram with 6TG as combination treatment on both USP2 and USP21 upregulated cancers.

Table of Contents
Acknowledgement i
Table of Contents ii
List of Figures iv
List of Tables v
List of abbreviations vi
Chinese Abstract vii
Abstract viii
Chapter 1 – Introduction 1
1.1 Ubiquitination 1
1.2 Deubiquitinating enzymes, DUBs 1
1.3 Ubiquitin specific proteases, USP 2
1.4 Structure correlation between USP2 and USP21 2
1.5 Roles of USP2 and USP21 in cancers 3
1.6 Old drugs as USP2 and USP21 inhibitors 4
1.7 Study aims 5
Chapter 2 - Materials and methods 6
2.1 Chemicals, enzymes, and reagents 6
2.2 Protein preparation 8
2.2.1 Construction of USP2 and USP21 expression vectors 8
2.2.2 Site-Directed mutagenesis 8
2.2.3 Expression and purification of human USP2 and USP21 9
2.3 Functional and inhibition studies 10
2.3.1 Deubiquitination (DUB) assay 10
2.3.2 Steady-state kinetic analysis 10
2.3.3 Multiple inhibition assay 11
2.3.4 Inactivation mechanism 12
2.3.5 Thermostability assay 12
Chapter 3 - Results and discussion 13
3.1 Production of recombinant human USP2 and USP21 13
3.2 DUB activity of USP2 and USP21 14
3.3 Inhibitory effect of 6MP and MPA on USP21 14
3.4 The inhibition of USP2 and USP21 by disulfiram 15
3.5 6TG inhibits USP2 and USP21 through different binding sites 16
3.6 Disulfiram and 6TG can synergistically inhibit USP2 and USP21 17
3.7 Time-dependent inhibition of USP2 and USP21 by disulfiram 18
3.8 Disulfiram is a potential broad-spectrum inhibitor for USP family 19
3.9 Disulfiram targeting USPs as potential treatment on cancers 20
References 22
Figures 27
Tables 46
List of Figures
FIGURE 1. Production of recombinant USP2. 27
FIGURE 2. Production of recombinant USP21. 28
FIGURE 3. Production of recombinant USP21 co-expressed with chaperone 5. 29
FIGURE 4. The DUB activity of USP2 and USP21. 30
FIGURE 5. The structure of USP2 and USP21 inhibitors. 31
FIGURE 6. Inhibitory effect of disulfiram on USP2 and USP21. 32
FIGURE 7. Structure alignment of USP2 and USP21. 33
FIGURE 8. Production of recombinant USP21 C398S. 34
FIGURE 9. Inhibitory effect of disulfiram on USP21 C398S. 35
FIGURE 10. Inhibition of disulfiram on USP2. 36
FIGURE 11. Inhibition of disulfiram on USP21. 37
FIGURE 12. Inhibitory effect of 6TG on USP2 and USP21. 38
FIGURE 13. Inhibition of 6TG on USP21. 39
FIGURE 14. Synergistic effect of disulfiram and 6TG on USP2 and USP21. 40
FIGURE 15. Secondary structure change of USP2 and USP21. 41
FIGURE 16. The recoverability of DUB activity by βME. 42
FIGURE 17. Slow-binding inhibition of disulfiram on USP2 and USP21. 43
FIGURE 18. Time-dependent inactivation of USP2 and USP21 by disulfiram. 44
FIGURE 19. Time-dependent inactivation of USP21 by 6TG. 45
List of Tables
TABLE 1. IC50 comparison of disulfiram, 6TG, and combined inhibition on USP2 and USP21 46
TABLE 2. Kinetic parameters of disulfiram and 6TG inhibition of USP2 and USP21 47

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