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研究生:王信杰
研究生(外文):Wang, Hsin-Chieh
論文名稱:人類組織蛋白酶S其新穎抑制劑篩選及鑑定
論文名稱(外文):Screening and Identification of Novel Human Cathepsin S Inhibitors
指導教授:張大慈
指導教授(外文):Chang, Margaret Dah-Tsyr
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:101
中文關鍵詞:組織蛋白酶S抑制劑癌症侵襲轉移
外文關鍵詞:CTSSinhibitorcancerinvasionmetastasis
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人類半胱胺酸組織蛋白酶S (cathepsin S, CTSS)為位於溶小體(lysosome)中的蛋白質分解酵素,其分子量為24 kD、無醣基化位點(non-glycosyated),於中性pH值相當穩定且具有高度活性。組織蛋白酶S主要功能包括細胞內恆定鏈處理(intracellular invariantchain processing) 、抗原呈現(antigen presentation)相關及降解細胞外基質 (extracellular matrix degradation)。目前研究顯示在某些癌症或具有高度侵襲(invasion)及轉移(metastasis)能力的腫瘤(tumor)細胞中,組織蛋白酶S的表現量大幅提高,因此CTSS其功能與癌症的侵襲與轉移關係密切。許多藥廠已積極發展組織蛋白酶S的抑制劑(inhibitor),其用途包括治療自主免疫疾病如類風濕症狀關節炎(rheumatoid arthritis)、多發性硬化症(multiple sclerosis)、哮喘(asthma)和重症肌無力(myasthenia gravis)等,也可發展成新型的抗癌藥物以應用於治療具有高度侵襲和轉移能力的腫瘤。
本論文以人類組織蛋白酶S為抗癌的分子標靶(molecular target),與清華大學化學系合作取得許多新合成的小分子化合物以篩選具能降低組織蛋白酶S活性的新穎抑制劑。利用大腸桿菌蛋白質表現系統以取得大量的組織蛋白酶S,經純化及酸催化水解產生具有活性的重組組織蛋白酶S,再利用釋放螢光的受質及96孔盤快速篩選有效的抑制劑。本研究結果顯示將化合物的彈頭(warhead)置換成α-酮醯胺胜肽(α-ketoamide)之化合物都具有相當好的抑制效果,已發現62個化合物之IC50 數值均約為10 nM等級。此外,從Lineweaver-Burk雙倒數圖分析顯示此類抑制劑都屬於競爭型抑制劑(competitive inhibitor)。由國家衛生研究院癌症研究所的抗癌細胞轉移實驗中發現,大部分抑制劑都能抑制具高度轉移能力的人類肺腺癌細胞株(CL1-5)及人類內皮細胞(HUVECs cells),顯示α-酮醯胺胜肽抑制劑可應用於治療具有高度轉移能力的癌症,因此具備發展成新穎藥物的潛力。
Human cathepsin S (CTSS) is a lysosomal cysteine protease of the papain-like superfamily. It is a single chain, non-glycosylated protease with a molecular weight of 24 kDa and is highly active and stable at neutral pH. Like most papain-like cysteine proteases, CTSS is synthesized as an inactive zymogen, procathepsin S, and is converted to the mature form by limited proteolysis at acidic pH by other proteases, or by autocatalytic processing. The possible function of human CTSS has been identified and involved in antigen presentation with intracellular invariant chain processing and extracellular matrix degradation. Increased expression of CTSS mRNA and protein has been observed in tumor cells with high metastasis potential. Hence CTSS is considered as a novel molecular target for prevention/reduction of tumor metastasis. The aim of the present study is to evaluate and discovery of novel CTSS inhibitors. A microplate-based screening procedure was used to study the inhibitory effect of CTSS on several compounds. Recombinant human CTSS was produced by E. coli expression system followed by purification and activation. Functional characterization of such recombinant mature CTSS was carried out by spectroscopic determination of enzymatic activity employing fluorescent substrate (Z-Val-Val-Arg-AMC) and a rapid screening. Initial screening of 819 mixture compounds revealed 45 potential targets. Subsequently 121 pure compounds were synthesized and the IC50 values were determined. It appeared that the warhead modification of these CTSS inhibitor analogs increased inhibitory ability such that an IC50 value of 10 nM was achieved. The Lineweaver-Burk plot indicated that these novel synthetic inhibitors belonged to competitive inhibition type. Till now 940 synthetic small molecules have been screened and IC50 values of the top 62 potent inhibitors were found to be lower than or close to the known CTSS inhibitor. Many of these inhibitors showed great inhibitory ability of cell migration in both CL1-5 and HUVECs cells. These results indicate that α-ketoamide-based CTSS inhibitors may be employed to treat metastatic malignancies in human cancers, at least in part, by inhibiting specific molecular target, CTSS, in terms of tumor and endothelial cell migration. Our results provide important fundamental understanding for further in silico and in vivo CTSS inhibitor design.
Acknowledgement I
中文摘要 II
Abstract III
Table of Contents V
List of Tables VII
List of Figures VIII
Abbreviations IX

Chapter 1 Introduction 1
Chapter 2 Materials and Methods 9
Chapter 3 Results 22
Chapter 4 Discussion 35
References 42
Tables 49
Figures 72
Appendix 93
1. Turk, B., Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov, 2006. 5(9): p. 785-99.
2. Lopez-Otin, C. and C.M. Overall, Protease degradomics: a new challenge for proteomics. Nature Rev. Mol. Cell Biol., 2002. 3: p. 509-519.
3. Turk, B., D. Turk, and V. Turk, Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta, 2000. 1477(1-2): p. 98-111.
4. Fisher, A., Mechanism of the proteolytic activity of malignant tissue cells. Nature, 1946. 157: p. 442.
5. Lopez-Otin, C. and L.M. Matrisian, Emerging roles of proteases in tumour suppression. Nat Rev Cancer, 2007. 7(10): p. 800-8.
6. Steeg, P.S., Tumor metastasis: mechanistic insights and clinical challenges. Nat Med, 2006. 12(8): p. 895-904.
7. Egeblad, M. and Z. Werb, New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer, 2002. 2(3): p. 161-74.
8. Borgono, C.A. and E.P. Diamandis, The emerging roles of human tissue kallikreins in cancer. Nat Rev Cancer, 2004. 4(11): p. 876-90.
9. Mohamed, M.M. and B.F. Sloane, Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer, 2006. 6(10): p. 764-75.
10. Gocheva, V. and J.A. Joyce, Cysteine cathepsins and the cutting edge of cancer invasion. Cell Cycle, 2007. 6(1): p. 60-4.
11. Gupta, G.P. and J. Massague, Cancer metastasis: building a framework. Cell, 2006. 127(4): p. 679-95.
12. Rawlings, N.D., F.R. Morton, and A.J. Barrett, MEROPS: the peptidase database. Nucleic Acids Res, 2006. 34(Database issue): p. D270-2.
13. Hooper, N.M., Proteases: a primer. Essays Biochem, 2002. 38: p. 1-8.
14. Shaw, E., Cysteinyl proteinases and their selective inactivation. Adv Enzymol Relat Areas Mol Biol, 1990. 63: p. 271-347.
15. Kamphuis, I.G., J. Drenth, and E.N. Baker, Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. J Mol Biol, 1985. 182(2): p. 317-29.
16. Turk, V., B. Turk, and D. Turk, Lysosomal cysteine proteases: facts and opportunities. Embo J, 2001. 20(17): p. 4629-33.
17. Turk, D. and G. Guncar, Lysosomal cysteine proteases (cathepsins): promising drug targets. Acta Crystallogr D Biol Crystallogr, 2003. 59(Pt 2): p. 203-13.
18. Wun-Shaing W. Chang, et al., Lysosomal Cysteine Proteinase Cathepsin S as a Potential Target for Anti-Cancer Therapy. J. Cancer Mol. , 2007. 3(1): p. 5-14.
19. Palermo, C. and J.A. Joyce, Cysteine cathepsin proteases as pharmacological targets in cancer. Trends Pharmacol Sci, 2008. 29(1): p. 22-8.
20. Finley, E.M. and S. Kornfeld, Subcellular localization and targeting of cathepsin E. J Biol Chem, 1994. 269(49): p. 31259-66.
21. Avril, L.E., et al., Identification of the U-937 membrane-associated proteinase interacting with the V3 loop of HIV-1 gp120 as cathepsin G. FEBS Lett, 1994. 345(1): p. 81-6.
22. Grimm, J., et al., Use of gene expression profiling to direct in vivo molecular imaging of lung cancer. Proc Natl Acad Sci U S A, 2005. 102(40): p. 14404-9.
23. Joyce, J.A. and D. Hanahan, Multiple roles for cysteine cathepsins in cancer. Cell Cycle, 2004. 3(12): p. 1516-619.
24. Joyce, J.A., et al., Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell, 2004. 5(5): p. 443-53.
25. Jedeszko, C. and B.F. Sloane, Cysteine cathepsins in human cancer. Biol Chem, 2004. 385(11): p. 1017-27.
26. Berdowska, I., Cysteine proteases as disease markers. Clin Chim Acta, 2004. 342(1-2): p. 41-69.
27. Rao, J.S., Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer, 2003. 3(7): p. 489-501.
28. Caglic, D., et al., Glycosaminoglycans facilitate procathepsin B activation through disruption of propeptide-mature enzyme interactions. J Biol Chem, 2007. 282(45): p. 33076-85.
29. Vasiljeva, O., et al., Recombinant human procathepsin S is capable of autocatalytic processing at neutral pH in the presence of glycosaminoglycans. FEBS Lett, 2005. 579(5): p. 1285-90.
30. Kihara, M., et al., Chondroitin sulfate proteoglycan is a potent enhancer in the processing of procathepsin L. Biol Chem, 2002. 383(12): p. 1925-9.
31. Ishidoh, K. and E. Kominami, Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro. Biochem Biophys Res Commun, 1995. 217(2): p. 624-31.
32. Fairhead, M., S.M. Kelly, and C.F. van der Walle, A heparin binding motif on the pro-domain of human procathepsin L mediates zymogen destabilization and activation. Biochem Biophys Res Commun, 2008. 366(3): p. 862-7.
33. Nascimento, F.D., et al., Cathepsin X binds to cell surface heparan sulfate proteoglycans. Arch Biochem Biophys, 2005. 436(2): p. 323-32.
34. Almeida, P.C., et al., Cathepsin B activity regulation. Heparin-like glycosaminogylcans protect human cathepsin B from alkaline pH-induced inactivation. J Biol Chem, 2001. 276(2): p. 944-51.
35. Taleb, S., et al., Cathepsin s promotes human preadipocyte differentiation: possible involvement of fibronectin degradation. Endocrinology, 2006. 147(10): p. 4950-9.
36. Buck, M.R., et al., Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues. Biochem J, 1992. 282 ( Pt 1): p. 273-8.
37. Gocheva, V., et al., Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev, 2006. 20(5): p. 543-56.
38. Guo, M., et al., Phorbol ester activation of a proteolytic cascade capable of activating latent transforming growth factor-betaL a process initiated by the exocytosis of cathepsin B. J Biol Chem, 2002. 277(17): p. 14829-37.
39. Kobayashi, H., et al., Effects of membrane-associated cathepsin B on the activation of receptor-bound prourokinase and subsequent invasion of reconstituted basement membranes. Biochim Biophys Acta, 1993. 1178(1): p. 55-62.
40. Cardone, R.A., V. Casavola, and S.J. Reshkin, The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer, 2005. 5(10): p. 786-95.
41. Gatenby, R.A., et al., Acid-mediated tumor invasion: a multidisciplinary study. Cancer Res, 2006. 66(10): p. 5216-23.
42. Baker, S.M., L. Karlsson, and R.L. Thurmond, Cloning, expression, purification, and activity of dog (Canis familiaris) and monkey (Saimiri boliviensis) cathepsin S. Protein Expr Purif, 2003. 28(1): p. 93-101.
43. McGrath, M.E., The lysosomal cysteine proteases. Annu Rev Biophys Biomol Struct, 1999. 28: p. 181-204.
44. Maubach, G., et al., The inhibition of cathepsin S by its propeptide--specificity and mechanism of action. Eur J Biochem, 1997. 250(3): p. 745-50.
45. Bromme, D., et al., Functional expression of human cathepsin S in Saccharomyces cerevisiae. Purification and characterization of the recombinant enzyme. J Biol Chem, 1993. 268(7): p. 4832-8.
46. McGrath, M.E., et al., Crystal structure of human cathepsin S. Protein Sci, 1998. 7(6): p. 1294-302.
47. Kaulmann, G., et al., The crystal structure of a Cys25 -> Ala mutant of human procathepsin S elucidates enzyme-prosequence interactions. Protein Sci, 2006. 15(11): p. 2619-29.
48. Turkenburg, J.P., et al., Structure of a Cys25-->Ser mutant of human cathepsin S. Acta Crystallogr D Biol Crystallogr, 2002. 58(Pt 3): p. 451-5.
49. Rawlings, N.D. and A.J. Barrett, Evolutionary families of peptidases. Biochem J, 1993. 290 ( Pt 1): p. 205-18.
50. Shi, G.P., et al., Human cathepsin S: chromosomal localization, gene structure, and tissue distribution. J Biol Chem, 1994. 269(15): p. 11530-6.
51. Leroy, V. and S. Thurairatnam, Cathepsin S inhibitors. Expert Opinion on Therapeutic Patents, 2004. 14(3): p. 301-311.
52. Riese, R.J., et al., Cathepsin S activity regulates antigen presentation and immunity. J Clin Invest, 1998. 101(11): p. 2351-63.
53. Honey, K. and A.Y. Rudensky, Lysosomal cysteine proteases regulate antigen presentation. Nat Rev Immunol, 2003. 3(6): p. 472-82.
54. Riese, R.J. and H.A. Chapman, Cathepsins and compartmentalization in antigen presentation. Curr Opin Immunol, 2000. 12(1): p. 107-13.
55. Saegusa, K., et al., Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity. J Clin Invest, 2002. 110(3): p. 361-9.
56. Wiendl, H., et al., Antigen processing and presentation in human muscle: cathepsin S is critical for MHC class II expression and upregulated in inflammatory myopathies. J Neuroimmunol, 2003. 138(1-2): p. 132-43.
57. Gupta, S., et al., Cysteine cathepsin S as an immunomodulatory target: present and future trends. Expert Opinion on Therapeutic Targets, 2008. 12(3): p. 291-299.
58. Sukhova, G.K., et al., Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells. J Clin Invest, 1998. 102(3): p. 576-83.
59. Wang, B., et al., Cathepsin S controls angiogenesis and tumor growth via matrix-derived angiogenic factors. J Biol Chem, 2006. 281(9): p. 6020-9.
60. Flannery, T., et al., The clinical significance of cathepsin S expression in human astrocytomas. Am J Pathol, 2003. 163(1): p. 175-82.
61. Liuzzo, J.P., et al., Inflammatory mediators regulate cathepsin S in macrophages and microglia: A role in attenuating heparan sulfate interactions. Mol Med, 1999. 5(5): p. 320-33.
62. Fernandez, P.L., et al., Expression of cathepsins B and S in the progression of prostate carcinoma. Int J Cancer, 2001. 95(1): p. 51-5.
63. Flannery, T., et al., Detection of cathepsin S cysteine protease in human brain tumour microdialysates in vivo. Br J Neurosurg, 2007. 21(2): p. 204-9.
64. Liu, J., et al., Increased serum cathepsin S in patients with atherosclerosis and diabetes. Atherosclerosis, 2006. 186(2): p. 411-9.
65. Flannery, T., et al., Cathepsin S expression: An independent prognostic factor in glioblastoma tumours--A pilot study. Int J Cancer, 2006. 119(4): p. 854-60.
66. Kos, J., et al., Cathepsin S in tumours, regional lymph nodes and sera of patients with lung cancer: relation to prognosis. Br J Cancer, 2001. 85(8): p. 1193-200.
67. Link, J.O. and S. Zipfel, Advances in cathepsin S inhibitor design. Curr Opin Drug Discov Devel, 2006. 9(4): p. 471-82.
68. Mandel, M. and A. Higa, Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970. 53(1): p. 159-62.
69. Tobbell, D.A., et al., Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens. Protein Expr Purif, 2002. 24(2): p. 242-54.
70. D'Alessio K, J., et al., Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast-specific protease. Protein Expr Purif, 1999. 15(2): p. 213-20.
71. Pauly, T.A., et al., Specificity determinants of human cathepsin s revealed by crystal structures of complexes. Biochemistry, 2003. 42(11): p. 3203-13.
72. Shevchenko, A., et al., Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem, 1996. 68(5): p. 850-8.
73. Bromme, D., F.S. Nallaseth, and B. Turk, Production and activation of recombinant papain-like cysteine proteases. Methods, 2004. 32(2): p. 199-206.
74. Ward, Y.D., et al., Design and synthesis of dipeptide nitriles as reversible and potent Cathepsin S inhibitors. J Med Chem, 2002. 45(25): p. 5471-82.
75. McGovern, S.L., et al., A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem, 2002. 45(8): p. 1712-22.
76. Barrett, A.J., et al., L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J, 1982. 201(1): p. 189-98.
77. Barrett, A.J. and H. Kirschke, Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol, 1981. 80 Pt C: p. 535-61.
78. Lineweaver, H. and D. Burk, The Determination of Enzyme Dissociation Constants. J. Am. Chem. Soc., 1934. 56(3): p. 658-666.
79. Copeland, R.A., Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. Wiley-VCH Publishers, New York., 2000: p. pp273-277.
80. Viatcheslav Volotovsky, N.K., Multienzyme Inhibition Biosensor for Amygdalin Measurement. Electroanalysis, 1998. 10(7): p. 512-514.
81. Catalano, J.G., et al., Design of small molecule ketoamide-based inhibitors of cathepsin K. Bioorg Med Chem Lett, 2004. 14(3): p. 719-22.
82. Adang, A.E., et al., Unique overlap in the prerequisites for thrombin inhibition and oral bioavailability resulting in potent oral antithrombotics. J Med Chem, 2002. 45(20): p. 4419-32.
83. Liu, H., et al., Design and synthesis of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 1. Bioorg Med Chem Lett, 2005. 15(22): p. 4979-84.
84. Adams, J., et al., Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids. Bioorg Med Chem Lett, 1998. 8(4): p. 333-8.
85. Patrick, G.L., An Introduction to Medicinal Chemistry Oxford University Press, 2005. 3th Edition.
86. Ekici, O.D., et al., Aza-peptide Michael acceptors: a new class of inhibitors specific for caspases and other clan CD cysteine proteases. J Med Chem, 2004. 47(8): p. 1889-92.
87. Cywin, C.L., et al., The design of potent hydrazones and disulfides as cathepsin S inhibitors. Bioorg Med Chem, 2003. 11(5): p. 733-40.
88. Verhelst, S.H., et al., Novel aza peptide inhibitors and active-site probes of papain-family cysteine proteases. Chembiochem, 2006. 7(6): p. 943-50.
89. Loser, R., et al., Azadipeptide nitriles: highly potent and proteolytically stable inhibitors of papain-like cysteine proteases. Angew Chem Int Ed Engl, 2008. 47(23): p. 4331-4.
90. Santos, M.M. and R. Moreira, Michael acceptors as cysteine protease inhibitors. Mini Rev Med Chem, 2007. 7(10): p. 1040-50.
91. Lipinski, C.A., et al., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev, 2001. 46(1-3): p. 3-26.
92. Kontoyianni, M., L.M. McClellan, and G.S. Sokol, Evaluation of docking performance: comparative data on docking algorithms. J Med Chem, 2004. 47(3): p. 558-65.
93. Venkatachalam, C.M., et al., LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. J Mol Graph Model, 2003. 21(4): p. 289-307.
94. Morris, A.L., et al., Stereochemical quality of protein structure coordinates. Proteins, 1992. 12(4): p. 345-64.
95. Tully, D.C., et al., Synthesis and evaluation of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 3: heterocyclic P3. Bioorg Med Chem Lett, 2006. 16(7): p. 1975-80.
96. Menard, R., et al., The specificity of the S1' subsite of cysteine proteases. FEBS Lett, 1993. 328(1-2): p. 107-10.
97. Gillet, L., et al., Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells. J Biol Chem, 2009. 284(13): p. 8680-91.
98. Thurmond, R.L., et al., Identification of a potent and selective noncovalent cathepsin S inhibitor. J Pharmacol Exp Ther, 2004. 308(1): p. 268-76.
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