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.

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