Abstract
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.