Cathepsin S is a papain-like cysteine protease. It is expressed primarily in professional antigen presenting cells (e.g. dendritic cells, macrophages and lymphocytes B) where it has an important role in processing and presenting antigens. Like other cysteine cathepsins, it is found in lysosomes. With its low pH and reducing environment, lysosome is the perfect milieu for cysteine cathepsin activity. Unlike other cysteine cathepsins, cathepsin S can also function in extracellular milieu for prolonged periods as it remains stable at neutral pH. It is evolutionarily related to cathepsin K, which is expressed in various tissues. Most abundantly, however, is expressed in osteoclasts, where it has an important role in bone remodelling. It is secreted into resorption pit where it cleaves the triple helix of type I collagen. Elevated activity is connected with numerous diseases including osteoporosis. In recent years, there has been an increase in development of allosteric inhibitors of different proteases. In contrast to orthosteric inhibitors, they only partially inhibit protease activity, which makes them more suitable for prolonged therapy.
The aim of this thesis was to investigate effects of known allosteric inhibitors of cathepsin K on selected mutant forms of cathepsin S in order to verify their binding to the chosen allosteric site and the involvement of selected amino acid residues in binding of potential allosteric inhibitors.
Based on the structure comparison of the two enzymes we prepared selected mutant forms of human cathepsin S by means of site-specific mutagenesis, where substituted residues were identical to matching residues in allosteric site of cathepsin K. By testing the kinetic parameters of potential allosteric inhibitors, we defined their reaction mechanisms on selected mutant forms. We showed that compounds 2- [2- (Diethylamino) acetamido] benzoic acid, methyl (R)-(2,5-dioxopyrrolidin-3-yl)glycinate and 2-benzylmalonic acid act on selected mutant forms of cathepsin S as allosteric effectors and that they most likely bind to the examined site. All tested mutant forms are inhibited more efficiently than wild type cathepsin S in the presence of these compounds. However, we cannot determine the involvement of individual residues in specificity of binding between allosteric inhibitors and tested enzymes.
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