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UBC Theses and Dissertations

Characterization of glycosaminoglycan interaction sites and exosite inhibitors of cathepsin K Cui, Haoran


Cysteine cathepsins belong to the papain-like family and are critically involved in the pathogenesis of various diseases, such as osteoporosis and cardiovascular diseases. These disorders are associated with failures in the degradation of extracellular matrix proteins, collagens and elastins. Based on enzymatic and structural studies, the active site mechanism has been well elucidated and many active site inhibitors have been identified, especially inhibitors for cathepsin K. Cathepsin K is primarily expressed in osteoclasts and the enzyme is responsible for most of the bone resorption, and thus an important pharmaceutical target for the treatment of osteoporosis. However, as cathepsin K is expressed not only in osteoclasts but also has functions outside the skeletal system, active site inhibitors may lead to severe side effects when used for treatment. In cathepsins, there are certain binding sites that are distinct from the active site, termed exosites, which have been defined as important for the degradation of extracellular matrix proteins. The hypothesis is that exosites could block the degradation of matrix proteins but would not interfere with other biological functions of the target protease such as cathepsin K. My thesis is focused on the characterization of these exosites and the identification of selective exosite inhibitors. In Project 1, protein-GAG interaction sites were studied for their involvement in the collagenase activity. According to the X-ray structures of cathepsin K -chondroitin sulfate (CS) complex, two mechanistic models were built: cathepsin K/CS tetramer and dimer. The hypothesis is that the cathepsin K/CS dimer complex solubilizes collagen fibers into tropocollagen and the cathepsin K/CS tetramer complex further degrades tropocollagen into peptides. The objective is to elucidate the mechanism of cathepsin K/CS complex-mediated collagen degradation by generating GAG binding sites mutants. In project 2, potential exosite inhibitors of cathepsin K were identified by drug screening assays. Compounds which have been reported to be effective in osteoporosis animal models were studied. The hypothesis is that exosite inhibitors for cathepsins specifically inhibit the degradation of collagen but not that of other biological substrates. The objective is to determine IC50 values of the compounds for the inhibition of the degradation of collagen.

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