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Abstract

Cysteine cathepsins are involved in the development and progression of numerous inflammatory diseases, although the exact role of individual cathepsins is not always clear. Cathepsins B, K, L and S are the most important in cancer, inflammatory bone diseases and atherosclerosis. While these proteases are usually contained in the endolysosomal system within cells, they are translocated in part to the cell membrane and the extracellular space after the induction of inflammation that is also accompanied by their elevated activities.
In this thesis we focused on the development of new tools for following the localization of cathepsins B and S in vivo, which would enable characterization of their role in diseases. The technology also has an important translational value in medicine, since optical imaging with these tools can be used for disease diagnosis in the cases in which the target protein is upregulated. In the case of cathepsins, the development of selective probes has been difficult in the past due to the high structural similarity within the cathepsin family.
In our work we focused on two new techniques that allowed us to develop tools for differentiation among these highly conserved targets.
We targeted cathepsin B with small binder proteins DARPins that recognize extended three-dimensional epitopes at the surface of target proteins that enabled differentiation of cathepsin B from the other cathepsins. We developed DARPin 8h6 that binds cathepsin B with high affinity at a site of low structural conservation in the cathepsin family. Fluorescently labelled DARPin 8h6 interacted with cathepsin B on the surface of tumour cells and macrophages and in the inflammatory environment in two mouse models of mammary gland tumours as well as an arthritis model, suggesting that this DARPin is an appropriate tool for following the localization of cathepsin B. Due to the fast elimination of access DARPin from bloodstream after the systemic administration in mice, we could detect a high signal contrast at the site of tumour and arthritis, which points to a potential in a diagnostic tool development for inflammatory diseases. Upon binding of DARPin 8h6 to cathepsin B, the DARPin covered part of the enzyme's active site and inhibited cathepsin B activity. In the future the DARPin could also be used in therapeutic or theranostic applications.
In the second part of the dissertation, we targeted cathepsin S with smart substrate probes that were developed with reversed design. This methodology is based on transformation of a known selective inhibitor with good pharmacological properties into a fluorescent substrate, by preserving the selectivity for the target enzyme. The selective substrate probe 2 was further modified with a lipid or a polymer group for improved in vivo targeting. The modification did not change the selectivity of the probes for cathepsin S, neither did it limit the processing in a cell model of differentiated macrophages. This suggests to the applicability of the probes in preclinical research as well as disease diagnostics.