Increased expression and activity of lysosomal cysteine carboxypeptidases cathepsins B and X are associated with a variety of pathological processes, including development and progression of cancer. In cancer, they have an important role in degradation of extracellular matrix (ECM), tumor invasion, migration, metastasis and angiogenesis. In addition, epithelial-mesenchymal transition (EMT) has been recognized as an important step during tumor progression, however the role of cathepsins B and X in this process is less known. In the doctoral thesis our first goal was to investigate the involvement of cathepsins B and X in EMT and to elucidate their mechanisms of action. Using tumor cell lines differing in expression of epithelial and mesenchymal markers and cell morphology, we show that higher levels of cathepsins B and X promote EMT and are associated with mesenchymal-like cell phenotype. Additionally, a reverse process of mesenchymal to epithelial transition was triggered by their simultaneous knockdown. Among two cathepsins, cathepsin B appears to be stronger promotor of EMT. To gain insight into mechanistic role of cathepsins B and X in EMT, changes in their expression in transforming growth factor β1 (TGF-β1) signaling pathway, one of the key signaling mechanisms during EMT in cancer, were analyzed. While in epithelial breast adenocarcinoma cell line MCF7 cathepsin B expression and activity increased after triggering EMT with TGF-β1, no change in cathepsin X expression was observed. Our data thus define EMT as additional mechanism linking cathepsins B and X with tumor progression. Additionally, by silencing and inhibition of enzymatic activity tumor cells could revert to less aggressive epithelial-like cell phenotype. Cathepsins B and X are, because of their important role in cancer, validated as targets for anti-tumor therapy and are as such interesting in drug development. In the following sections of the thesis we therefore focused on identification of novel small synthetic compounds that selectively and reversibly inhibit cathepsin B and X activities and are suitable for clinical use. Cathepsin B can in addition to carboxypeptidase (exopeptidase) activity, acts as endopeptidase, due to the presence of additional structural element, termed the occluding loop. Endopeptidase activity was proposed to be predominantly responsible for the harmful cathepsin B proteolysis in cancer. In this study, using new selective synthetic inhibitors {[(8-hydroxy-5-nitroquinoline-7-yl)methyl]amino}-acetonitrile (1), 8-(4-methylpiperidin-1-yl)-5-nitroquinoline (2) and 7-[(4-methylpiperidin-1yl)methyl]-5-nitroquinolin-8-ol (3) we investigated effect of both cathepsin B activities on ECM degradation and tumor invasion. We show that endopeptidase and exopeptidase activities of cathepsin both contribute to degradation of ECM and tumor cell invasion. Taken together, this opens new strategies for regulation of its increased harmful activity by design of novel inhibitors that would simultaneously and potently impair both activities. In in vitro and in vivo tumor models, we evaluated anti-tumor properties of an established antimicrobial agent nitroxoline that was previously identified as potent and selective inhibitor of cathepsin B endopeptidase activity. On selected tumor and endothelial cell lines, expressing higher levels of active cathepsin B, nitroxoline significantly reduced extracellular DQ-collagen type IV degradation, tumor cell invasion, growth and invasion of multicellular tumor spheroids, and endothelial tube formation in the angiogenesis assay. Moreover, it significantly abrogated tumor growth, angiogenesis and metastasis in vivo on three independent tumor mouse models. Taken together, our results establish nitroxoline as promising new drug candidate for treatment of cancer. Additionally, we evaluated anti-tumor properties of the 7-aminomethylated nitroxoline derivative (compound 17). In the previous study compound 17 exhibited significantly improved kinetic properties over nitroxoline. Here we show that compound 17 more effectively reduced tumor cell invasion and migration in different cell-based models. Furthermore, it was more efficient than nitroxoline in reducing tumor growth in LPB mouse fibrosarcoma model. With the addition of 2-(ethylamino)acetonitrile group to nitroxoline, we therefore successfully improved its pharmacological properties and obtain novel candidate for anti-tumor therapy. Next, in our study, we have identified clioquinol-ruthenium complex [Ru(η6-p-cymene)(Cq)Cl] as a potent inhibitor of cathepsin B. Clioquinol-ruthenium complex did not affect cell viability in low micormolar concentrations, however it did significantly reduced degradation of ECM proteins and tumor cell invasion. Our findings for clioquinol-ruthenium complex reveal specific anti-tumor mechanism, not related to a general compound-induced toxicity, and establish it as a lead compound for development of new ruthenium based anti-tumor agents. In the final part of our work, we have focused on identifying novel cathepsin X inhibitors. By screening in-house compound library, we identified triazole-based compounds as reversible selective inhibitors of cathepsin X activity. Among them the best performing compound with the lowest constant of inhibition and at least hundred times improved selectivity for cathepsin X compared to other related cathepsins was (1-(2,3-dihydrobenzob1,4dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one) (Z9). Z9 significantly reduced tumor cell migration and in addition to anti-tumor activity, it increased also the neurite outgrowth in the nevrodegeneration model. Cathepsin X inhibitors with triazole ring are therefore promising candidates for further in vivo evaluation and represent lead compounds for design of new cathepsin X inhibitors.
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