Cathepsins are a group of enzymes that are responsible for hydrolysis of peptide bonds and are a vital part of many physiological processes in all living organisms. On the other hand, their mislocalization, absence or unregulated activity can result in different pathological conditions. Based on the amino-acid residues in active site we can further divide cathepsins into three groups: serine, aspartyl or cysteine proteases. The representative of the latter group is cathepsin L, a lysosomal endopeptidase that is responsible for degradation of intracellular proteins, bone resorption and release of thyroid hormones. The malfunction of this enzyme can result in myofibril necrosis, myopathy, myocardial ischemia and proteinuria as well as in development and metastasis of cancer cells. Recently it was discovered that it enables SARS-CoV-2 to enter the cell, which is why cathepsin L (and other cathepsins) represents a potential target for new therapeutic agents.
In Master thesis I studied the interactions between selected ligands and cathepsin L in the presence of zinc(II) ions and their effect on enzyme activity. All of the selected compounds are pharmacologically active O,O-, N,O-, N,N- and N-ligands that have already exhibited (potential) effect on cathepsin activity. The effect of metal complexes prepared from ligands and various zinc(II) salts was evaluated as well. Studies included stability tests and enzyme tests. Stability tests were performed by using UV/VIS spectrophotometry that indicated in situ formation of zinc(II)-clioquinol complex, and by 1H NMR spectroscopy that did not provide clear results (regarding the identity of species of nalidixic acid and isoniazid) besides the fact that all of the tested species are stable over time. Enzyme inhibition effect was studied by fluorimetric methods in MES and acetate buffer. Ligands (with the exception of isoniazid) exhibited only partial inhibition of enzyme, while total inhibition was observed when zinc(II) salts were added. The trend was observed in different buffers and zinc(II) salts. Even better inhibitory characteristics were displayed by zinc(II) salts, especially ZnCl2 in acetate buffer, which could indicate that by complexation, the ligands reduce the inhibitory characteristics of the zinc(II) ions. However, based on EC50 values the best inhibitor of cathepsin L turned out to be the synthesized complex of Zn(OAc)2·2H2O and clioquinol in acetate buffer. Hill's coefficient larger than 1 indicates positive cooperativeness of binding.
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