| Abstract [eng] |
The aim of this work was to study recombinant carbonic anhydrase (CA) VI as a model of native CA VI for the determination of inhibitor intrinsic binding reactions and to explore lead inhibitors of CA IX in biological systems as an initial step of their development towards anti-cancer drugs. This thesis provides a novel concept to characterize compound efficacies by combining biochemical, biophysical, and cell biology methods. The affinities of the inhibitors were essentially identical towards the native CA VI obtained from human saliva and recombinant CA VI purified from Escherichia coli, highlighting the suitability of recombinant CA VI as a model of native CA VI for biophysical inhibitor binding studies. Analysis of the intrinsic parameters showed that the observed values can be misleading for the understanding of the chemical basis of the binding affinity. Model systems, such as zebrafish, Xenopus laevis oocytes, and human cancer cells, were used together with the enzymatic and biophysical methods to characterize newly designed CA IX inhibitors. Studies using Xenopus oocytes for the first time revealed high selectivity and efficacy (IC50 = 15 nM) of the compound against hetereologous CA IX in a biological model system. Experiments employing human cancer cells showed CA IX-dependent functional activities of tested inhibitors. Moreover, the compound reduced clonogenic cell survival in hypoxia-dependent manner using 3D cell culture. |
