| Abstract [eng] |
A family of human carbonic anhydrases (CA) contains 12 catalytically active isozymes, that play a role in various diseases, including cancer, glaucoma, epilepsy, and altitude sickness. Unfortunately, CA inhibitors used as drugs lack selectivity for the target isoform and cause serious side effects. Since all 12 CA isozymes have a high percentage of amino acid sequence homology, it is challenging to design isoform-selective inhibitors. The main aim of this thesis was to understand the mechanism of inhibitor recognition and selectivity profiles of CAs. Thus, we generated chimeric carbonic anhydrases (chCA) resembling properties of CA VI and CA XII. We used the off-target isoform CA II as a core of the chimeric protein and mutated 5-6 amino acids in its active site to those specific for CA VI or CA XII. We determined the affinities of a series of benzenesulfonamides binding to the off-target CA II, target CA VI and CA XII, and target-resembling chimeric proteins using isothermal titration calorimetry, enzymatic activity inhibition, and fluorescent thermal shift assays. In this thesis, I also highlighted the advantages and limitations of each technique, especially for measuring picomolar interactions. The chimeric carbonic anhydrases switched inhibitor recognition and bound inhibitors with similar affinities and binding modes as target isozymes CA VI and CA XII but not as their core isoform CA II. Thus, we confirmed the chimeric carbonic anhydrases as valuable models for designing isoform-selective inhibitors. The general strategy of engineering chimeric isozymes could be applied to other drug discovery projects. . |
