Biophysical characterization of the DYRK family. Studies on the kinases DYRK1A and DYRK1B

Abstract

The dual-specificity tyrosine phosphorylation-regulated kinase family (DYRKs) has recently emerged as new therapeutic targets for different kinds of cancer and neurodegenerative diseases. In the latest studies it was shown that DYRK1B plays a key role in cancer cell survival, and its inhibition induces apoptosis of cancer cells. This thesis is about the biophysical characterization of DYRK1B and its comparison to DYRK1A, the closest homologue. This includes establishment of an expression and purification protocol for large-scale protein production of DYRK1B. Buffer optimization by screening with thermofluor was employed to further improve the stability and solubility of the protein. Enzyme kinetics of DYRK1B were characterized by determination of the Michaelis-Menten constant. Fifty small molecular weight molecules were screened for their ability to inhibit DYRK1B. The results of the screening were compared with DYRK1A to determine a selectivity profile of these inhibitors. A point mutation of DYRK1B to mimic DYRK1A was introduced to further investigate the selectivity profile. Finally, the crystal structure of DYRK1A in complex with PKC412 was solved by molecular replacement to a resolution of 2.6 Å. The structure shows for the first time the formation of a disulfide bridge between the catalytic loop and activation loop and it is the first structure published with the staurosporine analog PKC412.