Strukturbiologische Untersuchungen von Kinaseinhibitoren

Abstract

The main function of protein kinases is widely considered the activation of their substrates by catalytic phosphotransfer from adenosine-5‘-triphosphate (ATP), explaining the central role of kinases in cellular signaling cascades. Recently, more and more functions independent from this catalytic mechanism were described, that are involved in several biological processes, e.g., by regulation or localization of their substrates via protein-protein interactions or the formation of multi-enzyme complexes, respectively. This is usually realized by addressing alternative binding regions distant from the enzyme’s active site. Hence, the discovery of corresponding binding pockets and the development of small molecules capable of modulating scaffolding functions are of great interest in current scientific research. In the present work, the structure-based design and organic synthesis of 2-arylquinazolines, which were identified as ligands of a lipophilic binding pocket (LiPoLis) in p38α mitogen-activated protein kinase (MAPK), is described. Since the function of this pocket is not fully understood yet, the LiPoLis were thought to aid in elucidating its biological role. The generated compounds were characterized applying several biophysical methods to determine their affinity towards the enzyme and the suggested binding mode was validated by means of protein crystallography. Concluding these studies, the ligands were shown to positively address the lipophilic pocket (LP) but exhibiting a very low affinity, making them unsuitable to serve as molecular probes in biological systems. Consequently, electrophile-decorated LiPoLis were designed to target cysteine mutants of p38α MAPK, forming a covalent bond within the LP to maximize the residence time at the enzyme. Employing mass spectrometric analysis, specific ligand-protein pairs were identified and in tandem measurements and via co-crystallization, the introduced cysteines were verified as the actual labeling positions. Therefore, it could be shown that the thus found ligands exhibit a favorable reactivity and accordingly irreversible modifications of the target enzyme, rendering them suitable to be used in future studies to dissect the biological function of the described binding pocket in p38α MAPK. In another part of this thesis, various complex crystal structures of diverse ATP-competitive p38α MAPK inhibitors could be solved and were discussed, significantly contributing insightful results with respect to the underlying scientific questions.