Die Rolle des Kristallographen im strukturbasierten Wirkstoffdesign für die Pim-1 Kinase sowie den Kernrezeptor PPARβ/δ

In this work, the influence and potential of Xray-crystallography on different areas of structure based drug design is investigated and discussed. For this purpose, three projects were accompanied by crystallographic or alternative methods for structure determination. In the first project, fragment-based drug design was supported by X-ray crystallography. The aim was to design potent inhibitors of the Pim-1 kinase, which shows significant effect on cell proliferation and apoptosis in various tumor types. With the determination of the binding modes of various fragments, many interactions in the Pim-1 binding pocket were identified. The activity of these ligands was determined in a luminescence-based assay which allowed considerations to be drawn about the potential and impact of those interactions to the overall affinity. This project led to two promising compound-classes the quinoxalines and 2-amino-benzimidazoles and derivatives. Based on the findings of the crystallographic analysis, further considerations of structure-based optimization towards potential Pim-1 inhibitors could then be made. In a second project with Pim-1 as a model, it could be shown that rational and structure-based optimization of a hit towards fully active inhibitors go hand in hand. The computer-assisted modification of a fragment led to the promising starting compound 3.13 which was successfully crystallized. Based on the X-ray structure of this starting compound, two interesting inhibitor series could be developed in the course of the further optimizati-on processes: The indolin-2-ones and the stilbenes. The different derivatives of both series already showed high activity and effectively inhi-bited the kinase at single-digit micromolar to nanomolar concentrations. Compounds from both series could be crystallized with the Pim-1 and the structures were successfully de-termined. By analyzing the binding modes, interactions important for the inhibitory poten-tial of these compounds could be identified and are discussed. The stilbene series also revealed a conformational change of Pim-1: upon binding of the trans derivatives, it was observed that the glycine-rich loop above the binding pocket was shifted to the ligand, closing the binding pocket around the ligand. Based on the X-ray structures, this observa-tion was attributed to potential -interactions of the aromatics of the ligands with Phe49 in the loop. This conformational change could not be observed for the cis-derivatives. The target of the third project in this work was the nuclear receptor PPARβ/δ, which is a transcription factor that regulates the expression of several genes associated with metabo-lism, inflammatory responses, and tumor growth and metastasis. The aim of this project deals with the crystallization and structure determination of this receptor to investigate the binding modes of inverse agonists, which trigger the recruitment of a co-repressor and the associated reduction of receptor activity below a basal level. First, the ideal crystallization concentration was determined for this purpose. In addition to that, various literature-known conditions for the crystallization of the ligand binding do-main of PPARβ/δ and other subtypes were tested. Large-scale automated screens were also performed. The problems and difficulties encountered, as well as potential solutions were analyzed and discussed and can be used for further crystallization approaches. Al-ternative methods for structure determination were also considered: With the method of HDX-mass-spectrometry, the binding of the inverse agonists to the ligand-binding do-main of PPARβ/δ could be confirmed. Furthermore, this experiment showed that the bin-ding of an antagonist does not trigger the recruitment of the co-repressor motif SMRT, as is the case with the binding of inverse agonists. The obtained findings, in combination with other methods, will help to fully understand the mechanisms of PPARβ/δ regulation and allow further rational optimization of inhi-bitors.