Strukturelle und funktionelle Charakterisierung neuartiger Transkriptionsmodulatoren

To facilitate the development of potent protein modulators in drug discovery, the exact mechanism of action must be elucidated. As biological processes in organisms are very complex and many different components are involved, the identification of these is of outmost importance for the efficient optimisation of the selectivity and potency of the respective modulators. The transcriptional regulation of target gene expression is a very important mechanism to regulate in many biological functions and therefore the mode of action of several modulators was analysed in this thesis. The hedgehog signalling pathway is the essential process in the embryogenesis, but the exact mechanism and the cofactors being involved in this complex transcriptional regulation are still not completely understood. An identified and validated structural class of ligands of the hedgehog signalling pathway was analysed and the structure activity relationship of this class was further determined, to improve the potency of these novel modulators of the hedgehog signalling pathway. Based on the established structure activity relationship, new biotinylated substances bearing different linkers were developed in order to isolate the target in a pull down experiment, to enable target identification via mass spectrometry. Besides the hedgehog signalling pathway, the mode of action of the nuclear receptor PPARβ/δ was analysed, which acts as a ligand-induced transcription factor. Previous work has shown, that one compound (DG172) of the identified structural class, bearing a stilbene moiety, promotes differentiation of myeloic cells from murine bone marrow cells, an effect that was not PPARβ/δ related. In the presence of the substance, a higher amount of mature and fully differentiated dendritic cells was observed, which was determined via FACS analysis utilizing the antibodies against MHC II, CD11c, Ly-6B and Ly-6G. The PPAR-inactive substance LD253 with a biotinylated substituent at the PPAR-essential part of the molecule was tested and was shown to be less active than the substance DG172. As the differentiation into dendritic cells was not fully stimulated by LD253, the experiment must be further optimised to allow for the isolation of the target. DG172 is one of the most potent PPARβ/δ inverse agonists besides two other potent ligands ST247 and PT-S264 that were identified and optimised in a ligand-based approach for generating novel potential drug candidates. Up to date, the X-ray structure of the PPARβ/δ-ligand binding domain was published in complex with an agonist but the structure with an inverse agonist has still not been elucidated so far. As specific corepressors, which in both cases lead to a stabilisation of the respective complexes, are recruited to the PPARβ/δ-complex with an inverse agonist compared to the essential coactivator recruitment to the PPARβ/δ:agonist-complex, the whole transfer of the published crystallisation conditions will most likely not be successful. In order to find suitable crystallisation conditions, protein crystallisation screenings were either by co-crystallising the three substances with a corepressor peptide, or by directly adding this corepressor peptide during the screening. The protocol of protein isolation and purification was optimised to 26.7 mg L 1 medium or 1482.0 mg g 1 cells after affinity chromatography, because a huge protein amount is needed for performing the crystallisation screening. Unfortunately, no crystals formed so far. In summary, different modulators of gene expression were studied to get more insights into the mechanism and mode of action of these essential regulation processes. Based on the results of the performed structure-activity relationship study, selective substances were developed for identifying the target. Furthermore, a robust and reproducible protein isolation method was established, and protein purification and yield were substantially improved which allows for high-throughput screening for crystallisation conditions.