Proteindynamik - Flexibilität in Zielproteinen des strukturbasierten Wirkstoffdesigns

In this thesis different methods of computer-aided drug design have been applied to diverse problems in several projects. // Five different target proteins from three distinct target classes have been investigated. (1) From the super family of aldo-keto reductases, two members of the subfamily 1 have been examined. Firstly, aldose reductase which is of special interest due to its role during complications in advanced states of diabetes. Secondly, another member of the same family: AKR1B10 which is associated with various cancers. (2) Protein arginine methyltransferase 4 (PRMT4) and PRMT6 that represent an up-to-date and seminal topic due to their epigenetic function. (3) A bacterial target represented by tRNA-guanin transglycosylase (TGT). This protein fulfills an initial function in the activation cascade of the pathogenic phenotype of Shigella flexneri and, therefore, serves as an interesting target for the treatment of shigella induced bacillary dysentery. //// Aldose reductase // AR belongs to the super family of aldo-keto reductases. It is a NAD(P)(H) dependent oxidoreductase and, hence, mostly known for the conversion of glucose to sorbitol in the polyol pathway. Furthermore, it contributes to the detoxification of the organism by reducing toxic aldehydes and carbonyls. The Investigations on aldose reductase were conducted in cooperation with the Diederich group (Marburg) and GE Healthcare Bio-Sciences AB. The project was focused on characterizing a ligand series and examining the binding properties of these ligands with regard to opening events of a transient specificity pocket in this protein. This thesis contributed to this via an investigation of protein dynamics related to the pocket and an analysis and description of possible opening mechanisms. The necessary data for the analysis was obtained by conducting molecular dynamics simulations. Furthermore, MM-GBSA calculations were performed to evaluate binding preferences of two similar ligands to the specificity pocket. // AKR1B10 // In this cooperation project, comparative simulations of a ligand (JF0064) were executed to achieve two goals. (1) Validate the protonation state of the ligand in complex with the protein that has been observed in the x-ray structure. (2) Gaining structural insights on the binding conformation of the ligand to the wildtype protein. Since structure determination of the wildtype:JF0064 complex with x-ray crystallography was not possible, these molecular dynamic simulations allowed us to gain an insight on its structure. // Protein arginine methyltransferases // In this project several methods were used to accomplish two different tasks. (1) In cooperation with the Bauer group an homology model of Gallus gallus PRMT4 was built. To date, birds were the only class of vertebrates without experimental proof for an PRMT4 homologue. Hannah Berberich succeeded as the first to prove the existence of putative Gallus gallus PRMT4 (ggPRMT4). This thesis contributed by generating a feasible in silico model of ggPRMT4. Several in silico methods have been used in combination to accomplish this goal. To generate the ggPRMT4 model, homology modeling was combined with protein-protein docking. Furthermore, molecular dynamics simulations were used to validate the model and analyze the dynamics of the modeled complex further. (2) PRMT4 and PRMT6 were addressed as targets in a docking study using small organic molecules and known crystal structures of both proteins. All dockings were further evaluated by visual inspection subsequent to pre-selecting the molecules with a scoring function. Afterwards, interesting compounds were purchased and in cooperation with the BAUER group tested in a biological assay by Antje Repenning. As a result of this process, active compounds were identified. These compounds will now be further characterized and may be used as a starting point for rational drug development. // tRNA-guanin transglycosylase // This target was analyzed in two studies. (1) The dynamics of a, by x-ray crystallographic methods newly discovered, sub-pocket underneath the beta1alpha1-loop were analyzed. Therefore, firstly the pocket was examined via molecular dynamics methods. In a second step, the pocket was addressed by docking with organic molecules. (2) The opening event of the transient extended guanin/preQ1 binding pocket was analyzed with molecular dynamics simulations.