Design, Synthese und Evaluation von Inhibitoren der bakteriellen RNase P und der Dengue-Virus-Protease

The discovery of new targets and drugs to treat bacterial and viral infections in the future is an integral part in fighting infectious diseases. In the first part of this thesis a synthetic route for a screening hit, an inhibitor of the bacterial RNase P, was developed, in order to provide the screening hit in larger quantities and to allow an easy variation of its substituents. This synthetic route delivered compounds with better solubility and improved activity in the in vitro assay as well as in the agar diffusion test compared to the initial screeninghit, resulting in an increased diameter of the inhibition zone. The second part of this thesis aimed at the de novo design of inhibitors of the dengue virus protease. Inhibitors of the dengue virus protease with a core structure different from what has been published so far, were synthesized. Due to a sparsely pronounced SAR of the inhibitors synthesized an unspecific inhibition of the dengue virus protease by the inhibitors had to be ruled out. By increasing the solubility of the compounds in the fluorescence-based inhibition assay and by using dynamic light scattering to detect aggregates in solution, a specific inhibition of the dengue virus protease was finally proven. The increased solubility was achieved by increasing the concentration of DMSO in the assay. The increased DMSO concentration in the fluorescence-based inhibition assay was also successfully applied to the thermal shift assay and to the soaking conditions for dengue virus protease crystals. Both in the thermal shift assay and for the soaking of the dengue virus protease crystals the DMSO percentage was increased to >30%. This very high concentration of DMSO allows to test less potent or poorly soluble compounds at higher concentrations for an inhibition of the dengue virus protease and thus can serve as a starting point for the development of new inhibitors. In the third part of this thesis the effect of the non-ionic detergent Triton X-100, which is used to break up aggregates in solution in order to prevent unspecific inhibition, was studied. It could be shown that the effects observed when using Triton X-100 together with specific inhibitors of the dengue virus protease were not limited to this protease but also occurred with different proteases and assay setups. By using Pepstatin A, a peptidic and competitive inhibitor of aspartic proteases, it could be shown that Triton X-100 does not negatively affect the assay system, as the inhibition of Pepstatin A versus HIV-1 protease and endothiapepsin was not altered. When testing structurally different inhibitors of the HIV-1 protease and endothiapepsin it could be shown that Triton X-100 influences the inhibition of both proteases by some inhibitors, while other structurally related inhibitors remained entirely unaffected, consequently resulting in false negatives. In screenings it is assumed that the inhibitor acts non-specific, via aggregation, when showing a lower inhibition when used together with Trition X-100 as detergent. Consequently, the decline of inhibition observed for specific aspartyl protease inhibitors together with Trition X-100 as a detergent would lead to false negative results. In order to avoid false negative results it could be shown that CHAPS as a detergent in dengue virus protease, HIV-1 protease and endothiapepsin inhibition assays did not produce false-negative hits. In summary, by applying different medicinal chemistry methods, the detection and an optimization of inhibitors of bacterial and viral targets could be achieved. The DMSO concentration used in the assay systems when screening for inhibitors of the dengue virus protease was increased and it was shown, that Triton X-100 influences specific inhibitors of different proteases, leading to false-negatives and thus being not the optimal detergent for screening campaigns. Taking into account the results of this thesis, inhibitors of the bacterial RNase P and the dengue virus protease can be designed and further optimized and due to the results of the Triton X-100 studies the hit rate of screening campaigns can eventually be increased, leading to more starting points for further hit optimization processes.