Ansätze zur Entwicklung und Charakterisierung von Inhibitoren gegen die Serinprotease Thrombin, die Dengue-Virus-Protease und die bakterielle RNase P

Within the scope of this work, three projects were realized on the basis of different preliminary work. The methods of rational drug design were applied in all three projects. In the first subproject, a fragment library consisting of 96 different small molecules was scree-ned against the serine protease Thrombin. Seven different fragment molecules could subse-quently be assigned to their complementary electron density in the X-ray structures and their binding modes evaluated. This corresponds to a hit rate of 7.3%. During the evaluation, the fragments were found to address the specificity pockets (S1-S4) of the protease. In addition, target-directed interactions with the catalytically active amino acids were identified in the case of fragments J77, J80, and J89. Furthermore, contributions to the understanding of the selecti-vity-determining characteristics between the serine proteases Trypsin and Thrombin could be derived. The serine protease Thrombin plays a key role in the blood clotting cascade and inhi-bition is mainly targeted for the treatment of atrial ventricular fibrillation or the prevention of stroke. Thus, the identified fragment molecules can serve as a starting point for the develop-ment of new inhibitors with improved bioavailability, selectivity and efficacy. In addition, the knowledge gained can be transferred to the development of inhibitors against serine proteases with a similar folding pattern. As no specific therapeutic treatment for dengue fever, an arthropod-borne disease, is available to date, the second subproject of this work deals with the development and evaluation of allos-teric inhibitors against the dengue virus protease (serotype 3). Based on in silico docking, 15 different compounds were acquired and tested via fluorescence-based enzyme assay. The most potent of these compounds (KMH14, IC50=136±16 μM) showed a non-competitive binding mode and the core structure was used as a starting point for subsequent design cycles. In the second cycle, 17 additional compounds based on the core motif of KMH14 were tested in vitro. The most potent compound (KMD027, IC50=28±7.9 μM) showed significantly enhanced activity against dengue virus protease (serotype 3) and a general structural motif for allosteric inhibition could be deduced. Thus, in the final design cycle, activity was increased to IC50=12.3±3.5 μM by targeted SAR study. Concomitantly, an attempt was made to investigate the identified compounds crystallographically. In this regard, crystallization was successfully established in our laboratory and stable soaking conditions were identified. In addition, a bi-nary zika virus construct was mutated to increase the binding pocket and allow crystallogra-phic investigation of the developed dengue virus inhibitors. In addition, low cytotoxicity of the ligands was determined. All in all, the investigations of the second part of this work re-present a very good starting point for the development of further potent inhibitors against the dengue virus protease. The third and last part of this work served to identify new starting points for the development of antibiotically active substances. For this purpose, the P protein of the RNase P was selected as a new target. Since the development of bacterial resistance and the lack of new antibiotics are threatening an increase in infectious diseases with fatal consequences, the development of new active substances is also a goal of drug research in this area. First, the P-Protein oft he RNase P of the pathogenic organism Mycobacterium ulcerans was successfully isolated and purified in high yield. In addition, attempts were made to crystallize this protein and initial conditions were identified. An inhibitor of RNase P already known from literature could be identified as PAIN (Pan-Assay Interference Compound) in further experiments and its postu-lated reaction could be elucidated as aggregation. Thus, drug development based on incorrect measurement data is no longer possible. Further more, new inhibitors can now be developed based on the preliminary work.