Strukturbasiertes Wirkstoffdesign am Beispiel der Zielproteine HIV-1 Protease, Transglutaminase 2 und Faktor XIII

The thesis at hand uses the example of three different target proteins to describe struc-ture-based design of drugs, the first being HIV-1 protease, the second and third being the human proteins transglutaminase 2 and coagulation factor XIII. The aim of this the-sis was to crystallize the respective proteins in complex with their inhibitors and to de-termine the three-dimensional structure of the complexes by X-ray analysis. The result-ing information about the binding mode of the inhibitors could then be used for the de-sign of new molecules and the optimization of binding properties, for example the affin-ity to the protein. In this way, a contribution to the development of new drugs has been made. Inhibitors of HIV-1 protease are potential drugs for the treatment of HIV-infected pa-tients. They might help slowing down the ongoing infection and later relieve the symp-toms of AIDS. Even though a variety of drugs is already available, the increasing resis-tance development of the virus and the worldwide spreading of AIDS up to a pandemic require permanent efforts to develop new therapeutics. Among other things, human transglutaminase 2 contributes to the pathogenesis of celiac disease. At the moment, there are no drugs available to treat this gluten intolerance. An inhibitor of transglutaminase 2 would be a potential drug allowing affected patients making their gluten-free diet less strict and thus increasing their quality of life. Coagulation factor XIII is also a member of the family of human transglutaminases. Because of its function in blood coagulation, the protein could be a target for an antico-agulation therapy. Inhibitors of factor XIII are therefore potential drugs and a new op-tion in prophylaxis and therapy of several cardiovascular diseases like thrombosis and embolism. Section 2.3 of this thesis presents several crystal structures of complexes of HIV-1 pro-tease with inhibitors having a secondary amine as core structure. The observation of a significant difference in binding modes in a series of structurally similar ligands, in rela-tion to the crystal form and the space group of the crystals, is reported in section 2.3.1. The occurrence of two different binding modes of the same inhibitor to HIV-1 protease, once C2-symmetric, once without twofold symmetry, is shown in section 2.3.2. Here, the relationship between the inhibitor's binding mode, symmetry and space group, is discussed in detail. Section 2.3.3 and 2.3.4 describe the characterization of molecules with a fullerene as well as with a piperidine core scaffold as novel inhibitors of HIV-1 protease by enzyme kinetic measurements. It was not possible, however, to obtain struc-tural information about the binding mode of these inhibitors. Nevertheless, as shown in section 2.3.5, the binding mode of a series of three-armed, non-symmetric pyrrolidine-based inhibitors was determined by crystallization of the complexes and subsequent X-ray analysis. Pyrrolidine-based bicyclic inhibitors are an advancement of the amine-based HIV-1 protease inhibitors. Their binding properties are shown in section 2.3.6. Crystal structures of complexes with these inhibitors were the basis for a fragment-based approach, which is described in section 2.3.7. Human transglutaminase 2 was crystallized in complex with covalently bound inhibitors in the open conformation of the protein, as shown in section 3.3. The success of crystal-lization of these complexes turned out to be highly dependent on the type of the inhibi-tor. Nevertheless, crystal structures of three different complexes were determined fi-nally. The structural analysis provided useful information about the binding mode of the inhibitors, which proved of value for the design of new molecules to be developed into an applicable drug by the cooperation partner in this project, ZEDIRA GmbH. As with transglutaminase 2, the aim was to crystallize coagulation factor XIII in com-plex with a covalently bound inhibitor and to determine the three-dimensional structure of the protein in the open conformation for the first time ever. As described in section 4.3, the protein was crystallized successfully in the closed conformation at first. After-wards, a large number of complexes of factor XIIIa with covalently bound inhibitors were applied in extensive screenings to find crystallization conditions for the inhibited protein. To this end, promising crystals of a complex were obtained and tested for dif-fraction power. Thus, a great milestone on the way to a crystal structure has been accomplished.