Dokument

Summary:
Influenza is one of the commonest infectious diseases affecting millions of people every year including 290,000 – 650,000 heavy casualties. Influenza viruses undergo constant genetic changes and every 10 – 50 years new influenza virus strains emerge that potentially cause a severe pandemic. In this modern interconnected world, experts believe the next influenza pandemic will be a “devastating global health event with far-reaching consequences” [1]. Novel effective anti-influenza drugs are in need. One strategy of influenza research is to focus on host-specific proteases that are essential for virus activation and spread. Trypsin-like serine proteases are crucial for influenza activation by mediating the cleavage of the viral surface glycoprotein HA and hence promoting the fusion potential of the virus. Therefore, their inhibition provides a promising therapeutic approach. The present work focused on the characterization of two relevant HA cleaving type-II transmembrane serine proteases matriptase and TMPRSS2. Chapter 3 and chapter 4 of this thesis engaged with the recombinant production of matriptase (chapter 3) in order to obtain a pure functional enzyme of high quality for a SAR study with novel monobasic (hence potentially bioavailable) matriptase inhibitors of the 3-amidinophenylalanine type (chapter 4). Adequate amounts of high-quality matriptase enzymes were isolated using a new expression system and in total 5 matriptase crystals were available at the end of this thesis for structural analysis. The matriptase inhibitor design in this thesis focused on matriptase-affine compounds with a fair selectivity profile against the blood coagulation enzymes thrombin and fXa. In total, 18 new monobasic and potentially bioavailable, as well as four new dibasic compounds of the 3-amidinophenylalanine types were tested. Based on the last published crystal structure of this inhibitor type in complex with matriptase from 2006 (PDB code 2GV6) docking was used as a structure-based virtual screening method for lead optimization of the compounds N-terminus. Selected compounds were suggested to interact with the carbonyl side chain of Gln175 of matriptase to achieve a higher affinity of matriptase compared to fXa. The 4-tert-butylureido-piperidine could be identified as suitable C-terminus in combination with 3-fluoro-4-hydroxymethyl biphenylsulphonyl N-terminally in order to obtain excellent selectivity over thrombin. The binding mode of this compound (compound 55) was crystallographically determined in complex with matriptase as well as trypsin. Trypsin proved as a suitable alternative to matriptase for detailed binding mode analysis of the compounds N-terminus. However, different preferences were detected for the C-terminus. Dibasic compounds showed higher matriptase affinity and selectivity in comparison with the monobasic analogues. However, the tested monobasic compounds were still decent matriptase inhibitors that are additionally suitable for cell culture and animal studies in their benzamidine prodrug forms, which are well established from related inhibitors of thrombin. In addition, selected monobasic as well as dibasic compounds demonstrated strong suppression of the replication of certain H9N2 influenza viruses in a matriptase-expressing MDCK II cell model. These matriptase inhibitors could be potential lead structures for the development of new drugs against H9 strains for influenza. TMPRSS2 is widely discussed for its role in influenza activation. With a TMPRSS2 dependancy of HA-activation of certain subtypes, the characterization of this protease is an important prerequisite for being available as a target for influenza drug design. However, only little is known about the physiological function of TMPRSS2 and no experimental structure data are available at the moment to enable a structure-based drug development. Therefore, chapter 5 of this thesis focused on the characterization of TMPRSS2 in order to develop a strategy for the isolation of proteolytically active TMPRSS2 from cell culture. Even though, no functional TMPRSS2 could be recovered at the end of this work some new structural characteristics of TMPRSS2 were identified as crucial for functionality insight the cell. In general, TMPRSS2 without the cytosolic part, the transmembrane domain and the LDLRA domain is able to undergo autocatalytically activation if an artificial signal peptide was added N-terminal to enable entry into the endoplasmic reticulum. The presence of the cysteine-rich SRCR domain and the presence of the disulfide chain that connects the SPD and the stem region after activation cleavage have been identified as crucial for activity. N-terminal truncation of TMPRSS2 did not result in obvious dislocation within the cell: as the full-length positive control truncated TMPRSS2 was exclusively found in cell compartments surrounding the nucleus in immunofluorescence experiments. However, a reduced proteolytic cleavage activity towards H3-HA in co-expression experiments has been observed and might be a result of dislocation, since truncated TMPRSS2 is not bound to the biomembrane anymore. In addition, TMPRSS2 has been identified as a potential substrate of matriptase in vitro, which suggests possible participation in several zymogen cascades.

Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten