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Research activities

The research activities of the Pietsch group aim at the discovery and development of novel drug candidates that show higher potency and selectivity towards the target structure. We focus on the interaction of primarily low molecular weight compounds with disease-related enzymes and protein-protein interactions, with our research projects being based on a network of collaborations with groups from Medical and Science Faculties of various universities as well as external research facilities.

In our investigations, we use a variety of methods, such as cell and bacterial culture, protein expression, and quantification of the interaction of protein targets with low molecular weight compounds using mainly spectrophotometric and fluorometric methods, including the analysis of the results by means of various mathematical models.

 

  1. Development of inhibitors of tumor-relevant protein kinase CK2
    This project investigates the interaction of protein kinase CK2 subunits (CK2α/CK2α‘ and CK2β) and its manipulation by (small molecule) inhibitors. Protein kinase CK2 is over-expressed in various types of cancer and is beneficial for survival of the tumor cells due to its anti-apoptotic effects. The development of CK2 inhibitors follows an alternative approach which does not address the kinase’s active site but the CK2 subunit interaction site. This strategy might lead to the identification of inhibitors with higher selectivity that may be used to investigate the effects of a disturbed CK2 holoenzyme (CK2α2β2) structure in a cellular environment. To analyze the CK2α/CK2α‘-CK2β interaction, a microscale thermophoresis (MST)-based assay and a fluorescence anisotropy assay suitable for high-throughput screening (Hochscherf et al., Anal. Biochem. 2015) were developed. These assays allow for the quantification of the CK2 subunit interaction and the interaction of CK2α/CK2α‘ with a cyclic peptide (Pc) derived from human CK2β, respectively, with the human CK2 subunits being accessible by recombinant expression in E. coli. The crystal structure of the CK2α-Pc complex has been recently published by Raaf et al., ACS Chem. Biol. 2013 (PDB 4IB5). Both assay methods led already to the identification of new inhibitors with dissociation constants on CK2α and CK2α‘ in the high nanomolar and (low) micromolar range. Selected inhibitors were also shown to reduce the catalytic activity of the CK2 holoenzyme towards CK2β-dependent substrates (Hochscherf et al., Anal. Biochem. 2015).

  2. Development of inhibitors of tumor-relevant transglutaminase 2
    Aim of this project is the development of new inhibitors of the enzyme transglutaminase 2 (TGase 2). TGase 2 is over-expressed in a variety of cancers which correlates with the tumors’ sensitivity towards chemo- and radiotherapy as well as their invasive potential (Pietsch et al., Bioorg. Med. Chem. Lett. 2013). Therefore, inhibition of TGase 2 is considered a promising strategy for the development of an anti-cancer therapy. The project includes the synthesis of small molecules and their kinetic characterization as inhibitors of TGase 2 and other transglutaminases. For this purpose, we have already developed fluorescence- and fluorescence anisotropy-based assays which address the enzymes’ transamidase activity (Wodtke et al., ChemBioChem 2016, Hauser et al., Amino Acids 2017) and have been used to characterize new inhibitors of human TGase 2 and TGase 2 from guinea pig liver (Wodtke et al., J. Med. Chem. 2018). Ongoing studies deal with the optimization of already identified lead compounds targeting TGase 2‘s active site and with the development of novel allosteric inhibitors including assay methods for quantifying the inhibitory potency of these latter compounds.

  3. Development of cholesterol esterase inhibitors as anti-atherosclerotic agents and selectivity studies on monoacylglycerol lipase and fatty acid amide hydrolase
    This project investigates the bile salt-dependent serine hydrolase cholesterol esterase (CEase) which is involved in a variety of inflammatory processes, such as atherosclerosis and rheumatoid arthritis, and, thus, represents a promising target for the development of inhibitors. The hydrolytic activity of CEase is quantified by means of a new fluorometric assay which has been validated by means of literature-known inhibitors on the human, murine and bovine pancreatic enzymes, with the former two proteins being obtained by recombinant expression in HEK-293 EBNA cells (Dato et al., ChemMedChem 2018). To obtain information on the selectivity of identified CEase inhibitors, we investigate the recombinant human serine hydrolases monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) which cleave substrates similar to those of CEase and represent targets on their own in relation to pathological processes, such as cancer, pain, inflammation and cardiovascular diseases. The inhibition of MAGL and FAAH is investigated by means of new assays that have already been established (Dato et al., ChemMedChem 2018, Dato et al., Anal. Biochem. 2018). A first study on the three serine hydrolases revealed that ω-phthalimidoalkyl aryl ureas act as potent inhibitors of murine and human CEase, whereas MAGL and FAAH activities are affected to much lesser extent (Dato et al., ChemMedChem 2018).