Identifizierung und Charakterisierung von niedermolekularen Modulatoren der Protein-Arginin-Methyltransferasen

Protein arginine methyltransferases (PRMTs) comprise an enzyme family of nine members responsible for the methylation of arginine residues in a plethora of substrate proteins in mammalian cells. The enzymes catalyze the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the guanidino group in arginine side chains resulting in three distinct methylation states: monomethylation (MMA), asymmetric dimethylation (ADMA) and symmetric dimethylation (SDMA). Arginine methylation is involved in the regulation of essential cellular processes, including transcription, RNA processing, DNA damage response and signal transduction. Any dysregulation in expression level and activity of PRMTs can lead to diseases, including cancer. Therefore, PRMTs are considered as promising targets in the context of pharmaceutical drug development. However, the majority of so far reported small molecule modulators of PRMTs are neither selective nor cell-active. This thesis aimed to identify selective and bio-available compounds targeting PRMTs by screening a library of 88 small molecule modulators predicted to specifically target the active sites of a subset of PRMT family members. The efficacy of these compounds in either inhibiting or activating the catalysis of the respective PRMTs was initially evaluated through in vitro screening, by employing an established quantitative fluorescence-based enzymatic assay using an artificial substrate. For the first time, molecules enhancing the enzymatic activity of PRMTs, namely PRMT1, were identified among the tested compounds. Such PRMT1 activator compounds are regarded to provide a therapeutic benefit in tumor entities, such as subtypes of the pancreatic ductal adenocarcinoma (PDAC), in which the tumor suppressor function of PRMT1 prevails and promotes chemotherapy-induced apoptosis. Accordingly, I continued to characterize the PRMT1 activator lead compound named TR-07 and its derivatives in the course of this thesis with regard to (1.) their selectivity and potential impact on the enzymatic activity of other PRMT family members, (2.) their efficacy on enhancing PRMT1’s activity towards natural cellular substrates, (3.) their binding mode and mechanism of action (MOA) through Fluorescence Polarization (FP), Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) analysis and pull-down assays, and (4.) their bio-availability and in vivo efficacy in PDAC cell cultures and PDAC xenograft mouse models. In summary, the results of this thesis identified TR-07 as a selective and cell-active PRMT1 activator compound that augments gemcitabine-induced apoptosis of PDAC tumor cells, unravelling for the first time the therapeutic potential and benefit of this novel class of PRMT1 modulators.