In vitro analysis of drug‐induced protein mislocalization and accumulation

Unexpected protein inclusions in rat proximal tubule epithelial cells are a reoccurring phenomenon in non‐clinical safety studies that might interfere with the drug development outcome, even when relevance to humans has not been assessed. Propiverine, a frequently‐prescribed medication for diverse bladder control diseases, gained only limited marketing authorization due to large cytosolic and intranuclear protein inclusions in rat kidney. Although it was shown that these protein inclusions mainly consist of the peroxisomal enzyme D‐amino acid oxidase (DAAO), the role and relevance of DAAO accumulation in humans remain unknown. This thesis aimed to provide a better understanding of the principle mechanism(s) of propiverine‐induced DAAO mislocalization and accumulation. For that purpose, a new in vitro model of EYFP‐DAAO expressing human and rat renal cell lines was established, thoroughly characterized and successfully applied to analyze the protein interaction network of DAAO, manipulate subcellular trafficking, and monitor protein dynamics in vitro. Although nuclear localization of a peroxisomal protein appears to be exceptional, manipulation of diverse active nuclear transport mechanisms did not change DAAO localization per se. In contrast, interference with the peroxisomal trafficking, i.e. deletion of the peroxisomal targeting signal type 1 (PTS1) or PEX5 knock‐down, resulted in passive diffusion of EYFP‐DAAO into the nucleus. The latter not only contradicts the often‐cited nuclear diffusion limit of 40 kDa, but also supports the hypothesis that propiverine and/or its metabolite(s) may interfere with peroxisomal transport and/or import, thereby provoking nuclear (mis)localization. Subsequently, synthesizing a biotin‐modified propiverine derivative for a Co‐IP coupled LC‐MS/MS analysis allowed for the identification of propiverine‐specific interactors from rat kidney homogenate. Besides binding to chaperones, that are DAAO interaction partners and involved in peroxisomal trafficking, propiverine was found to ...