Abstract

P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP) are ATP-binding cassette (ABC) transporters expressed in the physiological barriers of the human body. Due to their localization and broad substrate specificity, they influence the absorption, distribution and excretion of chemically diverse drugs and small molecules. The disruption of their transport function by drug interactions can lead to altered exposure to their substrates and hence, to adverse effects. Therefore, it is necessary to identify transporter inhibitors at the early stage of drug development.

This thesis focuses on understanding the molecular basis of P-gp and BCRP inhibition by small molecules with an emphasis on in vitro methods. For this, cell-based fluorescent substrate accumulation assays were set up, optimized and employed in three consecutive studies. The first study explored the selectivity of propafenone-type inhibitors on P-gp and BCRP using a combined approach of chemical synthesis, in silico prediction and in vitro testing. Secondly, virtual screening of the Drugbank database for BCRP inhibition was performed and subsequent biological testing of the selected small molecules identified two new inhibitors. The third study utilized synthesis, in vitro testing, as well as quantitative structure-activity relationship (QSAR) and molecular docking analysis to investigate the influence of lipophilicity and H-bond acceptor properties on the P-gp inhibitory activity of propafenone analogues. Finally, in order to explore the relation between the dissociation kinetics of propafenone-type compounds and their inhibitory potential, we focused on developing a competitive radioligand binding assay for P-gp.

Taken together, in vitro methods helped to study the inhibition of P-gp and BCRP by small molecules. Furthermore, this thesis offers easy-to-implement biological assays for the community to test the inhibition of these transporters. Our studies confirmed previous findings as well as extended our knowledge on the chemical characteristics possessed by P-gp and/or BCRP inhibitors, however, the exact molecular mechanisms of ABC transporter inhibition still remain elusive.