Design, synthesis and biological evaluation of tool compounds for the cellular investigation of deubiquitinases

Abstract

The ubiquitin proteasomal system, is an important mediator of protein homeostasis through ubiquitination of substrates, but also coordinates various other functions throughout the cell. Deubiquitinases (DUBs) are essential regulators within this system. They are a specialized class of proteases that can cleave ubiquitin from its substrates and their dysregulation is involved in the development of various diseases. However, the understanding regarding their substrates, cellular localization, their involvement in cellular signaling and structural features remains limited. Despite the enormous therapeutical potential only a few DUB inhibitors are known to date. Therefore, novel tool compounds are of urgent need to enhance the understanding of DUBs in a cellular setting. This thesis describes the discovery, chemical synthesis and biological evaluation of such tool compounds for investigating DUBs in a living environment. To accomplish this, literature-known compounds were resynthesized to introduce alkyne tags to utilize their use as activity-based probes (ABPs) in cell-based systems. An intensive cellular characterization of a dedicated small molecule ABP library targeting DUBs resulted in the discovery of the chemogenomic pair of probes GK13S and GK16S, usable for the selective investigation of the DUB UCHL1 in a living environment. The probes were used to unravel potential substrates and a cellular phenotype after the inhibition of UCHL1. Furthermore, this thesis describes the synthesis and cellular evaluation of PROTAC molecules to study DUBs beyond inhibition. Altogether, this thesis should foster the understanding of DUBs and serve as a blueprint for the synthesis of further probe molecules to study these enzymes.