Utilization of Functional Degradation Pathways for the Characterization of Ubiquitin-Proteasome System Tool Compounds.

The ubiquitin-proteasome system (UPS) is a highly regulated enzymatic machinery responsible for selective protein degradation, which is an essential process for maintaining cellular proteostasis, signaling, and homeostasis. Dysregulation of the UPS has been implicated in a variety of human diseases, including cancer, neurodegeneration, and inflammatory disorders. Recently, small molecule degraders such as PROTACs and molecular glues emerged that exploit the UPS to selectively degrade disease-associated proteins, and these new modalities are increasingly interesting for drug development and target validation. However, due to the complex multistep pathway, validation of the molecular mechanism of action remains challenging. A number of tool compounds have been developed that can be used to demonstrate that observed PROTAC-induced target degradation relies on the UPS. Despite the availability of these tool compounds, there is no consensus in the community regarding the most appropriate use for these compounds, in particular their dosing and best timepoints used for such functional studies. In this chapter, we present a protocol for the best practice use of UPS-targeting tool compounds to further understand their molecular mechanism of action within the CRBN- and VHL-mediated degradation pathways in human cells. We make use of the HiBiT system supported by a cell viability assay to enable identification of effective, nontoxic concentrations for target degradation rescue experiments. This protocol establishes a framework for performing functional studies and guides the informed, effective use of UPS tool compounds in both research and therapeutic discovery.