The Search for Inhibitors of the Ubiquitin-proteasome System from Natural Sources by Cell-based Screening in Reporter-expressing Cells

The ubiquitin-proteasome system (UPS) regulates cellular protein degradation to maintain protein homeostasis (proteostasis). The UPS mainly consists of three steps, ubiquitination, deubiquitination, and protein degradation. In the ubiquitination process, a series of enzymes, the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3), catalyze the formation of a polyubiquitin chain on target proteins. Prior to the degradation of the polyubiquitinated proteins by the proteasome, deubiquitination enzymes remove this polyubiquitin chain and cleave it into monoubiquitin molecules. The UPS plays a key role in controlling proteostasis and multiple signaling pathways. Dysfunction of the UPS has been implicated in the development of various diseases, including cancer, neurodegenerative diseases, and autoimmune diseases. Therefore, UPS inhibitors that disrupt protein degradation are promising as drug leads. In our study, we searched for natural products (NPs) that inhibit UPS-dependent proteolysis using dual-reporter HeLa cells expressing UbG76V-green fluorescent protein (GFP) and the oxygen-dependent degradation domain of HIF1α fused to luciferase (ODD-Luc). Here we report our research on NPs that inhibit the UPS using these cell-based reporter assays.