Research summary

Since the development and clinical application of the proteasome inhibitor, bortezomib as an anticancer agent, attention has been focused on the potential efficacy of proteasome inhibitors. However, there have been some cases of resistance to bortezomib, and the development of proteasome inhibitors with new mechanisms of action (MOA) is eagerly awaited. Most of the conventional proteasome inhibitors target the β1, 2, and 5 subunits of the 20S core particle, which exhibit protease activity, and while it is essential to create new target proteins. However, there has been no way to efficiently screening such compounds that regulate novel targets.
In this study, we devised a completely new detection strategy in which the probe molecule itself is regarded as a substrate of the ubiquitin-proteasome pathway, and becomes fluorescent only when all of the following processes are in progress: i) ubiquitination, ii) incorporation into the 26S proteasome, and iii) proteasomal degradation. Such "fluorescent small molecule probe" can observe the entire proteasomal degradation process in live cells in a single step. We will also apply the developed fluorescent probes to in cellulo phenotypic screening to search for and develop proteasome inhibitors with unprecedented targets and MOA, and demonstrate their efficacy as anticancer drug leads.
In addition, a key question in the development of probes for this research is "does the substrate for ubiquitination have to be a protein? In other words, conventional ubiquitination substrates are usually proteins, however, peptide chains containing lysine residues, lysine residues alone, and even aliphatic amino groups may be sufficient as substrates. By clarifying this question in this study, we may be able to expand the range of application of degradation manipulation technology using PROTACs in chemical biology research, and also present the necessity and opportunity to broaden the view of molecules that are degraded by the ubiquitin-proteasome system in vivo. Furthermore, if we can identify target molecules and regulatory small molecules that have not been identified so far, we will be able to expand the scope of proteasome drug discovery and increase the probability of success in future drug discovery research.