A new method to identify interacting proteins in drug development


Protein degraders, which induce protein degradation of target proteins, are expected to be the “next generation drugs” because they can remove disease-related proteins from cells. Protein degraders, such as thalidomide and its derivatives (immunomodulating drugs/IMiDs), induce the degradation of specific proteins by binding to cereblon (CRBN), which is a component of the E3 ubiquitin ligase complex, a proteolytic enzyme. IMiDs function as “molecular glue”, inducing protein degradation by recruiting target proteins to the E3 ubiquitin ligase and are referred to as molecular glue-like proteolytic inducers. Due to the clinical success of IMiDs, molecular glue-like protein degraders have become attractive compounds for use in the fight against many diseases. Additionally, proteolysis-targeting chimeras (PROTAC), which are chimeric compounds consisting of a compound that binds to an E3 ubiquitin ligase (E3 binder), such as IMiDs, and a compound that binds to a target protein (target binder), are now under development. The development of PROTACs should allow the targeting of proteins that have been difficult to target by therapeutic agents in the past. Therefore, the analysis of proteins that interact with E3 ubiquitin ligase and are induced to degrade is important for the development and clinical application of protein degraders. In 2020, this research group developed AirID (ancestral BirA for identification of proximity-dependent biotin) as a marker enzyme of proximal-dependent biotinylation useful for the analysis of protein-protein interactions. Therefore, we sought to develop a comprehensive analytical method to identify interacting proteins in a protein degrader-dependent manner.

In this study, we have shown that AirID-fused E3 ubiquitin ligase, such as AirID-CRBN, can biotinylate target proteins of protein degraders in cells. Additionally, we have shown that enrichment of biotinylated peptides using the avidin-like protein tamabidine 2-REV is useful for the analysis of protein degrader-dependent interactions by mass spectrometry. Using this method of analysis, we identified ZMYM2 (MYM zinc finger-like protein 2) as a target protein of pomalidomide, which is a derivative of thalidomide. In addition, the ZMYM2-FGFR1 fusion protein responsible for hematological cancer is also degraded by pomalidomide. Importantly, we have confirmed that this analytical method can be used for known protein degraders such as Indisulam and PROTACs. These results indicate that the fusion of AirID with the E3 ubiquitin ligase allows the comprehensive analysis of interacting proteins of protein degraders.

This analysis method using AirID can be used for the analysis of various protein degraders that will be developed in the future. Currently, various protein degraders are being actively developed for various diseases, and some of them are currently in the clinical stage. However, given the potent effects of protein degraders, there is a need to assess their interactions with proteins that are not intended to cause side effects. In fact, the results of this study indicated that various proteins interact with E3 ligase in a protein degrader-dependent manner. Based on these results, it is expected that the use of this analytical method will lead to the elucidation of the mechanism of action of protein degraders and the development of compounds that avoid side effects.

– This press release was provided by Ehime University


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