Signal Transduction and Targeted Therapy - From probe to prodrug: immunoproteasome activity as a trigger for disease-selective therapeutics

Targeted protein degradation (TPD) has emerged as a powerful strategy to eliminate disease-relevant proteins, yet current approaches remain largely constrained to hijacking ubiquitin ligases. We previously introduced ByeTACs, bifunctional molecules that directly recruit proteins to the proteasome for E-ligase independent degradation. Here, we report “Truly” degraders, a new class of dual-mechanism molecules that combine a ligand for the proteasomal receptor Rpn13 with a ligand for cereblon (CRBN) to simultaneously engage both ubiquitin-independent and ubiquitin-dependent degradation pathways. Structure-guided design identified an optimal linker length that supports efficient substrate processing, with the PEG4 derivative (Truly-4) inducing robust depletion of both Rpn13 and CRBN in several cancer cell types. Remarkably, Truly-4 is the first noncovalent small molecule shown to degrade full-length Rpn13, a target previously approached using covalent or domain-restricted strategies. Mechanistic studies confirmed that degradation of Rpn13 proceeds via CRBN-dependent E3 ligase activity, whereas CRBN degradation occurs through an E-ligase independent process, consistent with a ByeTAC mechanism. Importantly, Truly-4 induces selective cytotoxicity in hematologic and solid cancer cell lines but not in healthy cells, despite comparable Rpn13 depletion, indicating that dual degradation can uncouple target engagement from toxicity. These findings establish a generalizable framework for engineering bifunctional degraders that program the proteasome to execute parallel degradation mechanisms and highlight proteasome receptors as druggable nodes for selective destruction of disease-relevant proteins.

Targeted protein degradation is an emerging therapeutic strategy that leverages the cell’s natural protein clearance pathways to eliminate proteins of interest (POIs). One common approach involves bif...

Ligand discovery of nonenzymatic proteins can be accomplished through screening methods utilizing libraries comprising small molecules, peptides, and peptidomimetics. Incorporating peptoids, which are...

The development of targeted protein degradation by recruiting a protein of interest to a ubiquitin ligase to facilitate its degradation has become a powerful therapeutic tool. The potential of this approach is limited to proteins that can be readily ubiquitinated and relies on having a ligand with the various E3 ligases. Here, we describe a new methodology for targeted protein degradation that directly recruits a protein of interest to the proteasome for degradation. We generated bifunctional molecules that incorporate a small molecule ligand into a subunit on the 26S proteasome that recruits the protein directly for degradation. ByeTAC degradation requires binding to Rpn-13, a nonessential ubiquitin receptor of the 26S proteasome, and the protein of interest and does not have to rely on the E ligase cascade for ubiquitination. The ByeTAC methodology demonstrates the application of directly recruiting a protein to the proteasome via interactions with Rpn-13 for degradation.

Regulators of G protein signaling (RGS) proteins negatively regulate signaling through G protein-coupled receptors, and reduced RGS protein function i…

IRE1α is an ER protein involved in the unfolded protein response (UPR) and dysregulation of the ER stress pathway has been implicated in several diseases. Inhibitors of the cytoplasmic endonuclease or...

Immunoproteasome (iCP) is a proteasome isoform that is expressed under inflammatory conditions such as cytokine interferon-γ exposure. The iCP has different catalytic subunits other than the standard CP (standard core particle), allowing the production of major histocompatibility complex class I (MHC-I) compatible peptides for eventual T-cell activation. We have previously reported the design of a fluorescent probe that monitors iCP activity in cells called TBZ-1, and we applied TBZ-1’s iCP recognition sequence for prodrug release into iCP-active cells. Here, we demonstrate a proof-of-concept of the iCP as a prodrug release enzyme. The “payload” we utilized was a toxic moiety, doxorubicin, and a degrader for transcription factor, BRD4. Both examples show that iCP activity is required to elicit cell death or degradation of BRD4. This report highlights that the iCP is a viable prodrug target, and its activity can be used to release a variety of cargo in cells expressing the iCP.

National Institutes of Health grants generated almost $95 billion in economic activity nationwide in FY 2024 according to a new report by United for Medical Research.

Microbial infections lead to drastic changes in the architecture and metabolism of the host cell. The inaugural FEBS EMBO Cell Biology of Host-Pathogen Interaction Advanced Course explores the mechan…

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Degrons are short amino acids sequences that can facilitate the degradation of protein substrates. They can be classified as either ubiquitin-dependent or -independent based on their interactions with...

The ONX-0914-alkyne probe described here allows for the conjugation to a variety of fluorophores through click chemistry. It can then be used to visualize immunoproteasome activity in gel-based, micr....