Two Mechanisms One Molecule: Developing a 'Truly' Bifunctional Degrader Targeting Rpn13 and CRBN

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Targeted protein degradation (TPD) is a key therapeutic strategy, but current methods often rely on hijacking ubiquitin ligases.
• Previous work introduced ByeTACs for E-ligase independent protein degradation.
• Existing TPD approaches have limitations in scope and mechanism.

Purpose of the Study:

• To develop a new class of bifunctional molecules, termed "Truly" degraders, that utilize dual degradation mechanisms.
• To investigate the efficacy of Truly degraders in simultaneously engaging ubiquitin-dependent and -independent degradation pathways.
• To explore the potential of Truly degraders in selective cancer therapy.

Main Methods:

• Design and synthesis of "Truly" degraders, bifunctional molecules with ligands for Rpn13 and cereblon (CRBN).
• Structure-guided optimization of linker length for efficient substrate processing.
• Evaluation of Truly-4's degradation activity in various cancer cell lines.
• Mechanistic studies to elucidate degradation pathways for Rpn13 and CRBN.
• Assessment of selective cytotoxicity in cancer versus healthy cells.

Main Results:

• Truly degraders, specifically Truly-4, effectively depleted Rpn13 and CRBN in cancer cells.
• Truly-4 achieved noncovalent degradation of full-length Rpn13, a novel approach.
• Degradation of Rpn13 was CRBN-dependent, while CRBN degradation followed an E-ligase independent pathway.
• Truly-4 exhibited selective cytotoxicity in cancer cells without harming healthy cells, despite similar Rpn13 depletion.

Conclusions:

• Truly degraders represent a new class of bifunctional molecules enabling parallel proteasomal degradation mechanisms.
• This dual-mechanism approach can uncouple target engagement from toxicity, enhancing therapeutic selectivity.
• Proteasome receptors like Rpn13 are viable targets for developing novel, selective protein degraders.