Jonathan Benn

Selective degradation of pathological protein aggregates while sparing monomeric forms is of major therapeutic interest. The E3 ligase tripartite motif-containing protein 21 (TRIM21) degrades antibody-bound proteins in an assembly state-specific manner due to the requirement of TRIM21 RING domain clustering for activation, yet effective targeting of intracellular assemblies remains challenging. Here, we fused the RING domain of TRIM21 to a target-specific nanobody to create intracellularly expressed constructs capable of selectively degrading assembled proteins. We evaluated this approach against green fluorescent protein-tagged histone 2B (H2B-GFP) and tau, a protein that undergoes pathological aggregation in Alzheimer's and other neurodegenerative diseases. RING-nanobody degraders prevented or reversed tau aggregation in culture and in vivo, with minimal impact on monomeric tau. This approach may have therapeutic potential for the many disorders driven by intracellular protein aggregation.

Protein aggregation causes a wide range of neurodegenerative diseases. Targeting and removing aggregates, but not the functional protein, is a considerable therapeutic challenge. Here, we describe a therapeutic strategy called "RING-Bait," which employs an aggregating protein sequence combined with an E3 ubiquitin ligase. RING-Bait is recruited into aggregates, whereupon clustering dimerizes the RING domain and activates its E3 function, resulting in the degradation of the aggregate complex. We exemplify this concept by demonstrating the specific degradation of tau aggregates while sparing soluble tau. Unlike immunotherapy, RING-Bait is effective against both seeded and cell-autonomous aggregation. RING-Bait removed tau aggregates seeded from Alzheimer's disease (AD) and progressive supranuclear palsy (PSP) brain extracts and was also effective in primary neurons. We used a brain-penetrant adeno-associated virus (AAV) to treat P301S tau transgenic mice, reducing tau pathology and improving motor function. A RING-Bait strategy could be applied to other neurodegenerative proteinopathies by replacing the Bait sequence to match the target aggregate.

TRIM21 is a unique E3 ligase that uses a clustering-based activation mechanism to degrade complex multimeric substrates. This activity underpins the targeted protein degradation technology Trim-Away and genetically encoded degraders that selectively target aggregated tau protein and prevent tauopathy. Here we describe small molecules that mimic TRIM21's natural epitope and function as either effective inhibitors or potent and selective degraders called TRIMTACs. TRIMTACs mediate degradation as rapidly as PROTACs but can also selectively degrade specific protein pools depending on assembly state. We demonstrate the utility of this state-specific degradation by selectively removing the pro-inflammatory signalling protein Myd88 when assembled into the Myddosome and the cell-death protein RIPK3 when polymerised into the Necrosome. We further show that TRIMTACs can inhibit seeded tau aggregation under conditions where a PROTAC is ineffective. These results highlight that TRIM21's clustering-based activation can be exploited by small molecule degraders to carry out state-selective degradation of therapeutic targets.