Development of cellular HTS for 20S proteasome enhancers

This project details the development and application of a cellular high-throughput screening (HTS) strategy aimed at discovering small-molecule enhancers of the 20S proteasome to counter pathological α-synuclein aggregation driven by tubulin polymerization-promoting protein (TP/p25α). The work addresses a pressing unmet need in synucleinopathies such as Multiple System Atrophy (MSA) and Parkinson's disease, where α-synuclein accumulation and aggregation disrupt proteostasis and lead to neurodegeneration. Recognizing that the uncapped 20S proteasome degrades unfolded proteins independently of ubiquitination, the COM developed an approach to identify compounds that enhance 20S activity in cells, thereby promoting clearance of intrinsically disordered proteins (IDPs) implicated in disease.

The research builds on prior design of small-molecule 20S enhancers inspired by the neuroleptic Chlorpromazine (CPZ). That predecessor compound demonstrated in vitro activation but limited cellular efficacy, a shortcoming attributed to the phenothiazine core's metabolic liabilities and promiscuity. To overcome these limitations, the COM synthesized novel small molecules that retained the N,N-bis(4-fluorophenyl)amide tail of the optimized CPZ derivative while replacing the problematic phenothiazine core with a variety of heterocyclic scaffolds. These new molecules exhibited substantially improved cellular potency, preventing accumulation of pathological A53T α-synuclein at submicromolar concentrations (<1 μM).

A complementary proteasomal impairment model was developed to evaluate the pathological interaction between p25α and α-synuclein in cells. Using this model, the COM identified p25α as a substrate for 20S proteasome-mediated degradation and demonstrated that p25α promotes rapid co-aggregation with α-synuclein, driving proteasome impairment and cell death. The small-molecule 20S proteasome enhancers discovered through the cellular HTS prevented p25α-induced α-synuclein fibrillization, restored proteasome activity compromised by aggregation, and improved cell viability in these models. These findings were supported by assays showing reduced α-synuclein fibrillization and restoration of proteasome function following treatment with the enhancers.

Collectively, the COM's work provides an integrated discovery-to-validation pathway: rational small-molecule design to improve cellular effectiveness, a cellular HTS paradigm for identifying 20S enhancers, and a disease-relevant cellular model linking p25α-driven aggregation to proteasome impairment and toxicity. The results demonstrate that enhancing 20S proteasome activity can mitigate key pathological processes in synucleinopathies clearing aggregation-prone proteins, restoring proteasomal capacity, and preventing aggregation-driven cytotoxicity. This approach offers a promising therapeutic strategy distinct from direct targeting of α-synuclein, which has proven difficult due to the protein's conformational flexibility. The COM's cellular HTS and the identified heterocyclic 20S enhancers thus represent valuable tools and leads for further preclinical development aimed at proteostasis restoration in neurodegenerative disease.