20S proteasome enhancers prevent cytotoxic tubulin polymerization-promoting protein induced α-synuclein aggregation.

Synucleinopathies are a class of neurodegenerative diseases defined by the presence of α-synuclein inclusions. The location and composition of these α-synuclein inclusions directly correlate to the disease pattern. The inclusions in Multiple System Atrophy are located predominantly in oligodendrocytes and are rich in a second protein, p25α. P25α plays a key role in neuronal myelination by oligodendrocytes. In healthy oligodendrocytes, there is little to no α-synuclein present. If aberrant α-synuclein is present, p25α leaves the myelin sheaths and quickly co-aggregates with α-synuclein, resulting in the disruption of the cellular process and ultimately cell death. Herein, we report that p25α is susceptible for 20S proteasome-mediated degradation and that p25α induces α-synuclein aggregation, resulting in proteasome impairment and cell death. In addition, we identified small molecules 20S proteasome enhancers that prevent p25α induced α-synuclein fibrilization, restore proteasome impairment, and enhance cell viability.

Development of a Cell-Based AlphaLISA Assay for High-Throughput Screening for Small Molecule Proteasome Modulators.

The balance between protein degradation and protein synthesis is a highly choreographed process generally called proteostasis. Most intracellular protein degradation occurs through the ubiquitin-proteasome system (UPS). This degradation takes place through either a ubiquitin-dependent or a ubiquitin-independent proteasomal pathway. The ubiquitin-independent pathway selectively targets unfolded proteins, including intrinsically disordered proteins (IDPs). Dysregulation of proteolysis can lead to the accumulation of IDPs, seen in the pathogenesis of various diseases, including cancer and neurodegeneration. Therefore, the enhancement of the proteolytic activity of the 20S proteasome using small molecules has been identified as a promising pathway to combat IDP accumulation. Currently, there are a limited number of known small molecules that enhance the activity of the 20S proteasome, and few are observed to exhibit enhanced proteasome activity in cell culture. Herein, we describe the development of a high-throughput screening assay to identify cell-permeable proteasome enhancers by utilizing an AlphaLISA platform that measures the degradation of a GFP conjugated intrinsically disordered protein, ornithine decarboxylase (ODC). Through the screening of the Prestwick and NIH Clinical Libraries, a kinase inhibitor, erlotinib, was identified as a new 20S proteasome enhancer, which enhances the degradation of ODC in cells and α-synuclein in vitro.

Design, Synthesis, and Biological Evaluation of Potent 20S Proteasome Activators for the Potential Treatment of α-Synucleinopathies.

While neurodegenerative diseases affect millions of patients worldwide, there are insufficient available therapeutics to halt or slow down the progression of these diseases. A key pathological feature of several neurodegenerative diseases is the oligomerization and aggregation of specific intrinsically disordered proteins (IDPs) creating neuronal deposits, such as Lewy bodies in Parkinson's disease. Clearance of these pathogenic, aggregation-prone IDPs is mediated by the 20S isoform of the human proteasome. Thus, enhancing the 20S proteasome-mediated proteolysis could be a very useful therapeutic pathway to prevent neurotoxicity. Here, we report the successful development of sub-microM 20S proteasome activators based on a phenothiazine scaffold. This class of compounds prevented the accumulation of pathologically relevant IDPs, such as the pathogenic A53T mutated α-synuclein, in vitro and in mammalian cell lines.