A novel high-throughput screening strategy for targeting alpha-synuclein and other long-lived proteins

Small-molecule high-throughput screening (HTS) campaigns have frequently been used to identify lead molecules that can alter expression of disease-relevant proteins in cell-based assays. However, most cell-based HTS assays require short compound exposure periods to avoid toxicity and ensure that compounds are stable in media for the duration of the exposure. This limits the ability of HTS assays to detect inhibitors of the synthesis of target proteins with long half-lives, which can often exceed the exposure times utilized in most HTS campaigns. One such target is alpha-synuclein (α-syn)-a protein well-known for its pathological aggregation in Parkinson's Disease (PD) and other forms of neurodegeneration known collectively as synucleinopathies. Here, we report the development of an HTS assay using a CRISPR-engineered neuroblastoma cell line expressing a destabilized luciferase reporter inserted at the end of the coding region of the SNCA locus. The resultant destabilized fusion protein exhibited a significant reduction in half-life compared to the endogenous, unmodified α-syn protein, and accurately reported reductions in α-syn levels due to known protein translation inhibitors and specific α-syn siRNAs. The robustness and utility of this approach was shown by using the resulting cell line (dsLuc-Syn) to screen a focused library of 3,192 compounds for reduction of α-syn. These data demonstrate the general utility of converting endogenous loci into destabilized reporter genes capable of identifying inhibitors of gene expression of highly stable proteins even in short-term assays.