Cell based high-throughput screening for small molecule inhibitors of ATE1

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Arginyltransferase 1 (ATE1) mediates post-translational arginylation, influencing protein stability and cellular functions.
• Dysregulated arginylation is linked to neurodegenerative diseases, cancer, and inflammation, with elevated ATE1 activity causing cell death under stress.
• Current challenges in ATE1 inhibitor development stem from a lack of selective compounds and suitable high-throughput screening (HTS) assays.

Purpose of the Study:

• To develop a robust and scalable cell-based HTS assay for identifying selective small-molecule inhibitors of Arginyltransferase 1 (ATE1).
• To establish a reliable platform for discovering novel ATE1 inhibitors, addressing the limitations of previous screening efforts.

Main Methods:

• Developed a fluorescent reporter system using an ATE1 substrate peptide fused to a reporter protein and a normalization protein.
• Quantified ATE1 activity in real-time by measuring arginylation-dependent protein degradation via fluorescence ratios in intact cells.
• Validated the assay in 96-well and 1536-well formats, assessing scalability and performance using Z'-factor and signal-to-background ratios.

Main Results:

• Successfully established a cell-based HTS assay for quantifying ATE1 activity.
• Demonstrated the assay's scalability, robustness, and suitability for large-scale screening.
• Validated the assay's performance through key metrics and a pilot screen of the LOPAC®1280 library.

Conclusions:

• This study presents a validated, scalable, and selective platform for discovering novel Arginyltransferase 1 (ATE1) inhibitors.
• The developed assay facilitates the identification of effective and safe ATE1 inhibitors, crucial for therapeutic development.
• This platform opens new avenues for targeting ATE1-mediated pathways in various disease contexts.