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Updated: Sep 11, 2025
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
Cell based high-throughput screening for small molecule inhibitors of ATE1
Claudia McCown1, Evan Ambrose2, Devang Patel3
1Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, USA.
Abstract:
Arginyltransferase 1 (ATE1) catalyzes post-translational arginylation, a process implicated in protein stability, cellular function, and disease pathology. Dysregulated arginylation is associated with neurodegenerative disorders, cancer, and inflammation. Particularly, the increase of ATE1 activity has been shown to cause cell death in response to acute stress, highlighting ATE1 as a promising therapeutic target. Despite its therapeutic relevance, no selective small-molecule inhibitors of ATE1 have been FDA-approved at this time, with previous screening efforts yielding compounds with high promiscuity and toxicity. This, in part, is due to the lack of assays that would accommodate large-scale screening for effective and safe ATE1-inhibitors. To address this challenge, we developed a cell-based high-throughput screening (HTS) assay utilizing a fluorescent reporter system based on an ATE1 substrate peptide fused to a fluorescence protein and co-expressed alongside another fluorescence protein for normalization. The assay enables real-time quantification of ATE1 activity by monitoring arginylation-dependent protein degradation within intact cells, measured by the ratio of the two fluorescence signals. We validated the assay in 96-well and 1536-well plate formats, demonstrating its scalability and robustness through key performance metrics, including Z'-factor and signal-to-background ratio. A pilot screen of a Library of Pharmacologically Active Compounds (LOPAC®1280) was performed to evaluate this approach. This study establishes a scalable and selective platform for discovering ATE1 inhibitors, paving the way for future therapeutic development targeting ATE1-mediated disease pathways.
Insights
Researchers developed a new cell-based assay to find safe and effective Arginyltransferase 1 (ATE1) inhibitors. This high-throughput screening platform advances the discovery of therapeutics for diseases linked to dysregulated arginylation.
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.
