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.