Hijacking Post-translational Arginylation for Targeted Protein Degradation

Project summary Posttranslational arginylation on proteins installed by arginyltransferase ATE1 is a critical modification for cancer progression and metastasis. ATE1 is a beneficiary regulator, inhibition of which often results in elevated metastasis (e.g., prostate cancer) and poorer outcomes. Unfortunately, the physiological roles of ATE1 (and its

arginylation activity) in cancers remain poorly understood, mostly due to the lack of analytical methods to discover the protein substrates of ATE1 from cancer samples. Arginylation regulates protein half-lives in cellular systems through the Arg/N-degron pathway and represents one of the three (arginine, proline, and acetylation)

N-terminal degradation signals (N-degrons) for protein turnover. Here, we aim to discover the protein substrates of ATE1 in cancers for the development of targeted protein degradation (TPD) by hijacking ATE1 and its arginylation activity. We have developed an unbiased proteomic profiling method to discover the ATE1

substrates and their precise arginylation sites, the method is termed activity-based arginylation profiling (ABAP). Using ABAP, we have successfully profiled arginylation from 12 samples including 4 cancer cell lines. In this proposal, we would like to establish a library of cancerous substrates from commonly used cancer cell

lines using the NCI60 pellets. To take advantage of arginylation for targeted protein degradation against cancers, we have engaged ATE1 with protein of interest (POI) through our model Halo-FKBP system. We discovered, for the first time, that engagement of ATE1 with cancer targets (e.g., SHOC2 and RAD52) successfully induced POI

degradation in cellular models. We termed this technology arginylation targeting chimera (ArgTAC). Our preliminary data on degradation data from the first few POIs encouraged us to further develop this technology as a novel approach in the TPD field. We will apply a series of state-of-the-art approaches including proteomics

and molecular biology to characterize the mechanism of action of induced POI degradation after engagement with ATE1. A series of molecular events will be characterized including cellular proximity between ATE1 and POI, the arginylation and polyubiquitination of POI, and the POI degradation dependency on UBR E3 ligases

and proteasome. To achieve optimal degradation of broad cancer targets, we will expand our tool compounds designed for the Halo-FKBP system. We will also further develop new ATE1 binders/recruiters through peptide optimization based on LIAT1 (ligand of ATE1) binding domain to ATE1 using screening and structure/computer

modeling-based drug discovery. More ArgTACs targeting other POIs will be synthesized and tested for degradation activities in cell models to expand the application of ArgTACs. The proposed ABAP and ArgTAC platform here will likely generate a catalog of oncoproteins regulated by arginylation, and offer a general and

revolutionary TPD technology targeting oncoprotein degradation as a therapeutic strategy for cancers.