A Mammalian High-Throughput Screen for AI-Designed Peptide-Guided Protein Degraders
Targeted protein degradation (TPD) offers a route to eliminate disease-driving proteins that remain inaccessible to conventional inhibitors. However, degrader discovery remains low-throughput, labor-intensive, and dependent on randomized libraries or non-human display systems, limiting functional selection in mammalian cells. Here, we present a high-throughput, human cell-based platform for…
A new high-throughput screening method has been developed for identifying peptide-guided ubiquibodies (uAbs) that can degrader targeted proteins within mammalian cells. This platform aims to overcome the limitations of current low-throughput, labor-intensive methods reliant on randomized libraries or non-human display systems.
The system employs genetically encodable, doxycycline-inducible degraders that fuse peptide guides created by protein language models to the CHIP{Delta}TPR E3 ligase domain. This forms a modular, CRISPR-like setup for precise targeted protein degradation (TPD).
In the screening process, a pooled uAb library is introduced into fluorescent reporter cell lines specific to each target. Cells with decreased target abundance are isolated using fluorescence-activated cell sorting (FACS), and enriched peptide guides are recovered through sequencing. This approach was tested on several targets, including beta-catenin, GFAP, and EWS::FLI1.
For beta-catenin, enriched uAbs successfully reduced endogenous beta-catenin abundance and Wnt signaling in DLD1 cells. When GFAP-directed uAbs were used, they decreased endogenous GFAP abundance and cell viability in U251 glioblastoma cells. Additionally, EWS::FLI1-directed uAbs reduced the fusion oncoprotein abundance, suppressed EWSAT1 expression, and increased apoptosis in Ewing sarcoma models.
Lastly, a screen using endogenously tagged GATA2 identified uAbs that can lower GATA2 under native genomic regulation. This high-throughput, human cell-based platform successfully connects generative peptide design to functional mammalian selection, providing a scalable strategy for CRISPR-like proteome perturbation.
Written by urgent.news from bioRxiv's reporting — not their text. Machine-written — may contain errors; check the original before relying on it.
