Small-molecule degraders promote interactions between a protein of interest (POI) and an E3 ubiquitin ligase, leading to degradation of the POI. Degrader compounds such as molecular glues were identified through phenotypic screening in a target-agnostic manner. Although fluorescent or luminescent readouts have been previously used for high-throughput screening campaigns, the subtle quantitative changes make selection of potential candidate degraders difficult. Chu et al. developed an assay called DEFUSE (‘death fusion escaper’) that uses the rescue of cell death as a qualitative readout for compound-mediated protein degradation. DEFUSE relies on construction of a POI target fused with FKBP12–caspase 9 that promotes rapid cell death by AP1903-mediated caspase 9 activation. Compounds that induce degradation of the POI should be resistant to the effects of AP1903 due to the loss of FKBP12–caspase 9, creating a clear cellular survival–death readout. The design of DEFUSE enables it to be applied to a variety of cytoplasmic, nuclear and viral proteins. Chu et al. used DEFUSE to screen a library of natural and synthetic compounds for degraders of SKP2, identifying SKPer1 as a selective degrader in cells. SKPer1 required the SKP2 F-box for degradation, with mutation of three critical lysine residues in the F-box domain producing an SKPer1-resistant variant. A ten amino acid sequence from the SKP2 F-box domain acted as a degradation tag, with fusion to KRAS, MYC and CREPT resulting in SKPer1-mediated degradation. DEFUSE screening using an RNA interference library against genes involved in ubiquitin-mediated degradation identified STUB1 as the E3 ligase responsible for SKPer1-induced SKP2 degradation. SKPer1 was determined to act as a molecular glue, inducing a complex containing SKP2 and STUB1. Overall, DEFUSE offers a powerful tool to enable high-throughput identification of degraders in a target-defined manner.
Original reference: Nat. Biotechnol. https://doi.org/10.1038/s41587-025-02793-8 (2025)
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