Identification of Piperlongumine (PL) as a new E3 ligase ligand to induce targeted protein degradation

PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. Through competitive activity-based protein profiling, we found that piperlongumine (PL), a natural product, binds multiple E3 ligases. To evaluate whether PL can be used as an E3 ligase ligand, we generated a series of PL and SNS-032 (a selective CDK9 inhibitor) conjugates and found that the lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome dependent manner. In addition, 955 is more potent than SNS-032 against various tumor cells in vitro. Through TurboID-based proteomics and mechanistic studies, we identified KEAP1 as the E3 ligase recruited by PL to degrade CDK9. These findings demonstrate that PL is a novel E3 ligase ligand that can be used to generate potent anticancer PROTACs. ### Competing Interest Statement J.P., Y.X., X.L., W.H., Y.Y., G.Z., D.Z., and D.L. are co-inventors of a pending patent application for the discovery of PL as an E3 ligase ligand. G.Z. and D.Z. are co-founders and have equity of Dialectic Therapeutics that develops BCL-xL/2 PROTACs for cancer treatment. J.D.L. is a scientific advisor to Dialectic Therapeutics. All other authors declare no conflict of interest.