ngaging the mostly undruggable proteome to uncover new disease therapies not only requires technological innovations that facilitate rapid discovery of ligandable hotspots across the proteome but also demands new therapeutic modalities that alter protein function through novel mechanisms 1,2 . Targeted protein degradation (TPD) tackles the undruggable proteome by targeting specific proteins for ubiquitination and proteasomal degradation. One major class of small-molecule effectors of TPD, proteolysis-targeting chimeras (PROTACs), are heterobifunctional molecules that consist of an E3 ligase recruiter linked to a protein-targeting ligand to induce the formation of ternary complexes that bring together an E3 ubiquitin ligase and the target protein as a neo-substrate [3][4][5] . PROTACs have enabled the targeted and specific degradation of numerous disease-causing proteins in cells 3,6 . New approaches for TPD have also arisen that exploit endosomal and lysosomal degradation pathways with lysosome-targeting chimeras or autophagy with autophagy-targeting chimeras 7,8 . New approaches for chemically induced proximity beyond degradation have also been developed in recent years, including targeted phosphorylation with phosphorylation-inducing chimeric small molecules and targeted dephosphorylation, but no small-molecule-based induced proximity approaches exist for targeted deubiquitination and subsequent stabilization of proteins 9,10 .Active ubiquitination and degradation of proteins is the root cause of several classes of diseases, including many tumor suppressors in cancer (for example, TP53, CDKN1A, CDN1C and BAX), and mutated and misfolded proteins, such as ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis or glucokinase in pancreatic cells in maturity-onset diabetes of the young type 2. In these cases, a TPS therapeutic strategy, rather than degradation, would be beneficial [11][12][13][14] . Analogous to TPD, we hypothesized that TPS could be enabled by the discovery of a small-molecule recruiter of a deubiquitinase (DUB) that could be linked to a protein-targeting ligand to form a chimeric molecule, which would induce the deubiquitination and stabilization of proteins of interest. We call this heterobifunctional stabilizer a DUBTAC (Fig. 1a). In this study, we report the discovery of a covalent recruiter for the K48-ubiquitin chain-specific DUB OTUB1, which when linked to a protein-targeting ligand stabilizes an actively degraded target protein to demonstrate proof of concept for the DUBTAC platform. ResultsIdentifying allosteric ligandable sites within DUBs. To enable the DUBTAC platform, our first goal was to identify a small-molecule recruiter that targeted an allosteric site on a DUB without inhibiting DUB function, as the recruitment of a functional DUB would be required to deubiquitinate and stabilize the target protein. While many DUBs possess well-defined active sites bearing a catalytic and highly nucleophilic cysteine, there have not yet been systematic evaluations of ...

Proteolysis-targeting chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting C186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications by demonstrating that a PROTAC linking EN106 to the BET bromodomain inhibitor JQ1 or the kinase inhibitor dasatinib leads to the degradation of BRD4 and BCR-ABL, respectively. Our study showcases a covalent ligand that targets a natural E3 ligase−substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters suitable for TPD applications.

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific protein targets in a proteasome-dependent manner.However, a major limitation to broader TPD applications is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent ligand for the E3 ligase RNF114. When linked to the BET family inhibitor JQ1, the resulting heterobifunctional PROTAC molecule was capable of selectively degrading BRD4 in cancer cells. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or in some cases inactivity. Further contrasting from cereblon or VHL-recruiting degradation, we show that BT1 treatment not only leads to BCR-ABL degradation, but also stabilizes the endogenous RNF114 substrate and tumor suppressor substrate p21. This leads to additional anti-proliferative effects in leukemia cancer cells beyond those observed with cereblon or VHL-recruiting BCR-ABL PROTACs. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs with unique additional benefits for oncology applications. We also further demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing and unpredictable selectivity for the degradation of neo-substrate proteins.