Covalent drugs have been used to treat diseases for more than a century, but tools that facilitate the rational design of covalent drugs have emerged more recently. The purposeful addition of reactive functional groups to existing ligands can enable potent and selective inhibition of target proteins, as demonstrated by the covalent epidermal growth factor receptor (EGFR) and Bruton’s tyrosine kinase (BTK) inhibitors used to treat various cancers. Moreover, the identification of covalent ligands through ‘electrophile-first’ approaches has also led to the discovery of covalent drugs, such as covalent inhibitors for KRAS(G12C) and SARS-CoV-2 main protease. In particular, the discovery of KRAS(G12C) inhibitors validates the use of covalent screening technologies, which have become more powerful and widespread over the past decade. Chemoproteomics platforms have emerged to complement covalent ligand screening and assist in ligand discovery, selectivity profiling and target identification. This Review showcases covalent drug discovery milestones with emphasis on the lessons learned from these programmes and how an evolving toolbox of covalent drug discovery techniques facilitates success in this field.

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.