Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications

Abstract

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.

Chemoproteomics-Enabled Discovery of a Covalent Molecular Glue Degrader Targeting NF-κB

“…Through competitive profiling of EN450 covalent protein targeting in HAP1 cells against cysteine-reactive alkyne-functionalized iodoacetamide probe labeling, we identified 23 targets that showed significant engagement-control/EN450-treated ratio of >4 with adjusted pvalues <0.05-among 4501 cysteines quantified (Figure 2a; Table S2). Given that we and others have previously shown that the UPS is highly ligandable with covalent ligands, that even partial covalent occupancy of a UPS effector protein can lead to significant degradation of the target protein due to the catalytic nature of degraders, and that EN450 is a small-molecule fragment that is not likely to engage in high-affinity interactions, we conjectured that the covalent interaction of EN450 is likely occurring with a component of the ubiquitinproteasome system, rather than the target protein (Belcher et al, 2021;Henning et al, 2022bHenning et al, , 2022aSpradlin et al, 2019a;Zhang et al, 2018Zhang et al, , 2021. Among these targets, only one protein was involved in the UPS and the Cullin E3 ligase machinery-C111 of ubiquitin-conjugating enzyme E2D (UBE2D).…”

Advances in covalent drug discovery

“…Covalent ligands are not only useful as functional inhibitors, but also have important roles in emerging induced proximity modalities. Covalent drug discovery platforms have facilitated the expansion of targeted protein degradation approaches by enabling the discovery of covalent recruiters against E3 ubiquitin ligases 14 , 187 – 193 . Although most bifunctional degrader molecules (proteolysis-targeting chimeras (PROTACs)) recruit the E3 ligases cereblon (CRBN) or von Hippel–Lindau (VHL) protein to degrade target proteins, there are more than 600 E3 ligases with varying substrate scopes.…”

“…Although most bifunctional degrader molecules (proteolysis-targeting chimeras (PROTACs)) recruit the E3 ligases cereblon (CRBN) or von Hippel–Lindau (VHL) protein to degrade target proteins, there are more than 600 E3 ligases with varying substrate scopes. Since 2019, covalent recruiters have been used to validate a large proportion of the E3 ligases that have been harnessed for targeted protein degradation, including RING finger protein 114 (RNF114), RNF4, DDB1 and CUL4-associated factor 16 (DCAF16), DCAF11, KEAP1 and, most recently, fem-1 homologue B (FEM1B) 187 – 193 . In 2019, isoTOP-ABPP was used to identify RNF114 as the target of the enone-containing natural product nimbolide, which was used to make bifunctional degraders of bromodomain-containing protein 4 (BRD4) and BCR–ABL 187 .…”

Major Advances in Emerging Degrader Technologies

“…Their results showed potent BRD4 degradation depended on the covalent binding to KEAP1. Very recently, Henning et al ( Henning et al, 2022 ) identified the covalent ligand 24 ( Figure 7 ) that targeting FEM1B E3 ligase, and they transformed compound 24 to potential BRD4 and BCR-ABL degraders respectively. So far, only few E3 ligase was used for constructing PROTACs.…”