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 Cys186 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, in which we demonstrate that a PROTAC linking EN106 to the BET Bromodomain inhibitor JQ1 leads to specific FEM1B- and proteasome-dependent degradation of BRD4 in cells. 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 that can be deployed for TPD applications.

PROTAC targeted protein degraders: the past is prologue

“…Accordingly, besides the aforementioned ligases (CRBN, VHL, MDM2, IAPs, DCAF15 and DCAF16), a few additional ‘generic’ E3 ligases have been shown to be exploitable by PROTACs, using small-molecule covalent tools that specifically recruit the ligases to degrade the prototypical TPD substrate, BRD4; these ligases include ring finger protein 4 (RNF4) 132 , RNF114 (refs 133 – 135 ), KEAP1 (refs 136 , 137 ) and most recently, fem-1 homologue B (FEM1B) 138 . How far initial chemical matter will be developed for these newly established degrader ligases, and how widely they can and will be adopted as in vivo-compatible ligases with a broad target reach, will be the subject of intense experimentation over the next few years.…”

E3 Ligase Ligands in Successful PROTACs: An Overview of Syntheses and Linker Attachment Points

“…The key cysteine residue C186 was noted to present a possible target for developing a FEM1B recruiter useful in the field of targeted protein degradation. Through screening a library of cysteine-reactive covalent ligands, compound EN106 was identified and its structure modified to be incorporated into BRD4-targeting PROTACs (Henning et al, 2021).…”

“…After the nitro group was reduced, amine 328 was alkylated with acrylonitrile, followed by acylation with chloroacetyl chloride to yield 329. After hydrolyzing the methyl ester, POI ligandlinker-NH 2 conjugate was attached through coupling to provide PROTAC 330 (Scheme 59) (Henning et al, 2021).…”

“…Reagents and conditions: a) K 2 CO 3 , MeI, DMF, 0°C to rt, 24 h, 99% yield; b) iodobenzoic acid, fluorobenzene, DMSO, 85°C, 24 h, 87% yield; c) i. mCPBA, CH 2 Cl 2 , 0°C to rt, 24 h; ii. HBr, Br 2 , acetic acid, rt to 35°C, 24 h, 82% yield; d) CuCN, KI, DMF, 120°C, 24 h, 73% yield (Fu and Gribble, 2013 (Henning et al, 2021).…”

Inhibitors, PROTACs and Molecular Glues as Diverse Therapeutic Modalities to Target Cyclin-Dependent Kinase

“…A typical PROTAC consists of three parts: a POI binding ligand, an E3 ubiquitin-binding ligand, and a linker connecting two ligands. Despite the presence of >600 putative E3 ligases in the human proteome, only a handful of E3 ligase ligands (CRBN [ 31 , 32 ], VHL [ 33 , 34 , 35 ], MDM [ 36 , 37 , 38 ], IAP [ 39 ], RNF114 [ 40 , 41 ], DCAF15 [ 42 ], DCAF 16 [ 43 ], and FEM1B [ 44 ]) are readily available for the design of a PROTAC ( Figure 4 ). The PROTAC approach has been successfully applied to degrade kinases, G protein-coupled receptors, nuclear receptors, membrane proteins, transcription factors, and neurodegenerative proteins aggregates [ 23 , 26 , 27 , 28 , 29 , 30 , 36 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ].…”