scholarly journals Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications

2021 ◽  
Author(s):  
Nathaniel J Henning ◽  
Andrew G Manford ◽  
Jessica N Spradlin ◽  
Scott M Brittain ◽  
Jeffrey M McKenna ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Cellular Response ◽  
Protein Targeting ◽  
E3 Ligase ◽  
Therapeutic Modality ◽  
Critical Component ◽  
E3 Ligases ◽  
Reductive Stress ◽  
Ligand Screening

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.

scholarly journals Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications

2018 ◽  
Author(s):  
Carl C. Ward ◽  
Jordan I. Kleinman ◽  
Scott M. Brittain ◽  
Patrick S. Lee ◽  
Clive Yik Sham Chung ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Protein Targeting ◽  
E3 Ligase ◽  
Proteasomal Degradation ◽  
Protein Profiling ◽  
Dependent Manner ◽  
E3 Ligases ◽  
Ligand Screening ◽  
Bromodomain Proteins ◽  
Screening Approaches

AbstractTargeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

scholarly journals Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

2020 ◽  
Author(s):  
Bingqi Tong ◽  
Mai Luo ◽  
Yi Xie ◽  
Jessica Spradlin ◽  
John A. Tallarico ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Binding ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Protein Levels ◽  
Nrf2 Activator

<p>Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules (i.e. PROTACs). E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the >500 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-NRF2 activator bardoxolone to a BRD4 inhibitor JQ1. Notably, this work reports the first covalent, reversible E3 ligase recruiter for TPD applications. </p>

scholarly journals A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

2020 ◽  
Author(s):  
Benika J. Pinch ◽  
Dennis L. Buckley ◽  
Scott Gleim ◽  
Scott M. Brittain ◽  
Laura Tandeske ◽  
...  
Keyword(s):  
Protein Degradation ◽  
E3 Ligase ◽  
Therapeutic Modality ◽  
Live Cells ◽  
Substrate Selectivity ◽  
Covalent Functionalization ◽  
E3 Ligases ◽  
Powerful Approach ◽  
Potential Applications ◽  
Socs Box

ABSTRACTTargeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While the well-characterized E3 ligases CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution; however, characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligases against neo-substrates.

scholarly journals Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

2020 ◽  
Author(s):  
Bingqi Tong ◽  
Mai Luo ◽  
Yi Xie ◽  
Jessica Spradlin ◽  
John A. Tallarico ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Binding ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Protein Levels ◽  
Nrf2 Activator

<p>Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules (i.e. PROTACs). E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the >500 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-NRF2 activator bardoxolone to a BRD4 inhibitor JQ1. Notably, this work reports the first covalent, reversible E3 ligase recruiter for TPD applications. </p>

scholarly journals Bardoxolone conjugation enables targeted protein degradation of BRD4

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bingqi Tong ◽  
Mai Luo ◽  
Yi Xie ◽  
Jessica N. Spradlin ◽  
John A. Tallarico ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Binding ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Protein Levels ◽  
Nrf2 Activator

Abstract Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3’s has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

scholarly journals Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications

2019 ◽  
Vol 14 (11) ◽  
pp. 2430-2440 ◽  
Author(s):  
Carl C. Ward ◽  
Jordan I. Kleinman ◽  
Scott M. Brittain ◽  
Patrick S. Lee ◽  
Clive Yik Sham Chung ◽  
...  
Keyword(s):  
Protein Degradation ◽  
E3 Ligase ◽  
Ligand Screening

scholarly journals Novel Approach for Characterizing Ubiquitin E3 Ligase Function

2010 ◽  
Vol 15 (10) ◽  
pp. 1220-1228 ◽  
Author(s):  
Jeffrey G. Marblestone ◽  
K. G. Suresh Kumar ◽  
Michael J. Eddins ◽  
Craig A. Leach ◽  
David E. Sterner ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput Screening ◽  
E3 Ligase ◽  
Cost Effective ◽  
Ubiquitin Proteasome System ◽  
Dependent Manner ◽  
Ubiquitin Chain ◽  
E3 Ligases ◽  
Effective Manner ◽  
Wide Range

The ubiquitin-proteasome system is central to the regulation of numerous cellular events, and dysregulation may lead to disease pathogenesis. E3 ubiquitin ligases typically function in concert with E1 and E2 enzymes to recruit specific substrates, thereby coordinating their ubiquitylation and subsequent proteasomal degradation or cellular activity. E3 ligases have been implicated in a wide range of pathologies, and monitoring their activity in a rapid and cost-effective manner would be advantageous in drug discovery. The relative lack of high-throughput screening (HTS)–compliant E3 ligase assays has significantly hindered the discovery of E3 inhibitors. Herein, the authors describe a novel HTS-compliant E3 ligase assay platform that takes advantage of a ubiquitin binding domain’s inherent affinity for polyubiquitin chains, permitting the analysis of ubiquitin chain formation in an E3 ligase-dependent manner. This assay has been used successfully with members of both the RING and HECT families, demonstrating the platform’s broad utility for analyzing a wide range of E3 ligases. The utility of the assay platform is demonstrated by the identification of inhibitors of the E3 ligase CARP2. As the number of E3 ligases associated with various disease states increases, the ability to quantitate the activity of these enzymes in an expeditious manner becomes imperative in drug discovery.

scholarly journals Expanding the Toolbox of E3 Ligases for Protein Degradation: Targeting the “Undruggable” Fbw7 E3 Ligase

Proceedings ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 101
Author(s):  
Carles Galdeano
Keyword(s):  
Protein Degradation ◽  
E3 Ligase ◽  
Intracellular Protein ◽  
E3 Ligases ◽  
Intracellular Protein Degradation

Proteolysis targeting chimera molecules (PROTACS) are heterobifunctional small moleculesdesigned to induce intracellular protein degradation [...]

The Present and Future of Novel Protein Degradation Technology

2019 ◽  
Vol 19 (20) ◽  
pp. 1784-1788 ◽  
Author(s):  
Liwen Xia ◽  
Wei Liu ◽  
Yinsen Song ◽  
Hailiang Zhu ◽  
Yongtao Duan
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Vital Role ◽  
Therapeutic Modality ◽  
Bifunctional Molecules ◽  
Subsequent Degradation ◽  
Ubiquitin Proteasome ◽  
Future Direction ◽  
Novel Protein

Proteolysis targeting chimeras (PROTACs), as a novel therapeutic modality, play a vital role in drug discovery. Each PROTAC contains three key parts; a protein-of-interest (POI) ligand, a E3 ligase ligand, and a linker. These bifunctional molecules could mediate the degradation of POIs by hijacking the activity of E3 ubiquitin ligases for POI ubiquitination and subsequent degradation via the ubiquitin proteasome system (UPS). With several advantages over other therapeutic strategies, PROTACs have set off a new upsurge of drug discovery in recent years. ENDTAC, as the development of PROTACs technology, is now receiving more attention. In this review, we aim to summarize the rapid progress from 2018 to 2019 in protein degradation and analyze the challenges and future direction that need to be addressed in order to efficiently develop potent protein degradation technology.

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