Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

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.

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Bardoxolone conjugation enables targeted protein degradation of BRD4

Scientific Reports ◽

10.1038/s41598-020-72491-9 ◽

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.

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Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors

Cancers ◽

10.3390/cancers13123079 ◽

2021 ◽

Vol 13 (12) ◽

pp. 3079

Author(s):

Gabriel LaPlante ◽

Wei Zhang

Keyword(s):

Protein Degradation ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Proteasome Inhibitors ◽

Cancer Therapeutics ◽

Ubiquitin Proteasome System ◽

Cellular Protein ◽

E3 Ligases ◽

Protein Levels ◽

Ubiquitin Proteasome

The ubiquitin-proteasome system (UPS) is a critical regulator of cellular protein levels and activity. It is, therefore, not surprising that its dysregulation is implicated in numerous human diseases, including many types of cancer. Moreover, since cancer cells exhibit increased rates of protein turnover, their heightened dependence on the UPS makes it an attractive target for inhibition via targeted therapeutics. Indeed, the clinical application of proteasome inhibitors in treatment of multiple myeloma has been very successful, stimulating the development of small-molecule inhibitors targeting other UPS components. On the other hand, while the discovery of potent and selective chemical compounds can be both challenging and time consuming, the area of targeted protein degradation through utilization of the UPS machinery has seen promising developments in recent years. The repertoire of proteolysis-targeting chimeras (PROTACs), which employ E3 ligases for the degradation of cancer-related proteins via the proteasome, continues to grow. In this review, we will provide a thorough overview of small-molecule UPS inhibitors and highlight advancements in the development of targeted protein degradation strategies for cancer therapeutics.

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A Phenotypic Approach for the Identification of New Molecules for Targeted Protein Degradation Applications

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211017517 ◽

2021 ◽

pp. 247255522110175

Author(s):

Peter Stacey ◽

Hannah Lithgow ◽

Xiao Lewell ◽

Agnieszka Konopacka ◽

Stephen Besley ◽

...

Keyword(s):

Fusion Protein ◽

Protein Degradation ◽

E3 Ligase ◽

Ubiquitin Proteasome System ◽

E3 Ligases ◽

Von Hippel Lindau ◽

Protein Levels ◽

New Strategy ◽

Cellular Context

Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin–proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel–Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.

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HaloTag-Targeted Sirtuin Rearranging Ligand (SirReal) for the Development of Proteolysis Targeting Chimeras (PROTACs) Against the Lysine Deacetylase Sirtuin 2 (Sirt2)

10.26434/chemrxiv.12370469.v1 ◽

2020 ◽

Author(s):

Matthias Schiedel ◽

Attila Lehotzky ◽

Sándor Szunyogh ◽

Judit Oláh ◽

Sören Hammelmann ◽

...

Keyword(s):

Protein Degradation ◽

Small Molecule ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Ubiquitin Ligases ◽

E3 Ubiquitin Ligases ◽

Selective Inhibitors ◽

E3 Ligases ◽

Lysine Deacetylase

We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

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HaloTag-Targeted Sirtuin Rearranging Ligand (SirReal) for the Development of Proteolysis Targeting Chimeras (PROTACs) Against the Lysine Deacetylase Sirtuin 2 (Sirt2)

10.26434/chemrxiv.12370469 ◽

2020 ◽

Author(s):

Matthias Schiedel ◽

Attila Lehotzky ◽

Sándor Szunyogh ◽

Judit Oláh ◽

Sören Hammelmann ◽

...

Keyword(s):

Protein Degradation ◽

Small Molecule ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

E3 Ligase ◽

Ubiquitin Ligases ◽

E3 Ubiquitin Ligases ◽

Selective Inhibitors ◽

E3 Ligases ◽

Lysine Deacetylase

We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

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Exploring DCAF15 for reprogrammable targeted protein degradation

10.1101/542506 ◽

2019 ◽

Cited By ~ 1

Author(s):

Seemon Coomar ◽

Dennis G. Gillingham

Keyword(s):

Protein Degradation ◽

Small Molecules ◽

Chemical Biology ◽

E3 Ligase ◽

Human Proteome ◽

Splicing Factor ◽

Chemical Modifications ◽

E3 Ligases ◽

Ring Type ◽

Exciting Area

AbstractThe targeted degradation of proteins by reprogramming E3 ligases with bifunctional small molecules is an exciting area of chemical biology because it promises a set of chemical tools for achieving the same task as siRNA or CRISPR/Cas9. Although there are hundreds of E3 ligases in the human proteome only a few have been shown to be reprogrammable to target new proteins. Recently various arylsulfonamides were shown to induce degradation of the splicing factor RBM39 via the RING type E3 ligase CRL4DCAF15. Here we identify the arylsulfonamide most amenable to chemical modifications and demonstrate its behaviour in bifunctional reprogramming.

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Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications

10.1101/2021.04.15.439993 ◽

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.

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PHOTACs Enable Optical Control of Protein Degradation

10.26434/chemrxiv.8206688 ◽

2019 ◽

Cited By ~ 2

Author(s):

Martin Reynders ◽

Bryan Matsuura ◽

Marleen Bérouti ◽

Daniele Simoneschi ◽

Antonio Marzio ◽

...

Keyword(s):

Protein Degradation ◽

E3 Ligase ◽

Optical Control ◽

Cellular Proteins ◽

Bifunctional Molecules ◽

Protein Levels ◽

Lead Compounds ◽

Subsequent Degradation ◽

Temporal And Spatial ◽

Precision Therapeutics

PROTACs (proteolysis targeting chimeras) are bifunctional molecules that tag proteins for ubiquitylation by an E3 ligase complex and subsequent degradation by the proteasome. They have emerged as powerful tools to control the levels of specific cellular proteins and are on the verge of being clinically used. We now introduce photoswitchable PROTACs that can be activated with the temporal and spatial precision that light provides. These trifunctional molecules, which we named PHOTACs, consist of a ligand for an E3 ligase, a photoswitch, and a ligand for a protein of interest. We demonstrate this concept by using PHOTACs that target either BET family proteins (BRD2,3,4) or FKBP12. Our lead compounds display little or no activity in the dark but can be reversibly activated to varying degrees with different wavelengths of light. Our modular and generalizable approach provides a method for the optical control of protein levels with photopharmacology and could lead to new types of precision therapeutics that avoid undesired systemic toxicity.

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Targeted Protein Degradation: "The Gold Rush is On!"

Technology Transfer and Entrepreneurship ◽

10.2174/2213809907666200130111436 ◽

2020 ◽

Vol 7 (1) ◽

pp. 4-16

Author(s):

Daria Kotlarek ◽

Agata Pawlik ◽

Maria Sagan ◽

Marta Sowała ◽

Alina Zawiślak-Architek ◽

...

Keyword(s):

Drug Discovery ◽

Protein Degradation ◽

Small Molecule ◽

Gold Rush ◽

European Company ◽

Drug Candidates ◽

Protein Inhibition ◽

Small Molecule Drug ◽

Therapeutic Benefits ◽

Talent Pool

Targeted Protein Degradation (TPD) is an emerging new modality of drug discovery that offers unprecedented therapeutic benefits over traditional protein inhibition. Most importantly, TPD unlocks the untapped pool of the proteome that to date has been considered undruggable. Captor Therapeutics (Captor) is the fourth global, and first European, company that develops small molecule drug candidates based on the principles of targeted protein degradation. Captor is located in Basel, Switzerland and Wroclaw, Poland and exploits the best opportunities of the two sites – experience and non-dilutive European grants, and talent pool, respectively. Through over $38 M of funding, Captor has been active in three areas of TPD: molecular glues, bi-specific degraders and direct degraders, ObteronsTM.

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