Cholesterol increases protein levels of the E3 ligase MARCH6 and thereby stimulates protein degradation

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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PHOTACs enable optical control of protein degradation

Science Advances ◽

10.1126/sciadv.aay5064 ◽

2020 ◽

Vol 6 (8) ◽

pp. eaay5064 ◽

Cited By ~ 23

Author(s):

Martin Reynders ◽

Bryan S. Matsuura ◽

Marleen Bérouti ◽

Daniele Simoneschi ◽

Antonio Marzio ◽

...

Keyword(s):

Protein Degradation ◽

E3 Ligase ◽

Optical Control ◽

Cellular Proteins ◽

Modular Approach ◽

Bifunctional Molecules ◽

Protein Levels ◽

Lead Compounds ◽

Subsequent Degradation ◽

Precision Therapeutics

PROTACs (PROteolysis TArgeting Chimeras) are bifunctional molecules that target 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. We now introduce photoswitchable PROTACs that can be activated with the spatiotemporal precision that light provides. These trifunctional molecules, which we named PHOTACs (PHOtochemically TArgeting Chimeras), 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 with different wavelengths of light. Our modular 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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PHOTACs Enable Optical Control of Protein Degradation

10.26434/chemrxiv.8206688.v2 ◽

2019 ◽

Cited By ~ 5

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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PHOTACs Enable Optical Control of Protein Degradation

10.26434/chemrxiv.8206688.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Martin Reynders ◽

Bryan Matsuura ◽

Daniele Simoneschi ◽

Antonio Marzio ◽

Michele Pagano ◽

...

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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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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Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

10.26434/chemrxiv.12055935 ◽

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

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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Targeted Protein Degradation via a Covalent Reversible Degrader Based on Bardoxolone

10.26434/chemrxiv.12055935.v1 ◽

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

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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A Caged E3 Ligase Ligand for PROTAC-Mediated Protein Degradation with Light

10.26434/chemrxiv.11350079 ◽

2019 ◽

Cited By ~ 1

Author(s):

Cyrille Kounde ◽

Maria M. Shchepinova ◽

Edward Tate

Keyword(s):

Protein Degradation ◽

Irradiation Time ◽

E3 Ligase ◽

Von Hippel Lindau ◽

Short Irradiation ◽

Short Irradiation Time ◽

Spatiotemporal Control

A caging group has been appended to a widely used Von Hippel Lindau (VHL) E3 ligase ligand for targeted protein degradation with PROTACs. Proteolysis is triggered only after a short irradiation time allowing spatiotemporal control of the protein’s fate.

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Pirh2, an E3 ligase, regulates the AIP4–p73 regulatory pathway by modulating AIP4 expression and ubiquitination

Carcinogenesis ◽

10.1093/carcin/bgab009 ◽

2021 ◽

Author(s):

Rami Abou Zeinab ◽

H Helena Wu ◽

Yasser Abuetabh ◽

Sarah Leng ◽

Consolato Sergi ◽

...

Keyword(s):

Regulatory Protein ◽

Ectopic Expression ◽

E3 Ligase ◽

Regulatory Pathway ◽

Hect Domain ◽

E3 Ligases ◽

Multiple Substrates ◽

Protein Levels ◽

Cycle Arrest ◽

Negative Regulatory Pathway

Abstract Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4–p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins.

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