3-Fluoro-4-hydroxyprolines: Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation

Ubiquitination is a post-translational modification that has pivotal roles in protein degradation and diversified cellular processes, and for more than two decades it has been a subject of interest in the biotech or biopharmaceutical industry. Tripartite motif (TRIM) family proteins are known to have proven E3 ubiquitin ligase activities and are involved in a multitude of cellular and physiological events and pathophysiological conditions ranging from cancers to rare genetic disorders. Although in recent years many kinds of E3 ubiquitin ligases have emerged as the preferred choices of big pharma and biotech startups in the context of protein degradation and disease biology, from a surface overview it appears that TRIM E3 ubiquitin ligases are not very well recognized yet in the realm of drug discovery. This article will review some of the blockbuster scientific discoveries and technological innovations from the world of ubiquitination and E3 ubiquitin ligases that have impacted the biopharma community, from biotech colossuses to startups, and will attempt to evaluate the future of TRIM family proteins in the province of E3 ubiquitin ligase-based drug discovery.

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Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

Molecular Biology of the Cell ◽

10.1091/mbc.e12-07-0548 ◽

2012 ◽

Vol 23 (21) ◽

pp. 4203-4211 ◽

Cited By ~ 9

Author(s):

Dong-Hwan Kim ◽

Deanna M. Koepp

Keyword(s):

Cell Cycle ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

S Phase ◽

Temperature Sensitive ◽

Dependent Manner ◽

Charged Residues ◽

Phase Progression ◽

Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

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Photocontrollable PROTAC molecules: Structure and mechanism of action

Arhiv za farmaciju ◽

10.5937/arhfarm71-30785 ◽

2021 ◽

Vol 71 (3) ◽

pp. 161-176

Author(s):

Mladen Koravović ◽

Gordana Tasić ◽

Milena Rmandić ◽

Bojan Marković

Keyword(s):

Protein Degradation ◽

Ubiquitin Ligase ◽

Binding Sites ◽

E3 Ubiquitin Ligase ◽

Drug Binding ◽

Post Translational Modification ◽

Protein Activity ◽

Target Drug ◽

Traditional Drug

Traditional drug discovery strategies are usually focused on occupancy of binding sites that directly affect functions of proteins. Hence, proteins that lack such binding sites are generally considered pharmacologically intractable. Modulators of protein activity, especially inhibitors, must be applied in appropriate dosage regimens that often lead to high systemic drug exposures in order to maintain sufficient protein inhibition in vivo. Consequently, there is a risk of undesirable off-target drug binding and side effects. Recently, PROteolysis TArgeting Chimera (PROTAC) technology has emerged as a new pharmacological modality that exploits PROTAC molecules for induced protein degradation. PROTAC molecule is a heterobifunctional structure consisting of a ligand that binds a protein of interest (POI), a ligand for recruiting an E3 ubiquitin ligase (an enzyme involved in the POI ubiquitination) and a linker that connects these two. After POI-PROTAC-E3 ubiquitin ligase ternary complex formation, the POI undergoes ubiquitination (an enzymatic post-translational modification in which ubiquitin is attached to the POI) and degradation. By merging the principles of photopharmacology and PROTAC technology, photocontrollable PROTACs for spatiotemporal control of induced protein degradation have recently emerged. The main advantage of photocontrollable over conventional PROTACs is the possible prevention of off-target toxicity thanks to local photoactivation.

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Oestrogen causes ATBF1 protein degradation through the oestrogen-responsive E3 ubiquitin ligase EFP

Biochemical Journal ◽

10.1042/bj20111890 ◽

2012 ◽

Vol 444 (3) ◽

pp. 581-590 ◽

Cited By ~ 12

Author(s):

Xue-Yuan Dong ◽

Xiaoying Fu ◽

Songqing Fan ◽

Peng Guo ◽

Dan Su ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Feedback Loop ◽

E3 Ubiquitin Ligase ◽

Breast Development ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Dependent Cell ◽

Proteasome Pathway

We reported previously that the tumour suppressor ATBF1 (AT motif-binding factor 1) formed an autoregulatory feedback loop with oestrogen–ERα (oestrogen receptor α) signalling to regulate oestrogen-dependent cell proliferation in breast cancer cells. In this loop ATBF1 inhibits the function of oestrogen–ERα signalling, whereas ATBF1 protein levels are fine-tuned by oestrogen-induced transcriptional up-regulation as well as UPP (ubiquitin–proteasome pathway)-mediated protein degradation. In the present study we show that EFP (oestrogen-responsive finger protein) is an E3 ubiquitin ligase mediating oestrogen-induced ATBF1 protein degradation. Knockdown of EFP increases ATBF1 protein levels, whereas overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in oestrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumours the levels of ATBF1 protein are positively correlated with the levels of EFP protein, as both are directly up-regulated ERα target gene products. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for oestrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and oestrogen–ERα signalling and thus implicate ATBF1 in oestrogen-dependent breast development and carcinogenesis.

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Proteolysis targeting chimeras (PROTACs) in cancer therapy

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01672-1 ◽

2020 ◽

Vol 39 (1) ◽

Author(s):

Alberto Ocaña ◽

Atanasio Pandiella

Keyword(s):

Cancer Therapy ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Mechanism Of Action ◽

Clinical Setting ◽

Therapeutic Strategy ◽

E3 Ubiquitin Ligase ◽

Proteasomal Degradation ◽

Molecular Medicine ◽

Recent Developments

Abstract Exploitation of the protein degradation machinery as a therapeutic strategy to degrade oncogenic proteins is experiencing revolutionary advances with the development of proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional structures consisting of a ligand that binds a protein to be degraded and a ligand for an E3 ubiquitin ligase. The bridging between the protein of interest and the E3 ligase mediated by the PROTAC facilitates ubiquitination of the protein and its proteasomal degradation. In this review we discuss the molecular medicine behind PROTAC mechanism of action, with special emphasis on recent developments and their potential translation to the clinical setting.

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The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

Molecules ◽

10.3390/molecules25245956 ◽

2020 ◽

Vol 25 (24) ◽

pp. 5956

Author(s):

Bernat Coll-Martínez ◽

Antonio Delgado ◽

Bernat Crosas

Keyword(s):

Protein Degradation ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Therapeutic Agents ◽

Highly Active ◽

Drug Doses ◽

Proof Of Principle

The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline.

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Abstract 1457: Cbl, E3 Ubiquitin Ligase, is a Regulator of Focal Adhesion Protein Proteasomal Degradation during Cardiomyocyte Anoikis

Circulation ◽

10.1161/circ.116.suppl_16.ii_300-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Khadija Rafiq ◽

Rachid Seqqat ◽

Marie Hanscom ◽

Steven R Houser ◽

Abdelkarim Sabri

Keyword(s):

Dilated Cardiomyopathy ◽

Protein Degradation ◽

Focal Adhesion ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Adaptor Protein ◽

Dominant Negative ◽

Cathepsin G ◽

Dominant Negative Mutant ◽

Myocyte Apoptosis

Proteasome degradation of ubiquitin-targeted proteins is an important mechanism that negatively controls activated signaling pathways. The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor tyrosine kinase signaling, resulting in their ubiquitination and down-regulation. We have shown previously that neutrophil derived protease cathepsin G (Cat.G) induced focal adhesion (FA) protein degradation and myocyte apoptosis by anoikis. We hypothesized that Cbl is involved in myocyte apoptosis in response to Cat.G through ubiquitination and downregulation of FA proteins. Results : Immunoprecitpitation and western blot studies showed increased Cbl tyrosine phosphorylation in response to Cat.G that was associated with its interaction with FA proteins, FAK and paxillin. This led to an increase in FAK and Paxillin ubiquitination and degradation at 2 hrs after Cat.G addition. Inhibition of the ubiquitin proteasome system, with MG132 or lactacystin, or adenoviral expression of dominant negative mutant Cbl significantly reduced FAK and paxillin degradation. In contrast, adenoviral expression of wild type Cbl enhanced FAK and paxillin ubiquitination and degradation in response to Cat.G. Interestingly, Cbl activation was involved in Cat.G-induced myocyte apoptosis as overexpression of dominant negative Cbl significantly reduced caspase-3 activation and DNA fragmentation induced by Cat.G. Concomitant with these changes in vitro, heart tissue samples from patients with ischemic or dilated cardiomyopathy showed a marked increase in Cbl accumulation and FAK and paxillin degradation compared to heart controls. Conclusion : These studies show that Cbl abundance is increased in human ischemic and dilated cardiomyopathy and that activation of Cbl increases FA protein degradation and myocyte apoptosis. These results indicate that Cbl is a positive regulator of FA protein degradation and myocyte apoptosis and may play an important role in mediating the effect of neutrophil derived proteases during the progression of congestive heart failure.

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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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