From Conception to Development: Investigating PROTACs Features for Improved Cell Permeability and Successful Protein Degradation

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional degraders that specifically eliminate targeted proteins by hijacking the ubiquitin-proteasome system (UPS). This modality has emerged as an orthogonal approach to the use of small-molecule inhibitors for knocking down classic targets and disease-related proteins classified, until now, as “undruggable.” In early 2019, the first targeted protein degraders reached the clinic, drawing attention to PROTACs as one of the most appealing technology in the drug discovery landscape. Despite these promising results, PROTACs are often affected by poor cellular permeability due to their high molecular weight (MW) and large exposed polar surface area (PSA). Herein, we report a comprehensive record of PROTAC design, pharmacology and thermodynamic challenges and solutions, as well as some of the available strategies to enhance cellular uptake, including suggestions of promising biological tools for the in vitro evaluation of PROTACs permeability toward successful protein degradation.

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The Present and Future of Novel Protein Degradation Technology

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666191011162955 ◽

2019 ◽

Vol 19 (20) ◽

pp. 1784-1788 ◽

Cited By ~ 5

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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Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration

Journal of Biological Chemistry ◽

10.1074/jbc.m116.733626 ◽

2016 ◽

Vol 291 (33) ◽

pp. 17209-17227 ◽

Cited By ~ 32

Author(s):

Dolores Del Prete ◽

Richard C. Rice ◽

Anjali M. Rajadhyaksha ◽

Luciano D'Adamio

Keyword(s):

Amyloid Precursor Protein ◽

Transmitter Release ◽

Ubiquitin Proteasome System ◽

Precursor Protein ◽

E3 Ligases ◽

Ubiquitin Proteasome ◽

Functional Pathway ◽

Related Proteins

The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4CRBN, which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4CRBN, pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys649/650/651/676/688. Deletion of Crbn reduces ubiquitination of Lys676 suggesting that Lys676 is physiologically ubiquitinated by CRL4CRBN. The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro. This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.

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Abstract 294: Atrogin-1 Is Required for Atrophic Remodeling of the Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a294 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Kedryn K Baskin ◽

Rebecca Salazar ◽

Wenhao Chen ◽

Heinrich Taegtmeyer

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Ubiquitin Proteasome System ◽

Skeletal Muscle Atrophy ◽

Heart Weight ◽

Cross Sectional ◽

Mechanical Unloading ◽

Ubiquitin Proteasome ◽

Specific Proteins

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

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Chemical-Mediated Targeted Protein Degradation in Neurodegenerative Diseases

Life ◽

10.3390/life11070607 ◽

2021 ◽

Vol 11 (7) ◽

pp. 607

Author(s):

Soonsil Hyun ◽

Dongyun Shin

Keyword(s):

Neurodegenerative Diseases ◽

Protein Degradation ◽

Drug Targets ◽

Ubiquitin Proteasome System ◽

Cellular Protein ◽

Therapeutic Modality ◽

Target Proteins ◽

Bifunctional Molecules ◽

Ubiquitin Proteasome ◽

Related Proteins

Neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease, are a class of diseases that lead to dysfunction of cognition and mobility. Aggregates of misfolded proteins such as β-amyloid, tau, α-synuclein, and polyglutamates are known to be among the main causes of neurodegenerative diseases; however, they are considered to be some of the most challenging drug targets because they cannot be modulated by conventional small-molecule agents. Recently, the degradation of target proteins by small molecules has emerged as a new therapeutic modality and has garnered the interest of the researchers in the pharmaceutical industry. Bifunctional molecules that recruit target proteins to a cellular protein degradation machinery, such as the ubiquitin–proteasome system and autophagy–lysosome pathway, have been designed. The representative targeted protein degradation technologies include molecular glues, proteolysis-targeting chimeras, hydrophobic tagging, autophagy-targeting chimeras, and autophagosome-tethering compounds. Although these modalities have been shown to degrade many disease-related proteins, such technologies are expected to be potentially important for neurogenerative diseases caused by protein aggregation. Herein, we review the recent progress in chemical-mediated targeted protein degradation toward the discovery of drugs for neurogenerative diseases.

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Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽

10.3390/biom11010110 ◽

2021 ◽

Vol 11 (1) ◽

pp. 110

Author(s):

Dina Aweida ◽

Shenhav Cohen

Keyword(s):

Protein Quality ◽

Protein Structures ◽

Ubiquitin Proteasome System ◽

Surface Proteins ◽

Catalytic Core ◽

Complex Protein ◽

Ubiquitin Proteasome ◽

Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

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DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.254 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii63-ii63

Author(s):

Lakshmi Bollu ◽

Derek Wainwright ◽

Lijie Zhai ◽

Erik Ladomersky ◽

Kristen Lauing ◽

...

Keyword(s):

Protein Degradation ◽

Time Course ◽

Immune Cell ◽

Enzyme Inhibitors ◽

Tumor Development ◽

Ubiquitin Proteasome System ◽

Degradation Pathway ◽

Specific Protein ◽

Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

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A Yeast-Based Functional Assay to Study Plant N-Degron – N-Recognin Interactions

Frontiers in Plant Science ◽

10.3389/fpls.2021.806129 ◽

2022 ◽

Vol 12 ◽

Author(s):

Aida Kozlic ◽

Nikola Winter ◽

Theresia Telser ◽

Jakob Reimann ◽

Katrin Rose ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Functional Assay ◽

The Novel ◽

In Planta ◽

Amino Terminal ◽

Weak Binding ◽

Ubiquitin Conjugating Enzyme ◽

Ubiquitin Proteasome ◽

The Stability

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

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Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

10.1101/2020.03.30.015370 ◽

2020 ◽

Author(s):

Ganapathi Kandasamy ◽

Ashis Kumar Pradhan ◽

R Palanimurugan

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Degradation ◽

Ubiquitin Proteasome System ◽

Proteasomal Degradation ◽

Rna Degradation ◽

26S Proteasome ◽

Cellular Proteins ◽

Cellular Homeostasis ◽

Substrate Degradation ◽

Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

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E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

Oncogene ◽

10.1038/sj.onc.1209793 ◽

2006 ◽

Vol 26 (3) ◽

pp. 441-448 ◽

Cited By ~ 11

Author(s):

L Sun ◽

J S Trausch-Azar ◽

A Ciechanover ◽

A L Schwartz

Keyword(s):

Protein Degradation ◽

Ubiquitin Proteasome System ◽

Muscle Differentiation ◽

Ubiquitin Proteasome

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Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.37 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Kellie R Machlus ◽

Prakrith Vijey ◽

Thomas Soussou ◽

Joseph E Italiano

Keyword(s):

Protein Degradation ◽

Proteasome Inhibitors ◽

Aurora Kinase ◽

Proteasome Activity ◽

Major Pathway ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Proplatelet Formation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

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