Patented small molecule inhibitors in the ubiquitin proteasome system

Deregulation of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. The recent approval of the proteasome inhibitor Velcade(R) (bortezomib) for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. We review here new patented proteasome inhibitors and patented small molecule inhibitors targeting more specific UPS components, such as E3 ubiquitin ligases and deubiquitylating enzymes. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Small‐Molecule Control of Intracellular Protein Levels through Modulation of the Ubiquitin Proteasome System

Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be readily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small‐molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activity, can be targeted instead, thus increasing the number of druggable targets. Whereas targeting of the proteasome itself can lead to a global increase in protein levels, the targeting of other components of the UPS (e.g., the E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small‐molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small‐molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets, such as enzymes and receptors.     

Ubiquitin Ligases at the Heart of Skeletal Muscle Atrophy Control

Skeletal muscle loss is a detrimental side-effect of numerous chronic diseases that dramatically increases mortality and morbidity. The alteration of protein homeostasis is generally due to increased protein breakdown while, protein synthesis may also be down-regulated. The ubiquitin proteasome system (UPS) is a master regulator of skeletal muscle that impacts muscle contractile properties and metabolism through multiple levers like signaling pathways, contractile apparatus degradation, etc. Among the different actors of the UPS, the E3 ubiquitin ligases specifically target key proteins for either degradation or activity modulation, thus controlling both pro-anabolic or pro-catabolic factors. The atrogenes MuRF1/TRIM63 and MAFbx/Atrogin-1 encode for key E3 ligases that target contractile proteins and key actors of protein synthesis respectively. However, several other E3 ligases are involved upstream in the atrophy program, from signal transduction control to modulation of energy balance. Controlling E3 ligases activity is thus a tempting approach for preserving muscle mass. While indirect modulation of E3 ligases may prove beneficial in some situations of muscle atrophy, some drugs directly inhibiting their activity have started to appear. This review summarizes the main signaling pathways involved in muscle atrophy and the E3 ligases implicated, but also the molecules potentially usable for future therapies.     

An inhibitor of ubiquitin conjugation and aggresome formation

Proteasome inhibitors have revolutionized the treatment of multiple myeloma, and validated the therapeutic potential of the ubiquitin proteasome system (UPS). It is believed that in part, proteasome inhibitors elicit their therapeutic effect by inhibiting the degradation of misfolded proteins, which is proteotoxic and causes cell death. In spite of these successes, proteasome inhibitors are not effective against solid tumors, thus necessitating the need to explore alternative approaches. Furthermore, proteasome inhibitors lead to the formation of aggresomes that clear misfolded proteins via the autophagy–lysosome degradation pathway. Importantly, aggresome formation depends on the presence of polyubiquitin tags on misfolded proteins. We therefore hypothesized that inhibitors of ubiquitin conjugation should inhibit both degradation of misfolded proteins, and ubiquitin dependent aggresome formation, thus outlining the path forward toward more effective anticancer therapeutics. To explore the therapeutic potential of targeting the UPS to treat solid cancers, we have developed an inhibitor of ubiquitin conjugation (ABP A3) that targets ubiquitin and Nedd8 E1 enzymes, enzymes that are required to maintain the activity of the entire ubiquitin system. We have shown that ABP A3 inhibits conjugation of ubiquitin to intracellular proteins and prevents the formation of cytoprotective aggresomes in A549 lung cancer cells. Furthermore, ABP A3 induces activation of the unfolded protein response and apoptosis. Thus, similar to proteasome inhibitors MG132, bortezomib, and carfilzomib, ABP A3 can serve as a novel probe to explore the therapeutic potential of the UPS in solid and hematological malignancies.     

The Molecular and Pathophysiological Functions of Members of the LNX/PDZRN E3 Ubiquitin Ligase Family

The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.     

Specific interaction between Smad1 and CHIP: a surface plasmon resonance study

The TGF-β superfamily signaling pathway regulates many important biological processes, including cell growth, differentiation and embryonic pattern formation. Smad1, a member of this signaling pathway that functions downstream of serine/threonine kinase receptors, has ability to interact with carboxyl terminus of Hsc70-interacting protein (CHIP), which is an E3 ubiquitin ligase in other cases. It has been reported that Smurf1, a member of the Hect family E3 ubiquitin ligases, can target Smad1 to 26S proteasome for degradation. In this paper, we studied the interaction of Smad1 and CHIP by combination of surface plasmon resonance and supported monolayer approach. The specific binding of Smad1 to CHIP indicates that the degradation of Smad1 may also be mediated by CHIP, and CHIP may play an essential role in the TGF-β signaling pathway.     

Roles of p97-Associated Deubiquitinases in Protein Quality Control at the Endoplasmic Reticulum

To maintain protein homeostasis in the ER, an ER protein quality control system retains unfolded polypeptides and misassembled membrane proteins, allowing only properly folded proteins to exit the ER. Misfolded proteins held in the ER are retrotranslocated into the cytosol, ubiquitinated, and degraded by the proteasome through the ER-associated degradation pathway (ERAD). By timely eliminating misfolded proteins, the ERAD system alleviates cytotoxic stress imposed by protein misfolding. It is well established that ER-associated ubiquitin ligases play pivotal roles in ERAD by assembling ubiquitin conjugates on retrotranslocation substrates, which serve as degradation signals for the proteasome. Surprisingly, recent studies have revealed an equally important function for deubiquitinases (DUBs), enzymes that disassemble ubiquitin chains, in ERAD. Intriguingly, many ERAD specific DUBs are physically associated with the retrotranslocation- driving ATPase p97. Here we discuss the potential functions of p97-associated DUBs including ataxin-3 and YOD1. Our goal is to integrate the emerging evidence into models that may explain how protein quality control could benefit from deubiquitination, a process previously deemed destructive for proteasomal degradation.     

Modular PROTAC Design for the Degradation of Oncogenic BCR‐ABL

Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c‐ABL and BCR‐ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.     

Synthetic approaches to constructing proteolysis targeting chimeras (PROTACs)

The development of various heterobifunctional constructs dubbed PRoteolysis-TArgeting Chimeras (PROTACs) has gained a significant impetus in the last few years. A viable alternative to the traditional occupancy-based inhibition of aberrantly hyperactive proteins, PROTACs operate by an event-based catalytic mechanism bringing together the protein of interest (POI, to be degraded) and E3 ubiquitin ligases. The formation of the ternary complex ‘POI–PROTAC–E3 ubiquitin ligase’ is the critical step which leads to the ubiquitination of the POI and its proteasomal degradation. The current Focused Review aims to highlight the syntheses of selected innovative PROTAC-type degraders of the therapeutically important protein targets as well as some notable chemical aspects of PROTAC construction. The overview is focusing on PROTACs aimed at recruiting Cereblon, the most exploited E3 ligase for targeted protein degradation.     

The role of the UPS in cystic fibrosis

CF is an inherited autosomal recessive disease whose lethality arises from malfunction of CFTR, a single chloride (Cl-) ion channel protein. CF patients harbor mutations in the CFTR gene that lead to misfolding of the resulting CFTR protein, rendering it inactive and mislocalized. Hundreds of CF-related mutations have been identified, many of which abrogate CFTR folding in the endoplasmic reticulum (ER). More than 70% of patients harbor the DeltaF508 CFTR mutation that causes misfolding of the CFTR proteins. Consequently, mutant CFTR is unable to reach the apical plasma membrane of epithelial cells that line the lungs and gut, and is instead targeted for degradation by the UPS. Proteins located in both the cytoplasm and ER membrane are believed to identify misfolded CFTR for UPS-mediated degradation. The aberrantly folded CFTR protein then undergoes polyubiquitylation, carried out by an E1-E2-E3 ubiquitin ligase system, leading to degradation by the 26S proteasome. This ubiquitin-dependent loss of misfolded CFTR protein can be inhibited by the application of 'corrector' drugs that aid CFTR folding, shielding it from the UPS machinery. Corrector molecules elevate cellular CFTR protein levels by protecting the protein from degradation and aiding folding, promoting its maturation and localization to the apical plasma membrane. Combinatory application of corrector drugs with activator molecules that enhance CFTR Cl- ion channel activity offers significant potential for treatment of CF patients. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Structural Diversity of Ubiquitin E3 Ligase

The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.     

Chemical tools for E3 ubiquitin ligase study

E3 ubiquitin ligases catalyze the final step of ubiquitylation, a crucial post-translational modification involved in almost every process in eukaryotic cells. E3 ubiquitin ligases are key regulators of cellular events, and the investigation into their functions and functioning mechanisms are research areas with great importance. Synthetic or semi-synthetic tools have greatly facilitated the research about the enzyme activity, distribution in different physiological events, and catalytic mechanism of E3 ubiquitin ligase. In this review, we summarize the development of chemical tools for E3 ubiquitin ligases with an emphasis on the synthetic routes. We show the utility of these chemical tools by briefly discussing their applications in biological research.     

Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies

Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into β-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington''s disease (HD), Alzheimer''s disease (AD), Parkinson''s disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.     

Role of the ubiquitin proteasome system in Alzheimer's disease

Though Alzheimer's disease (AD) is a syndrome with well-defined clinical and neuropathological manifestations, an array of molecular defects underlies its pathology. A role for the ubiquitin proteasome system (UPS) was suspected in the pathogenesis of AD since the presence of ubiquitin immunoreactivity in AD-related neuronal inclusions, such as neurofibrillary tangles, is seen in all AD cases. Recent studies have indicated that components of the UPS could be linked to the early phase of AD, which is marked by synaptic dysfunction, as well as to the late stages of the disease, characterized by neurodegeneration. Insoluble protein aggregates in the brain of AD patients could result from malfunction or overload of the UPS, or from structural changes in the protein substrates, which prevent their recognition and degradation by the UPS. Defective proteolysis could cause the synaptic dysfunction observed early in AD since the UPS is known to play a role in the normal functioning of synapses. In this review, we discuss recent observations on possible links between the UPS and AD, and the potential for utilizing UPS components as targets for treatment of this disease. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability

Background  

The COP9 signalosome (CSN) is a conserved protein complex in eukaryotic cells consisting of eight subunits (CSN1 to CSN8). Recent data demonstrate that the CSN is a regulator of the ubiquitin (Ub) proteasome system (UPS). It controls substrate ubiquitination by cullin-RING Ub ligases (CRLs), a process that determines substrate specificity of the UPS. The intrinsic deneddylating activity localized to CSN5 as well as the associated kinases and deubiquitinating activity are involved in the regulatory function of CSN. The exact mechanisms are unclear. In this study we knocked down CSN1 (siCSN1), CSN3 (siCSN3) and CSN5 (siCSN5) by specific siRNA oligos permanently expressed in HeLa cells. The analysis and comparison of siRNA cells revealed differential impact of individual subunits on CSN structure and function.     

Role of the ubiquitin proteasome system in Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Although a subject of intense research, the etiology of PD remains poorly understood. Recently, several lines of evidence have implicated an intimate link between aberrations in the ubiquitin proteasome system (UPS) and PD pathogenesis. Derangements of the UPS, which normally functions as a type of protein degradation machinery, lead to alterations in protein homeostasis that could conceivably promote the toxic accumulation of proteins detrimental to neuronal survival. Not surprisingly, various cellular and animal models of PD that are based on direct disruption of UPS function reproduce the most prominent features of PD. Although persuasive, new developments in the past few years have in fact raised serious questions about the link between the UPS and PD. Here I review current thoughts and controversies about their relationship and discuss whether strategies aimed at mitigating UPS dysfunction could represent rational ways to intervene in the disease. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Cullin-based ubiquitin ligase and its control by NEDD8-conjugating system

Several studies have examined the importance of ubiquitin-like posttranslational modifiers (which consist of an unexpectedly large family). Of these, NEDD8 (also called Rub1, related to ubiquitin 1) with a high homology to ubiquitin is covalently linked to all members of cullin (Cul)-family proteins through an enzymatic cascade analogous to ubiquitylation. Cul-family proteins are scaffold proteins for a wide series of ubiquitin-protein ligase complexes, such as SCFs (Skp1, Cul-1, Roc1, and F-box proteins), which regulate the degradation of broad range of cellular proteins. Unlike ubiquitin, which mostly acts as a degradation signal for the target proteins, NEDD8 acts as an activation signal for Cul-family proteins; i.e., Cul-based ubiquitin-protein ligases. Accordingly, the NEDD8 conjugation pathway regulating Cul-protein function is responsible for a diverse array of biologically important processes, such as the cell cycle progression, signalling cascades and developmental programs. Furthermore, recent studies have revealed that the COP9/Signalosome complex interacts physically and genetically with Cul-family proteins, and catalyzes deconjugation of NEDD8 ligated to Cul-family proteins. This review summarizes recent advances in biochemical and genetic studies on how the NEDD8-modifying system regulates Cul-family proteins and their physiology.     

Quantitative analysis of a ubiquitin‐dependent substrate using capillary electrophoresis with dual laser‐induced fluorescence

Protein degradation by the ubiquitin‐proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS‐dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)‐fusion of the ubiquitin substrate ribophorin 1 (GFP‐RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP‐RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris‐CHES buffer (pH 9.0) containing 10 mM SDS. GFP‐RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP‐RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP‐RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS‐dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.