Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescence-based multiplex assay format

The reversible conjugation of ubiquitin and ubiquitin-like (UbL) proteins to protein substrates plays a critical role in the regulation of many cellular pathways. The removal of ubiquitin from target proteins is performed by ubiquitin proteases also known as deubiquitylases (DUBs). Owing to their substrate specificity and the central role ubiquitylation plays in cell signaling pathways, DUB are attractive targets for therapeutic development. The development of DUB inhibitors requires assays that are amenable to high-throughput screening and provide rapid assessment of inhibitor selectivity. Determination of inhibitor selectivity at an early stage of drug discovery will reduce drug failure in the clinic as well as reduce overall drug development costs. We have developed two novel assays, UbL-Enterokinase light chain and UbL-Granzyme B, for quantifying ubiquitin and UbL protease activity. In our quest to discover and characterize novel chemical entities, we have combined these assays with a previously developed assay in a multiplex format. This multiplex format allows for the detection of three distinct protease activities simultaneously, in a single well. We have demonstrated that the multiplex format is able to distinguish between selective and nonselective protease inhibitors. Specifically, we have used this assay format to characterize P022077, a selective ubiquitin-specific protease 7 inhibitor discovered at Progenra.     

DUBs and disease: activity assays for inhibitor development

Deubiquitinating enzymes (DUBs) cleave ubiquitin from substrate proteins and so influence the biochemical properties of these proteins, such as their half-life, by a reduction in proteasomal degradation. Several DUBs have been demonstrated to deubiquitinate oncogenic proteins and tumor suppressors, and their activities have been implicated in numerous processes related to disease, including cancer and neurodegeneration. The importance of various DUBs is well established, but the understanding of both their selectivity and reactivity in the ubiquitin-proteasome system is comparatively poor. In this review, the published methods that are available to study DUB action in vitro and their application in finding inhibitors are discussed.     

Protein cross-linking as a novel mechanism of action of a ubiquitin-activating enzyme inhibitor with anti-tumor activity

Ubiquitin-activating enzyme 1 (UBE1) is a critical regulator of the ubiquitination cycle and its targeted inhibition may be an appropriate therapeutic strategy as tumor cells are reported to have increased dependence on protein ubiquitination. PYR-41 is a small molecule with previously described UBE1 inhibitory activity. PYR-41 blocks ubiquitination reactions but paradoxically leads to the accumulation of high MW ubiquitinated proteins. Detailed evaluation of PYR-41 activity demonstrated that PYR-41 inhibited UBE1 activity but also had equal or greater inhibitory activity against several deubiquitinases (DUBs) in intact cells and purified USP5 in vitro. Both UBE1 and DUB inhibition were mediated through PYR-41-induced covalent protein cross-linking which paralleled the inhibition of the target proteins enzymatic activity. PYR-41 also mediated cross-linking of specific protein kinases (Bcr-Abl, Jak2) to inhibit their signaling activity. Chemical reactivity modeling provided some insight into the cross-linking potential and partial target selectivity of PYR-41. Overall, our results suggest a broader range of targets and a novel mechanism of action for this UBE1 inhibitor. In addition, since PYR-41-related compounds have demonstrated anti-tumor activity in animal studies, partially selective protein cross-linking may represent an alternate approach to affect signal transduction modules and ubiquitin cycle-regulatory proteins for cancer therapy.     

Synthesis and Evaluation of Derivatives of the Proteasome Deubiquitinase Inhibitor b‐AP15

The ubiquitin–proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b‐AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity. Building upon our previous data, we performed a structure–activity relationship (SAR) study on b‐AP15 and identified VLX1570 as an analog with promising properties, including enhanced potency and improved solubility in aqueous solution. In silico modeling was consistent with interaction of VLX1570 with key cysteine residues located at the active sites of the proteasome DUBs USP14 and UCHL5. VLX1570 was found to inhibit proteasome deubiquitinase activity in vitro in a manner consistent with competitive inhibition. Furthermore, using active‐site‐directed probes, VLX1570 also inhibited proteasome DUB activity in exposed cells. Importantly, VLX1570 did not show inhibitory activity on a panel of recombinant non‐proteasome DUBs, on recombinant kinases, or on caspase‐3 activity, suggesting that VLX1570 is not an overtly reactive general enzyme inhibitor. Taken together, our data shows the chemical and biological properties of VLX1570 as an optimized proteasome DUB inhibitor.     

Counteracting PINK/Parkin Deficiency in the Activation of Mitophagy: A Potential Therapeutic Intervention for Parkinson’s Disease

Parkinson’s Disease (PD) related genes PINK1, a protein kinase [1], and Parkin, an E3 ubiquitin ligase [2], operate within the same pathway [3-5], which controls, via specific elimination of dysfunctional mitochondria, the quality of the organelle network [6]. Parkin translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy [6]. PINK1 regulates Parkin translocation through a not yet completely understood mechanism [7, 8]. Mitochondrial outer membrane proteins Mitofusin (MFN), VDAC, Fis1 and TOM20 were found to be targets for Parkin mediated ubiquitination [9-11]. By adding ubiquitin molecules to its targets expressed on mitochondria, Parkin tags and selects dysfunctional mitochondria for clearance, contributing to maintain a functional and healthy mitochondrial network. Abnormal accumulation of misfolded proteins and unfunctional mitochondria is a characteristic hallmark of PD pathology. Therefore a therapeutic approach to enhance clearance of misfolded proteins and potentiate the ubiquitin-proteosome system (UPS) could be instrumental to ameliorate the progression of the disease. Recently, much effort has been put to identify specific de-ubiquitinating enzymes (DUBs) that oppose Parkin in the ubiquitination of its targets. Similar to other post-translational modifications, such as phosphorylation and acetylation, ubiquitination is also a reversible modification, mediated by a large family of DUBs [12]. DUBs inhibitors or activators can affect cellular response to stimuli that induce mitophagy via ubiquitination of mitochondrial outer membrane proteins MFN, VDAC, Fis1 and TOM20. In this respect, the identification of a Parkin-opposing DUB in the regulation of mitophagy, might be instrumental to develop specific isopeptidase inhibitors or activators that can modulate the fundamental biological process of mitochondria clearance and impact on cell survival.     

Blockade of deubiquitinase YOD1 degrades oncogenic PML/RARα and eradicates acute promyelocytic leukemia cells

In most acute promyelocytic leukemia (APL) cells, promyelocytic leukemia (PML) fuses to retinoic acid receptor α (RARα) due to chromosomal translocation, thus generating PML/RARα oncoprotein, which is a relatively stable oncoprotein for degradation in APL. Elucidating the mechanism regulating the stability of PML/RARα may help to degrade PML/RARα and eradicate APL cells. Here, we describe a deubiquitinase (DUB)-involved regulatory mechanism for the maintenance of PML/RARα stability and develop a novel pharmacological approach to degrading PML/RARα by inhibiting DUB. We utilized a DUB siRNA library to identify the ovarian tumor protease (OTU) family member deubiquitinase YOD1 as a critical DUB of PML/RARα. Suppression of YOD1 promoted the degradation of PML/RARα, thus inhibiting APL cells and prolonging the survival time of APL cell-bearing mice. Subsequent phenotypic screening of small molecules allowed us to identify ubiquitin isopeptidase inhibitor I (G5) as the first YOD1 pharmacological inhibitor. As expected, G5 notably degraded PML/RARα protein and eradicated APL, particularly drug-resistant APL cells. Importantly, G5 also showed a strong killing effect on primary patient-derived APL blasts. Overall, our study not only reveals the DUB-involved regulatory mechanism on PML/RARα stability and validates YOD1 as a potential therapeutic target for APL, but also identifies G5 as a YOD1 inhibitor and a promising candidate for APL, particularly drug-resistant APL treatment.     

Ubiquitin proteasome system in immune regulation and therapeutics

The ubiquitin proteasome system (UPS) is a proteolytic machinery for the degradation of protein substrates that are post-translationally conjugated with ubiquitin polymers through the enzymatic action of ubiquitin ligases, in a process termed ubiquitylation. Ubiquitylation of substrates precedes their proteolysis via proteasomes, a hierarchical feature of UPS. E3-ubiquitin ligases recruit protein substrates providing specificity for ubiquitylation. Innate and adaptive immune system networks are regulated by ubiquitylation and substrate degradation via E3-ligases/UPS. Deregulation of E3-ligases/UPS components in immune cells is involved in the development of lymphomas, neurodevelopmental abnormalities, and cancers. Targeting E3-ligases for therapeutic intervention provides opportunities to mitigate the unintended broad effects of 26S proteasome inhibition. Recently, bifunctional moieties such as PROTACs and molecular glues have been developed to re-purpose E3-ligases for targeted degradation of unwanted aberrant proteins, with a potential for clinical use. Here, we summarize the involvement of E3-ligases/UPS components in immune-related diseases with perspectives.     

Blockade of deubiquitinase YOD1 degrades oncogenic PML/RARα and eradicates acute promyelocytic leukemia cells

In most acute promyelocytic leukemia (APL) cells, promyelocytic leukemia (PML) fuses to retinoic acid receptor α (RARα) due to chromosomal translocation, thus generating PML/RARα oncoprotein, which is a relatively stable oncoprotein for degradation in APL. Elucidating the mechanism regulating the stability of PML/RARα may help to degrade PML/RARα and eradicate APL cells. Here, we describe a deubiquitinase (DUB)-involved regulatory mechanism for the maintenance of PML/RARα stability and develop a novel pharmacological approach to degrading PML/RARα by inhibiting DUB. We utilized a DUB siRNA library to identify the ovarian tumor protease (OTU) family member deubiquitinase YOD1 as a critical DUB of PML/RARα. Suppression of YOD1 promoted the degradation of PML/RARα, thus inhibiting APL cells and prolonging the survival time of APL cell-bearing mice. Subsequent phenotypic screening of small molecules allowed us to identify ubiquitin isopeptidase inhibitor I (G5) as the first YOD1 pharmacological inhibitor. As expected, G5 notably degraded PML/RARα protein and eradicated APL, particularly drug-resistant APL cells. Importantly, G5 also showed a strong killing effect on primary patient-derived APL blasts. Overall, our study not only reveals the DUB-involved regulatory mechanism on PML/RARα stability and validates YOD1 as a potential therapeutic target for APL, but also identifies G5 as a YOD1 inhibitor and a promising candidate for APL, particularly drug-resistant APL treatment.KEY WORDS: Acute promyelocytic leukemia, PML/RARα, Deubiquitinase, YOD1, Degradation, Drug resistance, Inhibitor, Therapy     

Scaffolding proteins in cardiac myocytes

Post-translational modification, such as protein phosphorylation, plays a critical role to reversibly amplify and modulate signaling pathways. Since kinases and phosphatases have broad substrate recognition motifs, compartmentalization and localization of signaling complexes are required to achieve specific signals. Scaffolds are proteins that associate with two or more binding partners and function to enhance the efficiency and/or specificity of cellular signaling pathways. The identification of scaffolding proteins that control the tempo and/or spatial organization of signaling pathways in cells has benefited enormously from recent technological advances that allow for the detection of protein-protein interactions, including in vivo in intact cells. This review will focus on scaffolding proteins that nucleate multi-protein complexes (and could represent novel entry points into signaling pathways that might be amenable to therapeutic manipulation) in cardiomyocytes.     

SCF E3 ubiquitin ligases as anticancer targets

The SCF multisubunit complex (Skp1, Cullins, F-box proteins) E3 ubiquitin ligase, also known as CRL (Cullin-RING ubiquitin Ligase) is the largest E3 ubiquitin ligase family that promotes the ubiquitination of various regulatory proteins for targeted degradation, thus regulating many biological processes, including cell cycle progression, signal transduction, and DNA replication. The efforts to discover small molecule inhibitors of a SCF-type ligase or its components were expedited by the FDA approval of Bortezomib (also known as Velcade or PS-341), the first (and only) class of general proteasome inhibitor, for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma. Although Bortezomib has demonstrated a certain degree of cancer cell selectivity with measurable therapeutic index, the drug is, in general, cytotoxic due to its inhibition of overall protein degradation. An alternative and ideal approach is to target a specific E3 ligase, known to be activated in human cancer, for a high level of specificity and selectivity with less associated toxicity, since such inhibitors would selectively stabilize a specific set of cellular proteins regulated by this E3. Here, we review recent advances in validation of SCF E3 ubiquitin ligase complex as an attractive anti-cancer target and discuss how MLN4924, a small molecule inhibitor of NEDD8-activating enzyme, can be developed as a novel class of anticancer agents by inhibiting SCF E3 ligase complex via removal of cullin neddylation. Finally, we discuss under future perspective how basic research on SCF biology will direct the drug discovery efforts surrounding this target.     

Controlling receptor downregulation by ubiquitination and deubiquitination

Growth factor-activated receptor tyrosine kinases (RTKs) undergo rapid endocytosis and degradation in lysosomes. This process, known as receptor downregulation, is essential to prevent the overgrowth of cells by terminating signal transduction from activated RTKs. Thus, defects in RTK downergulation lead to cell proliferative disorders such as cancer. Upon endocytosis, RTKs are delivered to endosomes, from where they are further transported to lysosomes. Ubiquitin serves as a sorting signal that is tagged on activated RTKs and directs their trafficking from endosomes to lysosomes. On the endosomal membrane, ubiquitinated RTKs are sorted by coordinated actions of the class E vacuolar protein sorting (Vps) proteins some of which form complexes that directly recognize the ubiquitin moieties of RTKs. UBPY and AMSH in mammals, as well as Doa4 in yeast, are deubiquitinating enzymes (DUBs) that associate with class E Vps proteins on endosomes. Here I review the recently unveiled roles and regulatory mechanisms of these DUBs in the endosomal sorting of ubiquitinated cargo proteins. These findings suggest that RTK downregulation is controlled not only by ubiquitination but also by deubiquitination of RTKs as well as other endosomal proteins. Therefore, elucidating the entire functions and regulation of the endosomal DUBs potentially provides novel molecular targets for the treatment of cancer accompanied by overexpression or constitutive activation of RTKs.     

Exploiting substrate recognition for selective inhibition of protein kinases

Protein kinases are potential targets of drugs to treat many human diseases. Intensive efforts have been made to develop protein kinase inhibitors, but a major challenge is achieving specificity. Exploiting regulatory elements outside the ATP binding pocket, such as the substrate binding site, may provide an alternative that allows generation of competitive inhibitors with improved selectivity. In-depth understanding of substrate recognition by protein kinase is essential for design and refinement of competitive inhibitors. Here we described strategies for specifically targeting protein kinases and highlight our current progress in the development of substrate competitive inhibitors for glycogen synthase kinase-3 (GSK-3).     

Common Mechanism for Target Specificity of Protein- and DNA-Targeting ADP-Ribosyltransferases

Many bacterial pathogens utilize ADP-ribosyltransferases (ARTs) as virulence factors. The critical aspect of ARTs is their target specificity. Each individual ART modifies a specific residue of its substrates, which could be proteins, DNA, or antibiotics. However, the mechanism underlying this specificity is poorly understood. Here, we review the substrate recognition mechanism and target residue specificity based on the available complex structures of ARTs and their substrates. We show that there are common mechanisms of target residue specificity among protein- and DNA-targeting ARTs.     

Identification of NAE Inhibitors Exhibiting Potent Activity in Leukemia Cells: Exploring the Structural Determinants of NAE Specificity

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The ubiquitin-mediated system for intracellular protein degradation.

Degradation of proteins by the ubiquitin system involves several discrete steps. Initially, multiple molecules of ubiquitin are covalently conjugated to the target substrate in an energy-requiring reaction. The protein thus marked is degraded by a specific ATP-dependent protease, and free and reutilizable ubiquitin is released. In this review we discuss the mechanisms involved in ubiquitin activation, selection of substrates for conjugation, and subsequent degradation of ubiquitin-conjugated proteins in the cell-free system. In addition, we summarize briefly what is currently known of the physiological roles of the ubiquitin system in vivo.     

A-kinase anchoring proteins as the basis for cAMP signaling

Common challenges to any cell are the processing of the extracellular stimuli it receives into intracellular signaling cascades that initiate a multitude of diverse biological functions. However, many of these stimuli act via a common signaling pathway, suggesting the cell must somehow discriminate between different stimuli and respond accordingly. Subcellular targeting through the association with adaptor and scaffolding proteins has emerged as a key mechanism by which cells maintain signaling specificity. Compartmentation of cAMP signaling is maintained by the clustering of cAMP signaling enzymes in discrete units by the scaffolding protein A-kinase anchoring proteins (AKAP). In doing so, AKAPs provide the molecular architecture for the cAMP micordomains that underlie the spacial-temporal control of cAMP signaling.     

Part I: parkin-associated proteins and Parkinson's disease

Parkin is an E3 ligase that plays an important role in the ubiquitin/proteosome pathway responsible for protein degradation events. Mutations in parkin result in a loss-of-function and lead to Parkinson's disease, a progressive neurological disorder of movement. Presumably, this occurs due to the toxic build-up of proteins that are no longer effectively cleared/degraded by the parkin-dependent ubiqutin/proteosome pathway. To date, three types of proteins have been shown to interact with parkin. Firstly, the E2 ubiquitin conjugating proteins called UbcH7 and UbcH8 interact with parkin. Secondly, putative substrates interacting with parkin include a synaptic vesicle associated GTPase named CDCrel-1; a G protein-coupled receptor named Pael; a novel from of alpha-synuclein; and an alpha-synuclein interacting protein synphilin-1. Thirdly and more recently, a PDZ domain containing scaffolding protein CASK/Lin2 has been shown to interact with the PDZ binding motif of parkin. A network of PDZ-interacting proteins has potential to form a complex web of molecules that surround parkin and regulate its subcellular localisation and function.     

Discovery of a potent and dual-selective bisubstrate inhibitor for protein arginine methyltransferase 4/5

Protein arginine methyltransferases (PRMTs) have been implicated in the progression of many diseases. Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery. Herein, we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine (SAM) analogue moiety and a tripeptide through an alkyl substituted guanidino group. Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration (IC₅₀) of 2.8 and 0.42 nmol/L, respectively. Computational studies provided a plausible explanation for the high potency and selectivity of AH237 for PRMT4/5 over other 40 methyltransferases. This proof-of-principle study outlines an applicable strategy to develop potent and selective bisubstrate inhibitors for PRMTs, providing valuable probes for future structural studies.     

Ubiquitin–proteasome system-targeted therapy for uveal melanoma: what is the evidence?

Uveal melanoma (UM) is a rare ocular tumor. The loss of BRCA1-associated protein 1 (BAP1) and the aberrant activation of G protein subunit alpha q (GNAQ)/G protein subunit alpha 11 (GNA11) contribute to the frequent metastasis of UM. Thus far, limited molecular-targeted therapies have been developed for the clinical treatment of UM. However, an increasing number of studies have revealed the close relationship between the ubiquitin proteasome system (UPS) and the malignancy of UM. UPS consists of a three-enzyme cascade, i.e. ubiquitin-activating enzymes (E1s); ubiquitin-conjugating enzymes (E2s); and ubiquitin-protein ligases (E3s), as well as 26S proteasome and deubiquitinases (DUBs), which work coordinately to dictate the fate of intracellular proteins through regulating ubiquitination, thus influencing cell viability. Due to the critical role of UPS in tumors, we here provide an overview of the crosstalk between UPS and the malignancy of UM, discuss the current UPS-targeted therapies in UM and highlight its potential in developing novel regimens for UM.