Inhibiting the deubiquitinating enzymes (DUBs).

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Inhibiting the Deubiquitinating Enzymes (DUBs) Miniperspective Chudi Ndubaku and Vickie Tsui* Department of Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ABSTRACT: The diverse roles of deubiquitinating enzymes, or DUBs, in determining the fate of specific proteins continue to unfold. Concurrent with the revelation of DUBs as potential therapeutic targets are publications of small molecule inhibitors of these enzymes. In this review, we summarize these molecules and their associated data and suggest additional experiments to further validate and characterize these compounds. We believe the field of drug discovery against DUBs is still in its infancy, but advances in assay development, biophysical techniques, and screening libraries hold promise for identifying suitable agents that could advance into the clinic.

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system as well as other biological processes.9 Figure 1 illustrates the main functions for which DUBs are responsible: (a) liberation of ubiquitin from protein substrates (e.g., to remove degradation signal), (b) editing of polyubiquitin signal on protein substrates to change the fate of the protein,10 (c) disassembling polyubiquitin chains to free up ubiquitin monomers, (d) cleaving ubiquitin precursors or adducts to regenerate active ubiquitin.11 The DUBs are subdivided into five families: ubiquitin Cterminal hydrolases (UCHs),12 ubiquitin-specific proteases (USPs),13 ovarian tumor proteases (OTUs),2,9,14,15 Josephins and JAB1/MPN/MOV34 metalloenzymes (JAMM/MPN +).10,12,13,16,17 The first four families (UCH, USP, OTU, and Josephin) are cysteine proteases, while JAMM/MPN+ are zinc metalloproteases. Among these, UCHs and USPs are the best characterized, and USPs represent more than half of the known human DUBs.18 A phylogenetic classification based on the cysteine protease catalytic domain, also known as the UCH domain, is shown in Figure 2. Because USP and UCH are also the only DUB families with published small molecule inhibitors, this review will focus on these two families. Figure 3 shows the reaction mechanism of the cysteine protease DUB families and is the same as that of the cysteine protease superfamily.19 Each enzyme active site requires the interplay between three conserved residues forming the catalytic triad: a cysteine, a histidine, and an aspartic acid. Three-dimensional structural differences between the positions of these residues among various DUBs will be discussed in the next section. As can be seen in Figure 3a,b, the initial attack creates a negatively charged transition state stabilized by the oxy-anion hole. The intermediate is a thiohemiacetal stabilized

BACKGROUND Ubiquitination is an important form of post-translational modification that can determine a protein’s fate. While ubiquitin itself is a small and conserved protein, its covalent conjugation to protein substrates and to other ubiquitin molecules is a tightly controlled process involving complex cellular machinery.1−3 Perhaps the most prominent and well-known function of ubiquitin is to target a protein for degradation by the 26S proteasome. This is done via isopeptide bond formation between the carboxy-terminal Gly on the ubiquitin and ε-amino group of lysine side chains of the protein substrate. The ubiquitin−substrate system is further diversified via the process of polyubiquitination, during which a ubiquitin molecule’s Cterminal Gly is conjugated with one of the seven Lys residues on another ubiquitin (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, or Lys63) or with the N-terminus to form linear chains.4 While Lys48-linked and Lys11-linked polyubiquitination has been shown mostly to target protein substrates for 26S-mediated degradation, other functions of ubiquitination continue to unfold. For example, Lys63-polyubiquitination is involved in DNA repair and endocytosis, while both Lys63-linked and linear polyubiquitination has been demonstrated to regulate immunity via NF-κB activation.4−8 The summary above merely scratches the surface of the biological complexity of ubiquitination and suggests that enzymes involved in this process must have remarkable specificity to correctly carry out their unique functions. E1, E2, and E3 are enzymes that together facilitate the multistep process of ubiquitin−substrate conjugation, and deubiquitinating enzymes (deubiquitinases or DUBs) carry out the reverse steps of breaking the isopeptide bond.2 There are ∼100 DUBs known in the human genome, any of which could be playing a key role in the ubiquitin−proteasome © XXXX American Chemical Society

Received: July 15, 2014

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dx.doi.org/10.1021/jm501061a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Functions of DUBs. (a) Mono- or polyubiquitin chains are cleaved from protein substrates, rescuing the protein substrate from degradation. (b) Deubiquitination of parts of a polyubiquitin chain allows for modified ubiquitin assembly on the protein substrate, hence changing the ubiquitin signal from one to another. (c) DUBs can disassemble polyubiquitin chains that are not conjugated to a protein substrate, creating ubiquitin monomers. (d) Polyubiquitin genes encode C-terminal amino acid extensions known as precursors, and these can be cleaved off to create free ubiquitin. Sometimes the precursor also refers to ribosomal proteins whose N-terminus is fused to ubiquitin.

will focus on the USP and UCH DUB families, especially on the relevance of their structural features to the binding of small molecule inhibitors. USP. X-ray structures have been determined for the catalytic domain of numerous USPs including, but not limited to, USP4, USP5, USP7, USP8, USP14, and USP21.11,18,19,23,24 These share a common fold containing three subdomains known as Palm, Thumb, and Fingers, in which the active-site cysteine sits between the Palm and Thumb while the Fingers “grip” the ubiquitin (Figure 4). In other words, structures of the enzyme/ substrate complex show that the C-terminal tail of ubiquitin extends down a narrow cleft to reach the catalytic cysteine, while the rest of the distal ubiquitin has a typical protein− protein contact area with the DUB, made up of shallow surfaces on both the ubiquitin and USP contact sites. For several of these members, structures exist both in apo and bound to ubiquitin such that the structural basis for enzyme−substrate interactions can be studied carefully. Komander et al. utilized comparisons of apo versus substratebound structures to distinguish USP7 from USP14 and USP8, in that USP7’s catalytic triad required ubiquitin binding in order to adopt the active conformation.11 Figure 5 shows the conformations of the catalytic triad in the apo structures of (a) USP14 and (b) USP7 in detail,23,25 illustrating that the distance between the catalytic cysteine and histidine in USP7, unlike that in USP14, is too far away for the reaction mechanism described in Figure 3 to occur. Even when their catalytic triad conformations are preorganized in the active state, USP14 and USP8 still exist in an autoinhibited state in which the folding of a loop close to the active site cysteine occludes the cleft which would need to open up for the C-terminal tail of ubiquitin. Recently, structures of USP catalytic domains bound to ubiquitin variants obtained from directed evolution specifically

through interactions with the active site residues as an incoming water liberates the lysine side chain from the conjugated ubiquitin or substrate. A nucleophilic attack of the water creates another negatively charged transition state, which rearranges to free up the carboxylate terminus of the N-terminal ubiquitin and restores the enzyme to its apo form. In the past 5−10 years, increased biological understanding has led to numerous DUBs being implicated in various diseases spanning oncology, neurodegeneration, hematology, and infectious diseases.20,21 Most recently, Bingol et al. carried out elegant experiments in vitro and in vivo to illustrate the role of USP30 as an antagonist of Parkin-mediated mitophagy, suggesting the inhibition of USP30 as a potential therapy for Parkinson’s disease (PD).22 The hunt for DUB antagonists is thus actively carried out by academic and pharmaceutical companies alike. This is illustrated through chemically diverse small molecules that have been reported to inhibit one or more of the UCH and USP family members. These molecules will be evaluated herein in terms of their chemical structures and supporting data; additional studies and characterizations to further validate or understand these molecules will also be suggested. Overall, DUBs serve as attractive and promising, albeit challenging, therapeutic targets. There is even a question of whether this class of enzymes is druggable. The development of small molecule inhibitors is still in its infancy as this review will show; however, we foresee breakthroughs in the near future as assay optimization, identification of protein-based inhibitors with improved potency and specificity, and more X-ray structures continue to demystify DUBs.

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STRUCTURES The three-dimensional structures of various families of DUBs have been summarized in a recent review.11 In this review, we B



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Figure 4. X-ray structure of USP14 bound to ubiquitin-aldehyde (PDB code 2AYO). The Thumb, Palm, and Fingers domains are colored in green, light brown, and cyan, respectively. Ubiquitin-aldehyde is colored and surfaced in light pink, with its C-terminal tail reaching USP14’s catalytic triad whose side chain carbons are depicted in yellow.

thermore, USP7-specific ubiquitin variants have been identified by Zhang et al. using computational design and phage display,27 along with crystal structures of the ubiquitin variants showing different backbone conformations of the ubiquitin variants. Both of these studies, which will be discussed in more detail later, indicate that identification and structure-based design of protein-based USP inhibitors are possible. Despite these advancements, no publicly available smallmolecule inhibitor bound to human DUB structure exists. As will be described in detail in the next section, numerous smallmolecule inhibitors have been reported as selective or multitargeted DUB inhibitors, and yet none of them have X-

Figure 2. Neighbor joining tree based on sequence alignment of human USP and UCH catalytic domains. This was generated using CLUSTALX104 for sequence alignment, followed by PFAAT105 for tree generation.

targeting USP21, USP8, and USP2a have been published, further illustrating the molecular basis for specificity of these ubiquitin variants as protein-based DUB inhibitors.26 Fur-

Figure 3. Reaction mechanism of the cysteine protease DUB families. The carboxy terminus of ubiquitin is shown in red, and the lysine side chain of the conjugated species is shown in blue. Shown in black are the catalytic triad of the DUB and (c) a water molecule involved in the acyl intermediate. C



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Figure 5. X-ray structures of apo (a) USP14 (PDB code 2AYN) and (b) USP7 (PDB code 1NB8), showing the catalytic triad in sticks with distances labeled between the Cys sulfur and His nitrogen and between the other His nitrogen and an oxygen on the Asp.

Figure 6. X-ray structure of UCH-L3 bound to Ub-VME (PDB code 1XD3). Ub-VME is colored and surfaced in light pink, with its Cterminal tail reaching UCH-L3’s catalytic triad whose side chain carbons are depicted in yellow. The active site crossover loop, which is disordered in structures of apo UCH family members, is observed when ubiquitin is bound and is shown in cyan. The rest of UCH-L3 is shown in green.

ray structures in complex with its target. It is worthwhile to point out that crystal structures of a series of small molecule inhibitors complexed with papain-like protease (PLpro), a viral DUB, have been published first in 200828 and most recently in 2014.29 However, comparison of both the structural overlay and sequence identity of the binding site suggests this pocket is not available to any of the USPs (the most homologous DUB family to PLpro), and assay data proved compounds in this series to be inactive against seven DUBs tested. A caveat to these crystal structures, which show a short loop with low Bfactor folded on top of the inhibitor, lies in that either the crystallographic symmetry mate or another molecule in the asymmetric subunit directly contacts the binding site protein as well as the small molecule, and this crystal packing could have a significant impact on the conformation and rigidity of the site. While this section has focused thus far on describing structures of the USP catalytic domain, small molecules could conceivably bind to other domains and inhibit the enzymatic activity via an allosteric mechanism. Several USPs contain additional domains or even protein partners that regulate their activities.30−32 For example, USP7 has an N-terminal TRAF domain that binds p53 and other targets plus five ubiquitin-like (UBL) domains C-terminal to the catalytic domain. X-ray structures have been published for the complex of TRAF and the catalytic domains of USP7,33 and in addition the UBLs were described in a more recent report.34 These structures increase the understanding of protein regulation and will potentially be important for structure-guided designs of any allosteric inhibitor. UCH. Compared to the USP family, the UCH family is simpler in that the only members are L1, L3, L5, and BAP1 and that each contains only a relatively short (200−300 amino acids) catalytic domain.11 X-ray structures have been solved for human UCH-L1, -L3, and -L5,19,35−37 with Figure 6 showing the structure of UCH-L3 with ubiquitin vinyl methyl ester (VME) bound (PDB code 1XD3).38 Perhaps the most interesting structural aspect of the UCH enzymes lies in the induced fit of the active-site “crossover loop” upon ubiquitin binding. This loop is disordered in the apo structures, and as ubiquitin approaches the enzyme, a number of correlated conformational changes occur to allow the C-terminal tail of ubiquitin to thread through the crossover loop to reach the active-site cysteine, while the bulk of the folded ubiquitin contacts a distal site of UCH on the other side of the crossover loop. This has implications on the biological functions of the UCH family as processors of short C-terminal extensions of ubiquitin precursors.39,40 It could also affect the kinetics of

small molecule inhibitors depending on their effect on the crossover loop conformation; however, more studies would be needed to test this hypothesis. Aside from the observed conformational differences in the crossover loop, different UCH members may have different degrees of preorganization of the catalytic triad. For example, Figure 7 shows that UCH-L5 in the apo form adopts a

Figure 7. X-ray structures of apo (a) UCH-L5 (PDB code 3RIS) and (b) UCH-L1 (PDB code 2ETL), showing the catalytic triad in sticks with distances labeled between the Cys sulfur and His nitrogen and between the His nitrogen and an oxygen on the Asp.

conformation in which the orientation of the cysteine side chain positions the sulfur in a catalytically unproductive form despite its proximity to the histidine in the catalytic triad.41 On the other hand, the apo UCH-L1 structure shows a longer distance between the cysteine and histidine and different rotamers relative to that of UCH-L5.36 This difference between family members offers a mechanism for distinguishing selectivity and kinetics between different DUBs in order for each to play its specific functional role. The degree of active site preorganization may have implications on the design of inhibitors. For example, a DUB whose unbound catalytic triad is in the active conformation may be more susceptible to covalent modifiers, whereas a DUB with an unbound and misaligned conformation may stay inhibited if a noncovalent small molecule could stabilize the inactive conformation. More studies and structural characterization involving small molecule inhibitors would be necessary to test this hypothesis. D



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Figure 8. Chemical structures of reported USP7 and USP47 inhibitors.

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PUBLISHED DUB INHIBITORS A number of reports have recently appeared that describe the identification and utility of small molecule DUB inhibitors. In this section, we will cover the biological rationale for targeting certain DUBs, and the reported inhibitors (for USPs and UCHs, in particular). In addition, we will offer general comments with regards to the chemical matter. Assays Available for Assessing DUB Activity. Prior to discussing the efforts directed toward identifying inhibitors of various DUBs, it is useful to introduce the types of assays that are typically employed. A number of platforms based on conjugation of ubiquitin to fluorescent tags have been developed. One such system is the ubiquitin C-terminal 7amido-4-methylcoumarin (or Ub-AMC)42,43 coupled with fluorescence polarization (FP). This was one of the first assays developed to measure DUB activity. An advantage of this assay is that it is relatively simple and is broadly applicable to various DUB enzymes. However, Ub-AMC is an unnatural substrate, and fluorescent artifacts (autofluorescence or fluorescent quenchers) could confound data interpretation. Subsequent optimization to the assay has involved replacing AMC with rhodamine 110 (Rho110) 44 and tetramethylrhodamine (TAMRA)45 that are red-shifted and thereby are less prone to artifacts. A reporter assay called the Ub-C/EBP homologous protein (Ub-CHOP) assay that addresses some of the drawbacks of the fluorescent-tagged assays introduced above has also been described.46,47 In this assay, ubiquitin is conjugated to an inactive precursor of a reporter enzyme (e.g., PLA2). Once cleaved, the reporter enzyme becomes active and proceeds to cleave its substrate. This activity can be measured in an orthogonal fashion. Ub-CHOP is also known as a “coupled” assay and, in effect, magnifies the activity of the DUB, allowing for screening at a lower concentration of enzyme. Attempts to increase the physiological relevance of the assays described above have involved inserting a substrate-based peptide linked through an isopeptide bond between ubiquitin and the dye. The substrate has been shown to work in FP48 and fluorescence resonance electron transfer (FRET)49 formats. An extension of this application is the use of diubiquitin (diUb) as substrates, and these can be linked through various lysine positions.50 The advantage with this approach is that the assay

substrate more closely mimics the innate substrate for certain DUBs. Since the advent of the Ub-AMC assay, numerous studies have introduced additional assays to aid in the measurement of DUBs, with more still under development. Characterization and validation of DUB inhibitors can be aided by assessing results from orthogonal assays. A very recent example is a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry method that has the capability of using unmodified substrate and offers an alternative for screening DUB inhibitors.51 Activity-based protein profiling has recently emerged as a useful tool to study DUB activity.52−56 It is a method used to assess protein activity in the native cellular environment with active-site directed functional probes. Its application to E3 ligases and DUBs has been reviewed by Lill and Wertz.1 Chemical Inhibitors of USP7. Of all the known DUBs, USP7 or herpes virus associated USP (HAUSP) has garnered a lot of attention and is the one for which there is the greatest number of inhibitors described. One of the main functions for USP7 identified using knockout experiments is the indirect regulation of the tumor suppressor p53.57,58 It does so by cleaving ubiquitin chains from Mdm2, thereby stabilizing this known oncoprotein. By elevation of cellular concentrations of Mdm2, USP7 promotes the reduction of the tumor suppressor p53.59,60 Inhibition of USP7 is thus expected to increase the levels of p53 in tumor cells, leading to anticancer activity. Separately, USP7 has been shown to deubiquitinate monoubiquitinated FOXO4, which results in deactivation of FOXO4 and thus up-regulation of the PI3K/Akt signaling pathway.33,61 Research into understanding the druggability of USP7 can be traced back to an enzymatic study performed by Faesen et al. and Wrigley et al.34,62 In Wrigley et al.’s study, it was found that the enzymatic activity of the full length USP7 was greater than that of the catalytic domain alone. This observation suggests that USP7 inhibitors can potentially bind outside the catalytic domain and still affect the enzymatic activity. This report also noted the effect of reducing agents on the catalytic activity of USP7. This suggests that DUBs, like other cysteine protease, possess active site thiols that are sensitive to reducing environment where minimizing disulfide bond formation and enzyme deactivation is important. Subsequent to this work, several inhibitors of USP7 have been described. The first documented example of a USP7 E



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experiment that could shed light on the nature of the enzyme− inhibitor covalent complex would be to acquire an X-ray crystal structure, but as described earlier in this review, this has not been achievable. In the absence of this piece of data, further structure−activity relationship (SAR) evaluation could be done to determine if tuning the reactivity of the electrophilic center in question can increase the activity of the inhibitor. The authors went on to carry out cellular experiments looking at modification of Mdm2 protein levels in HEK293 cells. They showed that 2 was able to partially reverse the deubiquitination of Mdm2. In a separate endeavor to identify small molecule inhibitors of USP7, Ub-CHOP reporter based screening was used to identify two compounds PR-619 (4) and P22077 (5), a 2arylthio-3-nitrothiophene (Figure 8).56 Subsequent potency optimization of 5 (EC50 = 8.0 ± 2.8 μM) generated additional analogs that led to the identification of P5091 (6, EC50 = 4.2 ± 0.9 μM), an inhibitor shown to be selective for USP47 and USP7 in a panel of DUB-related enzymes, as well as other cysteine proteases. Conversely, 4 exhibited broader inhibitory profile targeting multiple DUBs, which is not surprising given that the compound possesses two cyanosulfide reactive centers. In a follow-up effort on 5,68 Chauhan et al. utilized this compound to link USP7 with multiple myeloma (MM) by performing immunohistochemistry (IHC) analysis of MM cells from patient biopsies compared to healthy patients, showing a significantly higher usp7 gene expression in the diseased cells (also seen with protein extracts from these tumor cells vs normal cells). The SAR of closely related analogs around the compound was evaluated. However, in all cases the compounds had the 5-thia moiety present. Most compounds had an acetyl substituent, although a hydroxyethyl substitution at this position also had partial activity (EC50 = 20.1 μM). The presence of a halogenated phenylsulfide was critical for activity, as there was a 12-fold loss in potency going from 2,3dichlorophenyl to the unsubstituted phenylsulfide. In any case, it was shown that 5 (EC50 = 4.2 μM) had a noticeable effect in inhibiting USP7’s deubiquitinating ability in MM cells. It also had tumor suppressive activity that occurred in a pathwaydependent manner (HDM2/p53/p21). Next, 6 was demonstrated in vivo to suppress tumor growth in mouse models of MM, with bortezomib as a positive control. Interpretation of the in vivo effects observed with the compound could have been supported by pharmacokinetic studies to understand the half-life and clearance of the compound in the mouse models employed. Following the identification of 6, Weinstock et al. proceeded to identify compounds with improved USP7 inhibition, retention of DUB selectivity, increased solubility, and increased stability (with GSH incubation and plasma stability).69 Here the authors found that substituting the 5-pyridinylthiol moiety for the original 5-phenyl group resulted in increased potency. In addition, replacement of the acetyl group with a carboxamide generated compounds with enhanced solubility. Finally, replacement of the nitro group with a cyano on the thiophene ring resulted in reduced GSH adduct formation and enhanced stability in plasma. The authors also observed increased selectivity for USP7 and USP47 with little activity against caspase 3, calpain 1, 20S proteasome, and a panel of other USPs: USP2, -5, -8, -21, and -28. The dual inhibition of USP7 and -47 was deemed to be beneficial given the observed efficacy in cells. These enzymes have also been phylogenetically linked in their core catalytic domains showing the two have close

inhibitor was that of an optimized cyanoindenopyrazine derivative.63 The authors carried out a hit-to-lead effort that led to the identification of HBX 41,108 (1, Figure 8). An IC50 of 0.42 μM was determined for this compound using the UbAMC assay. The compound was also shown to preferentially inhibit USP7 after formation of the enzyme/substrate complex, indicating an uncompetitive mechanism. By use of gel filtration, it was determined that 1 was a reversible inhibitor of USP7 (enzyme activity was completely restored after filtration), whereas iodoacetamide (IA), a known thiol alkylating agent, showed irreversible inhibitory activity. Combining increasing amounts of 1 and IA showed decreased USP7 activity, suggesting that 1 did not mask the active site cysteine. Docking studies were then carried out to try to understand the possible binding modes to USP7. These studies suggested that 1 bound near the catalytic cysteine but modulated the enzyme activity from a location distal to the active site residues. To provide further evidence for this assertion, an X-ray crystal structure would be useful. Selectivity was determined against a panel of other representative proteases including aspartyl, serine, and cysteine proteases. Remarkably, the compound was selective for USP7. The authors demonstrated that 1 elicited the desired cellular effects including induction of p53, inhibition of USP7 and corresponding reduction of Mdm2 levels. Interestingly, in a follow-up effort, introduction of O-alkyloxime derivatives of compound 1 resulted in selective inhibitors of USP8 that lacked activity against USP7.64 Authors from the same group went on to describe the identification of a structurally distinct inhibitor.65 Once again, a high-throughput screen was conducted using the Ub-AMC assay platform from which they identified HBX 19,818 (2) and its close analog HBX 28,258 (3) (Figure 8). These compounds exhibited IC50 values of 28.1 and 22.6 μM, respectively. A discrepancy in the depiction of the chemical structures of the compounds was made between Figures 1A and Figure 2A in their original article (isomeric structures were drawn). We surmise the correct structures to be the one shown in Figure 8 in the present article. The structures of 2 and 3 contain a tetrahydroacridine core, which closely resembles the marketed drug tacrine (Cognex)66 but bears an electrophilic carbon center in the tetrahydroacridine core. It is conceivable that attack at this center could occur resulting in protein conjugation. Despite the potential for nonspecific protein conjugation of cysteine proteases, it was found that both compounds selectively targeted USP7 and not the other DUBs. Specificity was confirmed using human influenza hemagglutinin−ubiquitin−vinylsulfone (HA-Ub-VS), an activity-based probe, which binds covalently to the cysteine active site of DUBs.55,67 Compound 2 showed specific inhibition of USP7 labeling and not USP5, -10, and -8 or CYLD and UCH-L3. The mechanism of action of 2 was also studied using the catalytic domain of USP7 (K208-E560) and monitored by mass spectrometry. The compound formed a stoichiometric complex with USP7 with corresponding loss of a chloride atom and also showed time-dependent increases in IC50. Further confirmation of covalent binding to Cys223 by mass spectrometry under denaturing conditions was also demonstrated. A more in-depth analysis of labeling at the seven other cysteine residues in the catalytic domain or 19 others in the full-length USP7 was not described. Docking studies suggested that a conformational change occurs between the apo and ligand-bound forms, which they ascribe to binding to the active site near Cys223 that results in a displacement of Cl (−36 amu). An informative F



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formed has elevated deubiquitinating activity beyond the minimal rate offered by USP1 alone. A group at University of Delaware and NIH conducted an HTS using the USP1/UAF1 complex and identified potent and selective inhibitors of the complex.83 The best compounds inhibited through a noncompetitive mechanism and showed Ki < 1 μM. These authors cited issues with Ub-AMC substrate, and hence, they employed the Ub-Rho110 system instead. A library of 9525 small molecules were screened, and the active compounds were validated using an orthogonal diUb cleavage assay.84 They obtained quantitative kinetic data showing that USP1/UAF1 hydrolyzed K63-linked diUb using a gel-based assay (cleavage of K63-linked diUb was 5.5 times faster than K48-linked). Several compounds emerged after this confirmation assay, with pimozide (10, IC50 = 2 μM for diUb substrate) and GW7647 (11, IC50 = 5 μM for diUb substrate) (Figure 10)

familial ties (Figure 2). All of these optimizations resulted in a second-generation USP7 inhibitor 7 with an improved profile (IC50 = 0.42 μM for USP7 and IC50 = 1.0 μM for USP47), potentially useful for studying USP7/47 behavior in cells and in vivo. In this vein, additional pharmacokinetic characterization will be needed. USP30. USP30 was shown to be a mitochondria-localized DUB, which regulates mitochondrial morphology.70 More recently, as described in the section Background, the genetic experiments of Bingol et al. demonstrated that inhibition of USP30 could rescue defects associated with impaired mitophagy, providing potential for another approach for treating PD.22 Prior to that work, a group reported earlier in 2014 that 15oxospiramilactone S3 (9, Figure 9), a semisynthetic small

Figure 9. 15-Oxospiramilactone S3 (9) is derived from spiramine A (8) by semisynthesis.

molecule derived from the natural product spiramine A (8), was identified from a pool of 300 compounds because it induced mitochondrial elongation consistent with USP30 inhibition phenotype.71 This effect was achieved with 5 μM 9 and was found to be dose- and time-dependent. A 2 μM and lower dose was sufficient in inducing mitochondrial elongation without affecting cell viability, while concentrations higher than 5 μM killed the cells through apoptosis. The authors also reported that USP30 was the molecular target of 9 by pulling down the complex of the biotin-tagged version of 9 and myctagged USP30. Compound 9 was denoted as a “thiol Michael addition” molecule, and it was postulated that biotin-conjugated 9 bound to the cysteine in the catalytic domain of USP30. Support for this idea was gathered using mutants of USP30 and showing that the only variant that lost the binding by 9 was the C77 (the catalytic cysteine) mutation. Compound 9 was previously shown by the same group to induce apoptosis through inhibition of the Wnt pathway or up-regulation of Bim.72,73 Interestingly, in the USP30 study, the authors were able to control the activity toward USP30 by regulating the concentration of S3 with which cells were treated (400K compounds and subsequent medicinal chemistry optimization. However, the SAR covering this effort was not disclosed in the original publication. They found that 14 had IC50 = 76 nM against USP1/UAF1 complex in the Ub-Rho110 assay, IC50 = 174 nM against the K63-linked diUb assay, and IC50 = 820 nM against the Ub-PCNA substrate assay. Importantly, given that the chemical structure of 14 contains an aminopyrimidine, a possible kinase binding motif, it was screened against 451 kinases at 10 μM and showed only minimal activity against PIP5K, a lipid kinase belonging to the class II PI3-kinases. A negative control was also identified in which the isopropyl group was replaced by an oxetane. Compound 14 was shown to act as an allosteric and reversible inhibitor, and it was selective against several DUBs: USP2, USP5, USP7, USP8, USP10, USP11, USP14, USP21, and USP46/UAF1 complex. It also lacked activity (IC50 > 200 μM) against USP1 alone in the Ub-Rho110 assay. Furthermore, no activity was observed in the UCH, OTU, and Josephine families as well as other nonrelated proteases or post-translational processing proteins (e.g., deSUMOylases, SENP1 and deneddylase, NEDP1). To assess the activity of 14 in cells, the authors used the classical HA-Ub-VME as a probe in HEK293T cells. A noteworthy observation about this compound is that it targets two of the DNA-damage response pathways, TLS and FA, by inhibiting their common DUB (USP1/UAF1). On the basis of these data, the authors advanced this compound as a useful tool for understanding additional USP1/UAF1-related biology, including stem cell differentiation. Very recently, this group reported on the medicinal chemistry efforts leading to the identification of 14.87 UCH-L1. UCH-L1 is a 223 amino acid member of the UCH family of enzymes that is exclusively (and abundantly) expressed in neurons and testis in normal cells. Other members of this family include UCH-L3, UCH-L5, and BAP1. The exact physiological function of UCH-L1 is not well elucidated, but its

expression has been observed in many cancers; a link to PD has also been identified.88,89 Liu et al. investigated the relationship between UCH-L1 inhibition and tumor suppression using a forward chemical genetics approach.90 They began with screening compounds against UCH-L1 looking for novel inhibitors. From the confirmed hits in the screen, the acyloxime derivatives of isatin emerged (Figure 11). The derived compounds had affinity for UCH-L1 ranging from 6 to 51 μM and had reasonable (>5fold) selectivity over L3. The authors elucidated an SAR for substitution at R1, R2, and R3. Several other derivatives were prepared, and the SAR was further expanded. Various compounds were identified including 15, which exhibited a reversible mechanism of inhibition. Compounds also induced proliferation against certain tumor cell lines. It was inferred based on these results that UCH-L1 may affect the cell cycle and cell growth but not the cell viability. This study was significant in that it was the first reported class of UCH-L1 inhibitors. Thienopyridinone inhibitor 16 was later identified by Mermerian et al. during the course of screening for UCH-L1 inhibitors.91 Optimization efforts focused mainly on the aminothiophenes because of synthesis feasibility; however, additional thienopyridinones were also prepared and tested. Compounds were evaluated for UCH-L1 activity using the hydrolysis of Ub-AMC as readout. The carboxylate was found to be necessary as was the pyridone. Further work was conducted to probe the sensitivity of the phenyl ketone moiety. Nearly every modification of the phenyl-type substituent was detrimental to enzymatic potency except for the phenyl-tonaphthyl or phenyl-to-cyclohexyl changes. The most potent compound identified had Ki,app = 0.74 μM. The kinetic mechanism of these inhibitors was then investigated by determining the Vmax and Vmax/Km of the UCH-L1-catalyzed hydrolysis of Ub-AMC at different fixed concentrations of inhibitors. These experiments resulted in Vmax/Km shown to be independent of inhibitor concentration, while Vmax titrated with a Ki value of 2.8 μM (data reported for 16). These results indicate that the inhibitors were uncompetitive and were likely bound to a Michaelis complex of the enzyme and substrate. The selectivity of the inhibitors against a few other cysteine hydrolases such as UCH-L3, TGase 2, papain, caspase-3, and isopeptidase T was also demonstrated. USP14. In a paper published in 2010, Lee and co-workers set out to identify an inhibitor of USP14 because of its known function to increase proteosomal activity.92 A screen of >63K compounds was conducted using the Ub-AMC assay, and the resultant hits were counterscreened against a small panel of DUBs. After further characterization, a 3-ketopyrrole compound IU1 (17, Figure 12) was identified with an IC50 of 4−5 μM against USP14. The authors offered that 17 is suggestive of H



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was no correlation showing the relationship between the drug tumor or plasma concentrations with the reported observations. A limitation of this work is that the characterization of the activity of this compound relied heavily on the cellular and in vivo responses observed from treatment with the compound and less on the molecular mechanism of action. Pan-DUB Inhibitors. WP1130 (19) is a compound that was initially identified from cell-based screening as a JAK pathway transducer and inhibitor of STAT transcription factors.97 It was derived from extensive SAR analoging of tyrphostin AG490 to increase activity of STAT pathway inhibition. It does not, however, inhibit JAK2 directly but shows antiproliferative activity in chronic myeloid leukemia, melanoma, glioblastoma, and myeloproliferative disorders. It was reported to inhibit certain DUBs including USP9x, USP5, USP14, and USP37. The authors mentioned that 19 shares some chemical resemblance to other described DUB inhibitors such as dibenzylideneacetone (DBA) and curcumin. Structurally, 19 contains a α,β-unsaturated amide further substituted with a cyano group. This bears resemblance to the chemically tuned electrophiles recently described.98 The latter are cyanoesters and not cyanoamides, which are even slightly more electrophilic than 19. Since their report, derivatives of 19 have been recently described to have antiviral activity.99 Finally, in order to develop a specific small molecule that targets autophagy, the catabolic process in cells that controls the degradation of cytosolic proteins and organelles, Liu et al. conducted an imaging-based screen for inhibitors of autophagy.100 From this screen they derived 20 (Figure 12), which they termed “spautin 1” for specific and potent autophagy inhibitor 1. The source of compounds for the screen was the Harvard Institute of Chemistry and Chemical Biology compound library, which yielded the initial hit compound called MBCQ. Following the synthesis and analysis of over 100 analogs, they derived 20 and also identified a negative control. The authors observed that cells treated with 20 led to increased degradation of beclin 1 in Vps34 complexes and p53. This observation led them to investigate which DUBs were responsible for this activity and thereby identified USP10 and USP13 with IC50 values of ∼0.6−0.7 μM in the Ub-AMC assay. Unlike some of the other inhibitors described previously, the cross-reactivity of 20 with others DUBs was not evaluated (except for USP14, against which it was inactive). Furthermore, the structure of 20 contains a 4-aminoquinazoline scaffold, a motif often seen in potent kinase inhibitors such as erlotinib (Tarceva) and lapatinib (Tykerb).101 However, the authors make no mention of evaluating the activity of 20 against a panel of relevant kinases. It would be important to rule out that some of the activity of 20 could be due to kinase inhibition and somehow indirectly targeting Vps34-mediated autophagy independent of USP10 and -13 as proposed.

Figure 12. Chemical structures of other inhibitors targeting two or more DUBs.

an active site-directed thiol protease inhibitor. Perhaps this postulation was made because the hetaryl ketone present in the molecule may be susceptible to nucleophilic attack by an active site cysteine thiol. Interestingly, they also identified an inactive control compound, IU1C, which corresponds to the tertiary amide replacement for the ketone functional group of 17. It was observed that 17 did not act on USP14 in the absence of proteasome, suggesting that 17 only bound to the activated form of protein. A key piece of information included in this work was the investigation of the extent of cell permeability of 17. This was done using LC−MS and UV spectroscopy to contextualize the cellular activities observed. It was found that the compound was reasonably permeable, reaching a steadystate intracellular concentration of 13 μM after 1 h of treatment with 50 μM compound. In light of these data, the effects of 17 on cell viability, apoptosis, and proliferation were then evaluated with confidence. Overall, the authors were able to use the compound to elucidate the role of USP14 in ubiquitin chain trimming of substrates bound to the proteasome. Researchers at Uppsala University and the Karolinska Institute reported the identification of b-AP15 (18) in a screen for compounds that induced the lysosomal apoptosis pathway.93 In order to confirm that the compound was selectively targeting the intended pathway, they performed a gene expression signature of cells treated with 18. The caveat to this type of phenotypic profiling is that it can lead to false conclusions, especially since the compound contains three Michael acceptor sites that have the ability to conjugate to any number of cellular targets. Moreover, compounds belonging to this enone class have been flagged as pan-assay interference compounds, or PAINS.94−96 More detailed characterization of the compound is necessary to ensure that the activity of 18 was indeed operating exclusively through the reactive inhibition of USP14 and UCH-L5 alone as the authors claimed. Nevertheless, a number of cellular experiments were carried out where it was found that the compound caused effects that were akin to those expected from inhibition of deubiquitination (e.g., accumulation of polyubiquitin material when cells are treated with 18), and it was also shown that the compound indirectly blocked the deubiquitinating activity of the 19S regulatory particle. Furthermore, in vivo administration of 18 caused tumor growth suppression in mouse xenografts, although there

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PROTEIN-BASED DUB ANTAGONISTS The section above summarizes small molecule DUB inhibitors in the public domain. Another recent advancement in the field comes from protein engineering, including phage display techniques, computational modeling, and crystallography. These studies were motivated by the general lack of specific and potent inhibitors of DUBs, relative to other families such as kinases, as tool molecules to help manipulate and understand their functions. Two recent studies set the path. Zhang et al. utilized the RosettaDesign102 computational tool to identify seven residues I



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tion-based screening assays, and structure-guided design of small-molecule mimetics of the stabilized Ub−target interface”. Because such an interface is still large (≳1500 Å2), the smallmolecule mimetic approach as described is probably unlikely. A more realistic scenario is to utilize cocrystal structures of these selective variants with its DUB partner, along with cocrystal structures of screening hits to optimize for specificity and potency.

in wild-type ubiquitin with maximum potential to contribute to conformational variability and applied phage display to identify ubiquitin variants with high affinities for the catalytic domain of USP7.36 Among these variants, the tightest binder was used for another round of affinity maturation randomizing surface residues predicted to interact with USP7. The resulting best variant had a Kd of 56 nM for USP7’s catalytic domain and 68 nM for full-length USP7 and was selective against five other DUBs tested. X-ray structures were solved for the tightest binders from the first round and second round of affinity maturation, showing altered conformation of the β1−β2 region; nuclear magnetic resonance (NMR) and molecular dynamics simulations also showed that both variants were more rigid than wild-type ubiquitin. In the last section of the paper, a ubiquitin variant transfected into human cells and confirmed to specifically bind to USP7 (using immunoprecipitation and mass spectrometry analysis) enhanced Mdm2 ubiquitination and turnover, leading to stabilization of the p53 tumor suppressor. This is an illustration of an alternative method to achieve selective DUB inhibition. Such a ubiquitin variant can be useful to study the role of a specific DUB and gain understanding between macromolecular interactions as this paper has shown; it can also potentially help with assay development. In a subsequent report, Ernst et al. also utilized phage display as a tool to find variants that bound selectively to three USPs, an OTU member, and a JAMM member, in addition to other proteins beside DUBs.58 Here we will review the work on the three USPs. Unlike the Zhang et al. publication, Ernst et al. defined a ∼30-residue region as the binding site using a crystal structure of ubiquitin covalently conjugated to USP21 that they determined. Two phage-displayed libraries were used with a strategy that favored the wild-type sequence but allowed for diversity across the predefined binding site region. Selections using this method arrived at ubiquitin variants that bound selectively to USP8, USP21, and USP2a with IC50 values in the nanomolar range. Furthermore, they solved cocrystal structures of these variants with the respective USP binder and were able to compare the binding of these variants with that of wild-type for USP21 and USP2a. In both cases, the variant contained three mutations and contacted residues with their corresponding USPs that were not conserved in the USP family. On the other hand, the Ub-variant bound to USP8 contained 12 mutations. Even though the USP8 structure with wild-type ubiquitin bound has not been determined, comparison with other ubiquitin-bound USP structures showed a significant shift of the position of this variant, such that its C-terminal tail did not even reach into USP8’s active site cleft. As a follow-up, the USP21 and USP8 variants were expressed in mammalian cells, confirmed to retain specificity, and in vivo effects were examined. Upon cotransfection of the USP21specific variant, ubiquitination of RIP1 was observed as expected from prior literature, and NF-κB activation was restored as a result. To test USP8’s reported deubiquitinating activity on EGFR, cells expressing the USP8-specific variant were studied using confocal microscopy, showing greater localization of EGFR with the lysosome-associated protein 1 and hence accelerated degradation of EGFR in lysosomes due to ubiquitination. Similar to the results of Zhang et al., these selective ubiquitin variants designed using protein engineering techniques were also shown to be useful as genetic probes of the functions of specific DUBs. Ernst et al. also conclude that drug discovery applications include “target validation, competi-

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PERSPECTIVE A significant amount of literature supports the rationale for targeting DUBs for disease indications spanning from oncology to neurodegeneration and infectious diseases. These therapeutic opportunities have led to heightened enthusiasm to identify small molecule inhibitors as tool compounds for target validation and for further development into marketed drugs. Altogether, 19 distinct compounds were described in this review. These compounds were identified through various types of screening, some biochemical and some phenotypic, of libraries containing anywhere from 300 to >500K compounds. Just about one-third of the examples described herein involved any detailed medicinal chemistry hit-to-lead optimization prior to utilizing the screening hit directly for downstream studies. Perhaps one of the most advanced series, from a medicinal chemist’s point of view, involves the dual inhibitors of USP7 and USP47 from Weinstock et al. because of their attention to properties such as solubility, GSH adduct formation, and plasma stability during lead optimization.69 The recent studies from Dexheimer et al. also helped substantiate 14 as a USP1/ UAF1 inhibitor due to reported SAR and in vitro ADME profile.87 Aside from the lack of medicinal chemistry efforts, most of these small molecules suffered from insufficient characterization prior to being defined as tool compounds and arriving at conclusions upon being used in vitro or in vivo. In fact, none of the publications involving in vivo studies showed any pharmacokinetics data along with the pharmacodynamics or efficacy results. Also lacking for most of them are plasma protein binding, in vitro liver microsome or hepatocyte stabilities, solubility, and permeability measurements. The reported observations in animal models could be made more convincing with more detailed characterization of the specific compounds administered. In addition, there is a shortage of biophysical characterization as orthogonal validation for the DUB inhibitory activities of published compounds. Some recommended biophysical characterizations include isothermal titration calorimetry (ITC), NMR experiments, surface plasmon resonance (SPR), differential scanning fluorimetry (DSF), and crystallography. Of course, we recognize that obtaining these data may involve extensive assay development, and even true inhibitors may not give corroborating results between different techniques. However, the lack of any of these data for the 19 compounds reviewed here is a general concern. It is important to recognize with some of these seminal reports that improper or incomplete validation of the reported tool inhibitors can lead to a clutter of potentially false body of literature. The DUB inhibitors reported to date are typically derived from medium- or high-throughput screens, and the resulting hits would typically be championed as satisfactory tool compounds with little additional chemical optimization. In fact, the recent report on MALDI-TOF mass spectrometry method J



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opportunity to warn the scientific community against taking compounds from the literature without thorough evaluation and using these compounds for additional studies with the assumption that the reported potency and selectivity are truly as described. Many of the published DUB compounds came from a suboptimal assay, the Ub-AMC assay, and were administered to animals without assessment of pharmacokinetic and physicochemical properties. However, rather than being disappointed about the lack of promising molecules, we are optimistic that the improvement of assays being developed, coupled with increased DUB crystal structures and diversity of screening libraries, will bring forth well-characterized inhibitors in the near future.

illustrated that some of the published inhibitors behaved differently in an orthogonal assay.51 Despite the above challenges, the field has improved recently in terms of biochemical assay development. An example of how advancement in assay development affects inhibitor discovery can be seen in the Chen et al. paper.83 Previously, Ub-AMC had been the standard substrate for fluorescent-based biochemical assays, but the issue of interference led this group to utilize the Ub-Rho110 substrate instead. Furthermore, they validated the active compounds using K63-linked diUb as substrate. For DUBs whose main function resides in polyubiquitin cleavage, utilization of diUb linked at the most relevant lysine position presents the most physiologically relevant assay system. The mechanism of DUB−substrate recognition and its relationship in biological function is still unfolding. Proteomic methods using quantitative mass spectrometry have been utilized to characterize and quantitate ubiquitin sites, and these methods1 continue to be optimized to enhance our understanding of the architecture of the ubiquitosome. Studies on whether small molecule inhibitors exhibit differential effects cleaving substrates with different ubiquitin linkage would also be highly valuable, and such differential effects would not be surprising especially for inhibitors binding outside the active site. How these effects translate to biological phenotype could further elucidate a DUB’s mechanism of cleavage and its physiological role and widen our understanding of the complex pathway involving ubiquitination. The druggability of DUBs is still a question at large. This includes not just achieving potency but also selectivity. Covalent inhibitors have a greater likelihood of being identified because of cysteine protease-like activity. However, there the issue may lie in finding an inhibitor with the necessary selectivity profile against not only other DUBs but also the superfamily of cysteine proteases. Given that selectivity is needed in a compound both to serve as a tool for understanding the target biology and to exhibit adequate safety profile in the clinic, the path of optimizing and developing covalent inhibitors can be challenging. Other than the active site, there is no obvious binding pocket in the structures of the USP and UCH family members. This is in line with the dearth of druglike small molecule inhibitors for these family members in the literature despite numerous screening efforts. Alternative paths to screening of conventional collections could be to screen fragment libraries. This could be carried out biochemically at higher ligand concentrations or by using biophysical techniques such as SPR or NMR. On the other end of the molecular weight spectrum are macrocycles, and there exists now libraries of macrocycles for screening purposes (e.g., http://www.asinex.com/Libraries_Macrocyclic.html). Because these macrocycles are designed to mimic a patch of the protein surface, they are predicted to be more likely to inhibit protein− protein interactions.103 One could therefore imagine different macrocycles binding at the ubiquitin-binding surface on DUBs and then tweaking functional groups on the macrocycles to achieve desired potency and selectivity much like the ubiquitin variants described in the Zhang et al. and Ernst et al. reports.26,27 Our assessment of DUB inhibitors in the literature leads us to believe that more compounds of druglike quality need to be identified. We believe there is still a lack of high-quality tool compounds, either in vitro or in vivo, to study the functional effects of DUB inhibition. We also want to take this

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AUTHOR INFORMATION

Corresponding Author

*Phone: 650-534-8426. E-mail, [email protected]. Notes

The authors declare no competing financial interest. Biographies Chudi Ndubaku is a Senior Scientist in the Department of Discovery Chemistry at Genentech. He earned a B.S. in Chemistry from University of CaliforniaBerkeley and a Ph.D. in Organic Chemistry from the Massachusetts Institute of Technology, MA. In 2006, he joined the Discovery Chemistry group at Genentech where he has been involved in drug discovery against various disease targets including deubiquitinases. Vickie Tsui is a Senior Scientist in the Computational Drug Design group within the Department of Discovery Chemistry at Genentech. She received a B.S. in Chemistry from Yale, CT, and a Ph.D. under the direction of David Case at the Scripps Research Institute, CA. Since joining Genentech in 2002, she has been applying computational modeling to a variety of targets spanning from kinases to ones with less traditional binding sites such as deubiquitinases.

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ACKNOWLEDGMENTS The authors thank Jacob Corn, James Crawford, Nicholas Endres, and Jeff Blaney for useful discussions and critical reading of the review.

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ABBREVIATIONS USED DUB, deubiquitinating enzyme or deubiquitinase; UCH, ubiquitin C-terminal hydrolase; USP, ubiquitin-specific protease; OTU, ovarian tumor protease; JAMM/MPN+, JAB1/ MPN/MOV34 metalloenzymes; PD, Parkinson’s disease; PLpro, papain-like protease; UBL, ubiquitin-like; VME, vinyl methyl ester; Ub-AMC, ubiquitin C-terminal 7-amido-4methylcoumarin; FP, fluorescence polarization; Rho110, rhodamine 110; TAMRA, tetramethylrhodamine; Ub-CHOP, ubiquitin-C/EBP homologous protein; FRET, fluorescence resonance electron transfer; diUb, diubiquitin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HAUSP, herpes virus associated ubiquitin-specific protease; IA, iodoacetamide; HA-Ub-VS, human influenza hemagglutinin−ubiquitin−vinylsulfone; SAR, structure−activity relationship; MM, multiple myeloma; IHC, immune histochemistry; DDR, DNA damage response; TLS, translesion synthesis; FA, Fanconi anemia; UAF1, USP1-associated factor 1; DBA, dibenzylideneacetone; NMR, nuclear magnetic resonance; ITC, isothermal titration calorimetry; SPR, surface plasmon resonance; DSF, differential scanning fluorimetry K



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Mechanisms for regulating deubiquitinating enzymes.

Role of Deubiquitinating Enzymes in DNA Repair.

Deubiquitinating enzymes regulate PARK2-mediated mitophagy.

Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation.

Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease.

Regulation of pluripotency and differentiation by deubiquitinating enzymes.

Biological functions of hyaluronan and cytokine-inducible deubiquitinating enzymes.

Method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples.

The deubiquitinating enzymes USP4 and USP17 target hyaluronan synthase 2 and differentially affect its function.

Putative role of SUMOylation in controlling the activity of deubiquitinating enzymes in cancer.

Evolution of the highly networked deubiquitinating enzymes USP4, USP15, and USP11.

Deubiquitinating enzymes Ubp2 and Ubp15 regulate endocytosis by limiting ubiquitination and degradation of ARTs.

Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities.

Inhibitors of Deubiquitinating Enzymes Block HIV-1 Replication and Augment the Presentation of Gag-Derived MHC-I Epitopes.

H2A-DUBbing the mammalian epigenome: expanding frontiers for histone H2A deubiquitinating enzymes in cell biology and physiology.

DUBs, the regulation of cell identity and disease.

DUBs counteract parkin for efficient mitophagy.

Trial watch - inhibiting PARP enzymes for anticancer therapy.

Inhibiting epigenetic enzymes to improve atherogenic macrophage functions.

Deubiquitinases (DUBs) and DUB inhibitors: a patent review.

USP11 regulates p53 stability by deubiquitinating p53.

OTUD5 regulates p53 stability by deubiquitinating p53.

Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2.

Deubiquitinating c-Myc: USP36 steps up in the nucleolus.