Regulating the Master Regulator: Controlling Ubiquitination by

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Regulating the Master Regulator: Controlling Ubiquitination by Thinking Outside the Active Site Miniperspective Stacey-Lynn Paiva,†,§ Sara R. da Silva,‡,§ Elvin D. de Araujo,‡,§ and Patrick T. Gunning*,†,‡ †

Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, ON L5L 1C6, Canada

‡

ABSTRACT: The labeling of proteins with ubiquitin/ubiquitin-like (Ubl) proteins is crucial for several physiological processes and in the onset of various diseases. Recently, targeting ubiquitin protein labeling has shifted toward the use of allosteric mechanisms over classical activity-based approaches. Allosteric enzyme regulation offers the potential for greater selectivity and has demonstrated less susceptibility to acquired resistance often associated with active site inhibitors. Furthermore, the isoform diversity among E1 activating, E2 conjugating, E3 ligase, and deubiquitinating (DUB) enzymes offers an ideal platform for modulating activity via allostery. Herein, we have reviewed allosteric inhibitors of the ubiquitin E1-E2-E3 and DUB enzymatic cascade developed over the past decade with a focus on their mechanisms of action. We have highlighted the advantages as well as the challenges associated with designing allosteric modulators of the ubiquitin labeling machinery, and the future promise in targeting these systems using allosteric approaches.

1. INTRODUCTION Post-translational modifications (PTMs) extend the functional diversity of the human proteome and are critical for a diverse number of biological processes. One such PTM, ubiquitination, involves the covalent conjugation of ubiquitin or ubiquitin-like (Ubl) proteins to an intracellular protein substrate. The labeling of proteins with ubiquitin is implicated in both proteolytic and nonproteolytic functions including proteome turnover by the 26S proteasome, mediating immune responses, as well as regulating cell proliferation and survival.1−3 Protein ubiquitination is a multistep process whereby a ubiquitin/Ubl protein is successively relayed between different classes of enzymes (E1, E2, E3), in order to eventually tag a cellular substrate. Despite the broad diversity in ubiquitin/Ubl identity and substrate conjugation, the overall steps catalyzed by the canonical ubiquitin/Ubl conjugation enzyme cascade are conserved among different pathways and in different species.4,5 Initially, the C-terminal carboxylate of ubiquitin is adenylated by the E1 activating enzyme in an ATP-dependent step, accompanied by the release of pyrophosphate (Figure 1A). Subsequently, a conserved nucleophilic cysteine (Cys) residue of the E1 enzyme displaces the AMP from the ubiquitin/Ubladenylate, resulting in a covalent ubiquitin/Ubl-E1 thioester conjugate. The binding and ensuing adenylation of a second ubiquitin/Ubl molecule promote the recruitment of an E2 conjugating enzyme to this ternary complex (E1-S-ubiquitin/ Ubl*ubiquitin/Ubl-AMP; Figure 1B). An active site Cys on the E2 subsequently facilitates the transfer of the covalently linked ubiquitin/Ubl from the E1 to a Cys residue on the E2 through a transthioesterification (transthiolation) reaction. Concomitantly, an E3 ligase recruits a specific downstream target © 2017 American Chemical Society

protein and mediates the transfer of the ubiquitin/Ubl from the E2 enzyme to the terminal substrate through either a covalent or noncovalent mechanism (Figure 1C). To this end, each ubiquitin/Ubl is ligated to a protein through either a peptide bond with the N-terminal amino group or an isopeptide bond formed between a side chain ε-amino group of a select lysine (Lys) residue on the target protein and the ubiquitin or Ubl Cterminal Gly carboxylate (Figure 1D) .4,5 Deubiquitinating enzymes (DUBs) are enzymes that specifically cleave the ubiquitin/Ubl protein from the substrate, thereby offering additional mechanism of regulation over the entire labeling pathway (Figure 1D).6,7 The involvement of ubiquitin and Ubl labeling in a broad spectrum of physiological processes arises in part from the large diversity and assortment of enzymes within ubiquitin/Ubl pathways. In the human proteome, there currently exists 8 known human E1s, ∼40 E2s, >600 E3s, and >100 DUBs and Ubl proteases.8,9 Unsurprisingly, the dysregulation of various enzymes along ubiquitin/Ubl cascades, through either genetic mutations leading to aberrant expression or uncontrolled activity within the diverse pathways in which they are involved, is associated with several disorders including cancer and neurodegenerative diseases.10−12 For instance, the development of the clinical candidate pevonedistat (((1S,2S,4R)-4-(4-(((S)2,3-dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]Special Issue: Inducing Protein Degradation as a Therapeutic Strategy Received: September 8, 2016 Published: January 11, 2017 405

DOI: 10.1021/acs.jmedchem.6b01346 J. Med. Chem. 2018, 61, 405−421

Journal of Medicinal Chemistry

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Figure 1. Ubiquitin/Ubl enzymatic cascade for protein labeling with ubiquitin or other Ubls.

pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl sulfamate,1; Table 1) was inspired by the dysregulation of protein labeling with the Ubl, NEDD8 (neural precursor cell expressed developmentally down-regulated protein 8).13,14 Activation of cullin RING (really interesting new gene) E3 ubiquitin ligases via NEDD8 labeling has been associated with aberrant protein turnover often found in various malignancies such as acute myeloid leukemia (AML), multiple myeloma (MM), and diffuse large B-cell lymphoma (DLBCL), among others.14 Given the success of 1 at disrupting NEDD8 labeling through inhibition of the NEDD8 E1 activating enzyme, and inducing disease regression, a large amount of work has focused on investigating the normal and pathophysiology of these labeling

pathways and, more recently, in selectively antagonizing specific labeling processes for the mitigation of a selection of human diseases. The scope of illnesses that are involved with these enzymatic pathways offers several promising targets for developing inhibitors and therapies and has expanded the druggable portion of the proteome. As such, several parallels have been drawn between protein ubiquitination and phosphorylation, in terms of both their far-reaching implications in cellular physiology and their potential for inhibitor development. As illustrated in an insightful perspective,15 despite their importance, developing clinical drugs that target the ubiquitin/Ubl labeling system has trailed behind targeting 406



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Table 1. Select E1 Activating Enzyme Inhibitors for NAE, UAE, and UBA5a

a

Inhibitor represented by orange hexagon and ubiquitin/Ubl represented by light blue circle.

over other kinases is probably due to the proposed binding mode in an allosteric pocket adjacent to the ATP binding pocket.23,30,31 The rational design of allosteric inhibitors is limited by the availability of structural and biophysical information on some enzymes, in addition to the dynamic nature of most enzymes that typically precludes access to cryptic binding sites and pockets in the absence of a substrate or cofactor. This is probably why the few kinase drugs that operate purely through an allosteric mechanism were identified through screening strategies. Similarly, several allosteric inhibitors have been discovered that target protein ubiquitination, mainly against the E2, E3, and DUB enzymes. In this review, we focus on the discovery of allosteric inhibitors that modulate the multienzyme cascade of the ubiquitin pathway identified within this decade and discuss the potential use of these molecules in new therapeutic avenues.

protein phosphorylation, likely as a result of the increased complexity and discrete mechanisms of the protein ubiquitination enzymatic cascade.15 Since the early 2000s, drug discovery research aimed at targeting the ubiquitin proteasome system (UPS) has only afforded two clinical drugs, Velcade16 and Krypolis,17−21 while the kinase field has yielded ∼30 clinical agents since the emergence of the blockbuster drug Gleevec.15,22,23 The conventional inhibitory strategies for kinases has traditionally involved pursuing the orthosteric site, namely, the ATP-binding pocket.23 However, developing kinase inhibitors with high potency, selectivity, and minimal off-target activity has been challenging due to the necessity in outcompeting the millimolar intracellular concentrations of ATP in addition to the high sequence similarity among the ATP pockets and several other classes of ATP-binding proteins.23 Significant advancements in kinase inhibitors were realized by extending the targeted site to an allosteric region containing a hydrophobic pocket in close proximity to the ATP cleft.23−25 Interestingly, this strategy has yielded the first clinically approved lipid kinase drug, idelalisib, for use in the treatment of chronic lymphocytic leukemia (CLL).23,26 Identified from a kinome-wide screen, idelalisib selectively targets PI3Kδ (phosphatidylinositide 3-kinase δ) by simultaneously interacting with the ATP pocket and an adjacent unique hydrophobic pocket.23,27−29 Furthermore, efforts have also focused toward exclusively targeting allosteric sites remote from the ATP pocket. There are several advantages to targeting an allosteric site as opposed to the orthosteric site, including the greater specificity and selectivity that can be achieved through targeting sites unique to a specific kinase from a common family.23 For example, trametinib is one of the few allosteric clinical kinasetargeting drugs used in combination therapies to overcome resistance often observed in the treatment of melanoma using the B-Raf kinase drug dabrafenib.23,30−33 Trametinib was developed from a structure−activity relationship (SAR) conducted from a lead identified using a high-throughput screen (HTS), and its selective nature for MEK1/2 inhibition

2. ALLOSTERIC INHIBITION OF E1 ENZYMES All E1s are mechanistically related despite their structural differences and contain an ATP binding site, a ubiquitin/Ubl binding pocket, a catalytic Cys residue, and an oligomerization region. The first identified E1 inhibitors were ATP competitive and were composed of a nonhydrolyzable ubiquitin-nucleotide conjugate, namely, the ubiquitin-AMP conjugate adenosylphospho-ubiquitinol (APU, 2, Table 1).34 As a mimic of the ubiquitin-adenylate species, 2 binds to the ATP and ubiquitin pockets simultaneously and effectively prevents further ubiquitin activation (Table 1).34 An alternative strategy toward E1 inhibition has included the irreversible blockade of downstream thiolation reactions by covalent inhibitors that modify the active site Cys, such as the first cell-permeable E1 inhibitor PYR-41 (4[4-(5-nitrofuran-2-ylmethylene)-3,5-dioxopyrazolidin-1-yl]benzoic acid ethyl ester, 3, Table 1).35 Although effective, both modes of inhibition suffer from either the development of resistance, as was observed in cells treated with ATP-competitive inhibitors including compound 1, the 407



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Table 2. Allosteric Inhibitors of E2 Conjugating Enzymesa

a


NAE inhibitor,36 or by the covalent modification and crosslinking of off-target proteins associated with compound 3.35 Recently, compound 8.5 ((2S,3S,4R,5R)-N-(6-(1,4,7,10tetraazacyclododecan-1-yl)-6-oxohexyl)-5-(6-amino-9H-purin9-yl)-3,4-dihydroxytetrahydrofuran-2-carboxamide zinc(II) trifluoromethanesulfonate, 4, Table 1), which is composed of an adenosine scaffold appended to a zinc(II) coordination complex, was shown to block the transfer of the Ubl ubiquitin fold modifier 1 (UFM1) from its E1 (UBA5) to its respective E2 (UFC1).37 This compound was found to function through a noncompetitive mechanism with respect to ATP (Km= 24.9 ± 9.6 μM for ATP with a VMAX = 0.988 ± 0.137 pmol·min−1 in the presence of 5 μM 4; Km= 16.5 ± 2.5 μM for ATP with a VMAX = 1.90 ± 0.10 pmol·min−1 in the absence of 4).37 The authors hypothesized that 4 may operate by binding to the inactive ATP pocket of the UBA5 homodimer or to an alternative unidentified allosteric site (Table 1).37 Aside from this molecule, to date there are no other reported allosteric inhibitors that specifically target the E1 enzyme family. This may be due in part to a variety or combination of factors that underlie the mechanisms of these enzymes. Specifically, the dynamics required for the large conformational change indispensable in the formation of the E1−ubiquitin/Ubl thioester conjugate by the canonical E1 enzymes may preclude allosteric pockets that are only accessible throughout this reaction.38,39 While in contrast, the cross-talk between the monomers in the noncanonical E1 enzymes required for substrate binding and transformation may change the accessibility to potential allosteric sites throughout the reaction pathway.40−42 In general, the mechanistic complexity and highly dynamic nature of E1 enzymes are probably why there are few allosteric inhibitors reported to date. 2.1. Inhibitors of E2 Enzymes: Halting Ubiquitin Conjugation. Several studies have recently revealed the complex roles that E2 enzymes exert on ubiquitin labeling pathways. These include mediating substrate selection, ubiquitin chain initiation and elongation, and controlling the

processivity of ubiquitin chain transfer to a substrate protein for proper proteasomal recognition and degradation within the ubiquitin proteasome system (UPS).43 The majority of these E2-mediated processes are governed by protein−protein interactions (PPIs) of varying affinity and complexity between E1-E2, E2-E3, and E2-ubiquitin proteins. Thus, E2 enzymes are particularly equipped for allosteric regulation as a result of their modular domains. Functionally, E2s can be divided into two classes: “active” E2 enzymes, which contain a catalytic cysteine within their core ubiquitin conjugating (UBC) domain, and “regulatory” E2s that possess a noncatalytic UBC domain lacking an active-site Cys. These regulatory enzymes are labeled as ubiquitin E2 variant (UEV) proteins and often act as cofactors that bind to and regulate the activity or ubiquitin linkage specificity of an active E2.44 In total, there are over 40 E2s that process ubiquitin labeling within the human genome through interactions with the ubiquitin-specific E1 enzymes UAE and UBA6 and through interactions with hundreds of E3 ligases.44 The E2 scaffold has thus evolved several overlapping binding interfaces that are able to distinguish between protein binding partners so as to facilitate rapid ubiquitin labeling and maintain fidelity for protein substrates. In order to receive and process a ubiquitin molecule for labeling, the E2 must undergo a transient and noncovalent interaction with its cognate E1, which is either UAE or UBA6 for ubiquitin. The affinity of an E2 for an E1 increases significantly after the formation of the E1 ternary complex, described previously.45,46 The configuration of the ternary complex induces conformational changes in the E1 structure and reveals a negatively charged cryptic binding site within the E1 ubiquitin-fold domain (UFD). This site interacts with two highly conserved lysine residues present on the α-helix 1 of all ubiquitin E2s.47 Once associated, the E2 accepts the ubiquitin protein bound to the E1 active site cysteine through a transthiolation reaction, resulting in the release of the E1/ 408



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of 5 to Cdc34A increased in the presence of ubiquitin (EC50 = 267 μM to apo Cdc34A, cf. EC50= 19 μM in the presence of Ub). These results further support the cooperative binding of ubiquitin and 5 to Cdc34A. A 2.6 Å resolution crystal structure of the ternary Cdc34A-ubiquitin-5 complex revealed that 5 appears to bind within a pocket at the E2-ubiquitin interface.51 As such, 5 may act as a molecular bridge, strengthening nativelike interactions between Cdc34A and ubiquitin (Table 2). This may reduce ubiquitin-Cdc34A reactivity by impeding ubiquitin dissociation from the E2. Regulating the PPIs within ubiquitin labeling pathways may represent an alternative and effective strategy for abrogating protein ubiquitination through the use of small-molecule stabilizers of E2-ubiquitin interactions. Specifically, as demonstrated above, the targeted stabilization of the weak E2ubiquitin binding events may provide an interesting avenue for selective therapeutic development in the future.

ubiquitin-adenylate complex and an E2−ubiquitin thioester intermediate. The ubiquitin-charged E2 can then interact with several E3 ligase complexes through different subsets of residues and structural folds. Interestingly, E2s recognize and interact with E1 and E3 enzymes through overlapping surfaces, albeit with differing affinities.48 For example, E2s bind to E3 enzymes with weak affinity and often display micromolar dissociation constant (Kd) values in the apo-state compared with the E2ubiquitin bound state.48 These differing binding affinities have likely evolved in order to ensure the rapid response of protein ubiquitination and degradation to changes in cellular signaling. The higher E1/E2 affinity ensures that the majority of free intracellular E2s are loaded with mono-, di-, or polyubiquitin, whereas the lower affinity displayed by E2/E3 pairs may facilitate rapid E2 exchange that in turn mediates fast protein ubiquitination and chain formation.43 Recently, a high-throughput screen (HTS) was performed to identify small molecule inhibitors of the E3 subunit SCFSkp2, whose Skp2 component is often overexpressed in several human cancers leading to aberrant p27 ubiquitination, degradation and resulting in cancer cell proliferation.49 Unexpectedly, this study resulted in the discovery of CC0651 (((2R,3S,4S)-5-(3′,5′-dichlorobiphenyl-4-yl)-2,3-dihydroxy-4(2-methoxyacetamido)pentanoic acid), 5, Table 2), the first small-molecule inhibitor of the E2, hCdc34A.50 Compound 5 displayed IC50 = 1.72 μM when evaluated in vitro using an assay that monitored the ubiquitination of p27Kip1.50 Cocrystallization revealed that 5 localized in a hydrophobic region of hCdc34A, 19 Å away from the E2 active site Cys93.50 This resulted in a 2.1 Å outward displacement of α-helix 2, which induced secondary conformational changes at the active site, including a 2.0 Å shift of the 310 helix containing Cys93.50 5 did not disrupt E2-Ub conjugate formation (transthiolation reaction) and had no effect on hCdc34/SCFSkp2 binding in the absence or presence of the Sic1 ubiquitination substrate. However, 5 strongly reduced the rate of ubiquitin chain initiation on Sic1 in vitro.50 In contrast, the ability of hCdc34 to facilitate ubiquitin dimerization and auto-monoubiquitination was greatly increased, indicating that 5 affected the substrate selectivity and not the catalytic activity of hCdc34.50 A SAR of compound 5 was performed and provided some insight into the functional requirements for effective hCdc34 inhibition.50 Inhibitor activity, evaluated in a similar in vitro p27 ubiquitination assay, was reduced by protection or removal of the pentanoic acid on 5 and was completely abolished by separating the biphenyl rings with an amino group. Substituting both chlorine atoms in the dichlorobiphenyl scaffold with methyl groups demonstrated relatively no change in inhibitory activity (IC50 = 4.4 ± 2 μM cf. IC50 for 5 of 2.5 ± 1 μM), whereas substitution with fluoro, methoxy, or benzoyl groups completely abolished hCdc34 inhibition.50 Furthermore, derivatives CC7094 and CC8993 were able to antagonize p27 degradation within PC-3 prostate cancer cells, resulting in decreased cellular proliferation (EC50 ≃ 20 μM for both derivatives; Table 2).50 From the HSQC solution NMR studies with [15N]ubiquitin, it was observed that 5 induced chemical shifts and broadening of peaks corresponding to surface residues that interact with the core catalytic domain of the hCdc34 isoform, Cdc34A, indicating that 5 likely stabilizes the usually low-affinity E2/ ubiquitin interaction.51 Chemical shift changes from three surface ubiquitin residues also revealed that the binding affinity

3. E3 LIGASES: MODULATING UBIQUITIN CONJUGATION TO PROTEIN SUBSTRATES The term ligase, from the Latin verb “ligare” (to tie up), describes the functional role of the E3 enzymes, which is to facilitate the final conjugation step in protein ubiquitination.4,5 The E3 ligases are involved in a wide range of cellular pathways and significantly outnumber the cellular kinases.15 There are three major classes of E3 ligases, including the RING finger and U-box E3s, the HECT (homologous to the E6-AP carboxyl terminus) E3s, and the RING/HECT-hybrid type E3s known as the RING-between-RING E3 ligases, in addition to a minor class classified as the PHD (plant homeo domain)-finger type E3 ubiquitin ligases.52−56 With the exception of the active site Cys-containing HECT and RING-between-RING E3s, which act through a discrete two-step mechanism involving the formation of a transient thioester conjugate to ubiquitin (Figure 1C), E3 ubiquitin ligases generally promote substrate protein ubiquitination via mechanisms that involve facilitating the appropriate orientation and presentation of a charged E2 (E2− ubiquitin) to a target protein substrate amino group for subsequent ubiquitination via an aminolysis reaction (Figure 1C).4,5,57,58 RING finger-containing E3 ligases, the most abundant E3s, are composed of two conserved zinc fingers that contribute to the overall globular shape of these proteins necessary to promote the PPIs critical for substrate protein ubiquitination.59−62 Despite having a similar fold to RING E3s, an intricate network of hydrogen bonds and salt bridges contribute to the stability of the structure in U-box-containing E3 ligases, in the absence of the Zn2+-binding motifs found in the RING E3s.54 Both RING-type and U-box E3 ligases have been shown to play important roles in positioning the ubiquitin from E2− ubiquitin conjugate in an appropriate orientation for attack by a substrate protein amino group.54,59,62,63 Structural investigations of RING-type E3s have revealed these RING domains interact with a hydrophobic patch on ubiquitin, ultimately restricting the conformation of the E2− ubiquitin thioester conjugate.64−67 This binding event is thought to contribute to the appropriate positioning of the charged E2 for aminolysis by the acceptor amino group from the protein substrate.64−67 Additionally, structural data suggest RING-type E3 ubiquitin ligases also interact with E2 enzymes on the face opposite where the active site Cys resides, through discrete mechanisms depending on the RING E3 ligase.67−72 Currently, a popular area of research focuses on understanding 409



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Table 3. E3 Ligase Inhibitors That Modulate Substrate Binding Directly or through an Alternative Mechanisma

a

Inhibitor represented by orange hexagon.

protein binding sites, as well as (4) perturbing or inducing a conformational change in one of the several E3 protein subunits. As a result, the majority of molecules that affect ubiquitin labeling have been identified as E3 ligase inhibitors. To date, most of these compete for the protein substratebinding site with great success in targeting various E3 ligases including the von Hippel−Lindau (VHL) and MDM2/HDM2 (mouse/human double mutant 2 homolog) ubiquitin ligases, with some preclinical and clinical successes.82−86 For example, disrupting XIAP/cIAPs (X-linked and cellular inhibitor of apoptosis proteins, respectively) by mimicking their endogenous antagonist proteins, Smac/DIABLO (cellular second mitochondria-derived activator of caspases or direct IAP binding protein with low pI), has led to the development of several small molecule XIAP/cIAP inhibitors.87−90 Specifically, AT-406 ((5S,8S,10aR)-N-benzhydryl-5-((S)-2-(methylamino)propanamido)-3-(3-methylbutanoyl)-6-oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide, 6, Table 3), now licensed as Debio 1143, is currently being evaluated in phase I/II clinical trials in combination with concurrent cisplatin and radiotherapy (CRT) for patients with previously untreated stage III, IVa, or IVb head and neck cancer (ClinicalTrials.gov identifier: NCT02022098).89,91,92 Therefore, targeting E3 ligases by mimicking their protein substrate appears to be an effective strategy toward regulating their activity. However, due to their mechanism of action, these classes of competitive inhibitors are not described herein but are comprehensively reviewed elsewhere.93−96 Given the clinical successes of this approach, targeting E3 ligases via allostery could present new routes for

the intricate and discrete mechanisms by which RING-type E3 ligases promote substrate protein ubiquitination. Finally, since the protein substrate often binds about 50−60 Å away from the E2−ubiquitin binding site, orienting the substrate protein for ubiquitination of the appropriate amino group often requires large conformational change within RING E3 ligases.61,73−77 Meanwhile, the HECT domain, often located at the Cterminus of HECT E3s, is composed of two lobes separated by a flexible linker region where E2−ubiquitin recognition occurs at the N-lobe, while the catalytic Cys resides in the C-lobe for HECT E3−ubiquitin conjugate formation.52,53,63 The Nterminal domains of HECT E3s often mediate protein substrate recognition for subsequent ubiquitination, wherein a large conformational change is often necessary for protein ubiquitination.52,53,59,67,78,79 Alternatively, the canonical RING1 domain of RING-between-RING E3 ligases recruits the E2−ubiquitin conjugate just like that in RING-type E3 ligases. However, the noncanonical RING2 domain contains an active site Cys necessary for the transthiolation step which precedes protein ubiquitination.55,63 Interestingly, these RINGbetween-RING E3s typically operate through a variety of unique autoinhibitory mechanisms and are often involved in the formation of a linear ubiquitin chain assembly complex.55,80,81 Due to the many steps mediated by E3 ligases between E2ubiquitin/Ubl binding and protein substrate ubiquitination, there are several areas where E3 activity can be manipulated through small-molecule intervention, including (1) the E2ubiquitin/Ubl binding site, (2) accessory, or (3) substrate 410



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identify inhibitors of the mammalian target of rapamycin (mTOR) kinase.103 A library of 30 000 compounds was screened for synthetic lethality in yeast cell growth in combination with suboptimal levels of rapamycin. A small subset of structurally distinct small molecule enhancers of rapamycin (SMERs) that showed no effects on yeast cell growth alone was subjected to a yeast genome-wide expression profiling using DNA microarrays.103 The oxadiazolo-pyrazinone containing SMER3 (9H-indeno[1,2-b][1,2,5]oxadiazolo[3,4-e]pyrazin-9-one; 8, Table 3) was identified as an interesting candidate for mTOR inhibition as observed from hierarchical clustering and principal components analysis.103 The target of 8 was eventually identified as the E3 ligase SCFMet30, which monoubiquitinates and inhibits the activity of the Met4 transcriptional activator.103−105 The function of Met4 in genetic regulation includes the attenuation of cell proliferation in times of methionine depletion.104,105 Inhibition of SCFMet30 activity by 8 prevented Met4 ubiquitination, leading to the upregulation of MET gene expression and subsequently resulting in cell cycle arrest.103 Compound 8 inhibited SCFMet30mediated Met4 ubiquitination in a reconstituted in vitro ubiquitination assay while maintaining selectivity over the closely related SCFCDC4 and eliciting no effect on met4Δ cell viability.103 Similar to the genetic knockdown of SCFMet30 and its cognate E2 conjugating enzyme, Cdc34, compound 8 treatment also sensitized cells to the effects of rapamycin.103 Immunoprecipitation of an endogenously expressed Myctagged Met30 from yeast cell extracts indicated that 8 disrupted Skp1-Met30 interactions, which constitute a portion of the multisubunit SCF F-box complex. These results suggest that 8 inhibits SCFMet30 assembly or induces the dissociation of the SCF complex.103 Using stable isotopic labeling with amino acids in cell culture (SILAC), it was evident through Skp1 pulldown that the binding of Met30 to Skp1 was the only F-box PPI affected by 8, supporting the data obtained from immunoprecipitation.103 Furthermore, incubation of 8 with Skp1 and Met30 in vitro led to a dose-dependent decrease in the melting temperature (Tm) of the Met30−Skp1 heterodimer, but had no effect on Skp1 stability alone, as evaluated by differential scanning fluorimetry (DSF). The thermal data suggest that compound 8 induced a conformational destabilization and therefore lower melting temperature of the heterodimeric complex, which support previous observations recorded in both immunoprecipitation (IP) and SILAC pulldown assays.103 Lastly, data obtained from drug affinity responsive target stability (DARTS), used to assess the binding of 8 to Met30 and Skp1 in yeast cell lysates, suggest that 8 may mediate its activity by binding to the F-box motif on Met30 (Table 3).103 In addition to promoting the ubiquitin-mediated degradation of cell cycle regulators (SCF CDC4 ) or inhibiting the antiproliferative effects of Met4 via monoubiquitination by SCFMet30, cullin-RING ligases, such as APC (anaphasepromoting complex/cyclosome), are intimately involved in regulating the cell cycle. The activity of APC is responsible for facilitating cellular exit from mitosis.106,107 Many chemotherapeutics, namely, microtubule inhibitors, are responsible for disrupting mitosis and promoting the spindle assembly checkpoint, which effectively halts progression of mitosis at the metaphase stage.108 Since microtubule inhibitors are often associated with dramatic side effects such as peripheral neuropathy, developing an inhibitor of the APC E3 ligase

specific E3 inhibition or activation, considering the potential for achieving greater target selectivity. 3.1. Small Molecule Allosteric Modulators of CullinRING E3 Ligases. The cullin-RING E3 ligases were first discovered as components of the multisubunit Skp1-Cdc53/ cullin-F-box (SCF) protein complexes.97 Within the SCF ligase, the F-box subunit is responsible for substrate specificity and recruitment mediated by PPIs at Leu-rich motifs or WD40 repeats.97,98 These WD40 repeats form a β-propeller structure that recognizes protein substrates via a phosphorylated sequence, also known as a phosphodegron motif, which promotes substrate recruitment for ubiquitination and subsequent degradation.99 This RING E3 ligase family has been found to play a role in disease progression.11,12,58 For example, the human tumor suppressor cell division control protein 4 (CDC4), a substrate recognition component of an SCF complex, promotes the ubiquitination of a number of important regulatory substrates such as cyclin E, MYC, JUN, NOTCH, SREBP, and presenilin.100 However, it is a haploinsufficient tumor suppressor and is often mutated across various cancer types.100,101 Thus, this E3 complex presents a potential target that can be exploited in the continuous discovery of anticancer agents. In order to identify novel inhibitors of a CDC4-containing SCF ligase, a fluorescence polarization (FP) assay, evaluating the displacement of a fluorescein-labeled CDC4 phosphodegron (CPD) peptide from the yeast CDC4 ortholog, was employed to screen library of 50 000 compounds (compound source, Maybridge).102 Compound SCF-I2 ([1,1′-binaphthalene]-2,2′-dicarboxylic acid; 7, Table 3) was the only hit identified using FP (IC50 = 6.4 μM) and also inhibited the interaction between CDC4 and the phosphorylated Sic1 substrate in vitro.102 Furthermore, 7 inhibited SCFCDC4 binding and subsequent ubiquitination of both Sic1 and an additional substrate Far1 in vitro with IC50 ≈ 60 μM.102 Interestingly, 7 demonstrated no inhibition of the closely related SCFMet30 from yeast in vitro.102 The authors postulated that the lack of CDC4 inhibition by 7 in yeast cells is likely due to poor permeability as a result of its two carboxylic acids.102 Interestingly, only the R-(+) enantiomer of 7 cocrystallized with the Skp1CDC4 complex (2.6 Å resolution) and was found to bind within the WD40 domain at a distance of 25 Å from the CPD substrate binding pocket.102 Furthermore, it was identified that compound 7 bound between blades 5 and 6 of the WD40 blade structure, where it induced a 5 Å shift of the β21−β22 linker, displacing His631 outward by 13 Å.102 Altogether, this binding mode induced a conformational change in residues involved in CPD recognition, namely, Tyr574 and Leu634, which also destabilized key interactions with CDC4 residues that are essential for phosphate-group recognition on CPDs.102 Furthermore, 7 failed to inhibit the active CDC4 mutants Arg664Ala and Arg655Ala, highlighting the importance of the carboxylate groups on 7 in mediating SCF inhibition. Additionally, human CDC4 (FBW7), which contains Lys and Cys in place of Arg655 and Arg664, respectively, displayed resistance to 7, further supporting the role of carboxylate−protein interactions in mediating the inhibitory effects of this compound. Thus, it was apparent that 7 attenuated SCFCDC4-mediated ubiquitination of CPD substrates by disrupting CDC4−CPD binding through an allosteric mechanism (Table 3).102 Concurrently, an inhibitor for SCFMet30 was identified through a chemical genetics screen originally designed to 411



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HeLa cells from 1.0 to 1.6 and 4.8 h, respectively. Combining Cdc20 siRNA treatment with compound 10 resulted in a synergistic increase of mitotic duration to 19.4 h, surpassing the additive effect of the individual agents.113 Furthermore, there was an observed decrease in the degradation of various APC substrates in HeLa cells treated with 10, while mitotic spindle morphology was unaffected.113 This work highlighted the potential use of agents such as 10 to sensitize cells to mitotic arrest-inducing drugs and suggests that mitotic arrest can be achieved through the allosteric inhibition of specific E3 ligase complexes. 3.2. Thalidomide: A Story Spanning over 60 Years. Thalidomide ((S)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3dione, 11; Table 3) is famously known for its antiemetic properties. However, it is more infamously known by its effects at inducing the onset of debilitating teratogenic birth defects, as unexpectedly experienced by infants born to mothers who had taken this drug to treat morning sickness in the mid-20th century.115−117 Although 11 was swiftly discontinued as an antinausea treatment, it has since returned to the clinic for the treatment of multiple myeloma and erythema nodosum.118,119 Efforts to understand its molecular mechanism of action have revealed its cellular target as cereblon (CRBN), a protein that forms an E3 ubiquitin ligase complex with the cullin-RING 4a (Cul4a), resulting in a CRL4 complex.120 Interestingly, CRBN has been found to play a crucial role in fetal limb development, explaining the teratogenicity and abnormal limb formation that occurred in children born of mothers who had used this drug.120 Compound 11 and its derivatives, lenalidomide ((S)-3-(4amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione, 12; Table 3) and pomalidomide ((S)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione, 13; Table 3), are all known as immunomodulatory drugs (IMiDs) and have been effectively used in the treatment of several hematological cancers due to their immunomodulatory and anti-inflammatory properties.121,122 Recently, CRBN isolated from Gallus gallus domesticus was cocrystallized with human DNA damage-binding protein 1 (DDB1) bound to 11, 12, or 13. CRBN is composed of three domains: a seven-stranded β-sheet at the N-terminus (NTD), a seven α-helical bundle domain (HBD) that mediates DDB1 binding, and a C-terminal domain (CTD) composed of 8 β-sheets.123 This CTD hosts a conserved Zn2+-binding motif 18 Å distal from the ubiquitination substrate-binding site, which was identified as the motif to where the IMiDs were cocrystallized with similar binding modes and affinities (Kd = 250 nM for 11, Kd = 178 nM for 12, and Kd = 157 nM for 13).123 The glutarimide functionality is the main pharmacophore of the IMiD compounds and was found to bind in a cavity on CRBN between β10 and β13, where the carbonyls and the amide nitrogen of glutarimide engage in interactions with the proximal Trp382 and His380 through hydrogen bonds.123 Furthermore, the aliphatic ring of all three IMiDs appears to be buried in a hydrophobic pocket consisting of Trp382, Trp388, Trp402, and Phe404 in the crystal structure. Interestingly, when evaluated for displacement of a Cy5-labeled 11 from CRBN in an FP experiment, the binding of 11−13 to the Trp mutant variants CRBN Y384A, CRBN W386A, and CRBNYW/AA was not observed, suggesting that this interaction may be important for inhibitor activity.123 The C1 carbonyl of the phthalimide ring in all three molecules was observed to form a water-mediated H-bond to His359, while the phythaloyl ring was found to make favorable contacts with the aliphatic

could help evade these undesired responses while still preventing the proliferation of diseased cells.109,110 APC consists of 11 subunits including the proteins Cdh1 and Cdc20, which bind to and promote APC activity at various stages throughout the cell cycle.111 Cdh1 binds to APC during the G1 phase to promote substrate ubiquitination during interphase, whereas Cdc20 is required for the initiation of anaphase and exit from mitosis by promoting the ubiquitination and subsequent proteasome-mediated degradation of securin and mitotic cyclins such as cyclin B1.110,111 RNA interference (RNAi) experiments against Cdc20 showed that significant knockdown was required to achieve mitotic arrest, suggesting that APC inhibition might offer an alternative therapeutic avenue to conventional microtubule inhibitors such as the taxanes.110,111 Previous reports have demonstrated the ability of the small molecule TAME (methyl tosyl-L-argininate, 9; Table 3) to abrogate ubiquitin-dependent cyclin proteolysis in Xenopus extracts, isolated from mitotic and interphase cell cycle stages.112 Upon SAR analysis of compound 9 activity, it appeared that the tosyl group, Arg side chain, and the methyl ester were all important for APC inhibition. Compound 9 also inhibited APC E3 ligase activity in a reconstituted ubiquitination reaction while reducing proteolysis of cyclin B1 from interphase cell extracts.113 Furthermore, 9 dose-dependently decreased both Cdc20- and Cdh1-APC association in mitotic Xenopus extracts, suggesting that this molecule may impede the association between APC and its activator protein subunits.113 A radiolabeled 3H-9 analogue was subsequently incubated with Xenopus cells and was detected in IP pull-down of the APC subunit Cdc27 from Xenopus interphase extracts, as well as Cdc27 IP from HeLa cells.113 These findings support the hypothesis that compound 9 inhibits APC by preventing the association of activator proteins, possibly by interacting with Cdc27 and disrupting its PPI with other APC subunits. Since APC requires a C-terminal Ile-Arg tail on activator proteins for binding, pull-down experiments with a C-terminal Ile-Arg tail model were subsequently conducted with APC. It was found that 9 inhibited APC interactions with the Ile-Arg Cterminal motif, suggesting this as a possible binding region (Table 3).113 Furthermore, by use of cryoelectron microscopy, 9 was recently identified to bind areas on APC that are critical for activator binding including the previously described Ile-Arg tail binding site and the C-box binding site, a crucial conserved motif in both Cdc20 and Cdh1.114 Interestingly, despite mitigating APC association to both activators Cdc20 and Cdh1,113 Zhang et al. found that 9 is more potent against Cdc20-mediated APC activation.114 These results suggest that a small molecule approach, with inhibitor discovery facilitated by the use of HTS, can be exploited to selectively inhibit the association of one APC activator protein over another in order to elicit the desired downstream effect, such as mitigating cellular proliferation. Considering that 9 is not cell permeable, a prodrug, namely, proTAME ((S,E)-5-((5-methoxy-4-((4-methylphenyl)sulfonamido)-5-oxopentyl)amino)-3,7-dioxo-2,8-dioxa-4,6-diazanon-4-ene-1,9-diyl bis(2-phenylacetate), 10; Table 3), was synthesized by modifying the Arg side chain to a N,N′bis(acyloxymethycarbamate) derivative. Treatment of HeLa cells with 12 μM 10 led to the disruption of Cdh1-APC association and delayed mitotic entry.113 It was observed that using Cdc20 small interfering RNA (siRNA) or treatment with compound 10 (4 μM) increased the duration of mitosis in 412



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face of Pro354.123 It is possible that these additional interactions help mediate the tight binding of IMiDs to CRBN and enhance their subsequent effect on CRBNmediated ubiquitination. Interestingly, both the aniline-containing 12 and 13 agents promoted the ubiquitination and subsequent degradation of the transcription factors IKZF1 and IKZF3 by CRL4CRBN.123 Compound 13 alone and two derivatives of compound 11 (containing a substitution of the phthaloyl ring C4 position with -CH3 and -Cl) also promoted the recruitment and subsequent degradation of IKZF1.123 In contrast, the intracellular levels of MEIS2, an endogenous substrate of CRL4CRBN, were stabilized by treatment with 12, presumably by inhibiting MEIS2-CRBN binding and the ensuing CRBN-mediated MEIS2 polyubiquitination and eventual degradation.123 Thus, it is evident that IMiDs can either promote or inhibit protein substrate binding and subsequent ubiquitination (Table 3). Due to the plasticity of these phenotypes, IMiDs provide a prime example of how allosteric modulation of E3 ligases can either positively or negatively affect E3 enzymatic activity. 3.3. PROTACs: The Era of Molecular Machines. Fifteen years ago, Sakamoto et al. successfully hijacked the activity of E3 ligases in order to trigger specific protein degradation.124 They reported the first proteolysis-targeting chimera (PROTAC), consisting of a ligand capable of recruiting a specific E3 appended to a certain protein of interest (POI) binding motif, in order to promote POI ubiquitination and subsequent degradation in cellulo.124 Since this discovery, several advancements have been made in designing new PROTACs for recruiting various POIs to a specific E3, as well as varying the E3-recruiting motifs from peptides to small molecule derivatives.125−131 This has resulted in the discovery of PROTACs with promising activity in vivo.127−129 3.4. The Future of Modulating E3 Ligase Enzymatic Activity. As demonstrated by the E3 ligase inhibitors and activators reviewed, the allosteric modulation of E3 enzymes can be achieved through various mechanisms. We have highlighted the tempering of E3 ligase activity by (1) directly impeding or promoting substrate protein binding, (2) inducing a conformational change which precludes binding to substrate proteins, or (3) disrupting or enhancing the binding of activator protein subunits. Developing selective small molecule agents of E3 ligases that operate through allosteric mechanisms may provide mechanistic insight into the discrete steps involved in protein substrate ubiquitination among the various classes of E3 ligases. Furthermore, this mechanistic insight might lead to the development of novel therapeutic strategies when targeting E3 ubiquitin ligase. Given the success of the unique E3 ligase recruiters highlighted in PROTACs, it is apparent that the use and discovery of allosteric modulators can broaden the scope of future UPS drug development strategies through the production of artificial E3 ligase regulators.

4.1. The DUB Step of Ubiquitin Processing. Prior to its use in protein labeling, proubiquitin must be processed and transformed into its mature form to reveal Gly76 through the cleavage of a C-terminal peptide extension.132 Furthermore, single ubiquitin molecules or its polymers must eventually be removed from substrates, particularly those targeted for degradation by the 26S proteasome. This process ensures the reversibility of ubiquitin modification and maintains the homeostatic levels of free and bound ubiquitin within the cell.132 DUBs function by catalyzing the hydrolysis of the peptide, isopeptide, or thioester bonds made between ubiquitin Gly76 and other substrates. This includes removal of the proubiquitin terminal peptide, hydrolysis of bonds made between Gly76 and the ε-amino groups of lysine residues on protein substrates, and the removal of small intracellular nucleophiles such as glutathione that mislabel ubiquitin through the nucleophilic attack of ubiquitin thioester intermediates.6,133 These actions of DUBs ensure the proper activity, recycling, and extended lifetime of ubiquitin within the cell, which are vital processes that contribute to sustaining normal rates of proteolysis that mediate normal cell growth and survival. DUBs can be subdivided into 6 subfamilies based on their mode of action and sequence similarities. Five of these families, including the ubiquitin-specific processing protease group (UBP or USP), the ubiquitin carboxy-terminal hydrolase (UCH), the ovarian tumor-related protease (OTU), the ataxin-3/Josephin domain proteases, and the recently identified motif interacting with ubiquitin-containing novel family (MINDY), are cysteine proteases that promote hydrolysis through the action of a catalytic triad containing Cys, His, and Asp residues.79 Interestingly, the MINDY DUB enzymes preferentially catalyze the hydrolysis of polyubiquitin chains,134 as opposed to USP and UCH enzymes that operate on terminal ubiquitin monomers or cleaving small functional groups from the ubiquitin C-terminus, respectively. The sixth DUB family includes Jab1/Mov34/Mpr1 Pad1 N-terminal+ (JAMM/MPN +) proteases, which contain a metalloprotease motif composed of one Asp and two His residues that coordinate a Zn2+ ion crucial for their proteolytic activity.135 The USP category is the largest of these subfamilies, and USP proteases contain two conserved motifs, namely, Cys and His boxes, that are essential for ubiquitin recognition and chain/C-terminal proteolysis. USPs share common structural features with the UCH proteases, which are smaller in size and catalyze the hydrolysis of small ubiquitin C-terminal amides and esters.135 The active sites of both types of proteases assume catalytically incompetent conformations in the absence of ubiquitin, while ubiquitin binding stimulates the reorganization of residues within the active site to induce proteolysis. The revelation of cryptic active sites solely in the presence of ubiquitin ensures that apo-DUBs remain proteolytically inert toward other cellular proteins while promoting fine-tuned temporal and spatial regulation of signaling pathways, protein degradation, and correct ubiquitin chain formation on target substrates.135 Therefore, the conformational flexibility in the DUB active site may facilitate the design of molecules capable of regulating their enzymatic activity through the allosteric regulation of protein conformation, in either the presence or absence of ubiquitin. 4.2. Avoiding the Active Site for Selective DUB Inhibition. Although few inhibitors have been identified for UCH and USP enzymes that function through mechanisms of uncompetitive and noncompetitive inhibition, they generally

4. DEUBIQUITINATING ENZYMES (DUBS) AND UBIQUITIN-LIKE PROTEASES (ULPS) Protein labeling with ubiquitin and Ubls is largely regulated by the activity of DUBs and ULPs. These enzymes play a dual role in ubiquitin or Ubl conjugation pathways: they are responsible for the processing of proubiquitin and pro-Ubls into their mature polypeptide forms, as well as for the removal of ubiquitin/Ubl monomers or polymers from proteins once signaling is complete. 413



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Table 4. Deubiquitinating Enzyme Inhibitors That Mediate Their Activity outside the Active Sitea

a


misregulated activity of disease-inducing DUBs, such as the USP7 protease. USP7 mediates the deubiquitination of several cellular substrates, including the E3 mouse double minute 2 (MDM2) and its ubiquitination substrate p53. Silencing USP7 leads to antiproliferative effects in cancer cells, the induction of cell cycle arrest, an increase in steady-state WT p53 levels, and the promotion of ubiquitin-dependent MDM2 degradation.143 Furthermore, USP7 overexpression has been linked to prostate cancer proliferation, while its down-regulation has been implicated in the suppression of colon cancer cell division in vitro and tumor growth in vivo.144,145 Thus, the USP7 protease is an attractive drug target for the extenuation of cancer cell growth and disease progression. From an ubiquitin-7-amino-4-methylcoumarin (ubiquitinAMC) screen of 65 092 compounds against USP7 DUB activity, a lead inhibitor, HBX 41,108 (7-chloro-9-oxo-9Hindeno[1,2-b]pyrazine-2,3-dicarbonitrile, 15, Table 4), was derived through modifications performed on the most potent hits. The ubiquitin-AMC assay incorporates a 7-amido-4methylcoumarin C (AMC) chromophore appended to a ubiquitin molecule.146 Upon C-terminal hydrolysis by UCH enzymes, the AMC is released into solution and exhibits enhanced fluorescence intensity (λex/λem = 380/460 nm).146 Thus, the kinetic activity and inhibition of DUBs can be determined by measuring increases in AMC fluorescence intensity over time. Compound 15 contained a cyanoindenopyrazine scaffold and demonstrated inhibition of USP7 in vitro at an IC50 = 424 nM.147 Using Eadie−Hofstee enzyme kinetic analysis, it was found that compound 15 inactivates USP7 through a reversible uncompetitive mechanism. Thus, inhibitor binding likely occurs after formation of the enzyme− substrate (DUB*ubiquitin-AMC) complex and not to the apoenzyme alone.148 Using the molecular modeling and docking software Sybyl6.8 (Tripos) ParaDocks, it was observed that 15 preferentially

display impressive potency and moderate selectivity for their target protease. There are several excellent reviews and articles that discuss recent efforts in the discovery of small molecule and proteinbased DUB inhibitors.136−138 These agents vary in structure, mode of action, cellular effects, and selectivity among DUB enzymes and other proteases. The first reported cell-permeable DUB inhibitor was the cyclopentenone prostaglandin Δ12PGJ2 (((Z)-7-((S,E)-5-((S)-3-hydroxyoctylidene)-4-oxocyclopent-2-en-1-yl)hept-5-enoic acid, 14), which induced apoptosis as indicated by nucleosomal fragmentation and caspase-3 activation in RKO colon carcinoma cells.139 The cellular activity of 14 (EC50 between 20 and 60 μM) mirrored that of the proteasome inhibitor MG115, including the accumulation of polyubiquitinated substrates.139 These observations supported the hypothesis that the main target of 14 was in fact a protein or enzyme associated with the UPS. The mode of action of 14 inhibition likely resulted from Michael-type attack of the α,β-unsaturated carbonyl within its core scaffold by the cysteine sulfhydrl group of a DUB active site, classifying this compound as an irreversible competitive inhibitor.139,140 This compound was later found to also inhibit the DUB isoforms UCH-L1 and UCH-L3.141 The revelation of DUB inhibition by 14 stimulated the pursuit of novel competitive inhibitors and the development of drug-discovery screens specifically tailored for monitoring DUB inactivation. Although this review will not focus its discussion on competitive DUB inhibition, it should be noted that a large challenge presented in the discovery of isoform-specific competitive DUB inhibitors lies in ensuring selectivity among proteases within the same family, as a result of the conserved architecture of their active and substrate binding sites.142 Over the past decade, research efforts have started to focus on alternative approaches to DUB regulation by targeting pockets distal to the catalytic active site in order to counter the 414



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USP1/UAF1-mediated DDR may be involved in the onset of cellular chemoresistance to cisplatin. It should be noted, however, that a recent study by Ritorto et al. further evaluated the selectivity of select DUB inhibitors using a quantitative MALDI-TOF mass spectrometry based DUB assay, which measured the relative hydrolysis rates of 15Nlabeled diubiquitin.156 On the basis of these data, it was observed that 15 displayed strong promiscuous inhibitory activity against a panel of 32 DUBs, where over half of the DUBs assessed were inhibited at 10 and 1 μM. When evaluated in further detail, it was found that 15 displayed an IC50 of 0.21 ± 0.04 μM against USP8 cf. an IC50 of 5.97 ± 3.19 μM against its intended target, USP7.156 Similarly, using this method, it was observed that 16 was found to be considerably less selective for USP1/UAF1 than previously reported but displayed less activity against the 32 enzymes than 15. An advantage of using the MALDI-TOF method of screening for DUB inhibition includes the rapid sampling time (6−9 s per sample) and the ability to screen inhibitor activity against diubiquitin chains and ubiquitinated substrates instead of modified fluorophore-labeled ubiquitin substrates. Thus, it can be argued that the MALDI-TOF assay presents a more physiologically relevant environment for inhibitor assessment against DUB activity. Further work by Liang et al. led to the discovery of a third reversible inhibitor of USP1/UAF1, ML323 (N-(4-(1H-1,2,3triazol-1-yl)benzyl)-2-(2-isopropylphenyl)-5-methylpyrimidin4-amine, 18, Table 4; IC50 = 76 nM), which was obtained through the chemical modification of a hit identified by an ubiquitin-Rho qHTS (402 701 molecules screened).157 Compound 18 also displayed activity against diubiquitin hydrolysis (IC50 = 174 nM) and the deubiquitination of ubiquitin-PCNA (IC50 = 820 nM).157 Through kinetic analysis, it was discovered that 18 inhibits USP1/UAF1 activity by a mixed inhibition mechanism (Ki = 68 nM, free enzyme; K′i = 183 nM, enzyme− substrate complex).157 Furthermore, like compounds 16 and 17, compound 18 does not inhibit USP1/UAF1 by disrupting dimer formation, as assessed using native gel electrophoresis and hydrogen−deuterium exchange (HDX) experiments, which indicated that dimer assembly was not affected by 18. Using HDX, it was also observed that incubating USP1/UAF1 with 18 induced conformational changes to protein regions encompassing the ubiquitin-binding site, as well as rigidification of certain regions of the UAF1 subunit. These results support the hypothesis that 18, like 16 and 17, likely inhibits DUB activity by binding to an allosteric site (Table 4), possibly on the UAF1 subunit.150,157 The selectivity of 18 for USP1/UAF1 was evaluated against 18 DUBs, related de-SUMOylase (SENP1, for the Ubl small ubiquitin-like modifier, or SUMO) and de-NEDDylase (NEDP1) enzymes, 70 proteases, and 451 kinases through different screening platforms.157 Encouragingly, 18 had no noticeable effect on any other proteases or kinases, including the USP, UCH, OTU, or Machado−Joseph domain DUBs. This selectivity was also observed within HEK293T cells, where 18 prevented the labeling of USP1, but not other DUBs, by the covalent DUB modifier ubiquitin vinyl methyl ester (Ub-VME). These data indicate that 18 may induce conformational changes within USP1/UAF1, thereby discouraging ubiquitin binding. Furthermore, treatment of U2OS osteosarcoma, HEK293T, or H596 NSCLC cells with 18 resulted in the accumulation of monoubiquitinated PCNA and FANCD2, both of which are downstream substrates of USP1/UAF1.157

interacted with a hydrophobic pocket on USP7 in close proximity to the covalently bound ubiquitin substrate, indicating potential allosteric regulation of USP7 activity (Table 4). However, it should be noted that these docking studies were performed on X-ray crystal structures of the apoUSP7 and USP7 cocrystallized with ubiquitin-aldehyde covalently bound to the active site cysteine. Thus, it is possible that the conformation of the enzyme sampled in the docking studies does not accurately reflect the dynamic sampling of conformations that DUBs reportedly undergo when revealing cryptic binding sites for substrates. To this end, the hypothesized binding site for 15 on USP7 should be taken as a potential, and not absolute, binding conformation. However, despite some observed cellular activity, 15 also inhibited various other DUBs including USP5, USP8, UCH-L3, and the cysteine protease caspase-3, with IC50 values ranging between 70 and 200 nM.147 Recently, pimozide (1-(1-(4,4-bis(4-fluorophenyl)butyl)piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazol-2-one, 16, Table 4) and GW7647 (2-((4-(2-(3-cyclohexyl-1-(4cyclohexylbutyl)ureido)ethyl)phenyl)thio)-2-methylpropanoic acid, 17, Table 4) were identified as the first noncompetitive inhibitors of USP1/UAF1, a DUB involved in the DNA damage response (DDR) by regulating the deubiquitination of the proliferating cell nuclear antigen (PCNA) and the Fanconi anemia complementation group D2 (FANCD2) proteins.148,149 Compounds 16 and 17 were selected as top hits from a quantitative HTS (qHTS) of 9525 small molecules, in which the inhibition of USP1/UAF1 activity was measured in a ubiquitin-AMC-like assay wherein a rhodamine replaced the AMC group (ubiquitin-Rho).150,151 Kinetic studies of Lys63 diubiquitin hydrolysis revealed that 15 and 16 abrogated DUB activity through noncompetitive inhibition mechanisms (Ki = 0.5 and 0.7 μM, respectively).150 Both 16 and 17 displayed impressive selectivity for USP1/UAF1 over other USPs including USP7, USP2, USP5, and USP8, with IC50 values ranging from 44 to >114 μM.150 Furthermore, 16 did not inhibit the USP46 enzyme (IC50 > 114 μM), whose activity is also stimulated by forming a complex to the UAF1 protein; however, 17 induced modest inhibition of USP46/UAF1 (IC50 = 12 μM). Both compounds also displayed no activity against the UCH proteases UCH-L1 and UCH-L3 (IC50 > 200 μM) as well as against caspase-3 (IC50 > 250 μM).150 Treatment of HEK293T cells with either compound resulted in a significant increase in the levels of monoubiquitinated PCNA and FANCD2. These compounds were also evaluated within the cisplatin-resistant (H596) and cisplatin-sensitive (H460) non-small-cell lung cancer (NSCLC) cell lines. Cisplatin exerts its apoptotic and antiproliferative effects due, in part, to the induction of DNA lesions and the DDR through the formation of intra- and interstrand cross-links.152 However, prolonged treatment with cisplatin has resulted in acquired drug resistance through a variety of cellular mechanisms.153,154 Thus, as pathways involving PCNA and FANCD2 are involved in translesion synthesis and other facets of the DDR, the effects of 16 and 17 were evaluated on cell viability in combination with cisplatin. Synergy was observed between these USP1/ UAF1 inhibitors and cisplatin, as cisplatin/16 or cisplatin/17 (in a 1:4 ratio) led to a significant increase in cytotoxicity in H596 (EC50 = 5.1 and EC50 = 2.8 μM, respectively).150,155 This combination of inhibitors did not affect the cytotoxicity of the cisplatin-sensitive H460 cells, supporting the hypothesis that 415



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Similar to the work conducted by Chen et al.,150 the ability of compound 18 to sensitize cancer cells to the DDR induced by cisplatin was evaluated in the H596 cisplatin-resistant cell line. Treatment of H596 cells with a combination of cisplatin/18 (in a 1:4 ratio) resulted in a dramatic decrease in cell viability, with a resulting EC50 = 59 nM as compared to cisplatin or 18 alone (EC50 = 486 nM and EC50 = 10 μM, respectively).157 Analysis using the CalcuSyn software supports these data and suggests a strong synergistic effect of 18 and cisplatin on cytotoxicity.155,157 Furthermore, both siRNA-mediated knockdown of USP1 or treatment with 18 sensitized cells to lethal doses of UV-induced DNA damage. These data support that inhibition of USP1/UAF1 activity may only impair the DDR once damage has occurred, by either agents like cisplatin or UV damage. 4.3. The Future of DUB Inhibition. Several challenges need to be overcome in the pursuit of selective and potent inhibitors targeting DUB enzymes. The incomplete biophysical characterization of binding between identified small molecules and their respective enzyme targets has hampered quantitative comparisons of affinity and potency among inhibitors.137 Recent advances in DUB research have included the identification of molecules that target protease activity through alternative mechanisms. These molecules, specifically 18, display marked selectivity for USP1/UAF1 in vitro and within a cellular environment as compared to covalent DUB inhibitors and have demonstrated synergism with cisplatin against various cancer cell line viabilities. This cumulative in vitro evidence supports the therapeutic potential in achieving effective DUB inhibition by exploiting allosteric sites.

of modulating this enzymatic cascade in order to develop chemical tools to further study the roles of UPS enzymes in cellular physiology and to fuel the discovery and development of target- and disease-specific treatments.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (905) 828-5354. E-mail: patrick.gunning@utoronto. ca. ORCID

Patrick T. Gunning: 0000-0003-0654-735X Author Contributions §

S.-L.P., S.R.d.S., and E.D.d.A. contributed equally.

Notes

The authors declare no competing financial interest. Biographies Stacey-Lynn Paiva obtained her Honours Bachelor of Science in Biochemistry from Queen’s University (Kingston, ON, Canada) and is currently a Ph.D. student in Prof. Gunning’s laboratory at the University of Toronto (Toronto, ON, Canada). Her current research efforts include targeting E1 activating enzymes selectively through small-molecule inhibition with a particular focus on the SUMO activating enzyme (SAE) and the UFM1 activating enzyme (UBA5). Sara R. da Silva obtained an Honours Bachelor of Science in Biology and a Ph.D. in Chemistry from the University of Toronto. Her doctoral work in the Gunning research group focused on identifying novel inhibitors of ubiquitin-like activating enzymes, specifically of the NEDD8-activating enzyme (NAE) and the UFM1 activating enzyme, UBA5. Her work culminated in the discovery of the first presumed allosteric inhibitor of the UBA5 enzyme. Dr. da Silva is currently a Postdoctoral Fellow in the Department of Biology (University of Toronto Mississauga), where she is studying the roles of insulin on metabolic pathways in the Chagas disease vector, Rhodnius prolixus.

5. CONCLUSIONS The conventional pipeline for the discovery of allosteric inhibitors of the ubiquitin labeling pathways has mostly relied on structure-based approaches, HTS technologies, or simply serendipity. While structure-based design has driven the discovery of some clinically promising agents, as demonstrated by the activity of 6 against XIAP, limitations do exist. For example, the crystallographic data generated for E1, E2, E3, and DUB enzymes, from which de novo design typically has its origin, are limited in their usefulness, as they have been solved in either the apo or substrate-bound state, both of which potentially overlook a tractable allosteric pocket due to the dynamic nature of these enzymes. In contrast, although the use of HTS has historically given rise to hit compounds that lack target enzyme selectivity, several potent and target-specific molecules have been successfully derived from such screens for enzymes within the ubiquitin labeling cascade, such as the selective E2 inhibitor 5 or the DUB inhibitor 18. Additionally, increased efforts in developing allosteric inhibitors for the ubiquitin pathway have offered greater selectivity than some activity-based inhibitors, due to the inherent unique sequences in modulatory domains distal from the active site. Furthermore, a number of specific DUB inhibitors have demonstrated synergism with cisplatin, a commonly administered chemotherapeutic. Given the cases of acquired resistance to anticancer agents like cisplatin, modulating ubiquitin labeling through allosteric mechanisms may shift the paradigm to develop adjuvant therapies using this approach. Altogether, it is evident that targeting protein ubiquitination outside the orthosteric sites of enzymes involved in this labeling cascade is currently in its infancy. Thus, it is necessary for future research endeavors to explore novel modes

Elvin D. de Araujo received his Honours Bachelor of Science in Biological Chemistry in 2010 and a Ph.D. in Chemistry in 2015 from the University of Toronto (Toronto, Canada). After graduating, he pursued a postdoctoral fellowship in the laboratory of Prof. Patrick T. Gunning (Toronto, Canada) whose team develops small molecule inhibitors for cancer therapy. Currently, Elvin is developing strategies to optimize testing of these inhibitors with recombinant STAT3 and STAT5 protein targets. Patrick T. Gunning received his B.Sc. (2001) and Ph.D. (2005) from the University of Glasgow. He then pursued postdoctoral studies with Professor Andrew D. Hamilton (Yale University) before joining the University of Toronto, Department of Chemistry, as an Assistant Professor (Canada, 2007). Patrick was promoted to Associate Professor in 2012 and to Full Professor in 2016. Patrick’s medicinal chemistry research program investigates the development of small molecule inhibitors targeting Stat3 and Stat5; PTP1B; UBA1, -3, and -5 as well as development of SH2 domain mimetics and protein− membrane anchorage drug modalities. Patrick was the recipient of the 2010 Boehringer Ingelheim Young Investigator Award, the 2012 Royal Society for Chemistry MedChemComm Emerging Investigator Lectureship Award, and Canadian Society for Chemistry’s Bernard Bealleau Award for medicinal chemistry, among many others.

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ACKNOWLEDGMENTS The authors acknowledge financial support provided by grants from the Canadian Cancer Society Research Innovation to 416



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Impact Grant 703963 for S.-L.P. and S.R.d.S., Mitacs Accelerate for E.D.d.A., and Canada Research Chair for P.T.G.

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ABBREVIATIONS USED Ubl, ubiquitin-like protein; PTM, post-translational modification; DUB, deubiquitinating enzyme; NEDD8, neural precursor cell expressed developmentally down-regulated; RING, really interesting new gene; AML, acute myeloid leukemia; MM, multiple myeloma; DLBCL, diffuse large B-cell lymphoma; PI3Kδ, phosphatidylinositide 3-kinase δ; HTS, high-throughput screen; CLL, chronic lymphocytic leukemia; APU, adenosylphospho-ubiquitinol; UFM1, ubiquitin-fold modifier 1; UBA5, UFM1 E1 activating enzyme; UFC1, UFM1 E2 conjugating enzyme; UPS, ubiquitin proteasome system; PPI, protein− protein interaction; SAR, structure−activity relationship; HECT, homologous to the E6-AP carboxyl terminus; PHD, plant homeodomain; VHL, von Hippel−Lindau; MDM2/ HDM2, mouse/human double mutant homologue; XIAP/ cIAP, X-linked/cellular inhibitor of apoptosis protein; Smac/ DIABLO, cellular second mitochondria-derived activator of caspases/direct IAP binding protein with low pI; CRT, cisplatin and radiotherapy; SCF, Skp1-Cdc53/cullin-F-box; CDC4, cell division control protein 4; FP, fluorescence polarization; CPD, CDC4 phosphodegron; mTOR, mammalian target of rapamycin; SMER, small molecule enhancer of rapamycin; SILAC, stable isotopic labeling with amino acids in cell culture; Tm, melting temperature; DSF, differential scanning fluorimetry; IP, immunoprecipitation; DARTS, drug affinity responsive target stability; APC, anaphase-promoting complex/cyclosome; CRBN, cereblon; Cul4a, cullin-RING 4a; IMiD, immunomodulatory drug; DDB1, DNA damage-binding protein 1; NTD, N-terminus domain; HBD, α-helical domain; CTD, C-terminal domain; PROTAC, proteolysis-targeting chimera; POI, protein of interest; BCR-ABL, break point cluster-Abelson; TKI, tyrosine kinase inhibitor; CML, chronic myeloid leukemia; LSC, leukemic stem cell; ULP, ubiquitin-like protein protease; UBP/USP, ubiquitin-specific processing protease; UCH, ubiquitin carboxy-terminal hydrolase; OTU, ovarian tumorrelated protease; AMC, 7-amino-4-methylcoumarin C; DDR, DNA damage response; PCNA, proliferating cell nuclear antigen; FANCD2, Fanconi anemia complementation group D2; qHTS, quantitative high-througput screen; Rho, rhodamine; NSCLC, non-small-cell lung cancer; SENP1, deubiquitinating enzyme for the small ubiquitin-like modifier protein; NEDP1, deubiquitinating enzyme for NEDD8; VME, vinyl methyl ester; RNAi, RNA interference; siRNA, short interfering RNA

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