Exploring a New Frontier in Cancer Treatment: Targeting the Ubiquitin

Jan 29, 2013 - Exploring a New Frontier in Cancer Treatment: Targeting the Ubiquitin and Ubiquitin-like Activating Enzymes ... *Telephone: (905) 828-5...
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Exploring a New Frontier in Cancer Treatment: Targeting the Ubiquitin and Ubiquitin-like Activating Enzymes Sara R. da Silva, Stacey-Lynn Paiva, Julie L. Lukkarila, and Patrick T. Gunning* Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada ABSTRACT: The labeling of proteins with small ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers regulates a plethora of activities within the cell, such as protein recycling, cell cycle modifications, and protein translocation. These processes are often overactive in diseased cells, leading to unregulated cell growth and disease progression. Therefore, in systems where Ub/Ubl protein labeling is dysregulated, the development of drugs to selectively and potently disrupt Ub/Ubl protein labeling offers a targeted molecular approach for sensitizing these diseased cells. This Perspective outlines the progress that has been made in the context of inhibitor development for targeting Ub/Ubl pathways.

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of the E2 and E3 ligase enzymes.9,10 Once labeled, proteins are shuttled to different areas of the cell where they trigger or participate in certain cellular events.10 Cells showing aberrant activity by the UPS and a number of related pathways experience an increase in cell growth, proliferation, and progression into diseased states. For example, the UPS is hyperactive in a number of hematological malignancies such as acute myeloid leukemia (AML).11 Disrupting the UPS through the use of proteasomal inhibitors, such as the celebrated reversible proteasomal inhibitor Velcade (1, bortezomib; Millennium: The Takeda Oncology Company)12 and the more recently United States Food and Drug Association (FDA) approved irreversible proteasome inhibitor Kyprolis (2, carfilzomib; Onyx Pharmaceuticals),13 is one strategy currently used to treat leukemia, multiple myeloma, and some lymphomas. However, targeting the UPS upstream of the proteasome through the inhibition of the E1, E2, or E3 enzymes is an alternative strategy to disrupt cellular activity and can result in the development of a diverse arsenal of treatments for cancers in which the Ub/Ubl labeling pathways exhibit irregular activity. This Perspective will chronologically outline the discovery and development of inhibitors targeting enzymes of the E1 family. As detailed in the proceeding sections, the bulk of inhibitor discovery and design has focused on the Ub activating enzyme (UAE, PDB code 3CMM, yeast),7 the neural precursor cell expressed developmentally down-regulated 8 (NEDD8) activating enzyme (NAE, PDB code 3GZN, human),14 and the small Ub modifier 1 (SUMO-1) activating enzyme (SAE, PDB code 1Y8R, human).15 However, to date, no inhibitors have

he post-translational modification of proteins with ubiquitin (Ub) controls a number of fundamental processes within the cell, including protein degradation by the Ub-proteasome system (UPS). A protein destined for recycling by the 26S proteasome is labeled with Ub, a small modifier protein, by the concerted efforts of three enzymes: the Ub activating enzyme (E1), the Ub conjugating enzyme (E2), and the E3 Ub ligase, as shown in Figure 1.1 In addition to Ub, there are Ub-like (Ubl) proteins that are responsible for mediating critical cellular events, such as cell cycle regulation, cytokinesis, and cellular differentiation, through the conjugation of Ubls to target proteins substrates.2 Regardless of the Ub/Ubl, the general procedure of protein labeling by these small protein modifiers follows a similar process (Figure 1), which was first described in the literature for protein recycling through Ub labeling.3 First, ATP binds in the nucleotide-binding pocket of the E1 enzyme, followed by specific binding of Ub/Ubl to its respective E1. The E1 then catalyzes the adenylation of the bound Ub/Ubl to produce an AMP-Ub/Ubl intermediate that occupies both the nucleotide and Ub/Ubl pockets.4,5 Following Ub/Ubl adenlyation, the E1 undergoes a large conformational change in order to place a cysteine residue located in the E1 active site closer to the AMPUb/Ubl adenylate.6,7 The thiol moiety of the cysteine promotes nucleophilic attack of the AMP-Ub/Ubl phosphoester bond, forming a high-energy thioester bond and resulting in a covalently attached E1-S-Ub/Ubl intermediate. A second Ub protein is then adenylated, forming a ternary (Ub/Ubladenylate)−E1-S-Ub/Ubl intermediate complex.4,8 The E2 enzyme then binds to the E1, after which an active site cysteine on E2 attacks the E1-S-Ub/Ubl thioester in a crossprotein transthiolation reaction.9 Finally, the Ub/Ubl is transferred onto a target protein through the concerted action © XXXX American Chemical Society

Received: September 29, 2012

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Figure 1. Molecular mechanism of Ub/Ubl protein conjugation to protein substrates by the Ub-proteasome system and other related Ub/Ubl protein pathways.

Figure 2. Timeline representing the development of ubiquitin activating enzyme inhibitors since 1990. Inhibition of the UAE is achieved by interacting with the ATP pocket (green), targeting the active site cysteine (gray), through some unknown mechanism (orange) or by means of a substrate-assisted mechanism (blue).

been published for the five other E1 enzymes. The biological significance of these E1s and their therapeutic potential in targeting diseased cells will also be summarized in this Perspective.

inhibiting intracellular protein ubiquitination. The crystal structure of the entire human UAE protein (UBE1) has not yet been resolved; however, the crystal structure of UAE in Saccharomyces cerevisiae (UBA1) reveals a four building block canonical structure bound to Ub through the E1 active site cysteine.7 Researchers believe that based on the 50% sequence identity shared between UBE1 and UBA1, the homologous proteins should adopt similar architectures.7 UAE’s aberrant activity causes an overall increase in cellular protein turnover and is associated with many diseased states including leukemia

1. INHIBITORS TARGETING THE UBIQUITIN ACTIVATING ENZYME (UAE): A 20-YEAR HISTORY Since its discovery, researchers have pursued the inhibition of UAE in an attempt to understand the consequences of B

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Figure 3. Mechanisms of inhibitor activity against the E1s: UAE, SAE, and NAE. (a) Inhibition of the E1 is achieved by targeting the active site cysteine. (b) E1 inhibition is mediated by compounds interacting with the E1 ATP pocket. (c) Substrate-assisted inhibitors form a covalent bond to Ub or the Ubl protein and subsequently occupy both the ATP and the Ub/Ubl pocket. (d) The inhibitory activity of these compounds has not yet been elucidated.

dependently inhibited (IC50 ≈ 40 μM), while ATP-PPi exchange was disrupted at even lower concentrations when 4 was tested against purified UAE.17 Furthermore, the inhibitory activity of 4 was abolished in the presence of the sulfhydryl compounds dithiothreitol (DTT) and 2-mercaptoethanol, supporting the hypothesis that 4 mediates its inhibitory activity by reaction with the active site cysteine on UAE (see Figure 3a).17 The cytotoxicity of 4 was also measured against various tumor cell lines, with IC50 values ranging from 6 to 30 μM.17 However, 4 has additional cellular mechanisms of action as a chemotherapeutic agent, including DNA damage. The authors of this work also cautioned that in cells, 4 may have been interacting with the ubiquitin conjugating, ubiquitin ligase, or arginyl-tRNA protein transferase enzymes.17 As such, it is unlikely that UAE inhibition is a predominant mechanism by which 4 exerts its anticancer effects. Nonetheless, 4 was the first small molecule inhibitor of UAE identified in the movement toward the discovery and development of small molecules targeting UAE. A decade later, researchers at the Japanese Institute for Microbial Chemistry identified panepophenanthrin (5) isolated from the Panus radis mushroom, as the first natural product inhibitor of UAE (see Figure 2).18 Compound 5 inhibited the UAE-Ub intermediate formation dose-dependently, with an IC50 in vitro of 17.0 μg/mL, although no significant inhibitory effect was observed in cells treated with up to 50 μg/mL of this compound. 18 More recently, a complete enantio- and diastereoselective synthesis of (+)-5 has been reported,

and multiple myeloma.11 Therefore, UAE has become an attractive therapeutic target in diseases caused by irregular UPS activity. The first successful attempt at inhibiting UAE occurred in 1990, where Wilkinson et al. synthesized a nonhydrolyzable mimetic of the ubiquitin adenylate intermediate adenosylphospho-ubiquitinol (3, APU), shown in Figure 2.16 The inhibitor 3 was developed with the intention of acting as a proof-of-concept molecule that could target and bind to UAE selectively and was found to effectively prevent the conjugation of Ub to endogenous proteins (apparent Ki = 0.75 μM) by inhibiting the ATP-PPi exchange reaction catalyzed by UAE.16 The inhibition of UAE by 3 is ATP-competitive (Ki = 50 nM), and noncompetitive with Ub (Ki = 35 nM).16 The decrease in the ATP-PPi exchange induced by 3 was also observed in the presence of an excess of AMP, suggesting that UAE inhibition is mediated through binding of 3 to the free enzyme and not the UAE-Ub thioester intermediate (see Figure 3c).16 Although impractical as a potential drug molecule, the creation of 3 was the first successful attempt to impede protein ubiquitination by selectively targeting an enzyme upstream of the proteasome and demonstrated a therapeutic potential of developing compounds against UAE. Around this time, scientists observed a dose-dependent inhibition of cellular Ub-dependent proteolysis (IC50 = 90 μM) while studying the intracellular targets of the chemotherapeutic cisplatin (4; see Figure 2).17 In the presence of 4, E1 activity of a partially purified E1 sample (fraction II) was doseC

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the inhibition of the activation of tumor necrosis factor α induced NF-κB.11 Xu et al. further identified cytotoxic effects of 10 in leukemia and myeloma cells, with LD50 < 10 μM and LD50 ≤ 3 μM, respectively; however, 10 exhibited lower potency in solid tumor samples (LD50 = 15−20 μM).4 Biochemical evaluation of leukemia cells exposed to 10 revealed the induction of an unfolded protein response and endoplasmic reticulum stress, leading to cell death. Interestingly, systemic administration of 10 delayed leukemia progression without evidence of whole animal toxicity in leukemia mouse models. Although useful as chemical tools ex cellulo, one major drawback of the covalent nature of 9 and 10 as potential leads for drug development is the recently identified phenomenon of protein cross-linking in cells treated with 9.22 Around the time of the discovery of 9, the diazeniumdiolate prodrug JS-K (11, [O 2 -(2,4-dinitrophenyl) 1-[(4ethoxycarbonyl)piperazin-1-yl]deazen-1-ium-1,2-diolate]), designed to release the small radical nitric oxide (NO) when metabolized by glutathione S-transferase (GST) in cells, was tested as a potential UAE inhibitor (see Figure 2).23 Kitagaki et al. found that 11 inhibited UAE-Ub thioester formation dosedependently in RPE cell lysates (IC50 ≈ 2 μM) and believe that the NO released from 11 reacts with the UAE active site cysteine, resulting in S-nitrosylation and UAE inhibition (see Figure 3a).23 The incubation of UAE with the NO donor PABA/NO and a non-NO releasing analogue of 11 resulted in complete UAE-Ub thioester inhibition and no inhibitory activity against UAE, respectively, further corroborating that UAE inhibition by 11 is likely mediated through the reaction of the UAE cysteine with NO.23 Cells treated with 11 experienced increased levels of several UPS target proteins, including βcatenin (HEK293 cells), programmed cell death 4 (Pdcd4, A549 cells), and p53 and Hdm2 (RPE cells).23 Furthermore, HeLa cells incubated with 11 showed delayed IL-1α induced phosphorylation and degradation of IκBα, while 11 preferentially induced apoptosis in transformed cells overexpressing wild-type p53.23 A few years later, Ungermannova et al. identified NSC624206 (12), as shown in Figure 2, a covalent UAE inhibitor, through a compound library screen.21 Compound 12 inhibited the formation of the UAE-Ub conjugate with IC50 ≈ 9 μM and inhibited UAE nucleotide exchange reactions with IC50 = 13 μM.21 It was later determined that 12 inhibits the formation of the UAE-Ub thioester intermediate but had relatively no effect on Ub adenylation by UAE.21 The authors of this work believe that the inhibitory activity of 12 is due to its disulfide bond, which can react with the active site cysteine of UAE, as demonstrated in Figure 3a. Consistent with this hypothesis, no inhibition of UAE was observed in the presence of DTT or when a 12 derivative that lacked the disulfide bond was used.21 Kip16 and HepG2 cells incubated with increasing concentrations of 12 exhibited reduced polyubiquitination and concomitant increased levels of the UPS substrate p27.21 Both 11 and 12 were not tested against other E1s, and therefore, their specificity for UAE among other related enzymes remains unknown. Furthermore, although both inhibitors show impressive activity in cell-free and cellular assays, they have not yet been developed into more potent second-generation inhibitors en route to the development of a therapeutic lead. Over the past 3 years, progress toward developing UAEspecific inhibitors has largely focused on identifying compounds that target UAE-mediated Ub adenylation. Recently, Lu et al. re-evaluated the inhibition of UAE through the synthesis of

demonstrating similar activity as described in previous reports.19 The total syntheses of 5 derivatives RKTS-80 (6), RKTS-81 (7), and RKTS-82 (8) were also reported by Matsuzawa et al. and were found to inhibit the UAE-Ub intermediate more potently in cell-free assays than the originally isolated molecule (IC50 of 9.4, 3.5, and 90 μM, respectively).19 Interestingly, removing the 2,2-dimethyltetrahydrofuran found in 5 increased potency toward UAE, as seen by the activity of 6 and 7, shown in Figure 2, indicating that this moiety is not necessary for UAE inhibition.19 All three derivatives dose-dependently blocked the growth of breast cancer cells (MCF-7) with IC50 of 5.4, 1.0, and 3.6 μM, respectively.19 Unfortunately, the selectivity of 5 and its derivatives toward UAE is unknown (see Figure 3d), as these molecules have not been tested against other E1s or other related enzymes. It was after the development of 5 and its derivatives that several research groups independently discovered a series of electrophilic, covalent inhibitors that target the active site cysteine of UAE. The most celebrated of this group of compounds is 4-[4-(5-nitrofuran-2-ylmethylene)-3,5-dioxopyrazolidin-1-yl]benzoic acid ethyl ester, or PYR-41 (9; see Figure 2), developed by Yang et al. in 2007 and reported as the first cell-permeable UAE-selective inhibitor.20 Compound 9 inhibited the formation of the UAE-Ub thioester intermediate with IC50 < 10 μM in cell-free enzymatic assays, with similar results observed by Ungermannova et al. using ATP-AMP exchange reactions (IC50 = 6.4 μM).20,21 Both groups believe that 9 is susceptible to nucleophilic attack by the UAE active site cysteine to the exocyclic double bond through a Michaeltype addition or through reaction with the N-aryl bond, supported by the abolishment of inhibitor activity in the presence of reactive thiols such as glutathione (GSH) and DTT (see Figure 3a).20 Finally, through a small structure−activity relationship (SAR) study of the 9 backbone, it was established that the nitro group off the furan ring is crucial for inhibitor activity, as derivatives without this functionality were incapable of inhibiting UAE at any tested concentration.20 It was further discovered that 9 inhibits the UAE-Ub thioester formation in retinal pigment epithelial (RPE) cells (IC50 = 10−25 μM) and inhibited proteasomal and nonproteasomal target substrate ubiquitination in other cell lines, such as cyclin D3, ligandinduced EGFR, and TRAF6 in HeLa cells, as well as p53 in U2OS osteosarcoma cells.20 Furthermore, HeLa cells treated with 9 had reduced nuclear factor κB (NF-κB) activation as a result of disrupted signaling activity involving TRAF6 and decreased downstream proteasomal degradation of Iκβα.20 Finally, 9 selectively decreased protein ubiquitination over NEDDylation and SUMOylation and targeted transformed p53-expressing RPE cells for apoptosis.20 In 2010, 3 years after the identification of 9 as a novel inhibitor of UAE, Xu et al. screened a library of small molecules based on the 9 pyrazolidine scaffold and discovered a derivative, PYZD-4409 (10) as shown in Figure 2, which inhibited the formation of the UAE-Ub thioester intermediate and subsequent E1−E2 transthiolation, both in enzymatic based assays (IC50 ≈ 20 μM) and in K562 leukemia cells.11 A derivative of 10 lacking the nitro functionality was incapable of inhibiting UAE, further validating the importance of the nitro group in the activity of this family of molecules (see Figure 3a).11 Furthermore, 10 selectively inhibited UAE over the SAE, even at 100 μM. In leukemia cells treated with 10, there was an apparent increase in the levels of cyclin D3 and p53 along with D

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natural product inhibitor of UAE in 2012 (Figure 2).28,29 Interestingly, 17 induced cancer-cell-specific death and demonstrated antiproliferative activity in transformed mammary epithelial cells.28 As a result, 17 presented itself as an important synthetic target and, more importantly, as a potential cancer therapeutic. In 2012, the group revisited the study of 17 as an anticancer agent and postulated that the mechanism of its activity can be attributed to the inhibition of UAE by preventing the adenylation of Ub, as shown in Figure 3b.29 The authors of this work determined that 17, which consists of a macrocyclic structure and an aliphatic tail containing a thioester moiety, prevented the formation of the UAE-Ub thioester intermediate in enzymatic-based assays (IC50 = 29 μM). This activity was equipotent to that seen in the derivatives of 17 that contained an ester (25 μM) and ketone (30 μM) in place of the thioester.29 Therefore, while the macrocyclic backbone and aliphatic tail were important for inhibitor activity, it appeared that the thioester moiety was not necessary for effective UAE inhibition. 29 Furthermore, ATP:PPi and AMP:ATP exchange reactions were used to further corroborate that 17 and its derivatives inhibit Ub adenylation and have no effect on the UAE-Ub thioester formation.29 Treatment of Kip16 cells with 17 resulted in up-regulated levels of p27 expression, although Ungermannova et al. caution that this observed activity of 17 may be due to inhibition of histone deacetylase (HDAC).29 Furthermore, 17 effectively decreased the polyubiquitination of p27 and Trf1 in reconstituted systems, thereby blocking the UPS-mediated degradation of target substrates. Compound 17 was also selective for human over Schizosaccharomyces pombe UAE and was incapable of inhibiting the human SAE (IC50 ≈ 450 μM).29 The effect of 17 has not yet been evaluated against other E1 enzymes or other enzymes involved in the UPS. Regardless, there exists therapeutic potential in developing 17-based drugs that can selectively inhibit UAE in cancer cells exhibiting aberrant UPS activity. Lastly, from 2005 to 2012, two inhibitors were discovered to inhibit UAE with unknown mechanisms of action (Figure 3d). Himeic acid A (18, see Figure 2), extracted from the fungus Aspergillus sp., dose-dependently inhibited the formation of the UAE-Ub thioester, with densitometry analysis revealing apparent IC50 ≈ 50 μM.30 The authors of this work postulate that 18 undergoes attack by the UAE active site cysteine, resulting in irreversible UAE inhibition. Interestingly, an analogous compound containing the C-1 imide functionality of 18 was unsuccessful at inhibiting UAE, indicating that the complete structure of 18 is necessary for its inhibitory activity.30 However, in a recent study by Yamanokuchi et al., it was suggested that 18 inhibits UAE by blocking Ub binding to UAE, as shown through a UAE-Ub immunoprecipitation experiment in the presence of inhibitor.31 This inhibitor has not yet been tested against other E1 enzymes. More recently, Yamanokuchi et al. recently reported hyrtioreticulins A and B (19 and 20, respectively, see Figure 2), two novel natural product inhibitors of UAE isolated from the marine sponge Hyrtios reticulatus.31 Compounds 19 and 20 dose-dependently prevented the formation of the UAE-Ub conjugate (IC50 of 2.4 and 35 μM, respectively). A small SAR identified the “trans” stereoisomer at C1, along with the imidazole moiety, as structural features essential for UAE inhibition.31 Yamanokuchi et al. also used immunoprecipitation to determine that 19 and 20 do not prevent Ub binding to UAE.31 It is hypothesized that these inhibitors block either the formation of the Ub-adenylate

nonhydrolyzable Ub-AMP mimetics that, like Wilkinson’s inhibitor 3,16 inhibit UAE by occupying the AMP pocket and Ub binding pocket simultaneously.24 These inhibitors were constructed by ligating ubiquitin to the AMP mimetic AMSN or AVSN through native chemical ligation (Figure 2).24 UbAMSN (13) contains a sulfamide that acts as a nonhydrolyzable analogue of the phosphate in Ub-AMP and inhibits the first half-reaction of Ub-adenylate formation (Figure 3b), while UbAVSN (14) contains a vinylsulfonamide electrophile designed to trap the active site cysteine and inhibits the formation of the UAE-Ub thioester (Figure 3a).24 In cell-free assays, 13 dosedependently inhibited UAE-Ub intermediate formation by binding to UAE as an Ub-AMP mimetic, while 14 inhibited the UAE-Ub thioester through the formation of a 14−UAE conjugate.24 13 and 14 were selective for UAE over SAE; however, they were not further evaluated in cell-based assays. In 2011, researchers at Millennium: The Takeda Oncology Company successfully identified Compound 1 (15, see Figure 2),25 which was inspired by the discovery of the NAE-specific inhibitor MLN4924 (16).26 Compound 15 inhibits UAE through a novel substrate-assisted mechanism, as shown in Figure 3c. Through its mode of inhibition, 15 exploits the natural activity of its target enzyme in order to form a covalent bond to the native target substrate Ub. Therefore, unlike covalent inhibitors, 15 is classified as a mechanistic inhibitor, since its activity is dependent on the mechanism of Ub activation by UAE in order to form the final inhibitory 15−Ub complex in situ.25 The Ub−15 adduct mimics the Ub-adenylate intermediate and is formed through the nucleophilic attack of the UAE-Ub thioester by the sulfamate group of 15.25 Since nucleophilic attack of the intermediate thioester is a crucial step in the formation of the inhibitor adduct, the UAE active site cysteine is indispensable for the activity of 15, as confirmed by the abolished inhibitory activity of 15 in C632A mutants.25 The dissociation constant for the 15−Ub/UAE complex was determined to be KD < 50 pM by surface plasmon resonance (SPR) analysis in the absence of ATP, indicating a tight binding complex between 15−Ub and UAE. Furthermore, ATP is competitive with 15 inhibition, as shown by decreasing inhibitory activity (IC50 = 10.2 nM to IC50 = 5 μM) with increasing concentrations of ATP (10 μM to 1 mM).25 The E1−E2 transthiolation reaction between UAE and UbcH6 was also inhibited with IC50 = 1.4 ± 0.2 nM ([ATP] = 75 nM).25 Inhibitor 15 is therefore classified as the most potent UAE inhibitor developed to date; however, as Chen et al. reported, it is a nonselective inhibitor that also targets other E1s with varying degrees of potency (IC50 = 5.2 μM for UAE, 6.4 μM for SAE, 0.01 μM for NAE, 0.92 μM for Uba6, 4.0 μM for Uba7, and >100 μM for ATG7, at 1 mM ATP).18 Therefore, although 15 represents the most potent UAE inhibitor developed to date, 15 has not been pursued as a lead for therapeutic design against UAE because of its equipotent effect on other E1 enzymes. Subsequent work by Lukkarila et al. explored the gatekeeper residue of the ATP pocket on UAE through iterative modification of the adenosine C6 position on 15, in order to glean UAE specificity.27 However, regardless of the substituents appended at this position, all of the 15 analogues showed selectivity for NAE over other E1 enzymes, although the activity of the most potent compounds showed activity in the high nanomolar range against UAE.27 In 2008, the Leusch group at the University of Florida isolated the natural product largazole (17) from the cyanobacteria genus Symploca, which was later classified as a E

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Figure 4. Timeline representing the development of SUMO activating enzyme inhibitors since 2004. Inhibition of the SAE is achieved by interacting with the ATP pocket (green), targeting the active site cysteine (gray), or through some unknown mechanism (orange).

dependently inhibited SAE dimer activity, resulting in decreased protein SUMOylation in vitro.37 It is believed that 21 inhibits SAE by blocking Sae2-Uba2 heterodimerization and promoting complex destabilization, along with inducing the degradation of related E2 enzymes. The addition of proteasome inhibitors to in vitro assays replenished SAE and E2 levels, suggesting that 21 may also promote the UPS-mediated degradation of SAE subunits (Figure 3d).37 Molecule 21 also inhibited the SUMOylation of transcription factors associated with transcriptional repression, thereby acting as a transcriptional activator.37 However, although it is an interesting discovery in the E1 inhibitor field, no research has been conducted to further pursue 21 as an SAE inhibitor. Five years later, researchers from the Yoshida group isolated two natural products from the extracts of Gingko biloba leaves: ginkgolic acid (22) and anarcardic acid (23) (see Figure 4).38 Both 22 and 23 prevented SUMOylation of a target protein RanGAP1-C2 (C-terminal fragment of RanGAP1), with IC50 values of 3 and 2.2 μM; these compounds did not affect protein ubiquitination in vitro.38 Structurally, 22 and 23 are very simple molecules consisting of salicylic acid appended to long unsaturated and saturated alkyl chains, respectively.38 Researchers conducted a small SAR study and discovered that the alkyl chain is important for 22 and 23 inhibitory activity, since salicylic acid alone did not affect protein SUMOylation. Furthermore, 23 is a more potent inhibitor than 22, indicating that the double bond in 22 is likely not vital to inhibitory activity.38 The carboxylic acid on the salicylic acid moiety of 22 was subsequently converted to a methyl ester, yielding Me-22, which could not inhibit SUMOylation.38 Finally, the salicylic acid alcohol was acetylated producing Ac-22, capable of retaining SUMOylation inhibition. It is therefore suggested that the alcohol functionality could be eliminated entirely or

or the UAE-Ub thioester, although future experimentation is required to confirm these theories (Figure 3d). More importantly, both 19 and 20 exhibited limited cytotoxicity in HeLa cells (IC50 > 50 μg/mL) because of their cellular impermeability.31 Therefore, although reported as the most potent natural product inhibitor of UAE identified in the literature to date, it is unlikely that 19 will progress as a lead in the identification of novel therapeutics toward UPS-active cancers. The search for potent inhibitors targeting UAE spans more than 2 decades and encompasses a broad range of molecular scaffolds that target a variety of sites on UAE. Notably, the impressive inhibition of UAE by molecules such as 10 and 15 has reinforced the therapeutic potential of targeting UAE in cancer treatment. However, there exists a need for the development of more selective, reversible, cell-permeable UAE inhibitors in order to effectively treat human malignancies with aberrant UPS activity.

2. INHIBITING THE SUMO1-ACTIVATING ENZYME (SAE): WHERE NATURAL PRODUCTS DOMINATE Over 20 years ago, SAE was first discovered as a canonical heterodimer of Sae1 and Uba2 and has since been crystallized revealing a pseudosymmetric canonical structure.15 Researchers have determined that SUMOylation of proteins influences protein subcellular localization and overall protein stability.32 Dysregulation of SUMOylation in cells has been linked to many diseased states such as cancers, viral infections, and Alzheimer’s and Huntington’s diseases.33−36 These revelations have made SAE an attractive therapeutic target, where the prevalence of natural products dominates over rationally designed drugs. In 2004, the Gam1 (21, Figure 4) avian adenoviral protein was identified as the first SAE inhibitor. The inhibitor 21 doseF

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Figure 5. Timeline representing the development of NEDD8 activating enzyme inhibitors since 2009. Inhibition of NAE is achieved by interacting with the ATP pocket (green) or through a substrate-assisted mechanism (blue).

over UAE, neither inhibitor was evaluated for its activity against other E1 enzymes, and their inhibitory concentrations have not been established. Unlike the extensive research conducted on inhibiting UAE, SAE remains an elusive and yet necessary target for inhibition, as the SAE pathway is implicated in many ovarian cancers and carcinomas. The lack of inhibitors displaying potent activity against SAE allows for ample room to design and develop small molecules capable of disrupting SAE activity, increasing the novelty and attractiveness of SAE as a target for cancer therapy.

replaced by other functional groups in order to derive increased potency with second-generation molecules varied at that position.38 Lois et al. did not conduct further SAR studies on 23, considering the redundancy in their molecular composition (Figure 3). Later in 2009, the same group of authors unveiled a second natural product isolated from three atinomycete strains, kerriamycin B (24), as a novel inhibitor of SAE.39 This molecule completely inhibited RanGAP1-C2 SUMOylation at 20 μM (IC50 = 11.7 μM) without exerting any effect on protein ubiquitination.39 Furthermore, 293T cells incubated with 100 μM 24 experienced decreased poly-SUMOylation, and the SAE-SUMO-1 thioester complex was entirely blocked at 20 μM compound.39 Dating back to 1985, 24 (see Figure 4) was originally found to elicit antiproliferative effects on Ehrlich ascites carcinoma. Considering the increase in expression of the SUMO-1 E2 Ubc9 in some tumors, it was postulated that 24 could be targeting the SUMOylation pathway in these cells and therefore could have use as an anticancer agent in cancers where SAE and the SUMO pathway is more active.40,41 However, the selectivity of 22, 23, and 24 have not been determined for SAE; therefore, these molecules cannot yet be evaluated as leads for the development of effective therapeutics targeting SAE. The most recent and selective inhibitors of SAE were developed in 2010 by a similar technology used to develop the UAE inhibitors 13 and 14.24 Like the UAE inhibitors, SUMOAMSN and SUMO-AVSN (25 and 26, respectively, see Figure 4) inhibit SAE by simultaneously occupying the AMP and SUMO-1 binding pockets. Compound 25 contains a nonhydrolyzable sulfamide analogue of AMP and inhibits the first half-reaction of SUMO-1 activation, similar to the activity of 13 (see Figure 3b), while 26 traps the active site cysteine of SAE in a reaction with its vinylsulfonamide functionality, inhibiting the second half-reaction of SUMO-1 activation, as in the inhibition of UAE by 14 (see Figure 3a).24 Although selective for SAE

3. TARGETING THE NEDD8-ACTIVATING ENZYME (NAE): A PROMISING HISTORY OF CANCER TREATMENT THROUGH E1 INHIBITION NAE, like SAE, is a canonical heterodimer comprising APPBP1 and UBA3 and catalyzes the NEDD8ylation of protein substrates including the cullin proteins involved in forming the active E3 Ub ligases, the cullin-RING ligases (CRLs).42−44 Deregulation of NAE activity has been shown to correlate with the progression of various cancers.45 Because of the recent success of NAE-specific inhibitors currently in clinical trials, NAE has become an important target in the pursuit of novel E1 therapeutics. After the release of the successful proteasome inhibitor 1, Millennium: The Takeda Oncology Company turned their attention to the development of E1-specific inhibitors. Their efforts in functionalizing AMP for improved E1 specificity resulted in the identification of N6-benzyl adenosine through a high-throughput screen (HTS).26 This compound was iteratively functionalized to produce 16 in 2009 (Figure 5)26 and its derivative 15, the previously mentioned nonselective UAE inhibitor. 16 is a selective, mechanistic E1 inhibitor with impressive in vitro potency toward NAE (IC50 = 4.7 ± 1.5 nM) over UAE (IC50 = 1.5 ± 0.71 μM), SAE (IC50 = 8.2 ± 6.2 μM), the E1 Uba6 (IC50 = 1.8 μM), and the ISG-15 E1, ATG7 (IC50 G

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> 10 μM).26 The main differences between the structures of AMP and 16 include a deazapurine ring substituted with an aminoindane moiety at N6 in place of the adenine base, replacing the ribose sugar with a 2′-deoxycarbocycle, and a sulfamate moiety in place of the AMP phosphate group. Finally, the stereochemistry was inverted at the sulfamate position, producing the trans-isomer (relative to the deazapurine base), which was crucial to attain the observed specificity toward NAE over other E1s.26 Using X-ray crystallography, Brownell et al. observed the formation of a strong 2.7 Å hydrogen bond between one of the 16 sulfamate oxygens and the backbone amide of UBA3 Gly79, which was nearly identical to the interaction between one of the α-phosphate oxygens of ATP to Gly79.26 It is believed that this interaction reduces the pKa of the sulfamate amino group to ∼9.35 (measured in methanol/ water), which serves to increase its nucleophilicity, promoting the attack of the NAE-NEDD8 thioester bond by the 16 sulfamate and forming the covalent 16−NEDD8 inhibitory adduct, similar to what is observed for compound 1, as shown in Figure 3c.14 The existence of this adduct was further confirmed by detection through mass spectrometry and through Western blot analysis of trypsin digested protein, which showed a digylcine-16 fragment.14 16 inhibited protein NEDDylation in HCT116 cells at