Hostname: page-component-6898cfcb55-zsp5v Total loading time: 0 Render date: 2026-11-11T22:11:50.147Z Has data issue: false hasContentIssue false
  • English
  • Français

Pharmacological targets in the ubiquitin system offer new ways of treating cancer, neurodegenerative disorders and infectious diseases

Published online by Cambridge University Press:  17 November 2011

Mariola J. Edelmann,
Benjamin Nicholson and
Benedikt M. Kessler
Mariola J. Edelmann*
Affiliation:
Institute of Genomics, Biocomputing and Biotechnology, Mississippi Agricultural and Forestry Experimental Station, Mississippi State University, Mississippi State, MS 39762, USA
Benjamin Nicholson
Affiliation:
Progenra Inc., Malvern, PA 19355, USA
Benedikt M. Kessler*
Affiliation:
Henry Wellcome Building for Molecular Physiology, Nuffield Department of Medicine, University of Oxford, Oxford, OX3 7BN, UK
*
*Corresponding authors: Benedikt M. Kessler, Henry Wellcome Building for Molecular Physiology, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. E-mail: bmk@ccmp.ox.ac.uk and Mariola J. Edelmann, Institute for Genomics, Biocomputing and Biotechnology, Mississippi Agricultural and Forestry, Experimental Station, Pace Seed Lab, Rm 115, Mississippi State University, 650 Stone Boulevard, Mississippi State, MS 39762, USA. E-mail: mje100@mafes.msstate.edu
*Corresponding authors: Benedikt M. Kessler, Henry Wellcome Building for Molecular Physiology, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. E-mail: bmk@ccmp.ox.ac.uk and Mariola J. Edelmann, Institute for Genomics, Biocomputing and Biotechnology, Mississippi Agricultural and Forestry, Experimental Station, Pace Seed Lab, Rm 115, Mississippi State University, 650 Stone Boulevard, Mississippi State, MS 39762, USA. E-mail: mje100@mafes.msstate.edu
Rights & Permissions [Opens in a new window]

Abstract

Recent advances in the development and discovery of pharmacological interventions within the ubiquitin–proteasome system (UPS) have uncovered an enormous potential for possible novel treatments of neurodegenerative disease, cancer, immunological disorder and microbial infection. Interference with proteasome activity, although initially considered unlikely to be exploitable clinically, has already proved to be very effective against haematological malignancies, and more specific derivatives that target subsets of proteasomes are emerging. Recent small-molecule screens have revealed inhibitors against ubiquitin-conjugating and -deconjugating enzymes, many of which have been evaluated for their potential use as therapeutics, either as single agents or in synergy with other drugs. Here, we discuss recent advances in the characterisation of novel UPS modulators (in particular, inhibitors of ubiquitin-conjugating and -deconjugating enzymes) and how they pave the way towards new therapeutic approaches for the treatment of proteotoxic disease, cancer and microbial infection.

Information

Type
Review Article
Information
Expert Reviews in Molecular Medicine , Volume 13 , March 2011, e35
Copyright
Copyright © Cambridge University Press 2011. Re-use permitted under a Creative Commons Licence – by-nc-sa.

The ubiquitin–proteasome system (UPS) controls the turnover and biological function of most proteins within the cell, and alterations in this process can contribute to cancer progression, neurodegenerative disorders and pathogenicity associated with microbes. Therefore, pharmacological targeting of the UPS can potentially provide chemotherapeutics for the treatment of tumours, neurodegenerative conditions and infectious diseases. The widespread involvement of components of the UPS in many biological processes is reflected by the fact that several hundred genes have now been associated with this pathway (Refs Reference Behrends and Harper1, Reference Clague and Urbe2). Ubiquitin is a protein with 76 amino acids that can be covalently attached to other proteins, thereby influencing their fate and function. Protein ubiquitylation has numerous physiological functions. It can act as a recognition signal for proteasomal degradation (polyubiquitylation), serve as a signalling scaffold for protein–protein interactions (Lys63-poly- or monoubiquitylation) or represent a targeting signal for the lysosomal pathway or other cellular compartments (mostly monoubiquitylation). The ability of the ubiquitylation machinery to selectively target substrates is mediated by the specificity of ubiquitin ligation (E2 and E3 enzymes) and deconjugation, promoted by deubiquitylating enzymes (DUBs). Interference with either arm of this pathway should allow highly targeted pharmacological intervention, provided that compounds with sufficient selectivity can be identified (Refs Reference Bedford3, Reference Eldridge and O'Brien4, Reference Guedat and Colland5, Reference Kirkin and Dikic6, Reference Nalepa, Rolfe and Harper7, Reference Nicholson8, Reference Sippl, Collura and Colland9) (Fig. 1). Additional opportunities are provided by the discovery of pathogen-encoded factors that evolved to target the UPS of the host cell, representing attractive targets for treatments against infectious diseases (Refs Reference Edelmann and Kessler10, Reference Isaacson and Ploegh11, Reference Lindner12). Therefore, the UPS offers a source of novel pharmacological targets as the basis for the successful development of drugs to treat human diseases. However, the complexity of the ubiquitin system causes considerable challenges for high-throughput drug discovery because of extensive structural similarities. The generation of selective inhibitors is also impeded by the large number of DUBs (Refs Reference Nijman13, Reference Reyes-Turcu, Ventii and Wilkinson14), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) (Ref. Reference Pickart and Eddins15) that might have redundancies in their biological functions. All these enzymes possess affinity for ubiquitin and various ubiquitin conjugates. Therefore, their specificity is dependent on other structural subtleties and differences in protein–protein interactions unique to each enzyme species. To address this problem, an array of methodologies is used, such as the identification of ‘hits’ by high-throughput screening (HTS), the development of suitable assays for functional screening in vitro and in cells, and the use of protein structures to aid rational drug design. These approaches have already resulted in the discovery of a panel of inhibitory compounds against the proteasome, several ubiquitin-conjugating enzymes and DUBs, all of which have potential for further specific drug development, as discussed here.

Figure 1.

Small-molecule inhibitors in the ubiquitin–proteasome system (UPS). Schematic representation of components of the UPS including E1, E2–E3 ligases, DUBs and the proteasome complex (20Si: immunoproteasome). Ubiquitin is indicated as pink circle labelled U. The UPS pathway and different examples of E1, E2, E3s and DUBs are highlighted in blue boxes. Increasing numbers of small-molecule inhibitors that interfere at various steps of the UPS cascade are being discovered.

Targeting proteasome subsets for inhibition – reducing overall toxicity and overcoming drug resistance

Protein degradation by the proteasome, a multicatalytic proteinase complex, is at the centre of the UPS pathway (Fig. 1), and its pharmacological inhibition was originally considered lethal for all cell types. It was therefore rather surprising that bortezomib (Velcade) was approved as treatment for multiple myeloma in 2003 (Ref. Reference Chen16). Since then, bortezomib has also been approved for the treatment of mantle cell lymphoma (Ref. Reference Kouroukis17). More recently, other derivatives have been developed that are at various stages of clinical trials, such as carfizomib (Phase III against relapsed multiple myeloma), MLN9708 (Phase I), CEP18770 (Phase I) and the natural product NPI-0052 (Phase I) (Ref. Reference Bedford3) (Fig. 1). Ubistatins were also discovered to inhibit proteasomal proteolysis by interfering with the recognition of polyubiquitin chains by the proteasome (Ref. Reference Verma18). In addition to NPI-0052, further natural products with potential anticancer properties have been characterised to interfere with proteasomal proteolysis (reviewed in Ref. Reference Schneekloth and Crews19), such as celastrol (Ref. Reference Yang20), catechin(−), the component of green tea (Ref. Reference Landis-Piwowar21), disulfiram in combination with copper (Ref. Reference Chen22), a triterpenoid inhibitor (Ref. Reference Tiedemann23), curcumin (Ref. Reference Jana24) and JBIR-22, which inhibits homodimer formation of proteasome assembly factor 3 (Ref. Reference Izumikawa25). Many of these natural products have intrinsic antitumour properties, although it is not clear whether this is solely attributable to their proteasome inhibitory capacities. For instance, statins have pleiotropic effects and are used to treat a variety of different diseases, including prevention of cardiovascular events, although it is not entirely clear whether this is due to direct or indirect interference with proteasomal proteolysis (Ref. Reference Ludwig26). As expected, proteasome inhibition causes side effects such as peripheral neuropathy, myelosuppression, nausea, hypersensibility and increased susceptibility to infection (Ref. Reference Basler27). One problem with proteasome inhibitors is the emergence of resistance; however, the combination of several proteasome inhibitors that exert complementary specificities appears to overcome the problem of resistance and might have the added benefit of enabling reduced dosing of the individual drugs (Refs Reference Mirabella28, Reference Ruschak29). An alternative way of circumventing the general toxicity of these compounds is to develop inhibitors selective for immunoproteasomes (Refs Reference Ho30, Reference Singh31). Immunoproteasomes are predominantly expressed in B-cells, T-cells, macrophages, dendritic cells and other cell types of the haematopoietic lineage, and can be induced by cell exposure to interferon-γ and tumour necrosis factor -α. This leads to an exchange of the catalytic β-subunits, thereby enhancing antigen presentation and preserving cell viability during inflammation-induced oxidative stress (Refs Reference Kloetzel and Ossendorp32, Reference Seifert33). Based on the observation that the immunoproteasome promotes enhanced antigen processing and presentation, it is predicted that immunoproteasome inhibitors may have immunomodulatory effects, such as attenuating autoimmune-related pathologies. Indeed, a selective immunoproteasome inhibitor PR957 was shown to prevent experimental colitis (Ref. Reference Basler34) and interfere with arthritis in a mouse model (Ref. Reference Muchamuel35). This strategy appears to be less toxic and particularly promising for treating autoimmune disorders, and might be extended to targeting the thymoproteasome, a subform expressed in the thymus (Ref. Reference Groettrup, Kirk and Basler36).

Inhibition of DUBs as a novel approach to treat cancer, neurodegenerative diseases and viral infection

DUBs have also been molecular targets for inhibitor development in recent years (Fig. 1). Members of the DUB family known to contribute to neoplastic transformation include USP1 (Fanconi anaemia), USP2 (prostate cancer), DUB3 (breast cancer), USP4 (adenocarcinoma), USP7 (prostate cancer, non-small-cell lung adenocarcinoma), USP9X (leukaemias and myelomas) and BRCC36 (breast cancer) (Refs Reference Nijman13, Reference Reyes-Turcu, Ventii and Wilkinson14, Reference Hussain, Zhang and Galardy37, Reference Pereg38, Reference Schwickart39, Reference Song40). Mutations in the gene encoding the ubiquitin-specific protease CYLD can lead to the neoplastic condition cylindromatosis, and other DUBs are also expressed at lower levels in cancer, including A20 (B-cell and T-cell lymphomas), USP10 (carcinomas) (Ref. Reference Yuan41) and BAP1 (brain, lung and testicular cancers) (Ref. Reference Hussain, Zhang and Galardy37).

USP7 and cancer

USP7, also known as Herpes virus associated USP (HAUSP), is critical in cancer progression because of its destabilising effect on the tumour suppressor p53 (Refs Reference Nicholson8, Reference Cummins and Vogelstein42, Reference Li43). USP7 preferentially deubiquitylates the E3 ligase HDM2 and its binding partner HDMX, resulting in the destabilisation of p53 and the repression of p53 transactivation activity (Refs Reference Li43, Reference Cummins44, Reference Meulmeester45). Additional substrates of USP7 include claspin, FOXO4 and PTEN (Refs Reference Song40, Reference Faustrup46, Reference van der Horst47). Thus USP7 exerts both p53-dependent and p53-independent effects on the control of cell proliferation and apoptosis, making USP7 an attractive target for pharmaceutical intervention (Refs Reference Cheon and Baek48, Reference Nicholson and Suresh Kumar49). Recently, a high-throughput screen identified the small-molecule compound HBX 41,108 as an uncompetitive and reversible inhibitor of USP7 (Figs 1 and 2). HBX 41,108 (Fig. 2, compound 1) is a functionalised cyano-indenopyrazine derivative that inhibits several DUBs, including USP7, and stabilises p53 in a nongenotoxic manner, resulting in the induction of apoptosis (Refs Reference Guedat and Colland5, Reference Colland50). An independent screen recently identified compound P005091 and analogues such as P045204 and P022077 (Fig. 2, compound 2) as USP7 inhibitors in vitro and in living cells (Refs Reference Tian51, Reference Cao59). Proof of concept in cells was demonstrated by the stabilisation of p53 and the induction of p21 by a representative compound from the P005091 series (Ref. Reference Nicholson and Suresh Kumar49).

Figure 2.

Small-molecule inhibitors against deubiquitylating enzymes. Examples of DUB inhibitors characterised in the literature targeting the USP family: HBX41,108 (1) (Ref. Reference Colland50) and P022077 (2) (Ref. Reference Tian51) specific for USP7, HBX 90,397 inhibits USP8 (3) (Ref. Reference Colland52) and IU1 (7) inhibits USP14 (Ref. Reference Lee53). Inhibitors targeting the UCH family include 15d-PGJ2 (Ref. Reference Li54) (4) and isatin O-acyl oximes (5) (Ref. Reference Liu55) specific against UCH-L3 and other isatin derivatives (6) specific against UCH-L1 (Refs Reference Liu56, Reference Koharudin57). PR-619 (6) targets a broad range of DUBs (Ref. Reference Tian51) and GRL0617 (9) inhibits SARS virus encoded papain-like protease (PLpro) (Ref. Reference Ratia58).

USP8 (UBPY) and endocytosis

As a function of its key role in the regulation of receptor endocytosis and trafficking, USP8 (UBPY) interacts with a number of clinically relevant cancer targets, including the epidermal growth factor receptor (EGFR) (Ref. Reference Williams and Urbe60). Knockdown of USP8 results in the accumulation of ubiquitylated EGFR in endosomes (Ref. Reference Mizuno61). Hybrigenics reported the identification of the USP8 inhibitor HBX 90,397 (Fig. 2, compound 3). However, the clinical usefulness of USP8 inhibitors is questionable following the report that conditional knockout of USP8 in adult mice resulted in liver failure, probably as a result of pronounced decreases in receptor tyrosine kinases such as EGFR (Refs Reference Colombo62, Reference Niendorf63).

Prostaglandins as DUB inhibitors

Prostaglandins are also reported to have inhibitory activity against DUBs in cellular assays. This family of compounds form a group of lipid derivatives that serve as signalling molecules that affect diverse protein functions, depending on their localisation and physiological context. Prostaglandins have been characterised as important messengers in inflammation (Ref. Reference Scher and Pillinger64) and immune responses (Refs Reference Rocca and FitzGerald65, Reference Harris66), with emerging roles in cancer. The J-series prostaglandins are known to promote apoptosis in a p53-independent fashion (Refs Reference Clay67, Reference Mullally68). D12-prostaglandin J2 was shown to inhibit ubiquitin isopeptidase activity in cell lysates, owing to the presence of the cross-conjugated α,β-unsaturated ketones in their structure (Fig. 1). Similarly, compounds unrelated to prostaglandins, but also containing cross-conjugated α,β-unsaturated ketones and accessible β-carbons, also inhibit isopeptidase activity. By contrast, the A-series prostaglandins, which contain a single α,β-unsaturated ketone, are less efficacious as ubiquitin isopeptidase inhibitors (Refs Reference Mullally68, Reference Mullally and Fitzpatrick69). Hence, the mechanism of inhibition by J-series prostaglandins is most likely based on the nucleophilic addition of a DUB thiol to the endocyclic β-carbon of a compound and the electrophilic accessibility of prostaglandin resulting from olefin–ketone conjugation. The inhibitory effect of prostaglandins is exemplified by 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) (Fig. 2, compound 4), which inhibits the activities of UCHL-3 (Ref. Reference Li54) and UCH-L1 (Refs Reference Liu56, Reference Koharudin57) (Fig. 1). 15d-PGJ2 has a detrimental effect on UCH-L1 structure and therefore perturbs its activity, offering possibilities of interfering with progression of Parkinson disease associated with UCH-L1 mutations (Ref. Reference Koharudin57).

Punaglandins are cyclopentadienone and cyclopentenone prostaglandins chlorinated at the endocyclic α-carbon position. These compounds, originally isolated from Telesto riisei coral, exhibit anti-inflammatory and antitumour activities (Ref. Reference Baker and Scheuer70), with higher cytotoxic effects on cells than the J-series prostaglandins. Interestingly, their effect on apoptosis is independent of p53 (Ref. Reference Verbitski71). Consistent with this, punaglandins, such as punaglandin 4, are also more potent inhibitors of cellular DUB activity than the J-series prostaglandins. The proposed mechanism for this enhanced inhibitor activity is the presence of an electronegative Cl substituent at the α-position of α,β-unsaturated carbonyls that increases the reactivity towards the nucleophilic addition of the DUB catalytic cysteine thiol group. These chlorinated lipids could therefore represent a new class of cancer therapeutics (Ref. Reference Verbitski71).

Inhibitors based on other molecular scaffolds with preferential specificity towards UCHs have also been reported. For instance, Stein and colleagues have reported the discovery of isatin O-acyl oximes (Fig. 2, compounds 5 and 6) with inhibitory activity against UCH-L1 and UCH-L3 (Ref. Reference Liu55). More recently, the same group reported a second series of UCH-L1 inhibitors based on a pyridone scaffold (Ref. Reference Mermerian72). The authors speculate that these compounds might have potential in the treatment of neurodegenerative conditions.

USP14 and neurodegeneration

In HTS, the small-molecule compound IU1 was found to be a selective inhibitor of USP14. USP14 is a DUB associated with the proteasome that blocks the degradation of ubiquitylated substrates. Cellular data confirmed the hypothesis that IU1-mediated inhibition of USP14 indirectly accelerates proteasomal degradation of proteins, such as tau and ataxin-3, both of which are involved in neurodegenerative diseases (Ref. Reference Lee53). Because many neurodegenerative disorders such as Parkinson disease and Creutzfeldt–Jakob disease are associated with the accumulation of misfolded proteins, IU1 (Fig. 2, compound 7) or other USP14-directed small-molecule inhibitors could potentially be used to eliminate these toxic proteins and improve the prognosis in neurodegenerative diseases. In support of this, USP14 has been previously linked to neurodegenerative disease, and loss of USP14 leads to an ataxic neurological phenotype in mice (Refs Reference Wilson73, Reference Anderson74).

General DUB inhibitors

WP1130 (degrasyn) is a derivative of AG490, a small-molecule compound that blocks Janus-activated kinase 2 (JAK2) activity. In cells, WP1130 treatment induces accumulation of polyubiquitylated conjugates. This phenomenon has been attributed to the inhibitory activity of WP1130 towards several DUBs, including USP9x, USP5, USP14 and UCH-L5 (Ref. Reference Kapuria75). WP1130 might be of therapeutic value, and its proapoptotic properties have been recently described (Ref. Reference Bartholomeusz76). In support of this, treatment of cells with WP1130 results in the modulation of anti- and proapoptotic proteins, such as MCL-1 and p53 (Ref. Reference Kapuria75). Further work suggests that WP1130 could be administered with bortezomib because the combination resulted in synergistic inhibition of tumour cells, regulation of apoptosis and prolonged survival of the animals (Ref. Reference Pham77).

Recently, an additional broad-specificity DUB inhibitor, PR-619 (Fig. 2, compound 8), was discovered using ubiquitin–CHOP reporter technology (Refs Reference Tian51, Reference Nicholson78). Given its broad specificity, the utility of PR-619 probably lies in its role as a tool for recovering ubiquitylated proteins from both cell-free and cellular experimental systems.

Inhibitors of viral DUBs

Viruses also encode DUBs, and these can be targeted to block viral infection. Papain-like protease (PLpro) is a DUB encoded by severe acute respiratory syndrome coronavirus (SARS-CoV) (Refs Reference Lindner12, Reference Lindner79, Reference Barretto80). PLpro blocks IRF3-dependent antiviral responses, indicating its relevance to key infectious processes and viral evasion of host innate immune responses. GRL0617 is a noncovalent inhibitor of PLpro (Fig. 2, compound 9), which blocks SARS-CoV viral replication without measurable cytotoxic effects and hence is a promising antiviral drug candidate. GRL0617 induces a conformational change in PLpro, which effectively inactivates this DUB. Profiling of DUB activity in cells exposed to GRL0617 using ubiquitin-specific active-site probes demonstrated that this inhibitor is selective for PLpro, which might explain its low cytotoxicity (Ref. Reference Ratia58).

In summary, these studies illustrate the potential of many DUBs as suitable drug targets. Several challenges remain, but more recent developments in novel discovery platforms and enzyme substrates such as multi-ubiquitin chains of different type and length, CHOP reporter technology and other isopeptide-bond-based assays are now being used to identify novel DUB inhibitors with greater specificity and sensitivity, providing the framework for optimisation of more suitable drugs.

Antagonists of E3 ubiquitin ligases

E3 ubiquitin ligases represent a diverse group of proteins with significant roles in ubiquitin conjugation. First, the E3 ligases catalyse the covalent transfer of ubiquitin to a lysyl side chain of a substrate (Ref. Reference Hershko81). Second, the specificity of E3 ligases determines which substrates become ubiquitylated, based on the recognition signals on target proteins (Refs Reference Hershko and Ciechanover82, Reference Yaron83). Unsurprisingly, numerous functions in neurodegenerative disorders, autoimmune diseases, inflammation and cancer have been ascribed to E3 ligases (Refs Reference Melino84, Reference Gomez-Martin, Diaz-Zamudio and Alcocer-Varela85, Reference Nyhan, O'Sullivan and McKenna86). One of the most crucial E3 ligases in cancer cell physiology is the proto-oncogene HDM2 (or its murine homologue Mdm2), which has been reported to be amplified in many tumour cells (Ref. Reference Momand87). The best characterised substrate of HDM2 is p53, which is targeted to the proteasome by HDM2. Thus, inhibition of HDM2 leads to activation of the p53 pathway, providing an attractive therapeutic strategy for cancers that retain wild-type p53 expression. The crystal structure of HDM2 bound to p53-derived peptide reveals a deep hydrophobic cleft in HDM2 necessary for p53 binding. This feature can be exploited for anticancer treatment by a rational design of peptide- and nonpeptide-based antagonists of the HDM2–p53 interaction by targeting the HDM2 cleft.

HDM2 E3 ligase inhibitors in cancer

Nutlin-3a (Fig. 3, compound 10) belongs to a class of tetrasubstituted imidazolines and is a potent nonpeptide HDM2 antagonist. It acts as a competitive inhibitor, blocking the interaction of p53 with HDM2 (Ref. Reference Vassilev88). Consequently, Nutlin-3a stabilises p53 and its substrates p21 and Noxa, contributing to increased apoptosis and cell cycle arrest in the G1 phase. Its antitumour properties have been demonstrated for cancer cells expressing wild-type p53 (Refs Reference Endo97, Reference Tabe98). Nutlin-3a efficiently eliminates tumour xenografts in mice, causing no measurable abnormalities in animals (Ref. Reference Vassilev88). In other preclinical studies, the combination of Nutlin-3a and the proteasome inhibitor bortezomib induced additive cytotoxicity in malignant multiple myeloma cells. These synergistic antitumour activities might extend the clinical applications of bortezomib to neoplasias exhibiting reduced sensitivity to this proteasome inhibitor (Ref. Reference Ooi99). R7112, an analogue of Nutlin-3a, is currently in Phase I clinical trials for the treatment of solid tumours and haematological malignancies.

Figure 3.

Small-molecule inhibitors against ubiquitin/Ubl-conjugating enzymes. Examples of inhibitors against E3 ligases include Nutlin-3a (10), RITA (11), MI-219 (12), JNJ-26854165 (13), chlorofusin (14) and chalcone (AM114) (15), which specifically interfere with the HDM2–p53 or HDM2–proteasome interactions (Refs Reference Vassilev88, Reference Dickens, Fitzgerald and Fischer89, Reference Duncan90, Reference Tsukamoto91, Reference De Vincenzo92, Reference Stoll93), and thalidomide (16), which inhibits CRBN (Ref. Reference Ito94). The ubiquitin-activating enzyme E1 is targeted by PYR-41 (Ref. Reference Yang95) (17), and the NEDD8-activating enzyme NAE1 is inhibited by MLN4924 (18) (Ref. Reference Soucy96). All molecular targets are associated with disease pathologies, in particular cancer. See text for further details. Abbreviations: CRBN, cereblon; NEDD8, neural precursor cell expressed developmentally down-regulated 8; Ubl, ubiquitin-like protein.

The proof-of-concept data provided by the discovery of the nutlins spurred additional research in this area, including the identification of the HLI98 family of compounds (7-nitro-5-deazaflavin) and RITA (Fig. 3, compound 11) that also target the ubiquitin-ligase activity of HDM2, resulting in the activation of p53-dependent apoptosis (Refs Reference Issaeva100, Reference Yang101). In addition, the spiro-oxindoles exemplified by MI-63 and MI-219 (Fig. 3, compound 12) and the chromenotriazolopyrimidines were also reported to be effective nonpeptidomimetic small-molecule inhibitors of the HDM2–p53 interaction (Refs Reference Allen102, Reference Ding103, Reference Shangary104).

Another mode of inhibition of HDM2 is by blocking its association with the proteasome. JNJ-26854165 (Fig. 3, compound 13) is one of the first compounds found to induce p53 levels in tumour cell lines and activate p53 transcriptional activity (Ref. Reference Dickens, Fitzgerald and Fischer89). JNJ-26854165 is currently being investigated as an oral agent for the treatment of refractory solid tumours in clinical trials.

Natural products are also an important source of novel E3 ligase inhibitors. For example, 53 000 microbial extracts derived from fermented microorganisms, such as actinomycete bacteria and fungi, were screened to discover new compounds that antagonise the HDM2–p53 interaction. Among these, chlorofusin (Fig. 3, compound 14), a fungal metabolite isolated from Fusarium sp., was found to have the highest inhibitory activity towards HDM2 (Ref. Reference Duncan90). Chlorofusin has a nine-residue cyclic peptide containing an l-ornithine side chain linked to a highly functionalised tricyclic chromophore. Further studies established that neither the cyclic peptide, the chromophore of chlorofusin alone or simple derivatives thereof account for inhibition of the HDM2–p53 interaction, but that the whole structure of chlorofusin is required (Ref. Reference Clark105).

The second example of a natural product that exhibits inhibition of the p53–HDM2 interaction is the (−) enantiomer of hexylitaconic acid isolated from a culture of marine-sponge-derived fungus Arthrinium sp. (Ref. Reference Tsukamoto91). The (−) hexylitaconic acid impairs p53–HDM2 interactions in a dose-dependent manner, but its derivatives, including a monomethyl ester, a dihydro derivative and a dihydro derivative monomethyl ester, showed no inhibitory activity.

Chalcones are aromatic ketones previously characterised as potential antitumourigenic therapeutics in ovarian cancer (Ref. Reference De Vincenzo92), gastric cancer and other tumours (Ref. Reference Shibata106). Chalcone derivatives interfere with p53–HDM2 interactions by binding near the tryptophan-binding pocket of the HDM2 hydrophobic cleft (Ref. Reference Stoll93). Molecular modelling studies indicate that boronic acid binds to lysine residues Lys51 and Lys94 of HDM2 (Ref. Reference Modzelewska107). The detailed mechanism of the cytotoxic activity of chalcones remains to be determined. In some cases, the enhanced apoptosis is related to inhibition of the 20S proteasome and thus stabilisation of p53, as exemplified by boronic chalcone derivative AM114 (Fig. 3, compound 15) (Ref. Reference Achanta108). Regardless, it seems likely that chalcone-mediated inhibition of the p53–HDM2 interaction is a contributory mechanism to their reported antitumour properties (Ref. Reference Stoll93).

SCFSkp2 E3 ligase inhibitors and cancer

HDM2 is not the only ubiquitin E3 ligase that constitutes a potential therapeutic target. SCFSkp2 is an SCF (S-phase kinase-associated protein 1–cullin–F-box) ubiquitin E3 ligase containing Skp2, an F-box protein that determines substrate specificity. Upregulation of SCFSkp2 is associated with decreased p27Kip1 levels and is negatively correlated with a good prognosis in cancer (Ref. Reference Nakayama and Nakayama109); therefore, compounds directly targeting SCFSkp2 represent potential drugs for cancer therapy. Using HTS, Chen and colleagues identified compound A (CpdA) as a promising SCFSkp2 inhibitor that prevents incorporation of Skp2 F-box protein into the SCFSkp2 ligase complex. CpdA thus leads to ubiquitin-dependent accumulation of substrates for SCFSkp2 E3 ligase activity, such as p27, and consequently induces G1–S cell cycle arrest and apoptosis. Notably, CpdA works synergistically with the proteasome inhibitor bortezomib (Ref. Reference Chen110), probably by interfering with Cul1 neddylation.

Recently, two additional examples of E3 inhibitors were reported. First, using a fluorescence polarisation screen, the biplanar dicarboxylic acid compound SCF-I2 was shown to be an allosteric inhibitor of substrate recognition by the yeast F-box protein SCFCdc4. SCFCdc4 degrades many substrates, such as SIC1, in a phosphorylation-dependent manner, and the SCF-I2 inhibitor perturbs the phosphodegron binding pocket of SCFCdc4 (Ref. Reference Orlicky111). A second group used a yeast-based chemical genetics screen to identify modulators of SCFMet30 activity (Ref. Reference Aghajan112). Biochemical studies confirmed that SMER3 specifically inhibits SCFMet30-dependent ubiquitylation of the transcription factor Met4 by reducing the binding of Met30 to Skp1, which is probably due to its direct binding to Met30.

CRBN E3 ligase targeted by the teratogenic agent thalidomide

Cereblon (CRBN), damaged DNA-binding protein 1 (DDB1) and Cul4A form an E3 ligase complex that is important for embryonic development. This complex is targeted by thalidomide (Fig. 3, compound 16), a clinically approved drug for the treatment of multiple myeloma, leprosy and inflammatory bowel disease (Crohn disease) (Ref. Reference Bosch113). One of the enantiomers of thalidomide was found to have teratogenic side effects. Binding of thalidomide to the CRBN complex and inhibition of CRBN E3 ligase activity appear to be the underlying molecular mechanisms for thalidomide-induced teratogenicity by the perturbation of embryonic development (Ref. Reference Ito94).

MuRF1 E3 ligase inhibition and muscular atrophy

MuRF1 is a key effector enzyme of muscular atrophy, an area of unmet medical need for several different pathologies (Ref. Reference Bodine114). Using an ELISA-based HTS platform, Progenra identified a novel modulator of the E3 ubiquitin ligase, P013222, which inhibited MuRF1 autoubiquitylation and myosin heavy-chain ubiquitylation and protected myotubes from dexamethasone-induced muscle wasting (Ref. Reference Eddins115).

As outlined above, most of the identified E3 ligase inhibitors are directed towards protein–protein interactions, and their ‘druggability’ is therefore challenging. The complexity of this enzyme family, the lack of details on their precise molecular mechanism and the fact that most E3s rely on protein–protein interactions to mediate their activity makes the design of E3 ligase inhibitors difficult (Ref. Reference Eldridge and O'Brien4), but potentially offers the framework for translational applications by interfering with many different biological processes in a highly specific manner. Recent reports of the identification of specific SCF inhibitors increase our confidence that it will be possible to develop inhibitors of this emerging class of important drug targets.

Inhibition of ubiquitin-activating enzymes

Ubiquitin conjugation requires initial activation of ubiquitin by E1 enzyme, which adenylates the C-terminal carboxyl group of ubiquitin, forming a high-energy ubiquitin adenylate intermediate, followed by the formation of a thiol ester between the carboxyl group of Gly76 of ubiquitin and a thiol group of E1. This series of reactions activates the C-terminus of ubiquitin for a subsequent nucleophilic attack (Ref. Reference Ciechanover116). Blocking this reaction could therefore be used to inhibit ubiquitin conjugation. In vitro studies suggest that knockdown of E1 ligase results in lower levels of protein ubiquitylation and eventually induces cell death in malignant cells (Ref. Reference Xu117). To identify novel E1 inhibitors, Yang and colleagues screened a library of small compounds and identified 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first cell-permeable E1 inhibitor. PYR-41 efficiently reduces bulk protein ubiquitylation and sumoylation, and prevents degradation of p53, contributing to enhanced apoptosis. PYR-41 also attenuates cytokine-mediated nuclear factor-κB (NF-κB) activation by regulating proteasomal degradation of IκBα, an inhibitory subunit of NF-κB. Functionally, PYR-41 probably binds irreversibly to the active-site cysteine in E1 ligase, therefore preventing ubiquitin transfer (Ref. Reference Yang95). However, this compound also targets several DUBs, including USP5, and crosslinks to kinases and has antitumour activity in animals (Ref. Reference Kapuria118). PYZD-4409 (Fig. 3, compound 17), another small-molecule inhibitor, has been shown to interfere with the activity of E1 ligase, preferentially inducing tumour cell death in primary acute myeloid leukaemia cells. The effects of PYZD-4409 have also been studied in a mouse model of leukaemia, where it reduced tumour weight and volume. This study underlines the importance of E1 as a potential drug target in leukaemia and possibly other cancers, especially in cases where neoplastic cells are resistant to treatment with proteasome inhibitors such as bortezomib (Ref. Reference Xu117). Furthermore, two natural products have been identified to inhibit E1, panepophenanthrin isolated from the mushroom Panus rudis (Refs Reference Sekizawa119, Reference Matsuzawa120) and himeic acid A derived from the fungus Aspergillus sp. (Ref. Reference Tsukamoto121), both of which inhibit the E1-catalysed ubiquitin activation in vitro, but with unknown mechanisms.

Recently, small-molecule inhibitors of E2 enzymes were also discovered. Leucettamol A, a compound isolated from a marine sponge Leucetta aff. Microrhaphis, was identified as a novel inhibitor of the Ubc13–Uev1A interaction, thereby blocking the formation of the E1–E2 complex (Ref. Reference Tsukamoto122). Also, an allosteric inhibitor of the human Cdc34 E2 ligase, CC0651, was found through a small-molecule screen for inhibitors of SCFSkp2-dependent ubiquitylation of p27Kip1, and was shown to interfere with the proliferation of human cancer cell lines (Ref. Reference Ceccarelli123).

Generally, when targeting E1–E2 conjugating enzymes, several pathways that are dependent on ubiquitylation, such as DNA repair or endocytosis, are inhibited at the same time, potentially contributing to increased nonspecific cytotoxicity. Therefore, from the therapeutic standpoint, the use of ubiquitin E1 (or E2)-specific inhibitors is currently awaiting additional preclinical validation before advancing to clinical studies.

Targeted inhibition of NEDD8-activating enzymes

Neural precursor cell-expressed developmentally downregulated-8 (NEDD8) is a ubiquitin-like protein with the highest homology to ubiquitin. Its conjugation to substrates (neddylation) requires activation by the E1 APPBP1-UBA3 and transfer by the E2 UBC12 (Ref. Reference Xirodimas124). NEDD8 primarily functions in the regulation of E3 ubiquitin ligases, modifying most members of the cullin family. Cullins are scaffold components of the SCF E3 ubiquitin ligases that control the proteasomal degradation of proteins involved in the cell cycle, transcriptional regulation or signal transduction (Refs Reference Xirodimas124, Reference Herrmann, Lerman and Lerman125). Neddylation of cullins results in increased ubiquitylation of the SCF substrate proteins and their subsequent proteasomal degradation. SCF E3 ligases promote the ubiquitylation of proteins involved in inflammation and tumourigenesis (Ref. Reference Pan126), such as HIF-α and IκBα (Refs Reference Clifford127, Reference Soucy96); therefore, specific inhibition of NEDD8-activating enzymes (E1) and other components of the neddylation pathway represents an alternative approach to targeting the UPS for cancer treatment. MLN4924 (Fig. 3, compound 18) is a small-molecule inhibitor of the NEDD8-activating enzyme and is presently being evaluated in Phase I clinical trials. MLN4924 increases the apoptosis of several tumour cell lines and murine tumour xenografts and is considered a promising drug candidate for myeloid leukaemia (Refs Reference Soucy96, Reference Soucy, Smith and Rolfe128, Reference Swords129). In contrast to the proteasome inhibitor bortezomib, MLN4924 is more specific because it does not inhibit bulk proteasomal degradation (Ref. Reference Soucy96). The functional mechanism of MLN4924 involves formation of the MLN4924–NEDD8 covalent adduct, which is similar to the first intermediate of the reaction catalysed by the NEDD8-activating enzyme, thus efficiently inhibiting the NEDD8 E1 enzyme (Ref. Reference Brownell130).

Given that SCF E3 ligases represent several hundred of all known E3 ubiquitin ligases, there is concern that inhibition of ~300 E3 ligases might lead to serious side effects in a clinical setting. However, the impact of side effects must be taken in the context of proteasome inhibitors, which have been shown to exhibit acceptable clinical profiles for the treatment of cancer, yet modulate the stability of many more proteins (relative to SCF E3s). Ultimately, the evaluation of MLN4924 and any successor compounds in a clinical setting will determine whether this strategy is a therapeutically acceptable approach.

Conclusions and perspectives

The ubiquitin system or ‘ubiquitome’ has been compared with the well-characterised ‘kinome’ and has spurred an entire array of novel inhibitors against molecular targets that manipulate ubiquitin and ubiquitin-like molecules. Similarly to the kinase field, functional redundancy, the structural similarities of active sites (DUBs) and the diversity of protein–protein interaction domains (conjugating enzymes) render the discovery of specific inhibitors challenging. Several novel compounds are promising results in clinical trials, such as the proteasome inhibitors carfizomib (Phase III), MLN2238 (Phase I) and NPI-0052 (Phase I) or the NAE inhibitor MLN4924 (Phase I against AML and solid tumours). Thalidomide, which has been in clinical use for many years, has been recently identified as an E3 ligase inhibitor. Also, several E1 and E3 ligase inhibitors such as PYR-41, Nutlin-3a, Compound A, P013222 and SCF-I2 have proved successful in the preclinical stage. Generally, it remains to be determined whether small molecules are required to specifically target only one molecule to be clinically useful or whether, in a manner similar to medically relevant kinase inhibitors, molecules with broader specificities against subfamilies of enzymes might exhibit clinical efficacy. Clearly, the emergence of new inhibitors directed against UPS components supplements the activities of kinase inhibitors, cytotoxic agents and other compounds, and it is predicted that ultimately the combinatorial use of these drugs holds the greatest promise for future therapies against cancer, neurodegenerative disorders and infectious disease. These disease pathologies are highly complex, and substantial differences can occur between individuals. A broadened arsenal of small-molecule compounds, including drugs targeting components of the UPS, might provide the framework for individualised drug regimens as part of a trend towards personalised medicine.

Outstanding research questions

  • • Generate further understanding of the precise role of E1/E2/E3 ligases and DUBs and other components in the UPS in disease processes, to establish correlations between their dysfunction and properties of disease pathology.

  • • Develop promising lead compounds into more effective inhibitors with greater potency.

  • • Whereas many small-molecule compounds show antiproliferative activities in tumour cell lines, it is less clear how this can be translated into inhibiting tumour growth in vivo without affecting normal cells. Distinguishing between these two scenarios should drive the selection and development of more effective compounds in the future.

  • • Is it really necessary to chemically target one enzyme for optimal interference with disease progression? Many diseases, in particular cancer, exert aberrations in several biochemical pathways. The discovery and pharmacological targeting of all abnormally functioning networks will be necessary for better treatments in the future.

Acknowledgements

M.J.E. is supported by a USDA NIFA grant (Nr. 2009-34609-20222). B.N. is supported by grants DK07139 from NIDDK/NIH and CA115205 from NCI/NIH to Progenra Inc. B.M.K. is supported by the NIHR Biomedical Research Centre, Oxford, UK and by an Action Medical Research Grant (Charity Nr. 208701 and SC039284). We thank members of the Kessler group for helpful discussions and also the peer reviewers for useful comments on the manuscript.

References

References

1Behrends, C. and Harper, J.W. (2011) Constructing and decoding unconventional ubiquitin chains. Nature Structural and Molecular Biology 18, 520-528CrossRefGoogle ScholarPubMed
2Clague, M.J. and Urbe, S. (2010) Ubiquitin: same molecule, different degradation pathways. Cell 143, 682-685CrossRefGoogle ScholarPubMed
3Bedford, L. et al. (2011) Ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets. Nature Reviews. Drug Discovery 10, 29-46CrossRefGoogle ScholarPubMed
4Eldridge, A.G. and O'Brien, T. (2010) Therapeutic strategies within the ubiquitin proteasome system. Cell Death and Differentiation 17, 4-13CrossRefGoogle ScholarPubMed
5Guedat, P. and Colland, F. (2007) Patented small molecule inhibitors in the ubiquitin proteasome system. BMC Biochemistry 8 (Suppl 1), S14CrossRefGoogle ScholarPubMed
6Kirkin, V. and Dikic, I. (2011) Ubiquitin networks in cancer. Current Opinion in Genetics and Development 21, 21-28CrossRefGoogle ScholarPubMed
7Nalepa, G., Rolfe, M. and Harper, J.W. (2006) Drug discovery in the ubiquitin–proteasome system. Nature Reviews. Drug Discovery 5, 596-613CrossRefGoogle ScholarPubMed
8Nicholson, B. et al. (2007) Deubiquitinating enzymes as novel anticancer targets. Future Oncology 3, 191-199CrossRefGoogle ScholarPubMed
9Sippl, W., Collura, V. and Colland, F. (2011) Ubiquitin-specific proteases as cancer drug targets. Future Oncology 7, 619-632CrossRefGoogle ScholarPubMed
10Edelmann, M.J. and Kessler, B.M. (2008) Ubiquitin and ubiquitin-like specific proteases targeted by infectious pathogens: emerging patterns and molecular principles. Biochimica Biophysica et Acta 1782, 809-816CrossRefGoogle ScholarPubMed
11Isaacson, M.K. and Ploegh, H.L. (2009) Ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection. Cell Host and Microbe 5, 559-570CrossRefGoogle ScholarPubMed
12Lindner, H.A. (2007) Deubiquitination in virus infection. Virology 362, 245-256CrossRefGoogle ScholarPubMed
13Nijman, S.M. et al. (2005) A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773-786CrossRefGoogle ScholarPubMed
14Reyes-Turcu, F.E., Ventii, K.H. and Wilkinson, K.D. (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annual Review of Biochemistry 78, 363-397CrossRefGoogle ScholarPubMed
15Pickart, C.M. and Eddins, M.J. (2004) Ubiquitin: structures, functions, mechanisms. Biochimica Biophysica et Acta 1695, 55-72CrossRefGoogle ScholarPubMed
16Chen, D. et al. (2011) Bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives. Current Cancer Drug Targets 11, 239-53CrossRefGoogle ScholarPubMed
17Kouroukis, C.T. et al. (2011) A phase II study of bortezomib and gemcitabine in relapsed mantle cell lymphoma from the National Cancer Institute of Canada Clinical Trials Group (IND 172). Leukemia and Lymphoma 52, 394-399CrossRefGoogle Scholar
18Verma, R. et al. (2004) Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117-120CrossRefGoogle ScholarPubMed
19Schneekloth, J.S. Jr, and Crews, C.M. (2011) Natural product inhibitors of the ubiquitin-proteasome pathway. Current Drug Targets 12, 1581-94CrossRefGoogle ScholarPubMed
20Yang, H. et al. (2006) Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine,” is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Research 66, 4758-4765CrossRefGoogle ScholarPubMed
21Landis-Piwowar, K.R. et al. (2007) A novel prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate as a potential anticancer agent. Cancer Research 67, 4303-4310CrossRefGoogle ScholarPubMed
22Chen, D. et al. (2006) Disulfiram, a clinically used anti-alcoholism drug and copper-binding agent, induces apoptotic cell death in breast cancer cultures and xenografts via inhibition of the proteasome activity. Cancer Research 66, 10425-10433CrossRefGoogle ScholarPubMed
23Tiedemann, R.E. et al. (2009) Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-kappaB with antimyeloma activity in vitro and in vivo. Blood 113, 4027-4037CrossRefGoogle ScholarPubMed
24Jana, N.R. et al. (2004) Inhibition of proteasomal function by curcumin induces apoptosis through mitochondrial pathway. Journal of Biological Chemistry 279, 11680-11685Google Scholar
25Izumikawa, M. et al. (2011) JBIR-22, an inhibitor for protein–protein interaction of the homodimer of proteasome assembly factor 3. Journal of Natural Products 73, 628-631CrossRefGoogle Scholar
26Ludwig, A. et al. (2005) Effect of statins on the proteasomal activity in mammalian endothelial and vascular smooth muscle cells. Biochemical Pharmacology 70, 520-526Google Scholar
27Basler, M. et al. (2009) The proteasome inhibitor bortezomib enhances the susceptibility to viral infection. Journal Immunology 183, 6145-6150Google Scholar
28Mirabella, A.C. et al. (2011) Specific cell-permeable inhibitor of proteasome trypsin-like sites selectively sensitizes myeloma cells to bortezomib and Carfilzomib. Chemistry and Biology 18, 608-618Google Scholar
29Ruschak, A.M. et al. (2011) Novel proteasome inhibitors to overcome bortezomib resistance. Journal of the National Cancer Institute 103, 1007-1017CrossRefGoogle ScholarPubMed
30Ho, Y.K. et al. (2007) LMP2-specific inhibitors: chemical genetic tools for proteasome biology. Chemistry and Biology 14, 419-430CrossRefGoogle ScholarPubMed
31Singh, A.V. et al. (2011) PR-924, a selective inhibitor of the immunoproteasome subunit LMP-7, blocks multiple myeloma cell growth both in vitro and in vivo. British Journal of Haematology 152, 155-163Google Scholar
32Kloetzel, P.M. and Ossendorp, F. (2004) Proteasome and peptidase function in MHC-class-I-mediated antigen presentation. Current Opinion in Immunology 16, 76-81Google Scholar
33Seifert, U. et al. (2010) Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress. Cell 142, 613-624Google Scholar
34Basler, M. et al. (2010) Prevention of experimental colitis by a selective inhibitor of the immunoproteasome. Journal Immunology 185, 634-641CrossRefGoogle ScholarPubMed
35Muchamuel, T. et al. (2009) A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nature Medicine 15, 781-787CrossRefGoogle ScholarPubMed
36Groettrup, M., Kirk, C.J. and Basler, M. (2010) Proteasomes in immune cells: more than peptide producers? Nature Reviews. Immunology 10, 73-78CrossRefGoogle ScholarPubMed
37Hussain, S., Zhang, Y. and Galardy, P.J. (2009) DUBs and cancer: the role of deubiquitinating enzymes as oncogenes, non-oncogenes and tumor suppressors. Cell Cycle 8, 1688-1697CrossRefGoogle ScholarPubMed
38Pereg, Y. et al. (2010) Ubiquitin hydrolase Dub3 promotes oncogenic transformation by stabilizing Cdc25A. Nature Cell Biology 12, 400-406CrossRefGoogle ScholarPubMed
39Schwickart, M. et al. (2010) Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature 463, 103-107Google Scholar
40Song, M.S. et al. (2008) The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network. Nature 455, 813-817Google Scholar
41Yuan, J. et al. (2010) USP10 regulates p53 localization and stability by deubiquitinating p53. Cell 140, 384-396Google Scholar
42Cummins, J.M. and Vogelstein, B. (2004) HAUSP is required for p53 destabilization. Cell Cycle 3, 689-692CrossRefGoogle ScholarPubMed
43Li, M. et al. (2004) A dynamic role of HAUSP in the p53-Mdm2 pathway. Molecular Cell 13, 879-886Google Scholar
44Cummins, J.M. et al. (2004) Tumour suppression: disruption of HAUSP gene stabilizes p53. Nature 428, 1 p following 486Google Scholar
45Meulmeester, E. et al. (2005) Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2. Molecular Cell 18, 565-576CrossRefGoogle ScholarPubMed
46Faustrup, H. et al. (2009) USP7 counteracts SCFbetaTrCP- but not APCCdh1-mediated proteolysis of Claspin. Journal of Cell Biology 184, 13-19Google Scholar
47van der Horst, A. et al. (2006) FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nature Cell Biology 8, 1064-1073CrossRefGoogle ScholarPubMed
48Cheon, K.W. and Baek, K.H. (2006) HAUSP as a therapeutic target for hematopoietic tumors (review). International Journal of Oncology 28, 1209-1215Google Scholar
49Nicholson, B. and Suresh Kumar, K.G. (2011) The multifaceted roles of USP7; new therapeutic opportunities. Cell Biochemistry and Biophysics 60, 61-68CrossRefGoogle ScholarPubMed
50Colland, F. et al. (2009) Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells. Molecular Cancer Therapeutics 8, 2286-2295CrossRefGoogle ScholarPubMed
51Tian, X. et al. (2011) Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescence-based multiplex assay format. Assay and Drug Development Technologies 9, 165-173CrossRefGoogle ScholarPubMed
52Colland, F. (2010) The therapeutic potential of deubiquitinating enzyme inhibitors. Biochemical Society Transactions 38, 137-143Google Scholar
53Lee, B.H. et al. (2010) Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467, 179-184CrossRefGoogle ScholarPubMed
54Li, Z. et al. (2004) Delta12-prostaglandin J2 inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitin-protein aggregation without proteasome inhibition. Biochemical and Biophysical Research Communications 319, 1171-1180Google Scholar
55Liu, Y. et al. (2003) Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line. Chemistry and Biology 10, 837-846Google Scholar
56Liu, H. et al. (2010) Modification of ubiquitin-C-terminal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury. Neurobiology of Disease 41, 318-28Google Scholar
57Koharudin, L.M. et al. (2010) Cyclopentenone prostaglandin-induced unfolding and aggregation of the Parkinson disease-associated UCH-L1. Proceedings of the National Academy of Sciences of the United States of America 107, 6835-6840CrossRefGoogle ScholarPubMed
58Ratia, K. et al. (2008) A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication. Proceedings of the National Academy of Sciences of the United States of America 105, 16119-16124Google Scholar
59Cao, P. et al. (2011) Anti-neoplastic compounds, compositions and methods. WIPO Patent Application WO/2010/114881 http://www.sumobrain.com/patents/wipo/Anti-neoplastic-compounds-compositions-methods/WO2010114881.htmlGoogle Scholar
60Williams, R.L. and Urbe, S. (2007) The emerging shape of the ESCRT machinery. Nature Reviews. Molecular Cell Biology 8, 355-368CrossRefGoogle ScholarPubMed
61Mizuno, E. et al. (2006) A deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes. Traffic 7, 1017-1031Google Scholar
62Colombo, M. et al. (2010) Synthesis and biological evaluation of 9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile analogues as potential inhibitors of deubiquitinating enzymes. ChemMedChem 5, 552-558CrossRefGoogle ScholarPubMed
63Niendorf, S. et al. (2007) Essential role of ubiquitin-specific protease 8 for receptor tyrosine kinase stability and endocytic trafficking in vivo. Molecular and Cellular Biology 27, 5029-5039Google Scholar
64Scher, J.U. and Pillinger, M.H. (2009) The anti-inflammatory effects of prostaglandins. Journal of Investigative Medicine 57, 703-708CrossRefGoogle ScholarPubMed
65Rocca, B. and FitzGerald, G.A. (2002) Cyclooxygenases and prostaglandins: shaping up the immune response. International Immunopharmacology 2, 603-630Google Scholar
66Harris, S.G. et al. (2002) Prostaglandins as modulators of immunity. Trends in Immunology 23, 144-150CrossRefGoogle ScholarPubMed
67Clay, C.E. et al. (1999) Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells. Carcinogenesis 20, 1905-1911Google Scholar
68Mullally, J.E. et al. (2001) Cyclopentenone prostaglandins of the J series inhibit the ubiquitin isopeptidase activity of the proteasome pathway. Journal of Biological Chemistry 276, 30366-30373CrossRefGoogle Scholar
69Mullally, J.E. and Fitzpatrick, F.A. (2002) Pharmacophore model for novel inhibitors of ubiquitin isopeptidases that induce p53-independent cell death. Molecular Pharmacology 62, 351-358Google Scholar
70Baker, B.J. and Scheuer, P.J. (1994) The punaglandins: 10-chloroprostanoids from the octocoral Telesto riisei. Journal of Natural Products 57, 1346-1353CrossRefGoogle ScholarPubMed
71Verbitski, S.M. et al. (2004) Punaglandins, chlorinated prostaglandins, function as potent Michael receptors to inhibit ubiquitin isopeptidase activity. Journal of Medicinal Chemistry 47, 2062-2070CrossRefGoogle ScholarPubMed
72Mermerian, A.H. et al. (2007) Structure-activity relationship, kinetic mechanism, and selectivity for a new class of ubiquitin C-terminal hydrolase-L1 (UCH-L1) inhibitors. Bioorganic and Medicinal Chemistry Letters 17, 3729-3732CrossRefGoogle ScholarPubMed
73Wilson, S.M. et al. (2002) Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease. Nature Genetics 32, 420-425CrossRefGoogle ScholarPubMed
74Anderson, C. et al. (2005) Loss of Usp14 results in reduced levels of ubiquitin in ataxia mice. Journal of Neurochemistry 95, 724-731Google Scholar
75Kapuria, V. et al. (2010) Deubiquitinase inhibition by small-molecule WP1130 triggers aggresome formation and tumor cell apoptosis. Cancer Research 70, 9265-9276CrossRefGoogle ScholarPubMed
76Bartholomeusz, G.A. et al. (2007) Activation of a novel Bcr/Abl destruction pathway by WP1130 induces apoptosis of chronic myelogenous leukemia cells. Blood 109, 3470-3478Google Scholar
77Pham, L.V. et al. (2010) Degrasyn potentiates the antitumor effects of bortezomib in mantle cell lymphoma cells in vitro and in vivo: therapeutic implications. Molecular Cancer Therapeutics 9, 2026-2036Google Scholar
78Nicholson, B. et al. (2008) Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities. Protein Science 17, 1035-1043Google Scholar
79Lindner, H.A. et al. (2005) The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme. Journal of Virology 79, 15199-15208Google Scholar
80Barretto, N. et al. (2005) The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. Journal of Virology 79, 15189-15198CrossRefGoogle ScholarPubMed
81Hershko, A. et al. (1983) Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. Journal of Biological Chemistry 258, 8206-8214Google Scholar
82Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Annual Review of Biochemistry 67, 425-479Google Scholar
83Yaron, A. et al. (1997) Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase. EMBO Journal 16, 6486-6494Google Scholar
84Melino, G. et al. (2008) Itch: a HECT-type E3 ligase regulating immunity, skin and cancer. Cell Death and Differentiation 15, 1103-1112Google Scholar
85Gomez-Martin, D., Diaz-Zamudio, M. and Alcocer-Varela, J. (2008) Ubiquitination system and autoimmunity: the bridge towards the modulation of the immune response. Autoimmunity Reviews 7, 284-290Google Scholar
86Nyhan, M.J., O'Sullivan, G.C. and McKenna, S.L. (2008) Role of the VHL (von Hippel–Lindau) gene in renal cancer: a multifunctional tumour suppressor. Biochemical Society Transactions 36, 472-478CrossRefGoogle ScholarPubMed
87Momand, J. et al. (1998) The MDM2 gene amplification database. Nucleic Acids Research 26, 3453-3459Google Scholar
88Vassilev, L.T. et al. (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844-848Google Scholar
89Dickens, M.P., Fitzgerald, R. and Fischer, P.M. (2010) Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology 20, 10-18Google Scholar
90Duncan, S.J. et al. (2001) Isolation and structure elucidation of Chlorofusin, a novel p53-MDM2 antagonist from a Fusarium sp. Journal of the American Chemical Society 123, 554-560Google Scholar
91Tsukamoto, S. et al. (2006) Hexylitaconic acid: a new inhibitor of p53-HDM2 interaction isolated from a marine-derived fungus, Arthrinium sp. Bioorganic and Medicinal Chemistry Letters 16, 69-71Google Scholar
92De Vincenzo, R. et al. (1995) Effect of synthetic and naturally occurring chalcones on ovarian cancer cell growth: structure–activity relationships. Anticancer Drug Design 10, 481-490Google Scholar
93Stoll, R. et al. (2001) Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53. Biochemistry 40, 336-344Google Scholar
94Ito, T. et al. (2010) Identification of a primary target of thalidomide teratogenicity. Science 327, 1345-1350CrossRefGoogle ScholarPubMed
95Yang, Y. et al. (2007) Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Research 67, 9472-9481Google Scholar
96Soucy, T.A. et al. (2009) An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732-736CrossRefGoogle ScholarPubMed
97Endo, S. et al. (2010) Potent in vitro and in vivo antitumor effects of MDM2 inhibitor nutlin-3 in gastric cancer cells. Cancer Science 102, 605-13Google Scholar
98Tabe, Y. et al. (2009) MDM2 antagonist nutlin-3 displays antiproliferative and proapoptotic activity in mantle cell lymphoma. Clinical Cancer Research 15, 933-942Google Scholar
99Ooi, M.G. et al. (2009) Interactions of the Hdm2/p53 and proteasome pathways may enhance the antitumor activity of bortezomib. Clinical Cancer Research 15, 7153-7160CrossRefGoogle ScholarPubMed
100Issaeva, N. et al. (2004) Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nature Medicine 10, 1321-1328Google Scholar
101Yang, Y. et al. (2005) Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells. Cancer Cell 7, 547-559Google Scholar
102Allen, J.G. et al. (2009) Discovery and optimization of chromenotriazolopyrimidines as potent inhibitors of the mouse double minute 2-tumor protein 53 protein–protein interaction. Journal of Medicinal Chemistry 52, 7044-7053Google Scholar
103Ding, K. et al. (2006) Structure-based design of spiro-oxindoles as potent, specific small-molecule inhibitors of the MDM2-p53 interaction. Journal of Medicinal Chemistry 49, 3432-3435Google Scholar
104Shangary, S. et al. (2008) Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. Proceedings of the National Academy of Sciences of the United States of America 105, 3933-3938Google Scholar
105Clark, R.C. et al. (2009) Evaluation of chlorofusin, its seven chromophore diastereomers, and key analogues. Tetrahedron Letters 50, 3151-3153CrossRefGoogle ScholarPubMed
106Shibata, S. (1994) Anti-tumorigenic chalcones. Stem Cells 12, 44-52Google Scholar
107Modzelewska, A. et al. (2006) Anticancer activities of novel chalcone and bis-chalcone derivatives. Bioorganic and Medicinal Chemistry 14, 3491-3495Google Scholar
108Achanta, G. et al. (2006) A boronic-chalcone derivative exhibits potent anticancer activity through inhibition of the proteasome. Molecular Pharmacology 70, 426-433Google Scholar
109Nakayama, K.I. and Nakayama, K. (2006) Ubiquitin ligases: cell-cycle control and cancer. Nature Reviews. Cancer 6, 369-381Google Scholar
110Chen, Q. et al. (2008) Targeting the p27 E3 ligase SCFSkp2 results in p27-and Skp2-mediated cell-cycle arrest and activation of autophagy. Blood 111, 4690-4699Google Scholar
111Orlicky, S. et al. (2010) An allosteric inhibitor of substrate recognition by the SCFCdc4 ubiquitin ligase. Nature Biotechnology 28, 733, U1743Google Scholar
112Aghajan, M. et al. (2010) Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nature Biotechnology 28, 738-742Google Scholar
113Bosch, M.E. et al. (2008) Recent advances in analytical determination of thalidomide and its metabolites. Journal of Pharmaceutical and Biomedical Analysis 46, 9-17Google Scholar
114Bodine, S.C. et al. (2001) Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294, 1704-1708Google Scholar
115Eddins, M.J. et al. (2011) Targeting the ubiquitin E3 ligase MuRF1 to inhibit muscle atrophy. Cell Biochemistry and Biophysics 60, 113-118CrossRefGoogle ScholarPubMed
116Ciechanover, A. et al. (1982) “Covalent affinity” purification of ubiquitin-activating enzyme. Journal of Biological Chemistry 257, 2537-2542Google Scholar
117Xu, G.W. et al. (2010) The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma. Blood 115, 2251-2259CrossRefGoogle ScholarPubMed
118Kapuria, V. et al. (2011) Protein cross-linking as a novel mechanism of action of a ubiquitin-activating enzyme inhibitor with anti-tumor activity. Biochemical Pharmacology 82, 341-9CrossRefGoogle ScholarPubMed
119Sekizawa, R. et al. (2002) Panepophenanthrin, from a mushroom strain, a novel inhibitor of the ubiquitin-activating enzyme. Journal of Natural Products 65, 1491-1493Google Scholar
120Matsuzawa, M. et al. (2006) Enantio- and diastereoselective total synthesis of (+)-panepophenanthrin, a ubiquitin-activating enzyme inhibitor, and biological properties of its new derivatives. Chemistry, an Asian Journal 1, 845-851Google Scholar
121Tsukamoto, S. et al. (2005) Himeic acid A: a new ubiquitin-activating enzyme inhibitor isolated from a marine-derived fungus, Aspergillus sp. Bioorganic and Medicinal Chemistry Letters 15, 191-194Google Scholar
122Tsukamoto, S. et al. (2008) Leucettamol A: a new inhibitor of Ubc13-Uev1A interaction isolated from a marine sponge, Leucetta aff. microrhaphis. Bioorganic and Medicinal Chemistry Letters 18, 6319-6320Google Scholar
123Ceccarelli, D.F. et al. (2011) An allosteric inhibitor of the human cdc34 ubiquitin-conjugating enzyme. Cell 145, 1075-1087Google Scholar
124Xirodimas, D.P. (2008) Novel substrates and functions for the ubiquitin-like molecule NEDD8. Biochemical Society Transactions 36, 802-806Google Scholar
125Herrmann, J., Lerman, L.O. and Lerman, A. (2007) Ubiquitin and ubiquitin-like proteins in protein regulation. Circulation Research 100, 1276-1291Google Scholar
126Pan, Z.Q. et al. (2004) Nedd8 on cullin: building an expressway to protein destruction. Oncogene 23, 1985-1997CrossRefGoogle ScholarPubMed
127Clifford, S.C. et al. (2001) The pVHL-associated SCF ubiquitin ligase complex: molecular genetic analysis of elongin B and C, Rbx1 and HIF-1alpha in renal cell carcinoma. Oncogene 20, 5067-5074Google Scholar
128Soucy, T.A., Smith, P.G. and Rolfe, M. (2009) Targeting NEDD8-activated cullin-RING ligases for the treatment of cancer. Clinical Cancer Research 15, 3912-3916Google Scholar
129Swords, R.T. et al. (2010) Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia. Blood 115, 3796-3800Google Scholar
130Brownell, J.E. et al. (2010) Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ. Molecular Cell 37, 102-111Google Scholar

Further reading, resources and contacts

Bedford, L. et al. (2011) Ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets. Nature Reviews. Drug Discovery 10, 29-46Google Scholar
Nalepa, G., Rolfe, M. and Harper, J.W. (2006) Drug discovery in the ubiquitin–proteasome system. Nature Reviews. Drug Discovery 5, 596-613Google Scholar
Eldridge, A.G. and O'Brien, T. (2010) Therapeutic strategies within the ubiquitin proteasome system. Cell Death and Differentiation 17, 4-13Google Scholar
Nicholson, B. et al. (2007) Deubiquitinating enzymes as novel anticancer targets. Future Oncology 3, 191-199CrossRefGoogle ScholarPubMed
Dickens, M.P., Fitzgerald, R. and Fischer, P.M. (2010) Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology 20, 10-18Google Scholar
Figure 0

Figure 1.Small-molecule inhibitors in the ubiquitin–proteasome system (UPS). Schematic representation of components of the UPS including E1, E2–E3 ligases, DUBs and the proteasome complex (20Si: immunoproteasome). Ubiquitin is indicated as pink circle labelled U. The UPS pathway and different examples of E1, E2, E3s and DUBs are highlighted in blue boxes. Increasing numbers of small-molecule inhibitors that interfere at various steps of the UPS cascade are being discovered.

Figure 1

Figure 2.Small-molecule inhibitors against deubiquitylating enzymes. Examples of DUB inhibitors characterised in the literature targeting the USP family: HBX41,108 (1) (Ref. 50) and P022077 (2) (Ref. 51) specific for USP7, HBX 90,397 inhibits USP8 (3) (Ref. 52) and IU1 (7) inhibits USP14 (Ref. 53). Inhibitors targeting the UCH family include 15d-PGJ2 (Ref. 54) (4) and isatin O-acyl oximes (5) (Ref. 55) specific against UCH-L3 and other isatin derivatives (6) specific against UCH-L1 (Refs 56, 57). PR-619 (6) targets a broad range of DUBs (Ref. 51) and GRL0617 (9) inhibits SARS virus encoded papain-like protease (PLpro) (Ref. 58).

Figure 2

Figure 3.Small-molecule inhibitors against ubiquitin/Ubl-conjugating enzymes. Examples of inhibitors against E3 ligases include Nutlin-3a (10), RITA (11), MI-219 (12), JNJ-26854165 (13), chlorofusin (14) and chalcone (AM114) (15), which specifically interfere with the HDM2–p53 or HDM2–proteasome interactions (Refs 88, 89, 90, 91, 92, 93), and thalidomide (16), which inhibits CRBN (Ref. 94). The ubiquitin-activating enzyme E1 is targeted by PYR-41 (Ref. 95) (17), and the NEDD8-activating enzyme NAE1 is inhibited by MLN4924 (18) (Ref. 96). All molecular targets are associated with disease pathologies, in particular cancer. See text for further details. Abbreviations: CRBN, cereblon; NEDD8, neural precursor cell expressed developmentally down-regulated 8; Ubl, ubiquitin-like protein.