Dysregulation of the UPS (ubiquitin–proteasome system) has been implicated in a wide range of pathologies including cancer, neurodegeneration and viral infection. Inhibiting the proteasome has been shown to be an effective therapeutic strategy in humans; yet toxicity with this target remains high. DUBs (deubiquitinating enzymes) represent an alternative target in the UPS with low predicted toxicity. Currently, there are no DUB inhibitors that have been used clinically. To address this situation, Progenra has developed a novel assay to measure the proteolytic cleavage of Ub (ubiquitin) or UBL (Ub-like protein) conjugates such as SUMO (small Ub-related modifier), NEDD8 (neural-precursor-cell-expressed, developmentally down-regulated 8) or ISG15 (interferon-stimulated gene 15) by isopeptidases. In this review, current platforms for detecting DUB inhibitors are discussed and the advantages and disadvantages of the approaches are underlined.

Introduction

The conjugation of Ub (ubiquitin) and UBLs (Ub-like proteins) is an important regulatory mechanism that is widespread in many biological processes [1]. The assorted diseases associated with these pathways make the pathway enzymes particularly interesting for therapeutic targets. The UPS (Ub–proteasome system) has been validated following the approval of the proteasome inhibitor Bortezomib for the treatment of multiple myeloma; however, significant toxicities were seen during clinical trials, suggesting the need for more selective targets [2]. Ub- and UBL-isopeptidases represent a unique set of drug targets in the UPS that are responsible for removing Ub and UBLs, such as SUMO (small Ub-related modifier), NEDD8 (neural-precursor-cell-expressed, developmentally down-regulated 8) and ISG15 (interferon-stimulated gene 15), from target proteins, thus affecting the targets' fate [3]. To develop therapeutic agents that target isopeptidases, we developed a readily quantifiable novel isopeptidase assay platform that is suitable for HTS (high-throughput screening). The assay platform consists of Ub or UBL fused to the reporter enzyme PLA2 (phospholipase A2). Isopeptidase activity releases PLA2 that cleaves its substrate, generating a signal that is linear with isopeptidase concentration and is able to discriminate DUB (deubiquitinating enzyme), deSUMOylase, deNEDDylase and deISGylase activities. The assay can be successfully employed to screen for inhibitors of isopeptidases.

Therapeutic targets in the UPS

The approval of the proteasome inhibitor Bortezomib (Velcade) for the treatment of multiple myeloma validated the targeting of the UPS for the treatment of cancer [4]. However, extended treatment with Bortezomib is associated with toxicity and drug resistance, limiting its efficacy [2]. In contrast, therapeutic strategies that target specific aspects of the UPS upstream of the proteasome would be predicted to have lower toxicity. While activating enzymes (E1) and conjugating enzymes (E2) are upstream of the proteasome, one must be aware of the consequences of targeting them, as disruption of the E1 leads to cell-cycle arrest [5] and E2s have been shown to be required for development [6]. Targeting the Ub-activating enzyme may be predicted to affect too many cellular functions for it to be tolerated by normal cells; yet targeting the NEDD8-activating enzyme for inhibition has been reported to be successful in preclinical studies [7]. The mechanism of action is most likely through inactivation of the cullin-based E3 ligases, many of which play a crucial role in cell-cycle checkpoints whose disruption would have a more immediate effect on rapidly dividing cancer cells.

E3 ligases, with only a limited number of substrates, represent attractive drug targets in the UPS. One of the most interesting E3 targets is SCF [Skp (S-phase kinase-associated protein) 1/cullin/F-box]. The SCF complex consists of many variable F-box adaptor proteins each of which target only a few substrates for ubiquitination [8]. Two therapeutically relevant F-box proteins are Skp2 [9] and β-TRCP (β-transducin repeat-containing protein) [10], which play key roles in cell-cycle progression. However, to inhibit these proteins one must disrupt a protein–protein interaction, which is considered a more difficult target than an enzymatic target.

Isopeptidases identified to date have been grouped into five subfamilies. Four of these five families are cysteine proteases, which have been shown to be good therapeutic targets. The UCHs (Ub C-terminal hydrolases), UBP/USP (Ub-specific proteases), MJD (Machado–Joseph domain) and OTU (ovarian tumour-related) isopeptidases are cysteine proteases, whereas the JAMM (JAB1/MPN/Mov34 metalloenzyme) motif DUBs are Zn metaloproteases. A total of 90 putative DUBs have been identified with 79 most likely being functional. There are also many UBL-isopeptidases that are good therapeutic targets [3,11].

The role of DUBs and ULPs (UBL-specific proteases) in disease

Several isopeptidases have been implicated in disease [12], in particular cancer (refer to Table 1). For example, USP7 [Ub-specific protease 7; also known as HAUSP (herpesvirus-associated Ub-specific protease)] regulates the ubiquitination state of the RING (really interesting new gene)-finger E3 ligase Mdm2 (murine double minute 2) (and its human homologue Hdm2) [13]. Hdm2 targets the tumour suppressor p53 for ubiquitination and facilitates its degradation by the proteasome [14,15]. Many other RING-finger E3 ligases are capable of autoubiquitination; Hdm2 is no exception and autoubiquitinates resulting in its own proteolytic degradation [16]. However, Hdm2 also ubiquitinates p53, resulting in degradation of p53 via the proteasome. Initially, USP7 was believed to primarily deubiquitinate p53, increasing the level of p53 [17]. However, more recent genetic and biochemical studies have found that with respect to p53 and Hdm2, the primary target of USP7 is Hdm2 [13,18]. These results were corroborated by structural biology studies which revealed that Hdm2 and p53 recognize the TRAF (tumour-necrosis-factor-receptor-associated factor) domain of USP7 in a mutually exclusive manner, but Hdm2 binds to the TRAF domain with a higher affinity than p53 [19].

Table 1
Isopeptidases implicated in various diseases
Pathology DUBs 
Cancer USP2a [34], USP7 (HAUSP) [13], CYLD [35], UCH-L1 [36], USP6 (Tre-2) [37], USP20 (VDU2) [38], USP8 (UBPY) [39], STAMBP [STAM (signal transducing adaptor molecule) binding protein; also called AMSH (associated molecule with the SH3 domain of STAM)] [40
Neurodegeneration USP14 [41], ataxin 3 [42], UCH-L1 [43
Haematological USP1 [44], DUB-1, DUB-2 [45
Viral infection UL36USP [46], HMWP (pUL48) [47], PLP2 [48
Bacterial infection SseL [49], ElaD [50
Pathology DUBs 
Cancer USP2a [34], USP7 (HAUSP) [13], CYLD [35], UCH-L1 [36], USP6 (Tre-2) [37], USP20 (VDU2) [38], USP8 (UBPY) [39], STAMBP [STAM (signal transducing adaptor molecule) binding protein; also called AMSH (associated molecule with the SH3 domain of STAM)] [40
Neurodegeneration USP14 [41], ataxin 3 [42], UCH-L1 [43
Haematological USP1 [44], DUB-1, DUB-2 [45
Viral infection UL36USP [46], HMWP (pUL48) [47], PLP2 [48
Bacterial infection SseL [49], ElaD [50

While DUBs have received the most attention, proteases that deconjugate UBLs from their target proteins have also been linked to various pathophysiologies, as they are critical to cellular localization, transcriptional regulation, signal transduction pathways and the regulation of some Ub E3 ligases [2024].

Current assay systems for DUBS and UBL-isopeptidases

Many assays currently in use rely on cleavage of linear Ub fusions, which can be produced in Escherichia coli [tetra-Ub, Ub-CEP52, Ub-GSTP1 (Ub-glutathione transferase P1), Ub-DHFR (Ub-dihydrofolate reductase), Ub-PESTc etc.] or synthesized chemically [2527]. For small-scale analysis of isopeptidase activity, reaction products are analysed by gel electrophoresis, or are selectively precipitated and analysed by liquid-scintillation spectrometry. Gel-based procedures are labour-intensive and expensive, and, although scintillation-counting approaches are quantitative and allow processing of larger numbers of samples than gel-based assays, they require centrifugation and recovery of supernatant. For HTS, a fluorogenic substrate, Ub-AMC (Ub-7-amino-4-methylcoumarin), has been employed in some cases, as well as a similar substrate, the tetrapeptide Z (benzyloxycarbonyl)-LRGG-AMC, which mimics the C-terminus of Ub [28]. A limiting factor with both of these fluorescent substrates is the fact that this small adduct cannot be hydrolysed efficiently by the largest class of DUBs, UBP/USP class enzymes. Moreover, the excitation wavelength of Ub-AMC is in the UV range, which is known to excite a number of screening compounds and give rise to up to 20% false positives [29]. FRET (fluorescence resonance energy transfer) has also been developed for HTS screens [30]. FRET, however, suffers from the need for specialized reagents and equipment, as well as from difficulty in adapting to a multiwell plate format from which the endpoints can be read directly. In many of these approaches, expensive double-labelled or tagged substrates must be generated specifically for each assay (refer to Table 2 for comparison of current technologies).

Table 2
Comparison of current DUB assays
Platform Summary Advantages Disadvantages 
Ub-AMC Fluorogenic substrate fused to Ub that only fluoresces once it is cleaved from Ub by an isopeptidase Sensitive reporter for the four enzymes in the UCH family of DUBs λex=340 nm is an unfavourable wavelength for drug discovery 
  Ub, SUMO, NEDD8, ISG15-AMC reagents commercially available A poor substrate for USPs such as USP2 core 
LanthaScreen™ (TR-FRET) TR-FRET (time-resolved FRET)-based assay. The LanthaScreen™ DUB substrate consists of an N-terminal YFP (yellow fluorescent protein) fusion of Ub and a C-terminal extension of a terbium (Tb)-labelled cysteine residue Less susceptible to compound interference than AMC assay format Loss of signal assay 
 In the presence of a DUB, the Tb-labelled C-terminal extension is cleaved from the substrate, resulting in a decrease in the TR-FRET signal Sensitive reporter for the four enzymes in the UCH family of DUBs A poor substrate for USPs such as USP7 
   ISG15 LanthaScreen™ reagent not commercially available 
Ub–PLA2 UBL–PLA2 consists of a linear fusion that is available for isopeptidase cleavage. After isopeptidase activity, PLA2 is free to act on its substrate, giving a readily quantifiable fluorescent response More physiologically relevant substrate for most isopeptidases Relative to assays with a small adduct at the C-terminus of Ub, Ub–PLA2 is a less sensitive reagent for measuring the activity of the four enzymes in the UCH family 
  Generates a robust signal within 1 h  
  Fluorophores excited outside the UV range  
  Ub, SUMO, NEDD8, ISG15–PLA2 reagents commercially available  
Platform Summary Advantages Disadvantages 
Ub-AMC Fluorogenic substrate fused to Ub that only fluoresces once it is cleaved from Ub by an isopeptidase Sensitive reporter for the four enzymes in the UCH family of DUBs λex=340 nm is an unfavourable wavelength for drug discovery 
  Ub, SUMO, NEDD8, ISG15-AMC reagents commercially available A poor substrate for USPs such as USP2 core 
LanthaScreen™ (TR-FRET) TR-FRET (time-resolved FRET)-based assay. The LanthaScreen™ DUB substrate consists of an N-terminal YFP (yellow fluorescent protein) fusion of Ub and a C-terminal extension of a terbium (Tb)-labelled cysteine residue Less susceptible to compound interference than AMC assay format Loss of signal assay 
 In the presence of a DUB, the Tb-labelled C-terminal extension is cleaved from the substrate, resulting in a decrease in the TR-FRET signal Sensitive reporter for the four enzymes in the UCH family of DUBs A poor substrate for USPs such as USP7 
   ISG15 LanthaScreen™ reagent not commercially available 
Ub–PLA2 UBL–PLA2 consists of a linear fusion that is available for isopeptidase cleavage. After isopeptidase activity, PLA2 is free to act on its substrate, giving a readily quantifiable fluorescent response More physiologically relevant substrate for most isopeptidases Relative to assays with a small adduct at the C-terminus of Ub, Ub–PLA2 is a less sensitive reagent for measuring the activity of the four enzymes in the UCH family 
  Generates a robust signal within 1 h  
  Fluorophores excited outside the UV range  
  Ub, SUMO, NEDD8, ISG15–PLA2 reagents commercially available  

An improved paradigm for detection of DUB and UBL-isopeptidase inhibitors, the UBL–PLA2 reporter assay

While isopeptidases have been of interest for some time, to our knowledge no compounds have entered clinical trials. One reason for this could be the assays employed in drug-discovery campaigns. All of the previously described platforms are based on Ub linked to small chemical adducts and are not related to the physiological target of most isopeptidases, mono- or poly-Ub fused to a protein. To fulfil the need for a convenient and physiologically relevant assay that is suitable for HTS, Progenra has developed an isopeptidase assay, based on the observation that most USPs can hydrolyse linear Ub fusions (α-NH bond) as well as ϵ-NH2–isopeptide linkages, that exploits the requirement of certain proteins for a free N-terminus to be active. This assay can be used for either DUBs or ULPs.

The UBL–PLA2 assay is based on the concept that PLA2 requires a free N-terminus to be catalytically active. PLA2 cleaves phospholipids to produce lysophospholipids and non-esterified fatty acids and requires a free N-terminus for catalytic activity [31]. When PLA2 is fused to a UBL, it is inactive and cannot cleave its substrate. When a DUB or other UBL-isopeptidase is present, it cleaves the UBL from PLA2, freeing PLA2 to act on its substrate. There are a number of commercial substrates for PLA2 including the fluorescent phospholipid β-BODIPY C5-HPC [2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine] and NBD C6-HPC {2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine}. The fluorescence response produced by these substrates occurs over a wavelength range that is better suited for drug discovery than that employed in the Ub-AMC assay, and it is amplified by the coupling of DUB catalytic activity with PLA2, resulting in enhanced sensitivity. Importantly, the Ub/UBL–PLA2 fusion proteins represent more physiological substrates than the short C-terminal adducts exemplified by the commercially available AMC or TR-FRET (time-resolved FRET) reagents.

Future of DUB therapies

To our knowledge, no DUB inhibitors have entered clinical trials. However, modulation of the DUB CYLD (cylindromatosis) pathway with aspirin has been shown to be therapeutically viable in humans [32,33]. Progenra's proprietary assay technology has been used by multiple groups to screen more than 100000 compounds to date, with that number expected to double by the end of 2008. Owing to the increasing screening that is being performed against isopeptidases by ourselves and other researchers, we anticipate that multiple DUB inhibitors will be used clinically in the near future.

Third Intracellular Proteolysis Meeting: A joint Biochemical Society and INPROTEOLYS Network Focused Meeting held at Auditorio de Tenerife, Santa Cruz de Tenerife, Canary Islands, Spain, 5–7 March 2008. Organized and Edited by Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (Barcelona, Spain), Manuel Rodríguez (CICbioGUNE, Derio, Spain), Eduardo Salido (La Laguna, Tenerife, Spain) and Dimitris Xirodimas (Dundee, U.K.).

Abbreviations

     
  • DUB

    deubiquitinating enzyme

  •  
  • FRET

    fluorescence resonance energy transfer

  •  
  • HTS

    high-throughput screening

  •  
  • ISG15

    interferon-stimulated gene 15

  •  
  • NEDD8

    neural-precursor-cell-expressed, developmentally down-regulated 8

  •  
  • PLA2

    phospholipase A2

  •  
  • RING

    really interesting new gene

  •  
  • SCF

    Skp1/cullin/F-box

  •  
  • Skp

    S-phase kinase-associated protein

  •  
  • Ub

    ubiquitin

  •  
  • SUMO

    small Ub-related modifier

  •  
  • TRAF

    tumour-necrosis-factor-receptor-associated factor

  •  
  • TR-FRET

    time-resolved FRET

  •  
  • Ub-AMC

    Ub-7-amino-4-methylcoumarin

  •  
  • UBL

    Ub-like protein

  •  
  • UBP/USP

    Ub-specific proteases

  •  
  • ULP

    UBL-specific protease

  •  
  • UPS

    Ub–proteasome system

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