REVIEWS Deubiquitylating enzymes and drug discovery: emerging opportunities Jeanine A. Harrigan1*, Xavier Jacq1*, Niall M. Martin1,2, and Stephen P. Jackson1,3 Abstract | More than a decade after a Nobel Prize was awarded for the discovery of the ubiquitin– proteasome system and clinical approval of proteasome and ubiquitin E3 ligase inhibitors, first-generation deubiquitylating enzyme (DUB) inhibitors are now approaching clinical trials. However, although our knowledge of the physiological and pathophysiological roles of DUBs has evolved tremendously, the clinical development of selective DUB inhibitors has been challenging. In this Review, we discuss these issues and highlight recent advances in our understanding of DUB enzymology and biology as well as technological improvements that have contributed to the current interest in DUBs as therapeutic targets in diseases ranging from oncology to neurodegeneration. 10 Ubiquitin The sequential enzymatic processes that covalently exist in eukaryotic cells . The recent demonstration of A small protein that is attach ubiquitin, a 76‑residue polypeptide, to target post‑translational modification of ubiquitin itself pro‑ conjugated to other proteins proteins — a process known as ubiquitylation — are vides an additional layer of regulation that affects various (including itself) as a now well understood1 (FIG. 1a). In some cases, a single cellular processes11. post-translational modification, ubiquitin is attached to the target protein, whereas in Like other post‑translational modifications, ubiquity‑ often to control cellular signalling or degradation of the others, multiple monoubiquitin adducts are conjugated lation is reversible: peptidases termed deubiquitylating modified protein. to different residues of the target. In many instances, enzymes (DUBs) can cleave ubiquitin from substrate pro‑ various types of ubiquitin chains are produced, wherein teins, edit ubiquitin chains and process ubiquitin precur‑ Ubiquitin-like proteins one ubiquitin moiety is attached to a free amino group sors12. Some DUBs and related enzymes are involved in (UBLs). Proteins such as small 13 ubiquitin-like modifier (SUMO) of another. This leads to linear ubiquitin chains and editing or processing UBLs and their conjugates ; prime or interferon-stimulated gene chains involving internal ubiquitin lysine residues K6, examples of these being the SENP (sentrin/SUMO‑ 15 (ISG15) that adopt K11, K27, K29, K33, K48, K63, as well as mixed ubiq‑ specific protease) proteins that process SUMO precursors a β-grasp fold, which is uitin chains containing different linkages, or linkages and SUMO conjugates14. DUBs are classified into six fam‑ characteristic of ubiquitin and between ubiquitin and ubiquitin-like proteins (UBLs) ilies based on sequence and domain conservation (FIG. 1b): related proteins. that include small ubiquitin‑like modifier (SUMO) USPs (ubiquitin‑specific proteases), UCHs (ubiquitin and neuronal precursor cell‑expressed developmentally carboxy‑terminal hydrolases), MJDs (Machado–Josephin downregulated protein 8 (NEDD8). domain‑containing proteases), OTUs (ovarian tumour 1Mission Therapeutics Ltd, Moneta, Babraham Research These different types of ubiquitin and UBL modi‑ proteases), MINDYs (motif‑ interacting with ubiquitin-­ Campus, Cambridge CB22 fications, sometimes referred to as ‘the ubiquitin code’, containing novel DUB family) and JAMMs (JAB1, MPN, 3AT, UK have specific and diverse effects on protein and cell MOV34 family). SENPs and the first five DUB fami‑ 2Present address: Artios physiology. For example, such modifications can target lies are cysteine peptidases, whereas JAMMs are zinc Pharmaceuticals Ltd, Maia, proteins that are damaged or improperly folded, or that metallopeptidases. Babraham Research Campus, Cambridge CB22 3AT, UK have intrinsically short half‑lives for degradation via the Ubiquitylation and related processes control myr‑ 3The Wellcome Trust and ubiquitin–proteasome system (UPS)2. Appropriately iad aspects of human cell biology and physiology, and Cancer Research UK Gurdon polyubiquitylated proteins are recognized and degraded defects in such processes contribute to many diseases. Institute, and Department of by the 26S macromolecular proteasome complex3 via Accordingly, DUB deregulation contributes to vari‑ Biochemistry, Tennis Court Road, University of Cambridge, mechanisms that have been extensively reviewed else‑ ous sporadic and genetic disorders. Notable examples 4,5 Cambridge CB2 1QN, UK. where . In other instances, ubiquitylation regulates include: the UCH family member BRCA1‑associated *These authors contributed protein interactions, localization and enzymatic activ‑ protein 1 (BAP1), mutated in melanoma, meso thelioma equally to this work. ities, thereby affecting cellular processes, including and renal cell carcinoma15; USP6, translocated in aneu‑ 16 Correspondence to S.P.J. transcription, DNA damage signalling and DNA repair, rysmal bone cysts ; USP7, mutated in neurological [email protected] cell cycle progression, endocytosis, apoptosis and vari‑ disorders17; USP8, whose mutations cause Cushing 6–9 18,19 doi:10.1038/nrd.2017.152 ous other processes . Such control mechanisms often disease ; USP9X, whose mutations cause develop‑ Published online 29 Sep 2017 involve ubiquitin‑binding proteins, many of which mental disorders20 and whose expression is dysregulated NATURE REVIEWS | DRUG DISCOVERY VOLUME 17 | JANUARY 2018 | 57 ©2017 Mac millan Publishers Li mited, part of Spri nger Nature. All ri ghts reserved. REVIEWS a in cancer21; USP15, amplified in certain glioblastoma, Ub Ub Ub Ub Ub Ub Ub Ub Ub breast and ovarian cancers22; and CYLD, commonly Ub Ub Ub Ubiquitin mutated in cylindromatosis23. Deregulation of MJD- Ub L precursors family DUBs has also been linked to diseases associated Ub S with polyglutamine amplification. For example, expan‑ sion of DNA ‘CAG’ trinucleotide repeats in ataxin 3 (ATXN3) causes Machado–Joseph disease (also known DUB as spinocerebellar ataxia 3)24. Furthermore, mutations in the JAMM family member associated molecule with the SH3 domain of STAM (AMSH; also known as STAMBP) Ub Ub E1 25 C cause microcephaly–capillary malformation syndrome . Ub Ub There has been growing interest in exploiting com‑ DUB ATP ADP + P i ponents of the ubiquitylation machinery as therapeutic targets26. Although there has been strong progress in developing small-molecule inhibitors of ubiquitin and Ub Ub 27 Ub Ub Ub UBL E1 enzymes , the highly pleiotropic nature of E1s Substrate means that such drugs will likely be confined to acute K E2 K settings, such as in the treatment of aggressive cancers. C Substrate Ub Given their greater numbers and diversity, E2s, E3s and DUBs offer the potential for developing drugs with more specific effects. In particular, being a group of diverse enzymes with well-defined catalytic clefts, DUBs are intrinsically attractive as potential drug targets26. E3 E3 E2 E2 However — as we discuss further below — until recently, C C the development of selective DUB inhibitors has been Ub Ub Substrate Figure 1 | The ubiquitylation cascade and the deubiquitylase family of proteins. a | Schematic of key events in ubiquitylation and deubiquitylation. The E1 enzyme activates ubiquitin in an ATP-dependent manner, resulting in a covalent thioester linkage between ubiquitin and the E1 cysteine residue. Ubiquitin is then b USP37 USP20 USP33 USP48 USP28 USP25 transferred to an E2 conjugating enzyme, forming a USP9Y USP9X USP24 thioester linkage with the catalytic cysteine. Finally, an E3 USP26 USP29 USP5 USP34 USP13 USP39 USP47 USP44 ligase assists or directly catalyses the transfer of ubiquitin USP49 USP36 USP7 USP42 USP40 USP17-like from the E2 to a substrate, usually via a lysine side chain. CYLD DUB3 USP52 USP30 An example of a HECT (homologous to the E6AP carboxyl USP10 USP53 USP18 terminus) or RBR (RING-between-RING) E3 ligase is shown. USP54 USP41 USP15 USP8 In subsequent rounds, ubiquitin molecules can be USP21 USP4 USP14 conjugated to the N‑terminal amino group or lysines on USP11 USP50 ubiquitin itself to form chains. Deubiquitylating enzymes USP19 USP51 USP43 USP22 (DUBs) remove ubiquitin molecules from substrates or USP31 USP27X process ubiquitin precursors to generate free ubiquitin USP45 USP3 USP2 pools. b | DUB phylogenetic tree. Sequences for full-length USP16 USP12 DUB and SENP (sentrin/SUMO-specific protease) proteins USP1 USP46 USP38 USPL1 were aligned with COBALT (constraint-based multiple USP35 DESI1 alignment tool), a computational tool for multiple protein USP32 DESI2 sequences, and subsequently visualized with FigTree v1.4.3. USP6 SENP6 SENP7 In regard to USP17-like, note that various related human UCHL3 SENP3 USP17-like DUBs exist. AMSH, associated molecule with UCHL1 SENP5 UCHL5 SENP8 the SH3 domain of STAM; AMSHLP, AMSH-like protease; BAP1 SENP1 ATXN3, ataxin 3; BAP1, BRCA1‑associated protein 1; SENP2 MINDY2 MYSM1 CEZANNE, cellular zinc finger anti-NF-κB protein; CSN, MINDY1 MPND PRPF8 COP9 signalosome complex subunit; CYLD, MINDY4 AMSH MINDY3 AMSHLP cylindromatosis; DESI, desumoylating isopeptidase; EIF3, JOSD2 BRCC3 JOSD1 CSN5 PSMD14 eukaryotic translation initiation factor 3; JAMM, JAB1, MPN, EIF3S3 ATXN3L ATXN3 CSN6EIF3S5 MOV34 family; JOSD, josephin domain; MINDY, PSMD7 OTUB1 OTUB2 A20 OTULIN FAM105A motif-interacting with ubiquitin-containing novel DUB OTUD6B YOD1 OTUD6A OTUD1 family; MJD, Machado–Josephin domain-containing OTUD5 OTUD3 VCPIP1 OTUD4 protease; OTUD, OTU domain-containing protein; PRPF8,