Is novel chemistry the final frontier for ubiquitin system drug discovery?

Although the widespread use of target-focused libraries has led to the rapid expansion of novel chemistry as research tools in drug discovery to exploit many classes of drug target, the ubiquitin system still remains a largely untapped medicinal chemistry opportunity.

Protein kinases, on the other hand, have become one of the most important classes of drug targets for the pharmaceutical industry over the last decade, following on from the exploitation of kinase-focused libraries for at least the last two decades.

Like protein phosphorylation by kinases, protein ubiquitylation regulates many aspects of cell function and provides a wealth of drug target opportunities across many therapeutic areas including cancer, cardiovascular, metabolism, inflammation, neurodegeneration and infectious diseases.

In this article, we propose that chemical libraries which target the ubiquitin system are the missing keys to unlock the therapeutic potential of ubiquitin system drug discovery.

As James Black is quoted as saying: “The most fruitful basis for the discovery of a new drug is to start with an old drug (1).” Consequently, small-molecule drug discovery usually involves an iterative process of molecular design, chemical synthesis, biological assay and data analysis feeding directly into the next cycle, but this process always needs a chemical starting point.

For two decades or more, the rate at which drugs against new targets are launched has been relatively constant but the rate of developing drugs against completely new classes of drug target has been significantly lower (2). However, over the same period protein kinases have rapidly become one of the most significant classes of drug targets for the pharmaceutical industry, with the global market for kinase therapies being about US$15 billion per annum in 2010 and this value is predicted to double by 2020 (3).

Research on protein kinases is now reported to account for approximately 30% of the drug discovery programmes in the pharmaceutical industry and more than 50% of cancer research and development (3).

Target-focused compound libraries have been a key enabling component of the tool kit opening up kinase drug discovery, consisting of collections of compounds designed to interact with a family of related kinase targets (4). Such libraries are used for screening against therapeutic targets in order to find hit compounds that may be further developed into drugs and/or used as research tools to better understand the underlying biology and its relevance to pharmacological intervention.

The design of targeted libraries can use structural information, when available, but can also employ a model that incorporates sequence and mutagenesis data to predict the properties of the binding site. The pharmacology of known ligands of the target can also be used to inform the development of libraries from chemical scaffolds. However, computational methods for the selection of molecules for drug discovery are constantly evolving and new approaches are now available that can assist the medicinal chemist in selecting new compounds for library synthesis.

These methods may also incorporate simple calculated properties, for example the so-called ‘Rule of 5’ (5) to ensure libraries stay within sufficiently druglike areas of chemical space as well as existing structure activity relationship data describing the interaction of the chemical matter with the principle biological target, as well as related targets.

Ubiquitylation and phosphorylation have striking biological parallels

Ubiquitylation describes the covalent attachment of a small 76-amino acid protein, ubiquitin, to other proteins. The ubiquitylation process involves three sequential steps, each of which is controlled by a different class of enzyme (Figure 1).

In the first step, a single ubiquitin molecule is ‘activated’ by the ubiquitin activating enzyme (E1) to which it is conjugated, in an ATP-dependent reaction. In the second step, the ubiquitin molecule is transferred from E1 to a ubiquitin conjugating enzyme (E2) via a transthiolation reaction. In the final step, ubiquitin is transferred to the protein substrate in a process mediated by an E3 ubiquitin ligase, which provides a binding platform for ubiquitin-charged E2 and the substrate.

However, in contrast to protein phosphorylation, ubiquitin can not only be attached to a substrate as a monomer (mono-ubiquitylation) but may be conjugated in the form of a polyubiquitin chain and these may be assembled into at least eight different types determined by which amine group the ‘activated’ C-terminus of a distal ubiquitin attaches itself to on the proximal ubiquitin; through the epsilon amino group of a Lysine side chain (K6, K11, K27, K29, K33, K48 or K63) or through the alpha amino group of the M1 residue (there are also reports of branched and mixed linkage chains) (6). Further, monoubiquitylated proteins may act as substrates themselves resulting in them becoming polyubiquitylated (Figure 1).

Even greater versatility is provided by further families of ubiquitin-like proteins (including NEDD8, SUMO, FAT10 and ISG15), which are also attached covalently to proteins via their own dedicated E1, E2, E3 pathways. The formation of polyubiquitin chains and the function of these ubiquitin-like-modifier proteins make the ubiquitin system a more complex and, potentially, more versatile intracellular signalling mechanism for pharmacological manipulation than phosphorylation (3).

As is the case with phosphorylation, ubiquitylation is a reversible process. Deubiquitylases (or deubiquitinases; DUBs) catalyse the cleavage of ubiquitin from proteins, a process known as deubiquitylation. While a DUB deconstructs ubiquitin chains, other enzymes recognise substrate-conjugated SUMO and NEDD8, for example, and are thus referred to as desumolase and deneddylase enzymes respectively. Together these families of enzymes are known as deconjugating enzymes or DCEs (Figure 1).

Interestingly, the total number of DUBs is comparable with the number of protein phosphatases but, taken together, the number of E1-activating enzymes, E2-conjugating enzymes and E3 ligases encoded by the human genome (~700-1,000) exceeds the total number of protein kinases. The sole function of the ubiquitin system was originally thought to be restricted to the regulation of protein turnover inside the cell, as attaching a ubiquitin chain of a specific linkage type to a protein directs it to the proteasome for destruction (7) (Figure 1).

The canonical chain type determining that the protein to which it is attached will be destined for proteasome-dependent destruction being K48-linked however, other forms of ubiquitylation (defined by different chain linkages or linkage to non-lysine residues on substrate proteins) are known to occur and these may regulate a broad range of biological processes. However, the roles of some of these modifications remain to be fully elucidated (6,8).

Just like phosphorylation, ubiquitylation can also induce conformational changes that alter the biological function of proteins. In common with phosphorylation, many effects of ubiquitylation are mediated by interactions with modification state-dependent binding proteins: in this case ‘ubiquitin-binding proteins’. Alternative polyubiquitin chain linkage types can adopt distinct three-dimensional structures and interact with different polyubiquitin-binding proteins to regulate specific cellular processes (9). 

Protein phosphorylation and ubiquitylation should not be thought of as two distinct and separate regulatory systems because interactions between them are critical for the normal control of many cell signalling processes. Given the ubiquitous nature of protein phosphorylation and ubiquitylation inside the cell, understanding the complex interplay between these two systems is likely to become increasingly important in basic research, drug discovery, small molecule screening and selectivity profiling.

Current drugs and compounds in clinical trials that modulate the ubiquitin system

The proteasome inhibitor Bortezomib (marketed as Velcade®) was the first approved drug to target a key element of the ubiquitin system. Bortezomib was approved as a treatment for multiple myeloma and is a dipeptidyl boronic acid derivative, which is given intravenously, and binds non-covalently to catalytic subunits of the proteasome to inhibit their chymotrypsin-like activity. The precise molecular basis for its efficacy had been somewhat uncertain.

However, it is now thought that its mechanism of action is due, in large part, to the induction of a selective increased proteotoxic stress load in multiple myeloma c ells, which express high levels of misfolded immunoglobulins (10). There is considerable interest in developing improved less-peptidic inhibitors that can be taken orally and a number of such compounds are already in clinical trials (Table 1).

Although Bortezomib demonstrates the clinical benefits of modulating a component of the ubiquitin system, the compound is a more ‘classical’ protease inhibitor and does not illustrate a mechanistic proof of concept for modulating either members of the ligase machinery, DUB families or other elements of the ubiquitin cascade.

There are two inhibitors of E1 enzymes in clinical trials (Table 2).

MLN4924 targets NAE1 (NEDD8 Activating Enzyme 1), the E1 activating enzyme for the ubiquitin-like protein NEDD8 (11). The key targets of ‘NEDDylation’ are the Cullin scaffold proteins of the Cullin Ring Ligase (CRL) sub-family of E3 ubiquitin ligases. CRLs – in particular the SKP1-Cullin1-F-box sub-family – are thought to be the key sub-family of E3 ligases controlling turnover of proteins involved in cellular proliferation (12). MLN4924 is currently in Phase I/II trials. More recently MLN7243, a UAE1 (Ubiquitin Activating Enzyme 1) entered Phase I dose escalation studies.

There are currently no inhibitors of E2s in clinical trials although there have been reports of a small molecule E2 inhibitor whose mechanism of action appears to involve the stabilisation of the interaction of ubiquitin with the E2 donor ubiquitin- binding site (13,14).

Unexpectedly, thalidomide has been found to bind and alter the substrate specificity of an E3 ligase substrate binding adaptor called cereblon (15) that is important for limb outgrowth and the expression of a fibroblast growth factor during embryonic development (16). This finding explains why thalidomide, which was originally prescribed as a sedative and then as a treatment for nausea, caused severe developmental defects in unborn children.

Nevertheless, thalidomide is still used for the treatment of numerous conditions, including leprosy, skin sores and cancer. Indeed Celgene has a number of thalidomide (IMiD®) analogues in clinical trials across a number of applications including multiple myeloma and acute myeloid leukaemia.

Discovering the molecular mechanism of the compound’s devastating side ef fects five decades after its first use facilitates both a better understanding of the mechanism of action of this family of molecules but also an intriguing aspect of ubiquitin system biology – namely the ability of a small molecule to modulate E3 substrate specificity, a phenomena previously identified in plants (17), and that could help to inform one approach to small molecule targeting of E3 ligases.

Thalidomide was not originally designed to target an E3 ligase but demonstrates that orally bioavailable ligase modulators are chemically possible. A number of inhibitors or modulators of E3 ligases are in clinical trials (Table 3) and several are under pre-clinical investigation including a small molecule activator of the E3 ligase Parkin (18,19).

Although the above list is significantly less than for kinase-targeted drugs, pre-clinical drug discovery interest and activity in the ubiquitin field is increasing.

Ubiquitylation/phosphorylation cross-talk

Intriguingly the close interplay and cross-talk between ubiquitylation and phosphorylation (Figure 1) enables a further approach to controlling ubiquitylation pathways, that being via modulation of the phosphorylation of ubiquitylation ligase machinery, ubiquitin ligase substrates or even ubiquitin itself.

Indeed, substrate phosphorylation is critical to the recognition of a number of substrates by E3 ligase substrate binding adaptors (20) and likewise E3 ligases may be activated in a phosphorylation dependent manner; for example, IRAK-catalysed phosphorylation of the E3 Pellino (21) and Src family kinase (SFK) phosphorylation of the E3 TRAF6 (22). Indeed, this cross-talk operates in both directions with TRAF6 ubiquitylating SFKs.

A further level of complexity and demonstration of the interplay between ubiquitylation and phosphorylation is exemplified by the recent intriguing discovery that ubiquitin itself may be phosphorylated and play a role in E3 ligase activation. Thus activation of the E3 ligase parkin is dependent upon both phosphorylation of a ubiquitin-like domain on the E3 ligase itself and interaction with phosphorylated ubiquitin; both parkin and the ubiquitin with which it interacts are phosphorylated by PINK1 (23-25).

These examples underline the fact that post-translational modifications, such as ubiquitylation and phosphorylation, may display significant cross-talk and interdependency and thus benefit from being studied through an integrated cross-specialty approach.

The opportunity for chemical libraries targeting the ubiquitin system

The parallels between the biology of protein phosphorylation and ubiquitylation, as well as their exploitation for the development of drugs to treat diseases have been well described previously by Philip Cohen and colleagues (3). In summary, both biological regulatory mechanisms were identified many years ago but interest in targeting them systematically for drug discovery only really started to take off in earnest over the last two decades.

The first compounds inhibiting components of these systems entered clinical trials at around the same time: Bortezomib in 1997 and Gleevec (the first kinase inhibitor) in 1998. Gleevec was approved in 2001 and overtook Bortezomib, which was approved later in 2003. Both Gleevec and Bortezomib have subsequently proved to be of significant clinical benefit in the treatment of cancer. However, kinase inhibitors have subsequently shot ahead.

Following on from the development of Gleevec, about 25 drugs targeting protein kinases have been clinically approved for use, mostly in cancer, whereas only one other drug (Kyprolis®; Carfilzomib) targeting the ubiquitin system has been approved since Bortezomib; both of these molecules target catalytic subunits of the proteasome. In addition, kinase inhibitors currently undergoing clinical trials also outnumber the inhibitors of the ubiquitin system by more than 10 to one (3).

As pointed out by Philip Cohen, a key factor driving the kinase field forward at such a rapid pace is the ease with which targeted chemical libraries can be synthesised and exploited to develop inhibitors of many protein kinases from the same subfamily (3). This observation probably explains the stark disparity between the two different classes of drug target.

Although E3 ubiquitin ligases outnumber protein kinases, medicinal chemists have still not developed a widely available targeted library approach for identifying inhibitors of E3s. To date, chemists have mostly focused on disrupting the interaction between E3 ligases and their substrates, interactions which are likely to be specific to particular E3 ligase-substrate pairs.

One explanation of this discrepancy is that finding compounds to disrupt the interface of two proteins can be intrinsically more difficult to achieve – and possibly less generically applicable across other members of the protein family – than searching for small molecules that block enzyme catalytic activity, particularly where one of the substrates is common to all enzyme sub-family members as with ATP in the case of kinases.

Surprisingly, less effort has been devoted to developing compounds that disrupt the interactions between E2-conjugating enzymes and E3 ligases or at targeting E2s directly. E2-E3 interactions are usually relatively weak and may therefore be relatively easy to disrupt. Moreover, compounds that disturb the interaction between an E2-conjugating enzyme and an E3 ligase could, in principle, exert their effects by binding to the E2, the E3, the E2-E3 interface or via an allosteric mechanism, creating the potential to identify three or four types of inhibitors from a single screen.

There are ~40 E2-conjugating enzymes encoded by the human genome; therefore, on average, each E2 might be predicted to interact productively with ~15 E3 ligases although the reality is likely to be more complicated than this with certain E2s having distinct roles (26-29) whose interactions with E3s may be regulated as much by spatial and temporal factors as binding affinities. Compounds that disrupt E2-E3 interactions by binding specifically to the E3 ligase could be identified by counter screening with another E3 that also forms a productive interaction with the same E2.

Focusing efforts on large families of E3 ligases may lead to the development of chemical libraries with the capability of disrupting many E2-E3 interactions. By analogy with kinases, perhaps the key to developing inhibitors of specific E2-E3 interactions is to find compounds that bind to small hydrophobic pockets on the E2 or E3 located proximal to the E2-E3 interface itself or to identify allosteric inhibitors that disrupt the E2-E3 interaction by inducing long-range conformational changes.

The determination of such three-dimensional structures of E2-ubiquitin/E3 ligase complexes will be critical to such efforts. For example, the solving of the structure of the E3 ligase CBLB/ E2~ubiquitin complex may not only help to inform such approaches but has also revealed how the phosphorylation of Tyrosine-363 in the E3 induces a structural event that enhances catalytic efficiency by ~200-fold, providing a further example of the close interplay between these two key signalling systems (30). The CBL family of E3 ligases attenuate non-receptor and receptor tyrosine kinase signalling by ubiquitylating and thereby directing these kinases for degradation through the endocytic or proteasomal pathway (31).

Another area where more effort will probably be fruitful is the design and generation of chemical libraries to target the large and diverse families of DUBs (Figure 2).

Deubiquitylases are attracting increasing attention as drug targets across many therapeutic areas, including cancer (32-34) and infectious diseases (35). Inhibitors of many DUBs are already in pre-clinical development. Learning from experience with kinase-targeted libraries has taught us that compounds developed as inhibitors of one protein kinase commonly turn out to inhibit other protein kinases, sometimes with even greater potency, and can kick start completely new drug discovery projects.

Therefore, developing chemical libraries that target DUBs is likely to yield similar surprises and generate drug leads targeting a number of these enzymes. Iterative hit-to-lead medicinal chemistry optimisation after such library screening and hit identification is commonly supported by selectivity profiling. Such activities inform us which chemical series deliver the required balance between target affinity and selectivity versus enzymes of the same family (eg related DUBs; see Figure 2).

To be effective, targeted libraries need to deliver useful Structure Activity Relationship (SAR) data to the medicinal chemist. Such libraries also provide opportunities for the identification of potent tool molecules to probe target tractability and mechanism of action. This offers significant scope for impacting early stage biology where research efforts may be hampered by inadequate tools to elucidate the biology and, at the same time, kick start hit-to-lead optimisation.

Currently any one of a number of hit identification methods may identify a good number of interesting looking molecules which then go through a rigorous triage process to ensure only the most tractable are actually resourced in the laboratory. Evaluation of a wider range of chemical hits though targeted libraries, coupled with stringent design criteria, will enable a more thorough evaluation of the accessible chemical space, which would be expected to impact positively on the final quality of candidates being produced.

A key element of the fully integrated approach to drug discovery is the rapid cycle time, whereby biological assays are integrated with the targeted libraries. Consequently, integrated platforms, and assay kits combining biology and chemistry, will enable a range of chemical structures and corresponding biological activity data to potentially transform ubiquitin system drug discovery. However, the keys to unlocking the potential of ubiquitin system drug discovery are the introduction of chemical libraries targeting DUBs and other enzyme classes of the ubiquitin system.

As Ubiquigent is based in the Sir James Black Centre we are now following his advice and, even if there are few existing ‘old drugs’ to start from, we are planning to fill this chemical void with new libraries as ‘the most fruitful basis for the discovery of new drugs’ targeting the ubiquitin system. Ultimately, the acid test for our prediction of targeted chemical libraries being the keys to unlock ubiquitin system drug discovery will be when development candidates targeting the ubiquitin system approach or even overtake those targeting protein kinases. DDW

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This article originally featured in the DDW Summer 2014 Issue

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Dr Jason Brown co-founded Ubiquigent in 2009 as its Managing and Scientific Director in collaboration with the University of Dundee, the Medical Research Council and Stemgent Inc. Before starting Ubiquigent he was part of a biotech investment and operations group and involved in supporting a molecular diagnostics, kinase drug discovery and various other drug discovery-focused service companies as well as evaluating investment opportunities. Prior to this he built and ran a kinase-focused assay development and drug discovery service facility for Upstate Biotechnology, a leading provider of cell signalling research products and services which grew out of a close collaboration with Sir Philip Cohen and his MRC Protein Phosphorylation Unit (now the MRC Protein Phosphorylation and Ubiquitylation Unit where Ubiquigent is based). Jason received his MPhil and DPhil from the University of Cambridge in association with Parke-Davis/Warner-Lambert (Pfizer), during which he identified a voltage-dependent calcium channel subunit as the molecular target of the blockbuster epilepsy and neuropathic pain drugs Neurontin and Lyrica. After his DPhil Jason worked in and subsequently ran an assay development group for Parke-Davis.

Dr Mark Treherne has been actively involved in the biopharmaceutical industry for more than 25 years and previously led the neurodegeneration research group at Pfizer’s research facility in Sandwich, where he initiated a number kinase drug discovery projects with Sir Philip Cohen in Dundee. In 1997, he co-founded Cambridge Drug Discovery as Chief Executive, leading the company’s subsequent acquisition by BioFocus plc, where he became Commercial Director and drove significant growth of the profitable services business, again in collaboration with Sir Philip Cohen. BioFocus pioneered the commercial exploitation of kinase-focused libraries. Dr Treherne joined Ubiquigent as Chairman in 2013 to help unlock the potential of ubiquitin system drug discovery from the Sir James Black Centre in Dundee. He has a BSc in Physiology and Pharmacology from the University St Andrews and a PhD in Pharmacology from the University of Cambridge.

References
1 Raju, TN. The Nobel Chronicles. 1988: James Whyte Black, (b 1924), Gertrude Elion (1918-99), and George H Hitchings (1905-98). Lancet, 2000. 355(9208): p. 1022.

2 Overington, JP, Al-Lazikani, B and Hopkins, AL. How many drug targets are there? Nat Rev Drug Discov, 2006. 5(12): p. 993-6.

3 Cohen, P and Tcherpakov, M. Will the ubiquitin system furnish as many drug targets as protein kinases? Cell, 2010. 143(5): p. 686-93.

4 Harris, CJ et al. The design and application of target-focused compound libraries. Comb Chem High Throughput Screen, 2011. 14(6): p. 521-31.

5 Lipinski, CA et al. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Delivery Rev., 1997. 23: p. 3-25.

6 Komander, D. The emerging complexity of protein ubiquitination. Biochem Soc Trans, 2009. 37(Pt 5): p. 937-53.

7 Ciechanover, A and Stanhill, A. The complexity of recognition of ubiquitinated substrates by the 26S proteasome. Biochim Biophys Acta, 2014. 1843(1): p. 86-96.

8 McDowell, GS and Philpott, A. Non-canonical ubiquitylation: Mechanisms and consequences. Int J Biochem Cell Biol, 2013. 45(8): p. 1833-42.

9 Husnjak, K and Dikic, I. Ubiquitin-binding proteins: Decoders of ubiquitin-mediated cellular functions. Annu Rev Biochem, 2012. 81: p. 291-322.

10 Kubiczkova, L et al. Proteasome inhibitors – molecular basis and current perspectives in multiple myeloma. J Cell Mol Med, 2014. [Epub ahead of print].

11 Nawrocki, ST et al. MLN4924: A novel first-in-class inhibitor of NEDD8-activating enzyme for cancer therapy. Expert Opin Investig Drugs, 2012. 21(10): p. 1563-73.

12 Zhou, W, Wei, W and Sun, Y. Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases. Cell Research, 2013. 23(5): p. 599-619.

13 Ceccarelli, DF et al. An allosteric inhibitor of the human cdc34 ubiquitin-conjugating enzyme. Cell, 2011. 145(7): p. 1075-87.

14 Huang, H et al. E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin. Nat Chem Biol, 2014. 10(2): p. 156-63.

15 Licht, JD, Shortt, J and Johnstone, R. From anecdote to targeted therapy: The curious case of thalidomide in multiple myeloma. Cancer Cell, 2014. 25(1): p. 9-11.

16 Ito, T et al. Identification of a primary target of thalidomide teratogenicity. Science, 2010. 327(5971): p. 1345-50.

17 Tan, X and Zheng, N. Hormone signaling through protein destruction: A lesson from plants. Am J Physiol Endocrinol Metab, 2009. 296(2): p. E223-7.

18 Johnston, JA. Ubiquitin Drug Discovery and Diagnostics Conference – targeting E3 ligases. IDrugs, 2010. 13(10): p. 695-7.

19 Regnstrom, K et al. Label free fragment screening using surface plasmon resonance as a tool for fragment finding – analyzing parkin, a difficult CNS target. PLoS One, 2013. 8(7): p. e66879.

20 Evrard-Todeschi, N et al. Structure of the complex between phosphorylated substrates and the SCF beta- TrCP ubiquitin ligase receptor: A combined NMR, molecular modeling, and docking approach. J Chem Inf Model, 2008. 48(12): p. 2350-61.

21 Ordureau, A et al. The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1. Biochem J, 2008. 409(1): p. 43-52.

22 Liu, A et al. TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia. J Biol Chem, 2012. 287(20): p. 16132-45.

23 Kazlauskaite, A et al. Parkin is activated by PINK1- dependent phosphorylation of ubiquitin at Ser65. Biochem J, 2014. 460(1): p. 127-39.

24 Koyano, F et al. Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature, 2014. 510(7503): p. 162-6.

25 Shaw, GS. Switching on ubiquitylation by phosphorylating a ubiquitous activator. Biochem J, 2014. 460(3): p. e1-3.

26 Christensen, DE, Brzovic, PS and Klevit, RE. E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages. Nat Struct Mol Biol, 2007. 14(10): p. 941-8.

27 Wenzel, DM, Stoll, KE and Klevit, RE. E2s: Structurally economical and functionally replete. Biochem J, 2010. 433(1): p. 31-42.

28 Pruneda, JN et al. Ubiquitin in motion: Structural studies of the ubiquitin-conjugating enzyme approximately ubiquitin conjugate. Biochemistry, 2011. 50(10): p. 1624-33.

29 Metzger, MB et al. RINGtype E3 ligases: Master manipulators of E2 ubiquitinconjugating enzymes and ubiquitination. Biochim Biophys Acta, 2014. 1843(1): p. 47-60.

30 Dou, H et al. Essentiality of a non-RING element in priming donor ubiquitin for catalysis by a monomeric E3. Nat Struct Mol Biol, 2013. 20(8): p. 982-6.

31 Mohapatra, B et al. Protein tyrosine kinase regulation by ubiquitination: Critical roles of Cbl-family ubiquitin ligases. Biochim Biophys Acta, 2013. 1833(1): p. 122-39.

32 Lim, KH and Baek, KH. Deubiquitinating enzymes as therapeutic targets in cancer. Current Pharmaceutical Design, 2013. 19(22): p. 4039-52.

33 Jacq, X et al. Deubiquitylating enzymes and DNA damage response pathways. Cell Biochem Biophys, 2013. 67(1): p. 25-43.

34 Fraile, JM et al. Deubiquitinases in cancer: New functions and therapeutic options. Oncogene, 2012. 31(19): p. 2373-88.

35 Nanduri, B et al. Deubiquitinating enzymes as promising drug targets for infectious diseases. Current Pharmaceutical Design, 2013. 19(18): p. 3234-47