Drug discovery in the ubiquitin proteasome system

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1 Drug discovery in the ubiquitin proteasome system Grzegorz Nalepa*, Mark Rolfe and J. Wade Harper* Abstract Regulated protein turnover via the ubiquitin proteasome system (UP) underlies a wide variety of signalling pathways, from cell-cycle control and transcription to development. Recent evidence that pharmacological inhibition of the proteasome can be efficacious in the treatment of human cancers has set the stage for attempts to selectively inhibit the activities of disease-specific components of the UP. Here, we review recent advances linking UP components with specific human diseases, most prominently cancer and neurodegenerative disorders, and emphasize potential sites of therapeutic intervention along the regulated protein-degradation pathway. *Department of Pathology, Harvard Medical chool, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, UA. Current address: Herman B. Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, 1044 West Walnut treet, R4/402A, Indianapolis, Indiana 46202, UA. Millennium Pharmaceuticals Inc., 40 Landsdowne treet, Cambridge, Massachusetts 02139, UA. Correspondence to J.W.H. wade_harper@hms. harvard.edu doi: /nrd2056 Regulated protein degradation is an essential aspect of cell signalling 1. Cells must be able to respond immediately to environmental changes to maintain homeostasis or to undergo specified developmental decisions. Alterations in the transcriptome provide a means by which to buffer rapid shifts in extra- or intracellular signals, but posttranslational modifications of the proteome provide a much faster mechanism for activation or inhibition of signalling pathways. Much of the control of signalling pathways occurs via protein phosphorylation or protein turnover, and in many cases these two mechanisms are interwoven. Protein phosphorylation is useful because it is reversible via protein phosphatases, thereby allowing a particular protein to be in two or more states and to interchange between them, depending on the signals being received. By contrast, protein degradation provides an irreversible means by which to alter the flux through a particular pathway 2 4. The ubiquitin proteasome system (UP) is responsible for much of the regulated proteolysis in the cell, although it is now appreciated that ubiquitin and ubiquitin-like proteins (Ulps) can, under some circumstances, function in reversible systems, aided by the activity of de-ubiquityl ating enzymes. Our expanding understanding of the UP and its role in human diseases has elicited significant interest in the development of small molecules that target specific components of the pathway. The role of the UP in disease is really in its infancy, as only a small fraction (perhaps <20%) of the genes with potential links to the pathway based on known sequence motifs have been studied in any detail. Moreover, there has been no comprehensive analysis of mutations in the UP in human diseases. Nevertheless, there is accumulating evidence of altered functions for components of the UP in cancer, as well as neurodegenerative diseases. Examples of alterations in UP genes will be elaborated below but it is clear that mutational inactivation of the machinery involved in linking ubiquitin to specific substrates (or removing it) can occur in tumour-suppressor proteins (for example, F-box and WD40 domain protein 7 (FBW7)), in proteins involved in DNA repair and genome integrity (for example, breast cancer type 1 susceptibility protein (BRCA1)), or in proteins that are important for proper neuronal homeostasis (for example, parkin) Moreover, it is also clear that alterations in the expression of UP genes (most notably overexpression) can have a dramatic impact on the levels of their target genes, and this is sometimes linked to disease. This is most clearly understood for double minute 2 (MDM2) and -phase kinase-associated protein 2 (KP2), two proto-oncogenes that are often overexpressed in cancer and which promote the degradation of proteins that negatively regulate the cell-division cycle (p53 (REF 14 21) and p27 (REF 22 26)); however, other genes, such as the de-ubiquitylating enzymes CYLD, ubiquitin-specific peptidase 4 (UP4) and UP6, have also been linked to transformation 27. Likewise, it is becoming appreciated that targets of the UP are also frequently mutated in disease. In these cases, the target protein, normally destined for degradation, becomes mutated in residues that contribute to recognition by the UP, making these proteins immune to the action of their cognate degradation machinery. This typically leads to aberrant expression and altered cellular properties, including transformation in the case of mutations in the c-myc and β-catenin transcription factors Although there are potentially many different components of the UP that might be targeted for inhibition in the context of a particular disease, the first success 596 JULY 2006 VOLUME 5

2 in the clinic has come from the inhibition of the proteasome itself. Bortezomib (Velcade; Millennium), a peptide boronic-acid inhibitor of the proteasome 31 36, is the first drug targeting the UP to be approved for human applications, including treatment of relapsed or refractory multiple myeloma 34,37. The relative selectivity of proteasome inhibition for killing tumour cells as opposed to normal cells, coupled with the manageable side effects of the drug, were somewhat surprising initially. However, this selectivity can now be rationalized based on the idea that tumour cells generate higher concentrations of aberrant proteins as well as higher amounts of oncoproteins, making them more sensitive to the effects of proteasome inhibition. Nevertheless, the success with proteasome inhibition, at least for certain types of cancer, suggests that the development of more specific compounds targeting the UP could be clinically important. In considering the UP for drug development, it is important to keep several issues in mind. First, what is the disease to be targeted and what is the nature of the alteration in the pathway that one is trying to modify with a small-molecule therapeutic? For example, it might be easier to inhibit a hyper-active or overexpressed component of the pathway than to resurrect activity from a mutant protein with reduced or negligible activity, such as occurs with familial mutations in the BRCA1 or parkin ubiquitin ligases. econd, it is important to consider how particular classes of proteins within the UP can be targeted. ome components of the UP including the ubiquitin-activation machinery and de-ubiquitylating enzymes are conventional enzymes and might therefore be more amenable to inhibition with small molecules than other components of the system, such as adaptor proteins, which typically lack active-site pockets. Moreover, because many components in the pathway are multi-gene families, some attention to specificity is required, as off-target effects can reduce the suitability of particular classes of inhibitors. In this review, we describe the structures and functions of major components in the UP and summarize our current state of knowledge for those UP components that have been implicated in disease. We also discuss published efforts to identify inhibitors of the UP pathway and suggest possible additional points of therapeutic intervention. Targeting ubiquitin activation Although most post-translational protein modifications involve attachment of relatively small functional tags, such as phosphates or methyl groups, the regulated protein-destruction pathway is different. Here, a protein targeted for degradation is marked with a chain of ubiquitin molecules iquitin is a 76-amino-acid, evolutionarily conserved polypeptide that adopts a tightly packed, nearly globular conformation 43. In the canonical pathway, a lysine residue in a protein is attached to the C-terminal carboxylate of ubiquitin through an isopeptide linkage [ -CO-NH 2 ε- ]. This ubiquitin molecule then serves as the point of further chain extension via formation of additional isopeptide linkages with the C terminus of ubiquitin through lysine residues located on the ubiquitin molecule itself. iquitin contains seven lysine residues. The most prominent site of ubiquitin chain extension is lysine-48 (K48); K48 polyubiquitin chains linked to a target protein are thought to be the primary form that is recognized by the proteasome However, other chain linkages, including K63-linked polymers, have also been identified and these chains seem to be regulatory as opposed to promoting degradation (BOX 1). FIGURE 1 explains the five major steps of the protein polyubiquitylation pathway. The most upstream components of the ubiquitin () and ubiquitin-like protein (Ulp) conjugation machinery (generically referred to as E1 ubiquitinactivating enzymes) perform the activation step (FIG. 2), in which the /Ulp first becomes adenylated on its C-terminal glycine residue and then becomes charged as a thiol ester, again at its C terminus. The or Ulp is then transferred to one of several distinct ubiquitinconjugating enzymes, also through a thiol ester bond. Although the E1-activating enzyme for was the first to be identified, we now know that there are several related enzymes, which serve to catalyse activation of different Ulps. Prominent among these are the E1 for the Ulps small ubiquitin modifier (UMO; E1 UMO ), NEDD8 (neural precursor cell expressed, developmentally downregulated 8; E1 NEDD8 ) and IG15 (E1 IG15 ) (BOX 1). The mechanism of action of these enzymes is thought to be very similar, although each of these Ulp E1s seems to be able to charge only a single : ubiquitin-conjugating enzyme C9 (UBC9), UBC12 and UBC8, respectively. By contrast, E1 can charge a wide variety of s. In addition, these Ulps generally form mono-conjugates, and unlike ubiquitin do not generally form polymeric chains upon conjugation to targets. Although crystal structures of E1 have not been reported, detailed structural analysis of E1 NEDD8 (REF 47,48) and E1 UMO (REF. 49) have been reported and provide a framework for contemplating inhibitors of and Ulp activation (FIG. 3). Both E1 NEDD8 and E1 UMO are heterodimeric complexes in which each of the two subunits (amyloid-β precursor protein binding protein 1 (APPBP1)/ubiquitin-activating enzyme 3 (UBA3) and UMO-1-activating enzyme subunit 1 (AE1)/A, respectively) contains domains related to the bacterial thiamine biosynthesis protein ThiF. The ThiF domain contributes substantially to the ATP-binding site responsible for adenylation of ubiquitin (FIG. 3b,c). By contrast, E1 represents a fusion of these two proteins to form a single polypeptide which is expected to form a similar three-dimensional structure. In addition to the adenylate pocket, two additional functional domains are found in the complex. Both A and UBA3 share a domain containing a conserved cysteine residue, which is the site of thiol ester formation with the Ulp. This domain is referred to as the catalytic cysteine domain (CC) (FIG. 3b,c). In addition, the C terminus of these subunits also contains a small domain that forms a ubiquitin-like fold and is important for interaction with (FIG. 3d). tructural analysis of E1 NEDD8 with NEDD8 and with its cognate, UBC12, have provided insight into NATURE REVIEW DRUG DICOVERY VOLUME 5 JULY

3 Box 1 Polyubiquitylation, monoubiquitylation and ubiquitin-like proteins In addition to ubiquitylation, eukaryotic cells have evolved additional signalling systems utilizing other polypeptides that share the same fold as ubiquitin. Examples of such ubiquitin-like proteins (ls) 169 are shown in the table and include NEDD8, UMO and IG15. The ls are conjugated to their target proteins in a manner that mechanistically resembles ubiquitylation but have dramatically different functional consequences. Instead of promoting proteasomal degradation, l conjugation modifies target activity or subcellular localization. Moreover, conjugation of a single ubiquitin molecule (that is, monoubiquitylation) by contrast with polyubiquitylation has been shown to regulate target activity instead of enforcing its destruction. everal classes of ubiquitin-binding proteins have been identified, and many of these interact with mono-ubiquitylated forms of ubiquitin (for example, Cue domain containing proteins). Thus, mono-ubiquitylation and l conjugation can be conceptualized as a canonical posttranslational modification that employs proteins (ubiquitin or l) instead of a small functional group such as phosphate or acetyl groups. In addition to K48-linked polyubiquitin chains, other classes of conjugation products, such as K64-linked chains and branched polyubiquitin chains, can also be formed on targets. To date, K63-chains have been implicated in controlling protein activity and appear to have no role in protein degradation. Type II ls contain other functional domains in addition to the ubiquitin-fold motif. They are generally larger protein molecules that are not conjugated to other proteins. Instead, they perform multiple and incompletely understood cellular functions that might or might not intersect with the ubiquitin proteasome pathway of protein degradation, such as DNA repair, cell cycle control or serving as E3 ligases. One of the type II ubiquitin-like proteins, parkin, has been implicated in familial Parkinson s disease as a putative ubiquitin ligase. UBL Target examples Outcome iquitin (as a K48-linked polyubiquitin chain) iquitin (as a K63-linked polyubiquitin chain) iquitin (as a monomer) NEDD8 UMO IG15 Multiple unstable proteins TRAF2/TRAF6; NEMO; PCNA; synphilin-1 FANCD2; PCNA; p53; EGFR Cullins PML; p53; IκBα, RanGAP; caspase 8; c-jun; ER; p300 Proteasomal degradation 38 Regulation of kinase activity in NF-κB pathway (TRAF2/TRAF6/NEMO) Error-free DNA replication (PCNA) Unknown (synphilin-1; possible role in Lewy body formation) ubnuclear targeting and function of a Fanconi anaemia-associated protein FANCD2 Error-prone DNA replication in the presence of DNA damage (PCNA) Nuclear export (p53) Membrane receptor internalization (EGFR) Activation of the CF ubiquitin ligase Known consequences of UMO conjugation include: Dynamic target shuttling to other subcellular compartments (PML, RanGAP1, caspase 8) Activation (p53) or inactivation (AR, p300, c-jun) of the target tabilization of a UMO-conjugated protein by preventing its ubiquitination (IκBα) Innate immunity against viral infections EGFR, epidermal growth factor receptor; FANCD, Fanconi anaemia complementation group D; IκB, inhibitor of NF-κB; NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κb; PCNA, proliferating cell nuclear antigen; PML, promyelocytic leukaemia; TNF, tumournecrosis factor, TRAF, TNF-associated factor. recognition of s by E1s 47. The interaction of E1 NEDD8 with UBC12 is bipartite in character it involves interactions between two separate domains on each of these proteins. Insertion of 13 N-terminal residues of UBC12 into a surface groove on UBA3 is specific for the NEDD8 pathway, as there is no similarity between the N terminus of UBC12 and other s. However, the general mechanism of interaction between a ubiquitin-like fold of UBA3 and a core domain of UBC12 seems (FIG. 3b,d) to be widely conserved in various E1/ pairs, including those involved in ubiquitin activation and transfer 47. Current models suggest that large conformational changes are necessary for delivery of the NEDD8 C terminus to the catalytic cysteine of UBC12. The ubiquitin-fold domain of E1 NEDD8 was proposed to undergo a dramatic rotation around a flexible linker 50 found between the -fold domain and the NEDD8-binding site. This large conformational reorganization would bring the ubiquitin-fold domain of E1 NEDD8 (together with bound UBC12) closer to the NEDD8-preloaded E1 NEDD8 catalytic cysteine to facilitate NEDD8 transfer to UBC12. Importantly, the length and flexibility of this linker were shown to be crucial for transfer to UBC12 (REF 48 50). These and other studies suggest two possible approaches to inhibiting E1 proteins. The first involves the identification of inhibitors of or Ulp adenylation by blocking either access of the /Ulp to the adenylate site or by blocking access of ATP. There is a long history in the pharmaceutical industry of successful targeting of ATP-binding sites, most notably protein kinases. For instance, imatinib (Gleevec; Novartis), a potent inhibitor of the oncogenic BCR ABL fusion kinase and several other tyrosine kinases, is approved for treatment of chronic myelogenous leukaemia 51. Multiple clinical trials that address the potential role of imatinib in other malignancies, such as gastrointestinal stromal tumours, ovarian carcinomas and childhood gliomas, are currently under way, and there is some additional information suggesting that the scope of ATP pockets targeted by imatinib might be broader than previously anticipated. The experience gained in the identification of protein kinase inhibitors could have major benefits 598 JULY 2006 VOLUME 5

4 in efforts to identify inhibitors of E1s, although the aforementioned example of kinase inhibitors indicates that the small molecules targeting ATP-binding pockets are not always as specific as predicted. The second potential site for drug development involves the E1 interaction (FIG. 3d). It is important to keep in mind that such protein protein interaction surfaces might make structure-based design of novel drugs E1 e ubstrate destruction Bortezomib; other proteasome inhibitors ATP Inhibitors of ubiquitin activation/ transfer ADP ADP E1 ATP a iquitin activation d Death-tag binding 20 proteasome E1 istatins E3 E3 E1/ complex b Building ligase substrate complex... polyubiquitin chain E3 KP2 E3 p53 MDM2 interface RBX1 CF ubiquitin ligase KP1 c Polyubiquitylation E3 Cullin Targeting specific ligases by small-molecule compounds Figure 1 Overview of the ubiquitin proteasome system (UP). Protein degradation through the UP is a highly regulated process, involving several steps 3,38. The first step in the cascade is ubiquitin activation by E1 (ubiquitinactivating enzyme) followed by ubiquitin delivery to (ubiquitin-conjugating enzyme) (a). The second step involves complex formation by -Cys~, E3 (ubiquitin ligase) and the substrate (b). These initial steps involve formation of thiol esters between the active-site cysteines of E1 and enzymes and the carboxy-terminal carboxylate of ubiquitin. The third step (c) comprises transfer of ubiquitins to the substrate lysine(s) to earmark the substrate with a polyubiquitin chain. In the fourth step of the pathway (d), a polyubiquitylated substrate is released from the E3. Proteasomes recognize the polyubiquitin chain as a signal to de-ubiquitylate and destroy the substrate. The fifth step seals the fate of a doomed protein (e). The proteasome unfolds the substrate in ATP-dependent manner, removes the ubiquitin chain through a proteasome-associated ubiquitin hydrolase activity, and threads the unfolded protein into the proteasome chamber, where the protease active sites are located. The ubiquitin molecules are recycled, and the peptides generated are used in major histocompatibility class I-coupled antigen presentation or degraded to amino acids that are recycled for new protein synthesis. MDM2, double minute 2; CF, KP1 Cullin F-box; KP, -phase kinase-associated protein; RBX, RING-box protein. NATURE REVIEW DRUG DICOVERY VOLUME 5 JULY

5 Neddylation Covalent attachment of the ubiquitin-like protein NEDD8 (RUB1) to another protein. challenging because of difficulties with identification of small-molecule-binding pockets 52. Although it might be difficult to custom-tailor a small molecule that would bind the E1 linker and prevent its rotation, this domain of E1 is localized on the molecular surface and therefore would be available to small molecules. A functional or biophysical screen for small-molecule inhibitors of E1 ubiquitin transfer could therefore possibly reveal stabilizers of the linker that could be utilized for nonspecific silencing of ubiquitylation pathway(s). Assuming that it is possible to develop a specific inhibitor of E1 enzymes, most likely through blocking access of ATP, the question arises as to the biological consequences of such inhibition. Previous studies in mammalian cells indicate that temperature-sensitive mutations in E1 arrest the cell cycle in late phase and G2, indicating that E1 is required for cell proliferation. imilarly, mutations in E1 NEDD8 effectively block cell division in Caenorhabditis elegans 53, and both UBC12 and UBC9 are required for development in mammals. In principle, therefore, such E1 inhibitors might provoke cell-cycle arrest and therefore could be useful in hyperproliferative disorders such as cancer. However, it is likely that multiple pathways would be affected by inhibitors targeting the E1 machinery, and many of these pathways will be important for the functions of normal cells. One potential advantage of inhibiting E1 NEDD8 over E1 is that the only validated function for NEDD8 is activation of the cullin class of ubiquitin ligases (FIG. 3a), several of which are candidate targets for drug discovery (see below); however, it is possible that other targets of NEDD8 conjugation (such as von Hippel-Lindau (VHL) tumour suppressor 54 and p53 (REF 55,56)) exist as well, and this could potentially complicate clinical use of neddylation inhibitors. Nevertheless, it is hoped that inhibition of E1 NEDD8 would inhibit cullin-based E3 conjugation of substrates but would presumably not affect the many hundreds of ubiquitylation reactions that are not cullin-dependent. till, it remains to be determined whether a sufficient therapeutic window can be attained with an inhibitor of the E1 class of enzymes. Targeting E3 ubiquitin ligases Overview of E3s. The organization of the polyubiquitylation cascade is hierarchical. One common E1 enzyme activates ubiquitin for all cellular polyubiquitylation networks. Then, multiple combinations of several s and at least several hundred E3s are used to catalyse ubiquitylation of many more substrates in a target-specific manner (FIG. 4a). It is this carefully regulated pattern of interactions between E3s and their targets that provides the specificity necessary for appropriate degradation of ubiquitylation substrates. The exact composition of an E3 substrate complex and the details of ubiquitylation biochemistry depend on the E3 class involved. E3s can be divided into three major classes: RING-finger E3s; HECT (homologous to E6-AP COOH-terminus)-domain E3s; and U-box E3s. Each of these three classes of E3s has a distinct protein interaction domain (RING-finger, HECT or U-box domain) to bind s, and other domains in H E1 NH 2 NH 2 COOH ATP a AMP Pi Pi H AMP E1 E1 E1 b c AMP ATP d Figure 2 Biochemical steps in the ubiquitin-activation reaction. This step of the ubiquitylation cascade requires ATP binding to the ATP-binding cleft of E1. This process can be divided into four major sub-steps: (a) adenylation of the carboxyl terminus of a free ubiquitin molecule by E1; (b) rapid cis transfer of the E1-bound ubiquitin molecule from AMP to the active-site cysteine in E1, with subsequent release of free AMP; (c) adenylation of another free ubiquitin residue by the same Cys~-loaded E1 molecule; and (d) recruitment of (ubiquitin-conjugating enzyme) followed by transfer of the activated ubiquitin from the active cysteine of E1 to the catalytic cysteine of. the protein or protein complex function to recruit substrates (FIG. 4). E3s therefore serve to bring s into proximity of their substrates. As discussed below, the molecular interactions involved in the interaction of s with these three classes of recruiters are highly conserved. Biochemically, the HECT class differs from the RING-finger class in that HECT domains function directly in ubiquitin transfer by forming a thiolester intermediate with ubiquitin, which is then transferred to substrate. By contrast, RING-finger E3s do not seem to function directly via a thiolester intermediate but simply function as adaptors 60. E3 specificity modules determine which substrate is to be ubiquitylated. It might therefore be feasible to regulate the activity of selected proteins by manipulating their specific ubiquitin ligases. Although bortezomib targets all ubiquitylated proteins destined for degradation by the proteasome, attacking a single ubiquitin ligase might allow for manipulation of distinct pathway components, leading to more selective stabilization of a subset of ubiquitylated proteins. This increase in the specificity of therapeutic intervention could potentially boost the effectiveness of the treatment and eliminate some nonspecific side effects at the same time. Here we 600 JULY 2006 VOLUME 5

6 a b UBA3 ATP UBA3 NEDD8 APPBP1 Catalytic cysteine domain Adenylate domain c APPBP1 NEDD8 APPBP1 APPBP1 UBA3 NEDD8 UBC12 UBC12 UBA3 s ubiquitin-like domain NEDD8 NEDD8 Cullin UBA3 s ubiquitinlike domain C632 A CC A A CC A l E1 C216 A CC A A CC A l ThiF APPBP1 domain ThiF UBA3 domain UBA3 s ubiquitin-like UBA3 domain Figure 3 iquitin-activating enzymes as potential pharmacological targets. a The NEDD8-activating enzyme (E1 NEDD8 ) is composed of UBA3 and APPBP1. In the presence of ATP, NEDD8 is activated through formation of a thiol ester, which is then transferred to UBC12. UBC12 transfers NEDD8 to a single lysine residue in the cullin proteins to form an isopeptide linkage. b I Organization of the APPBP1/UBA3 heterodimer and comparison of the domain structures of E1 NEDD8 with E1. c tructure of the E1 NEDD8 complex bound to NEDD8 (APPBP1 is shown in yellow, UBA3 in magenta, NEDD8 in pink). ATP (space-filled) is buried in the adenylation pocket and is located near the carboxy-terminal glycine residue of NEDD8. d The ubiquitin-like domain of E1 NEDD8 (magenta; see panel b) uses a β-sheet surface common to all ubiquitin folds to bind to UBC12 (yellow). The catalytic cysteine of UBC12 is shown in a space-filled model. APPB1, amyloid-β precursor protein binding protein 1; NEDD, neural precursor cell expressed, developmentally down-regulated; UBA, ubiquitin-activating enzyme. d Catalytic cysteine E1 NEDD8 UBA12 will discuss a few ubiquitin ligases that are potentially attractive from a pharmacological or disease standpoint, and describe current strategies of manipulating a specific ubiquitin ligase activity, including the small-molecule inhibitors of ligase target interactions. RING-finger E3s as drug discovery targets. The RINGfinger domain is a subtype of zinc-finger domain found in a large number of proteins in mammals (more than 300 RING-finger genes in humans) 60,61. RING-finger proteins constitute the largest class of ubiquitin ligases. There are two major sub-classes within the RING-finger class of E3s: simple RING-finger E3s in which the RINGfinger and the substrate-binding domain are located on the same polypeptide; and cullin-based RING-finger E3s, which utilize RING-box protein 1 (RBX1) and RBX2 in complexes with modular cullin-dependent substrate receptor proteins (FIG. 4b e). Because of the diversity of these substrate receptor families (FIG. 4d), there might be more than 200 different RBX1-/RBX2-dependent E3s in humans, as described below. RING-fingers contain seven crucial cysteines and one histidine (C 3 HC 4 ), or six cysteine and two histidine residues (C 3 H 2 C 3 ), that hold two zinc atoms in a characteristic spatial conformation referred to as the cross-brace motif 61 (FIG. 5). Although the sequence of all and length of some spacers separating the zinc-binding residues is quite diverse, there is significant three-dimensional similarity between different RING-finger domains, especially in the components known to interact with s (FIG. 5). everal RING-finger-containing E3s have been implicated in disease processes, including cancer and neurodegenerative diseases. In cases in which RING-finger E3s are overexpressed in disease (for example, cancer), development of inhibitory compounds might be warranted. There are two potential approaches for blocking the activity of RINGbased E3s. One is to develop molecules that disrupt the interaction of the RING-finger with s. To date, such molecules have not been reported for any RING-finger E3. Alternatively, the identification of molecules that interrupt the interaction between the ubiquitylation substrate and the substrate interaction domain on the RING-finger protein might provide a means by which to selectively block degradation of one or a small number of proteins. As described below, recent results in the p53 arena indicate that this is possible, at least with the MDM2 RING-finger protein. p53 and MDM2. Human cells contain intrinsic tumoursuppressor networks that dynamically respond to shifts in gene-expression patterns and monitor genome status to prevent neoplastic transformation. However, these mechanisms are not perfectly reliable, and their deterioration opens the way to tumorigenesis. The p53 tumour suppressor is amenable to experimental therapeutic approaches 62 as a highly regulated node of multiple proliferation- and apoptosis-related networks 63. Approximately 50% of all human tumours contain mutations in the p53 gene 64, and the neoplasms that retain wild-type p53 frequently derange other elements of the p53 network via promotion of p53 degradation, for instance 65. Therefore, p53 is a well-recognized guardian of the genome that prevents mutagenesis and, consequently, carcinogenesis by promoting cell-cycle arrest or apoptosis (FIG. 6a) (for a review see REF. 66). There are multiple potential drug targets in this pathway 62, the most attractive being the major ubiquitin ligase of p53, MDM2. MDM2 is an oncogenic RINGfinger protein whose expression is transcriptionally induced by p53 to generate a negative feedback loop by degrading the p53 protein via the UP (FIG. 6a). Logically, inhibition of the MDM2 p53 interaction or inhibition of the conjugation of ubiquitin to p53 might result in stimulation of the tumour-suppressor activity of p53. Moreover, inactivating MDM2 might prove beneficial not only in tumours carrying wild-type p53: MDM2 might work as a ubiquitin ligase for other antioncogenic proteins 67. MDM2 disarms p53 by at least three complementary mechanisms: physically blocking the N-terminal transactivation domain on p53; promoting nuclear NATURE REVIEW DRUG DICOVERY VOLUME 5 JULY

7 a b E1 s E3s?? 1 E3 Ligase RING E3 Ligase 2 HECT 3 F-box E3 ligase Cullin RBX??? RING-finger ubiquitin ligase HECT ubiquitin ligase CF ubiquitin ligase ubstrates?? c ubstrate iquitin pecificity module Ring-finger subunit Cullin Rbx iquitin-transferring enzyme CF caffold d Cullins pecificity modules e RBX1 3 F-box Cullin 1 RBX CF1 (FBW7, KP2, β-trcp) VHL EB EC Cullin 2 RBX BTB Cullin 3 RBX DDB1/2 Cullin 4 RBX CF2 CF3 CF4 CF5 (VHL) KP2 CF ubiquitin ligase KP1 OC EB EC Cullin 5 RBX Cullin Figure 4 iquitin ligases as ubiquitylation specificity modules. a The ubiquitin cascade is pyramidal in design. A single E1-activating enzyme transfers ubiquitin to roughly three dozen s, which function together with several hundred different E3 ubiquitin ligases to ubiquitylate thousands of substrates. b Classes of ubiquitin ligases: single RING-finger E3s; HECT E3s; and multi-subunit RING-finger E3s, exemplified by the CF complexes. Only HECT-domain E3s form a covalent bond with ubiquitin during polyubiquitylation of their target proteins. pecific E3s discussed in this review are indicated. c CF E3 complexes consists of a scaffold-like cullin molecule; a RING-finger-containing subunit (RBX1 or RBX2), which functions to bind s; and a substrate-specificity module, which binds substrates. d Distinct cullins utilize structurally related specificity factors that are specific to each cullin. e The crystal structure of CF KP2 reveals that the carboxy-terminal domain of CUL1 binds RBX1, while the amino-terminal recruits the substrate-specificity module (in this case, a KP1/KP2 heterodimer) 58. β-trcp, β-transducin repeat-containing protein; BTB, broad complex/tracktrack/bric-a-brac; DDB, DNA-damage binding protein; FBW, F-box and WD40 domain protein; HECT, homologous to E6-AP COOH-terminus; RBX, RING-box protein; CF, KP1 Cullin F-box; KP, -phase kinase-associated protein; VHL, von Hippel-Lindau. export of p53 to keep this transcription factor away from its target genes 21 ; and ubiquitin-dependent p53 degradation 15,16,21,63. It has therefore been proposed that the p53 protein could be reactivated in p53-positive cancer cells via disabling MDM2. The resurrected p53 would then promote cell-cycle arrest, enhance apoptosis and synergize with conventional anticancer treatment. This concept is being addressed experimentally with increasing success. Initial attempts to crack the MDM2 p53 regulatory loop included the generation of anti-mdm2 antisense oligonucleotides 68 70, scaffold-attached peptides 71 and proteins 72 that were useful as a proof of principle. But the major challenge was to discover bioavailable small molecules that could block the MDM2 p53 interaction and potentially find their way into the clinic. These efforts were supported by an understanding of the anatomy and physiology of the p53 MDM2 interaction. From a structural standpoint, the MDM2 p53 interface 19 looks inviting for experimental drug design: MDM2 contains an open p53-binding pocket that might be accessible for small molecules (FIG. 6c). The p53-binding pocket on MDM2 is approximately 25 Å long and 10 Å wide. Two α-helices supported by a short β-sheet 602 JULY 2006 VOLUME 5

8 a X4 48 b X2 C C H C Zn X2 X2 3 X1 3 C C C C X9 39 Zn X2 H2 H1 III 50 H3 N W408 W87 P Loop2 P II I Loop1 F418 L C 3 Figure 5 RING-finger domain. a chematic representation of a C 3 HC 4 RING finger. Most RING fingers contain two zinc atoms (yellow) coordinated with cysteine or cysteine/histidine-rich clusters (red). The general consensus sequence is: C-X 2 -C-X C-X 1 3 -H-X 2 3 -C-X 2 -C-X C-X 2 -C, although some variations exist. b Overlay of crystal structure of the RING-finger domains found in c-cbl (blue) and RBX1 (red) reveals a significant degree of structural similarity in their -binding components. RBX, RING-box protein. N C mark the boundaries of the groove; the bottom is formed by two shorter, antiparallel α-helices that are roughly perpendicular to the pocket margins 19. Most aminoacid residues lining the interior surface of the groove are hydrophobic. The MDM2-binding domain of p53, which overlaps the p53 transactivation domain, is flexible in solution but adopts a stable α-helical conformation upon binding to MDM2 as a result of generating a network of hydrogen bonds between the hydrophobic MDM2 pocket and a few crucial amino-acid side chains on the p53 N terminus, including Phe19, Trp23 and Leu26 (FIG. 6c) The first small-molecule MDM2 inhibitors Nutlins (cis-imidazoline derivatives) were identified in a chemical library screen 73 for small molecules able to block the interaction between p53 and MDM2. Based on their structural, biochemical and pharmacodynamical parameters, Nutlins seem to be bona fide anti-mdm2 compounds. tructurally, they mimic the spatial conformation of the crucial MDM2-interacting residues on p53 and therefore are able to occupy the p53-binding pocket and displace p53 from it 73 (FIG. 6c). As expected, Nutlins activate p53-dependent cell-cycle arrest and apoptosis in cancer cell lines. Importantly, Nutlins are effective in vivo upon oral administration: they halt the growth of nude mouse tumour xenografts without noticeable toxicity to healthy tissues 73. These findings indicate reasonable bioavailability of these small-molecule MDM2 inhibitors. Another p53-stabilizing small molecule (RITA; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) was initially found to have anticancer activity in the National Cancer Institute Anticancer Drug creen 74 and recently scored as a hit in another functional chemical library screen designed to identify compounds that specifically arrest growth of a p53-positive cancer cell line 75. RITA is unrelated to the Nutlins both structurally and functionally (FIG. 6b). In contrast to the Nutlins 73, RITA does not seem to bind MDM2. Instead, it binds the N terminus of p53 and, although no definitive structural data are currently available, it seems to either prevent the recognition of p53 by MDM2, stabilize the N-terminal α-helix domain of p53 in the MDM2 groove, or both 75. Interestingly, RITA seems to prevent p53 from interacting with other regulatory proteins 75, such as p53-associated parkin-like cytoplasmic protein (PARC, the p53 cytoplasmic anchor) and p300, which, in addition to acetylating p53 (REF 76,77), can promote p53 polyubiquitylation by MDM2 as p53 s E4 78,79. The RITA-stabilized p53 is transcriptionally active as it can promote expression of endogenous p53-target genes 75. Most excitingly, RITA slows down the growth of mice tumour xenografts in a dose-dependent manner 75, although oral bioavailability of RITA was not tested in this initial study. One question that has not been addressed is whether RITA and Nutlins might show synergistic antitumour activity in the mouse tumour xenograft model, because they attack different facets of the MDM2 p53 recognition process 73,75. Furthermore, the interaction between RITA and p53 should be looked at more closely by NMR or crystallography to help understand the mode of RITA-mediated p53 activation, especially given that the initial attempts to detect RITA p53 interaction by NMR produced ambiguous results 80,81. It is also necessary to understand why administration of RITA causes cell-cycle perturbations and slight increase of apoptosis in p53-negative cells 75. Does this mean that RITA targets other cellular proteins for example, other p53-related proteins such as p63 or p73? Finally, possible side effects of long-term Nutlin/RITA administration should be addressed in the animal model, especially because furan derivatives were previously claimed to be mutagenic o although it is clear that small molecules can be generated that have the desired effect of activating p53 via suppression of its degradation, further study is required to determine whether these molecules will be useful in the treatment of human cancer. NATURE REVIEW DRUG DICOVERY VOLUME 5 JULY

9 a b Br Br HO Oncogene activation ARF O N N N Nucleolar sequestration O N O Nutlin MDM2 OH O CH 3 RITA Phosphorylation by DNA-damage kinases -dependent degradation Transcriptional activation OH c p53 p53 MDM2 Cell-cycle arrest Apoptosis TOP Nutlin MDM2 Figure 6 p53 MDM2 interaction as a therapeutic target in human cancer. a implified view of p53 signalling pathway. Genotoxic stress activates a network of protein kinase pathways that converge on the p53 protein. Although unphosphorylated p53 is rapidly degraded via the ubiquitin proteasome system, phosphorylated p53 cannot bind its primary E3 ligase (MDM2) and is stabilized. tabilized p53 arrests the cell cycle, and promotes DNA repair and apoptosis, depending on the cellular context. Failure of the p53 response is the most common event in human cancers, and restoration of p53 stabilization is a well-established potential anticancer strategy. b tructure of Nutlin (left) and RITA (right), the first small-molecule inhibitors of the p53 MDM2 interaction 73,75. c tructure of the Nutlin p53 complex 19. Left: the sides of p53-binding groove in MDM2 (red) is limited by two α-helices and a short β-sheet while the bottom is formed by two shorter α-helices perpendicular to the pocket sides. The amino-terminal domain of p53 (green) is stabilized in α-helical conformation upon binding to MDM2 due to a network of hydrogen bonds trapping three crucial residues of p53, Phe19, Trp23 and Leu26. These crucial p53 residues point towards the bottom of the groove. Right: Nutlin prevents interaction between p53 and MDM2 by mimicking the conformation of Phe19, Trp23 and Leu26 of p53 to block the MDM2 p53-interacting domain 73. MDM2, double minute 2. Parkin. In Parkinson s disease, the dopaminergic neurons of substantia nigra degenerate and die. Loss of these neurons leads to partial denervation of the striatum. The clinical consequence is impaired extrapyramidal control of skeletal muscle movement on multiple levels, which manifests principally as rest tremor, muscular rigidity and bradykinesia. Although some of the Parkinson s symptoms are initially controlled by dopamine precursors and agonists that boost the activity of remaining dopaminergic neurons in the substantia nigra, the disease eventually progresses towards a patient s incapacitation. The malfunction of the ubiquitin proteasome pathway leading to improper handling of misfolded proteins likely contributes to the pathogenesis of Parkinson s disease. Lewy bodies, the histopathological hallmark of Parkinson s disease, contain ubiquitin and filamentous aggregates of denatured proteins including α-synuclein. There is ongoing debate as to whether this collapse of the UP in patients brains is a consequence of genetic insults, environmental influences such as oxidative stress or toxin exposure, or both. ince Parkinson s disease most probably represents a constellation of diverse neurodegenerative syndromes that result in clinically similar outcomes rather than a single pathological entity, both hypotheses seem to be true, at least in some cases. (For more a detailed discussion of diagnosis, pathogenesis and genetics of Parkinson s disease, see REF 9 13,86 92.) Approximately 50% of the patients with a specific type of juvenile Parkinson s disease carry mutations in a gene named parkin 93, which encodes a RING-finger E3. The parkin molecule contains an N-terminal l (ubiquitinlike) domain (which has been postulated to mediate parkin s interaction with the proteasome) and two C-terminal RING-finger domains that flank an in-between-ring (IBR) domain. The C-terminal parkin domain binds putative ubiquitylation substrates as well as other interacting molecules, such as U-box-containing E3 ligase, carboxyl terminus of the HC70-interacting protein (CHIP) and the chaperone heat-shock protein 70 (HP70). It seems that parkin protects neurons and glial cells from excitotoxic insults, presumably by acting as an E3 for as yet unknown proteins. Parkin-knockout mice show some evidence of neurotoxicity, although in a manner that is not reminiscent of Parkinson s disease patients. parkin-mutant Drosophila show muscle degeneration and decreased lifespan, which is possibly due to mitochondrial failure that results in oxidative damage of the tissues 94,95 ; the Drosophila phenotype can be rescued by expression of human parkin 96. Interestingly, parkin gene therapy has also been shown to rescue the rat model of Parkinson s disease (α-synucleinopathy) 97. Although it is not known whether and how abnormal parkin function contributes to the pathogenesis of sporadic Parkinson s disease in humans, the hope is that research on parkin could at least pinpoint the affected pathways and therefore provide targets for therapeutic manipulation. What is the mechanism of parkin s neuroprotective function in the central nervous system? A major limitation in our understanding of parkin is that we currently do not know what the relevant substrates of its E3 activity are. everal candidate proteins including Parkin-associated endothelin receptor-like receptor (PAEL-R, an endoplasmic reticulum-associated, misfolding-prone protein of unknown function), α-synuclein 98, misfolded dopamine transporters and polyglutamine-repeat proteins, cyclin E, synaptotagmin XI and tubulin have been suggested to be substrates, but the relevance of these proteins is in question because the abundance of none of the candidate substrates is increased in neurons of mice lacking parkin. Point mutations in both the first and second RING-finger domains of parkin, as well as mutations in the ubiquitinlike domain, are found in patient families. This genetic clue suggests a role for ubiquitin ligase activity in the function of parkin that promotes neuronal survival. As such, any small-molecule therapeutics targeting parkin would need to be capable of promoting activity of an otherwise non-functional or partially functional allele. With the existing understanding of RING-domains and their interaction with s, it is not clear how this would be accomplished, other than by gene therapy, which would be extremely challenging in the case of Parkinson s disease. In principle, it might be possible to identify small molecules 604 JULY 2006 VOLUME 5

10 Endoreduplication Duplication of the genome without mitosis, which results in an increase in the nuclear DNA content, permitting amplification of the genome of specialized cells. that facilitate recruitment of s to a defective parkin RING-domain, but such molecules would probably be allele-specific and would therefore be of limited utility. This exemplifies the situation for many E3s whose loss of function promotes disease, including BRCA1. CF ubiquitin ligases. The CF (KP1 Cullin F-box) complex is a multi-subunit ubiquitin ligase that uses interchangeable specificity factors (F-box proteins) to recognize specific substrates 99. These complexes contain four core subunits (FIG. 4c,e). Cullin 1 (CUL1) serves as a scaffold for assembling the ubiquitin-conjugating machinery and the substrate. The C terminus of CUL1 interacts with the RING-finger protein RBX1 (also called ROC1), which in turn interacts with ubiquitin-conjugating enzymes. The N terminus of CUL1 binds to KP1, which in turn binds to F-box proteins. The human genome contains at least 68 F-box proteins, and each of them is likely to target multiple substrates for degradation 100. Other cullins use different classes of substrate specific adaptors, including BTB proteins for CUL3 and suppressor of cytokine signalling (OC)-box proteins for CUL5 (FIG. 4d) 87,88. There are more than 170 BTB proteins and more than 45 OC proteins encoded by the human genome. However, we are only beginning to decipher the complicated molecular networks linking CF E3s to their substrates. Even for the most thoroughly studied CUL1-based CF complexes, only a small number of F-box protein substrate pairs have been identified. Most prominent among these are CF KP2, CF β-trcp and CF FBW7. For these widely studied pathways, it is clear that a single F-box protein can have multiple substrates, and in some cases such substrates can actually act antagonistically to each other. For example, β-transducin repeat-containing proteins (β-trcps) promote the degradation of distinct proteins that either activate proliferation and survival of mammalian cells (such as β-catenin 101,102 ), or inhibit such proliferation and survival (such as inhibitor of NF-κB (IkB) 103 ). Clearly, more research is needed to understand which proteins are targeted by specific CF E3s and, consequently, to determine which CF complexes deserve more attention from a therapeutic standpoint. The majority of F-box proteins interact with their targets in a manner that depends on post-translational modification of the target, most commonly phosphorylation 44,86. F-box proteins contain C-terminal protein-interaction domains that interact specifically with modified targets. These interaction domains include WD40 domains and leucine-rich repeats (LRRs) which are thought, in many cases, to represent phosphopeptide-interaction motifs. Indeed, WD40 motifs derived from F-box proteins have been shown to interact directly with small phosphopeptides referred to as phosphodegrons Within each class of WD40 or LRRs, the specificity for phosphopeptides differ, so these domains can be thought of as scaffolds that are used during evolution to adapt new phosphopeptide-binding surfaces with distinct specificities. Because the interaction surface of substrate and the F-box protein provides the most specific point for therapeutic intervention, understanding how substrates and F-box proteins interact has been an active area of research. CF KP2. KP2 is an F-box protein that specializes in the degradation of several negative cell-cycle regulators, such as the cyclin-dependent kinase inhibitor p27 (REF 22,26). p27 is phosphorylated on threonine-187 by cyclin-dependent kinases (CDKs) to generate a phosphodegron that is recognized by CF KP2. Interestingly, KP2-knockout mice are viable but have significantly decreased body mass 25 (which is a reverse phenocopy of p27 loss ), and the phenotype seen in KP2 / hepatocytes is reversed by p27 disruption 24. KP2 / cell lines show a reduced growth rate and a tendency to undergo endoreduplication of their genetic material in concordance with stabilization of p27 (REF. 25). Interestingly, the small CDK-interacting protein CK1 is a subunit of the CF KP2 complex. CK1, like KP2, is required for p27 degradation, and CK1 seems to be important for the interaction between KP2 and p27 (REF. 60). Mice and cells lacking CK1 have phenotypes quite similar to those produced by loss of KP2. These findings implicate a crucial role of this CDK-binding protein in the CF KP2 -dependent pathways. KP2 has also been implicated in ubiquitin-dependent degradation of other cell-cycle regulatory molecules, such as a retinoblastoma-family protein p130 (REF 107,108), the CDK inhibitors p21 (REF. 109) and p57 (REF. 110), and the cell-cycle inhibitory transcription factor forkhead box protein O (FoxO) 111,112. As expected from its role in the destruction of antiproliferatory molecules, KP2 is a putative proto-oncogene 113. Indeed, KP2 is overexpressed in many human cancer types 36. Forced overexpression of KP2 in mouse prostate gland 114 and T-lymphocyte progenitors 115 stimulates tumour formation, confirming the oncogenic potential of this F-box protein. In all cancers analysed to date, overexpression of KP2 correlates with loss of p27 expression. KP2 therefore seems to be a valid therapeutic target and several pharmaceutical companies have active drug discovery programmes targeting this ubiquitin ligase. Downregulating KP2 activity might prove useful in the therapy of cancers that gain a malignant phenotype resulting, in part, from KP2 overexpression (FIG. 7a). On the ex vivo level, it has been shown that inhibiting KP2 by RNA interference or intracellular injection of anti-kp2 antibodies slows down proliferation of cancer cell lines. These findings have been confirmed in vivo in a tumour xenograft model 116,117,119, further reinforcing the possibility that KP2 is a potential pharmacological target. The recent crystal structure of the KP1 KP2 CK1 phospho-p27 peptide complex 120 provides insight into structural features in the complex that might be exploited for the development of inhibitors of p27 degradation. KP2 contains an N-terminal F-box motif (three-helix bundle) that interacts with an antiparallel helical cluster in KP1 (FIG. 7b). KP2 also has C-terminal leucine-rich repeats, which function in CK1 and substrate recognition (FIG. 7b). Major contacts between phosphorylated T187 in the p27 peptide and the CF complex occur via CK1, which binds, in turn, to the concave surface of the KP2 leucine-rich repeats. Multiple interactions maintain the association of CK1 NATURE REVIEW DRUG DICOVERY VOLUME 5 JULY

11 a CDK inhibition b CK1 p27 CF KP2 p130 p27 KP1 KP2 Cell-cycle arrest d c R285 N P P DGLD IκB DGIH β-catenin DGY Emi1 DGFCL Cdc25A R521R474 R431 Figure 7 Recognition of phosphorylated substrates by CF ubiquitin ligases. a KP2 targets cell-cycle inhibitors (p27 and p130) for ubiquitin-dependent degradation and therefore can work as an oncogene when hyperactive. b tructure of the CF KP2 complex bound to the coactivator protein CK1 and a phosphodegron from the p27 protein, which is targeted for destruction by the CF KP2 complex. KP1 is shown in yellow, KP2 in magenta, CK1 in pink, and p27 phosphodegron in green. The position of phosphothreonine 187 in the p27 phosphodegron is shown in red (stick model). The phospho-t187 moiety interacts predominantly with a basic pocket in the CK1 protein. CK1, in turn, interacts with the concave surface of the leucine-rich repeats in the carboxy-terminus of KP2. c equences of doubly phosphorylated phosphodegrons found in targets of the CF β-trcp ubiquitin ligase. d tructure of the β-catenin phosphodegron bound to the WD40 repeats of β-trcp 123. The surface of β-trcp is displayed in an electrostatic view with basic surfaces in blue and acidic surfaces in red. The β-catenin phosphodegron (orange) is bound to a highly basic surface of β-trcp and the phosphate groups (green and red) interact directly with R285 and R431. β-trcp, β-transducin repeat-containing protein; CDK, cyclin-dependent kinase; CK, CDC kinase subunit; CF, KP1 Cullin F-box; KP, -phase kinase-associated protein. with KP2, and the p27 peptide with CK1. There are three potential targets for small molecules within the CF KP2/CK1 complexes: the KP1 [F-box]KP2 interaction surface 58 ; the cluster of CK1 residues involved in substrate recognition; and the cluster of KP2 residues that interact with CK1. Although disabling the KP1 KP2 interaction might be possible in theory, sequence conservation within the F-box might limit specificity. The interaction of the F-box with KP1 involves interactions between multiple helical bundles, and many of the key conserved residues in the F-box are hydrophobic and interact with a hydrophobic surface in KP1. It is unclear whether molecules could be designed that would specifically bind to one F-box but not the dozens of other F-box proteins in the cell. Perhaps it is more likely that molecules that disrupt the interaction of either p27 or CK1 with KP2 would be identified. However, the interaction between CK1 and KP2 occurs over a relatively flat surface and involves ~1,200 Å 2 of buried surface area. The identification of molecules blocking the KP2 CK1 interaction could therefore be challenging. Nevertheless, the biological rationale for developing an CF KP2 inhibitor is clear, making attempts to drug this unconventional target potentially worthwhile. C CF β TRC P. β-transducin repeat-containing proteins 1 and 2 (β-trcp1 and β-trcp2) are members of the F-box/ WD40 subfamily of F-box proteins and are arguably the best-understood mammalian F-box proteins. β-trcp1 and 2 are the products of distinct genes but are ~85% identical with each other and are thought to be largely redundant in function 43. Initial interest in CF β-trcp as a drug target came from its linkage with the nuclear factor-κb (NF-κB) pathway, which is crucial to both cellular survival functions as well as the response of cells to inflammatory agents. NF-κB is held in an inactive form within the cytoplasm through association with IκB. In response to cytokines and other extracellular signals, the IκB kinase complex phosphorylates IκB, thereby promoting its degradation through the UP; this allows re-localization of NF-κB into the nucleus, where it activates the expression of genes important for cytokine and survival responses. The identification of CF β-trcp as the E3 for IκB 60,103,121 suggested that it might be a target for molecules that act as anti-inflammatory agents by blocking IκB degradation. Early studies indicated that the interaction of IκB with β-trcp occurred directly through a phosphodegron generated on IκB upon phosphorylation of 32 and 36 by IκB kinase, and this interaction involved C-terminal WD40 repeats in β-trcp 102,121. This phosphodegron conforms to a canonical phosphodegron sequence recognized by β-trcp (DpGφXp, where φ is a hydrophobic residue) (FIG. 7c). The potential use of β-trcp as a target for antiinflammatory agents is complicated by the diverse biological settings in which it functions. It is now clear that β-trcp promotes the ubiquitylation of numerous other proteins, including the oncogenic transcription factor β-catenin, the cell-cycle regulatory proteins Early mitotic inhibitor 1 (EMI1) and cell division cycle protein 25A (CDC25A), and the progesterone receptor 60. Each of these proteins contains a phosphodegron that is quite similar to that found in IκB (FIG. 7c). Previous studies have demonstrated that an inability to destroy β-catenin can promote the transcription of genes such as c-myc and cyclin D1 that lead to cellular transformation (reviewed in REF. 60). Indeed, mutations in the phosphodegron of β-catenin have been identified in various types of human cancer and these proteins are highly stable, leading to inappropriate transcription of β-catenin target genes. o the question arises of how β-trcp recognizes its substrates and whether it is possible to identify small molecules that would inhibit ubiquitylation of IκB but not β-catenin. Given the conservation of the phosphodegron sequence in various β-trcp substrates, it seems likely that the binding site for β-trcp will be similar for different substrates. Indeed, previous work has revealed the structure of β-trcp bound to the phosphodegron from β-catenin 102 (FIG. 7d). Both phosphoserine residues in the phosphodegron, as well as the conserved aspartate residue at position 1, make crucial contacts with a basic surface of the WD40 repeats; furthermore, the hydrophobic residue at position 4 in the phosphodegron (FIG. 7c) is buried in a hydrophobic pocket at the central cavity of the WD40 repeat. The residue at position 5 in the 606 JULY 2006 VOLUME 5

12 a CBL b Catalytic domain Hinge WWP1 UBC7 100 E6AP UBC7 Catalytic domain E6AP Hinge UBC7 Figure 8 tructure of HECT-domain ubiquitin ligases. a The UBC7 (red) uses the same structural motif to interact with two different classes of E3s: the CBL RING-finger (green) and the E6-AP HECT domain (blue). b Comparison of the structures of the HECT domains of WWP1 and E6-AP indicate a possible conformational change important for ubiquitin transfer. An unstructured hinge region (red) links the catalytic domain (blue) containing the active site cysteine (yellow) to the UBC-binding domain (green). UBC7 bound to E6-AP is shown in pink with its catalytic cysteine in yellow 143. HECT, homologous to E6-AP COOH-terminus; UBC, ubiquitin C. phosphodegron (FIG. 7c) is the most variable, reflecting the fact that it is directed away from the β-trcp surface. Consistent with an overlapping binding site, mutations in key residues in β-trcp that contact the β-catenin phosphodegron block the ability of CF β TRCP to promote IκB ubiquitylation in vitro 102. Given the architecture of the β-trcp β-catenin phosphodegron complex and the structural relationship between phosphodegrons in diverse β-trcp substrates, the development of inhibitors that selectively affect one substrate and not one of several others is likely to be a challenge. CF FBW7. Like β-trcp, FBW7 is a WD40-containing F-box protein that functions within the CF complex to promote the degradation of proteins primarily involved in cell proliferation, including oncogenes. However, unlike β-trcp, FBW7 seems to be quite susceptible to mutation during transformation. The substrates of FBW7 include cyclin E, c-myc, c-jun, Notch and sterol regulatory element-binding proteins (REBPs) 99. Each of these proteins contains a doubly phosphorylated phosphodegron with the consensus sequence ptpxxp, which is thought to bind to the surface of FBW7 in a manner quite similar to that seen with the β-trcp β-catenin complex. This interface is central to FBW7 function and is quite revealing in what it tells us about transformation mechanisms. First, arginine residues in the FBW7 WD40 repeats make up the bulk of the interaction surface with the phosphodegron, based on structural analysis of the yeast orthologue of FBW7, CDC4, the structure of which has been solved bound to the cyclin E phosphodegron (reviewed in REF 60,99). Importantly, the arginines that make up this binding pocket are targeted for mutation in several types of human cancers 5,122, providing genetic evidence of the crucial nature of this binding site in promoting the degradation of these oncogenic proteins 99. Likewise, several targets of FBW7 have been shown to be mutated within the phosphodegron that is recognized by FBW7. These mutations either abolish the capacity of these sequences to be phosphorylated or block the capacity of the phosphorylated proteins to be recognized by FBW7. For example, the most frequent mutation of c-myc in cancer is seen in the T58 residue, which serves as the first phosphoacceptor residue in the c-myc phosphodegron that is bound by FBW7 (reviewed in REF. 99). Recent experiments in mice indicate that mutation of T58 in c-myc leads to increased stabilization of c-myc, as expected based on its resistance to destruction by FBW7, but also acquires new activity which reduces its apoptotic tendencies 123. This mutation in c-myc therefore becomes highly transforming. imilarly, the v-jun mutation represents mutations in the viral JUN protein that block its capacity to be phosphorylated in the degron, leading to stabilization and higher transforming activity 30. As with parkin, the loss of FBW7 seems to be detrimental to cells, leading to genomic instability 122. Despite our understanding of the biochemical requirements for its recognition of substrates and its role in the degradation of oncoproteins, it will remain a challenge to develop approaches that reactivate mutant alleles. Given that many oncoproteins, including c-myc, accumulate in cells containing FBW7 mutations, it seems plausible that drugs that are synthetically lethal with loss of FBW7 might be identified. However, because FBW7 mutations are relatively rare, it is not clear how much of an impact such molecules would have in the clinic. CUL2 VHL. The key role of angiogenesis in malignant tumour growth has been firmly established, and it is reasonable to expect that an anti-angiogenic protein such as VHL would have tumour-suppressor activity. Indeed, the VHL ubiquitin ligase, which inhibits angiogenesis under normoxic conditions, is mutated in a familial cancer susceptibility syndrome, von Hippel- Lindau syndrome. Moreover, somatic mutations of VHL genes are found in a fraction of some human cancers, such as sporadic clear-cell renal carcinomas 124. It is thought that loss of VHL activity forces normoxic cells to behave as if they were exposed to hypoxia, and the burst of vascular endothelial growth factor (VEGF) synthesis resulting from VHL mutations is thought to stimulate the formation of new blood vessels. Not surprisingly, an abnormally dense meshwork of capillaries are a feature of tumours found in the VHL patients, and sporadic renal-cell carcinomas are highly vascular as well. NATURE REVIEW DRUG DICOVERY VOLUME 5 JULY