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PhD Lit Review

  1. 1. ARTICLE IN PRESS G Model YSCBI-848; No. of Pages 9 Seminars in Cancer Biology xxx (2009) xxx–xxx Contents lists available at ScienceDirect Seminars in Cancer Biology journal homepage: Review Small-molecule inhibitors of MDM2 as new anticancer therapeutics Michael P. Dickens, Ross Fitzgerald, Peter M. Fischer ∗ School of Pharmacy & Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK a r t i c l e i n f o a b s t r a c t Keywords: It has long been known that traditional anticancer radio- and chemotherapies in part work through direct Cancer therapy or indirect activation of the p53 tumour suppressor pathway. However, many of these strategies are non- Drug discovery and development selective and genotoxic. The emerging understanding of the pathways that regulate p53 has led to the MDM2 inhibitor notion that it should be possible to activate the p53 pathway in ways that are inherently nongenotoxic. MDMX Important targets for pharmacological interference in this respect are MDM2 and MDMX, key negative Nongenotoxic p53 activation Protein–protein interaction regulators of p53. Genetic and pharmacologic studies suggest that blocking the physical interaction of E3 ubiquitin ligase inhibitor these proteins with p53, or inhibiting the catalytic role of MDM2 in tagging p53 for proteasomal degra- Proteasomal degradation dation, both of which lead to an increase in the transcriptional activity of p53, may indeed be an efficient Nutlin and safe way to eradicate tumour cells that retain wild-type p53. Here we review the rationale for such Benzodiazepinedione strategies, as well as the current state in the discovery and development of drugs that reactivate p53 Spiro-oxindole by inhibiting its inhibitors MDM2 and MDMX. The first compounds that have been shown in model systems to be able selectively to kill cancer cells in this way are now entering clinical trials and the promise of MDM2 inhibitors as a new therapeutic anticancer modality should therefore become clear in the not-too-distant future. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction While MDM2 controls the protein levels of p53, it is itself under transcriptional control of p53 and the two are thus linked in a tight The p53 tumour suppressor protein is a transcription factor that autoregulatory feedback loop [7,8]. Depending on the nature of is activated in response to cellular stress. Depending on the severity genotoxic or nongenotoxic stress a cell may experience, the nega- of the threat to genome integrity, p53 then imposes cell cycle arrest tive regulation of p53 by MDM2 is interrupted in several different or apoptosis. Because p53 has strong growth-suppressive activity, ways. Most importantly, the functions of MDM2 in p53 suppression it must be tightly regulated to allow normal cells to function. This are inhibited upon association with the ARF protein, an alterna- is achieved to a large extent by a protein known as murine double tive transcript of the INK4a/ARF tumour suppressor locus, which is minutes-2 (MDM2), so called because its gene was first discovered induced upon oncogenic stress (reviewed in [9]). Similarly, MDM2 in DNA associated with paired acentric chromatin bodies, termed is inhibited upon ribosomal stress by the ribosomal proteins L5, L11, double minutes, in spontaneously transformed mouse 3T3 fibrob- and L23 [10,11]. Furthermore, MDM2 is regulated through post- lasts [1]. The corresponding human protein is sometimes referred translational modifications, including autoubiquitinylation [12], to as HDM2 but here we shall use the abbreviation MDM2 regard- sumoylation, and multi-site phosphorylations by a range of kinases, less of species. especially the DNA damage-induced kinases (reviewed in [13]). MDM2 regulates p53 in at least three different ways, i.e. at the MDMX (also known as MDM4) is a nonredundant homologue levels of p53 function, protein stability, and subcellular location. of MDM2 that also regulates p53 [14] and is overexpressed in MDM2 forms a protein–protein interaction with the N-terminal many cancers [15]. Unlike MDM2, however, MDMX expression is transcription activation domain of p53, thus blocking p53 tran- not regulated by p53 and MDMX is thus not part of the negative scriptional activity [2,3]. Furthermore, MDM2 is the E3 ubiquitin feedback loop with p53. MDMX also lacks intrinsic ubiquitin ligase ligase that promotes ubiquitin-dependent proteasomal degrada- activity but is itself a target for MDM2 ubiquitinylation. It forms tion of p53 [4,5]. Finally, MDM2 causes nuclear export of p53 into heterodimers with MDM2, which enhances the ability of MDM2 to the cytoplasm of the cell, moving p53 away from its site of action induce p53 degradation [16]. MDMX binds p53 at the same site and [6]. with similar affinity as MDM2 and in so doing blocks p53 transcrip- tional activity. The functions of p53 are ablated in all cancers as a means of ∗ Corresponding author. Tel.: +44 0115 846 6242; fax: +44 0115 951 3412. evading apoptosis, either by disabling p53 directly through muta- E-mail address: (P.M. Fischer). tion or deletion, or indirectly by alterations of various components 1044-579X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2009.10.003 Please cite this article in press as: Dickens MP, et al. Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology (2009), doi:10.1016/j.semcancer.2009.10.003
  2. 2. ARTICLE IN PRESS G Model YSCBI-848; No. of Pages 9 2 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx of the pathways that regulate p53 [17]. About half of all cancers has helped to reshape our perception of protein–protein interac- retain wild-type p53 [18] and in these the normal regulation of p53 tions as drug targets, since in many cases these contain so-called is sometimes disrupted through direct overexpression of MDM2 hot spots, where the binding energy of protein–protein interactions (in ca. 7% of cancers [19]). MDM2 overexpression due to gene is concentrated [35]. amplification is especially frequent (ca. 30%) in human osteogenic sarcomas and soft tissue sarcomas [20]. 3.1. Early work with peptide antagonists of the p53–MDM2 Because of the central role of p53 in tumour suppression, interaction nongenotoxic therapeutic strategies that activate p53 in one way or another are highly desirable. Depending on p53 status this should A detailed discussion of peptide and peptidomimetic be able to be achieved in various ways. For example, proof-of- approaches to modulate the p53–MDM2 interaction has been concept studies have shown that mutant p53 might be able to be provided elsewhere [36–38] and we shall only summarise in stabilised or otherwise reactivated pharmacologically [21–23]. In outline some of the early peptide optimisation studies that defined tumours that retain a functional p53 pathway, on the other hand, the pharmacophore model which provided the platform for preventing p53 degradation is an attractive option. subsequent development of nonpeptide inhibitors. There are many potential therapeutic targets within the p53 Initially, screening of phage-displayed peptide libraries led to pathway, downstream of the stress response, which offer the the discovery of a 12mer peptide MPRFMDYWEGLN with 28-fold possibilities of nongenotoxic p53 activation and bypassing the par- potency increase compared to the corresponding p53 sequence ticular defects that could render an upstream target ineffective. 16 QETFSDLWKLLF27 [39]. Interestingly, only the three key inter- The MDM2–p53 regulatory system is one such target. Modula- acting residues (bold type in preceding sequences) were conserved tion of this system with small molecules is a very active area of between these peptides and the basic pharmacophore feature of research. Here we review current progress in the development of all potent ligands is indeed three suitably oriented hydrophobic small molecules that inhibit the MDM2–p53 protein–protein inter- groups. Next, artificial amino acids were used to explore conforma- action or the ubiquitin ligase activity of MDM2. tional features. This led to the development of a highly optimised 8mer peptide that inhibited the p53–MDM2 PPI with low nanomo- lar potency, which represented a >1700-fold increase in affinity 2. Target rationale and therapeutic window compared with the 12mer p53 peptide [40]. Incorporation of a chloro group at the indole C6 position of the The key question for any therapeutic strategy that aims to acti- key Trp residue showed that better occupancy of the binding site vate the p53 response is whether of not this will result in a selective compared to the cognate ligand could be achieved with substantial effect on tumour cells as opposed to the cells of healthy tissues. potency gains. The effect of introducing helix-stabilising residues Such specificity of p53 to kill tumour cells, but not normal cells, showed the importance of a rigid scaffold in presenting the key appears to underlie the safety of p53 gene therapy, which has residues in a way that results in optimal shape complementarity gained approval in China and is now being developed elsewhere with the binding site. Again this feature was subsequently recapit- [24–29]. It has been shown that mice with a hypomorphic MDM2 ulated with nonpeptidic inhibitors. The increases in affinity brought allele produce only about 30% of the normal level of MDM2 and about by inclusion of charged nonnative residues indicated that fur- exhibit increased transcriptional and functional activation of p53 ther polar contacts not present in the native system could be made [30]. The effects of p53 under these circumstances are not lethal outside of the main binding cleft. as one might expect, although the animals are small and show A complex crystal structure of this high-affinity 8mer peptide p53-dependent apoptosis of lymphoid cells. Nevertheless they are bound to MDM2 was solved recently [41] and shows that the pep- viable, do not age prematurely, and are resistant to tumour forma- tide does indeed bind in the expected manner (Fig. 1). The structural tion [31]. features of this peptide have been inherited by subsequent small- Similarly, in vivo suppression of MDM2 using antisense molecule inhibitors, the best of which are those that mimic the oligonucleotides has been demonstrated to result in therapeutic peptide most closely. The optimised peptide also provided pharma- antitumour effects without overt toxicity (reviewed in [32]). From cological target validation, since it was somewhat permeable and these and other results [33] it is clear that the p53 pathway dif- thus able to reach its target MDM2 in intact cells. It was observed to fers significantly in normal and p53 wild-type cancer cells and induce apoptosis selectively in MDM2-overexpressing cancer cells that the latter are selectively sensitive to increases in p53 effec- via nongenotoxic p53 activation [42]. tor functions. This notion is enhanced by the results of extensive pharmacological studies, especially those using the nutlin pioneer 3.2. Small-molecule p53–MDM2 antagonists MDM2 inhibitors (discussed in more detail below), which also sug- gest that cancer cells are more susceptible to proapoptotic effects Because of their central role as pioneers for protein–protein of p53 than noncancerous cells (reviewed in [34]). interaction drug target modulators in general, inhibitors of the p53–MDM2 interaction have been reviewed extensively. We do 3. The p53–MDM2 interaction not intend to duplicate these efforts here but direct the interested reader to some of the most recent reviews [43–47]. One of these Prior to elucidation of the structure of the p53–MDM2 inter- gives an up-to-date summary, covers some 20 distinct classes of action it was thought that protein–protein interactions could not small-molecule p53–MDM2 inhibitors, and assesses critically to be effectively inhibited with membrane-permeable and otherwise what extent these have been validated, i.e. which can be regarded as drug-like small molecules because of the extensive size and poor genuine p53–MDM2 inhibitors and which operate to block MDM2 definition of protein interfaces. The X-ray crystal structure of a functions by different mechanisms [45]. complex between the N-terminal domain of MDM2 and a 12mer Of the small-molecule inhibitor series described to date, three peptide encompassing residues 16–27 of the p53 transactivation are of particular importance. The nutlins [48], the benzodi- domain showed that the bulk of the p53–MDM2 interaction in fact azepinediones [49–53], and the spiro-oxindoles [54,55] (important involved just three lipophilic residues of p53, buried in a well- representative members from these series are shown in Fig. 2) all defined hydrophobic surface cleft in MDM2, of a size that could bind MDM2 with low nanomolar affinity and induce cancer cell clearly be fully occupied by a small molecule [2]. This observation apoptosis in a p53-dependent manner. Typically these compounds Please cite this article in press as: Dickens MP, et al. Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology (2009), doi:10.1016/j.semcancer.2009.10.003
  3. 3. ARTICLE IN PRESS G Model YSCBI-848; No. of Pages 9 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx 3 stituents that closely mimic the 6-Cl-Trp modification discussed above in the context of peptide inhibitors. Because the p53-binding cleft of MDM2 is highly hydrophobic, minimal nonpeptide inhibitors are by necessity very lipophilic and thus lack aqueous solubility. Throughout their development, all of the inhibitors have thus evolved to include solvent-exposed polar groups that aid solubility. In the benzodiazepinediones, addition of such solubilising groups improved cellular potency, but at the expense of some binding affinity. In the case of the spiro-oxindoles, however, addition of a solubilising group to the core structure resulted in increased affinity as well as cellular activity, and led to the development of the most potent inhibitors. No detailed medic- inal chemistry has been disclosed about the nutlins but the three compounds presented in the original report [48] vary mostly in the solubilising group and a review of the MDM2 inhibitor patent lit- erature suggests considerable dependence of biological activity on the nature of this group [56]. Whether solubilising groups just act as property-improving appendages or become an additional pharma- cophore feature depends largely on their attachment point. In the Fig. 1. MDM2-binding mode of an optimised p53-derived peptide. Residues of case of MI-219 the solubilising chain makes additional hydropho- the p53 peptide (grey CPK sticks) are labelled (Ac3 C, cyclopropylglycine; 6-Cl-Trp, bic contacts outside the main binding cleft and the polar groups are 6-chloro-tryptophan; Pmp, phosphonomethylphenylalanine; Aib, aminoisobutyric thought to mimic contacts made by the Pmp or Glu residues of the acid). MDM2 is shown as a green CPK surface. Constructed from PDB entry 2GV2 [41]. This and subsequent illustrations showing 3D structures were prepared with optimised p53 peptide (Fig. 1). The binding modes of nutlins and the PyMOL programme (DeLano, W.L. The PyMOL Molecular Graphics System (2002) benzodiazepinediones show that their solubilising groups project on the World Wide Web differently and cannot make similar interactions (Fig. 3). The fact that all proteins and their binding sites are flexible is also evident from structural studies with MDM2. When no ligand is bound, the p53-binding cleft of MDM2 exists in a closed confor- are only active in cells that express wild-type p53, and p53 tran- mation, which opens upon binding to p53 [57]. A flexible lid covers scriptional products can be observed to be upregulated as a result. the cleft in the unbound state and is displaced upon p53 peptide Importantly, the presence of posttranslationally unmodified p53 binding but not upon binding small molecules [58]. Comparison of following treatment of cells with these compounds shows that they the MDM2 conformations in the various bound structures shown act in a nongenotoxic manner. Furthermore, optimised analogues in Fig. 3 clearly demonstrates that the small-molecule inhibitors from these compound series have all been shown to cause tumour bind to MDM2 so that it adopts a similar conformation as it does regression in xenograft models. upon binding p53. The partially closed form is evident in the case Although chemically distinct, all adhere to the basic hydropho- of the benzodiazepinedione inhibitor complex, which is the only bic three-pronged pharmacophore model. Each uses a unique, rigid, small-molecule inhibitor complex where the lid region is present heterocyclic scaffold to project three lipophilic groups into the in the MDM2 construct employed. three subpockets in the binding site that are occupied by the F19 , W23 and L26 residue side chains in the case of p53 as the ligand (Fig. 3). As these interactions are predominantly hydrophobic, alter- 3.3. Inhibition of the p53–MDMX interaction ing the size of the lipophilic binding groups in order optimally to fill the site in a shape-complimentary manner greatly increases It has recently transpired that because of the nonredundant but affinity. Potent members of all three inhibitor chemotypes bear overlapping functions of MDM2 and MDMX in the regulation of halide-substituted aromatic groups positioned to maximise these p53, and because of the comparatively frequent amplification of contacts. Indeed the nutlins rely entirely on hydrophobic contacts MDMX in cancer cells, an ideal nongenotoxic p53 activator should for their affinity. Several compounds contain chlorophenyl sub- inhibit both MDM2 and MDMX. However, it has been shown that Fig. 2. Chemical structures of potent small-molecule p53–MDM2 interaction inhibitor compounds. The groups that interact with the F19 , W23 , and L26 subsites of the p53-binding cleft of MDM2 are shown in green, red, and purple, respectively. Compare Fig. 1. Solubilising groups are indicated in blue. Please cite this article in press as: Dickens MP, et al. Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology (2009), doi:10.1016/j.semcancer.2009.10.003
  4. 4. ARTICLE IN PRESS G Model YSCBI-848; No. of Pages 9 4 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx Fig. 3. Binding modes of p53 and small-molecule ligands with MDM2. (a) An -helix of p53 (green) interacts with a well-defined binding pocket in MDM2 (grey CPK surface) predominantly through three side chains (green CPK sticks, labelled). Complexes of nutlin-2 (cyan), a closely related compound (yellow), a benzodiazepinedione inhibitor (purple), and the spiro-oxindole MI-219 (blue) with MDM2 are shown in b–e. (f): The ligand–MDM2 complexes were aligned and the ligands are shown superimposed using the same colour schemes as in a–e. Constructed from PDB entries 1YCR [2] (a), 1RV1 [48] (b), 1TTV [98] (c), and 1T4E [99] (d). The predicted binding mode of MI-219 [59] (e) was obtained through docking of a multiconformer database of the compound into an MDM2 model derived from 1YCR (using the programmes OMEGA2 and FRED from OpenEye Scientific Software, the small-molecule MDM2 inhibitors nutlin-3 and MI-219 have Recently, a crystal structure of a complex between MDMX and a 260- and >10,000-fold lower affinity, respectively, for MDMX than p53 peptide was solved, which shows why MDM2 inhibitors have for MDM2 [59]. While MDMX overexpression can prevent p53 reac- such low affinity for MDMX [63,64] (Fig. 4). Mainly this is due to the tivation by nutlin-3 [60,61], an indiscriminate peptide inhibitor has fact that the L26 subsite in the p53-binding cleft is slightly smaller in been shown to activate p53 in cells that overexpress both MDM2 MDMX than in MDM2. The Y99 (MDMX) residue is oriented differ- and MDMX and to induce (expressed intracellularly in thioredoxin- ently to the corresponding Y100 (MDM2) and a larger M55 (MDMX) scaffolded form from an adenoviral construct) tumour regression replaces L54 (MDM2). Although the two proteins have very similar in corresponding xenograft models [62]. Up until the present, no secondary and tertiary structure, the presence of a unique and con- specific inhibitors of MDMX have been reported. formationally constrained 95 PSP97 sequence in MDMX at the start Fig. 4. Structural comparison of MDM2 and MDMX. (a) The coordinates of X-ray crystal structure complexes with bound p53 peptides of MDM2 (cyan; PDB entry 1YCR [2]) and MDMX (green; PDB entry 3DAB [63]) were aligned and are shown as secondary structure diagrams. The 2 helix in MDMX adopts a different orientation to that in MDMX, presumably due to the unique presence of the conformationally constrained 95 PSP97 sequence (shown as unlabelled stick model) in MDMX. This results in a different orientation of the Y99 (MDMX) side chain (labelled green CPK sticks) compared with the corresponding Y100 (MDM2) side chain (cyan CPK sticks from 1YCR). Intermediate Y99 (MDM2) positions are observed in other MDM2 complex structures such as 1RV1, 1T4E, 1T4F, 2AXI and 2GV2 (magenta CPK sticks) [41,48,99,100]. Together with the presence of the larger M53 (MDMX) residue compared to L54 (MDM2), the altered position of Y99 (MDMX) results in occlusion of part of the p53-binding site in MDMX. (b) The complex (1RV1) between nutlin-2 (grey CPK sticks) and MDM2 (grey surface with L54 and Y100 in cyan CPK). (c) The MDM2 (1RV1) and MDMX (3DAB) structures were aligned and one of the bromophenyl groups of the nutlin-2 ligand from the former can be observed to clash with the altered M53 ,Y99 (MDMX) region. Please cite this article in press as: Dickens MP, et al. Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology (2009), doi:10.1016/j.semcancer.2009.10.003
  5. 5. ARTICLE IN PRESS G Model YSCBI-848; No. of Pages 9 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx 5 of the 2 helix, which supports the Y99 residue, results in a dif- MDM2-mediated p53 ubiquitinylation screen of a chemical library ferent orientation of this helix compared to MDM2. In MDMX this [76]. It was shown that all three compounds behaved as simple brings Y99 into close proximity of the larger M55 , resulting in par- reversible inhibitors of MDM2 in vitro, that they bound to MDM2 tial obstruction of the p53-binding cleft. A recent computational in a mutually exclusive manner, and that inhibition was noncom- comparison of MDM2 and MDMX also suggests that differences petitive with respect to both E2 and p53 substrates. Furthermore, between the proteins may affect how they recognise p53, as well the compounds were selective insofar as they did not inhibit E3 as small-molecule inhibitors [65]. As the flexible N-terminal lid of ligases other than MDM2, and, surprisingly, did not inhibit MDM2 MDM2 is known to play a role in molecular recognition [58], it has autoubiquitinylation. been suggested that the significant differences in the lids of MDM2 It is known that while the isolated MDM2 RING domain that and MDMX may differentially influence ligand recognition and in includes the extreme C-terminus of MDM2 retains E3 ligase activ- turn selectivity [66]. ity, ubiquitinylation of p53 by MDM2 also requires the N-terminal The structure-activity relationships of MDM2 inhibitors show domain, where the main p53 recruitment site resides, as well as the that increasing the size of the lipophilic binding groups so that central acidic domain, which contains a secondary p53-binding site they fill the binding site better greatly increases potency. There- [77]. One could therefore imagine that the above compounds might fore optimised MDM2 inhibitors are probably too bulky to bind prevent p53 ubiquitinylation not at the level of the MDM2 catalytic MDMX, whereas p53 and peptides derived from it can bind both. activity but by preventing p53 binding. A lack of effects of the com- While the affinity for MDMX of only very few MDM2 inhibitors has pounds on the physical interaction between MDM2 and p53 was been reported, it appears that the more potent and optimised they demonstrated, however, suggesting that the mode of inhibition are for MDM2, the more selective they are for MDM2 over MDMX. may be allosteric, perhaps by blocking a structural rearrangement of MDM2 necessary for p53 ubiquitinylation but not for MDM2 autoubiquitinylation [72]. 4. MDM2 as an E3 ubiquitin ligase Regardless of the mechanism of MDM2 inhibition, the selectivity towards p53 ubiquitinylation as opposed to MDM2 autoubiquitiny- 4.1. Background lation by the arylsulfonamide, bisarylurea, and acylimidazolone compounds in Fig. 5 would be desirable from a therapeutic view- Protein ubiquitinylation involves three ATP-dependent point, since inhibition of both activities might lead to accumulation enzymes in a sequential reaction. A ubiquitin activating enzyme of MDM2, which in turn would be expected to limit inhibition of (E1) forms a thioester bond between its active site cysteine residue p53 ubiquitinylation and subsequent degradation. However, no cel- and the C-terminal glycine of ubiquitin. Activated ubiquitin is lular or in vivo activity data were presented for these compounds, then transferred from the E1-ubiquitin complex to a ubiquitin and apparently there has not been any follow-up since the original conjugating enzyme (E2) by transthioesterification. In the final report [76]. step a ubiquitin protein ligase (E3) binds the E2-ubiquitin complex The only other p53-selective MDM2 E3 ligase inhibitor in and aids in the formation of an isopeptide linkage between the the public domain concerns a compound (of undisclosed struc- C-terminus of ubiquitin and the -amino group of a lysine residue ture) that was also identified in a high through-put chemical in the target protein, or to a ubiquitin already attached [67]. Once library (>600,000 compounds) screen using an MDM2-mediated four or more ubiquitins are linked through lysine 48 (K48) of p53 ubiquitinylation assay, as well as an MDM2 autoubiquitiny- ubiquitin, the modified protein is recognised by the 26S protea- lation counter screen [78]. It was observed that although most of some, and is degraded. Specificity of the ubiquitinylation process the numerous screening hits identified showed similar activity in occurs mostly at the level of the E3 enzymes, of which over 1000 the p53 and autoubiquitinylation assays, a few chemotypes dis- are known in the human body, whereas there are only around 30 played some selectivity. The most selective compound inhibited different E2 enzymes, and a single E1 (two isoforms referred to as p53 ubiquitinylation with an IC50 value of 8 M but was inactive at E1a and E1b) [68]. concentrations up to 100 M in the autoubiquitinylation assay. MDM2 belongs to the family of E3 ubiquitin ligases that con- A family of closely related 7-nitro-5-deazaflavin compounds tain a RING (really interesting new gene) domain [69]. These are called HLI98 (deazaflavins 1–3 in Fig. 5) have been identified as structurally defined by cross-branched active site histidine and cys- inhibitors of MDM2 E3 ubiquitin ligase activity by high through- teine residues bound to two zinc ions. Although MDM2 can catalyse put screening of MDM2 autoubiquitinylation [79]. Using cell-based multiple monoubiquitinylation and polyubiquitinylation reactions assays, the lead compound HLI98C was demonstrated to inhibit on p53, it remains unclear to what extent MDM2 or other E3 lig- selectively p53 ubiquitinylation, to increase MDM2 and p53 pro- ases (such as p300) are responsible for the p53 polyubiquitinylation tein levels, to reactivate p53 function, and to induce p53-dependent required for efficient degradation in vivo [70–72]. apoptosis in cancer cells. However, the HLI98 compounds appear to Apart from MDM2, three other proteins can act as E3 ubiq- have low potency and to promote at least some p53-independent uitin ligases for p53. PIRH2 (p53-induced protein with RING-H2 cellular toxicity. Nevertheless, these compounds succeed in show- domain), like MDM2, is linked with p53 in an autoregulatory feed- ing proof of principle that small molecules can inhibit MDM2 E3 back loop that controls p53 function [73]. COP1 (constitutively ubiquitin ligases and may have potential for use in cancer therapy photomorphogenic 1) acts independently of MDM2 as a RING E3 [80]. ubiquitin ligase [74] and ARF-BP1 (ARF-binding protein) acts as a A potential problem with the HLI98 compounds results from HECT (homologous to E6-AP carboxyl terminus) E3 ubiquitin ligase the high redox potential of 5-deazaflavins. The nitro group is sus- towards p53 [75]. The roles of these E3 ubiquitin ligases in p53 reg- ceptible to one-electron reduction leading to the generation of ulation are not well understood and there is no evidence that they the nitro anion radical. The planar heteroaromatic system of the can replace MDM2 in the regulation of p53 stability [34]. HLI98 compounds can intercalate with DNA [81] and the pres- ence of the reactive radical can then result in cytotoxicity through 4.2. MDM2 E3 ligase inhibitors DNA damage [82,83]. More recent work investigated the ability of 5-deazaflavin analogues to stabilise and activate p53. Results The first report on MDM2 E3 ligase inhibitors dates back to show that the nitro group present in HLI98 compounds is in fact 2002 and concerns the arylsulfonamide, bisarylurea, and acylimi- not essential for 5-deazaflavins to reactive p53. Thus 6-chloro-5- dazolone compounds shown in Fig. 5, which were discovered in an deazaflavin compounds such as deazaflavin 4 in Fig. 5 were found Please cite this article in press as: Dickens MP, et al. Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology (2009), doi:10.1016/j.semcancer.2009.10.003
  6. 6. ARTICLE IN PRESS G Model YSCBI-848; No. of Pages 9 6 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx Fig. 5. Chemical structures of small-molecule MDM2 E3 ubiquitin ligase inhibitors. to induce elevation of p53 levels to a similar extent as HLI98 [84]. might block ATP binding or interfere with the E3 catalytic site in Very recently, a more soluble and potent deazaflavin some other way. The C-terminal tail of MDM2 was shown to be (deazaflavin 5 (HLI393) in Fig. 5) was discovered, which pos- critical for MDM2 E3 ligase activity [90,91] and a recent X-ray crys- sesses a 5-dimethylaminopropylamino side chain but lacks the tal structure of an MDM2–MDMX RING domain heterodimer [92] 10-aryl group of HLI98 compounds [85]. HLI393 was determined shows that this tail, by inserting into a groove of the partner protein, to be highly water-soluble and to inhibit MDM2-mediated p53 forms a composite binding site for the E2-ubiquitin complex (Fig. 6). ubiquitinylation with low micromolar cellular potency, resulting Since this interaction is apparently required for MDM2 E3 ligase in increased MDM2 and p53 protein levels, leading to selective activity both in trans and in cis (perhaps by a similar tail insertion p53-dependent apoptosis in a variety of different cancer cell lines intramolecularly), it is possible that nonselective inhibitors target containing wild-type p53. the E2-binding site or the tail-binding groove directly. Sempervirine was discovered as an inhibitor of MDM2 E3 ubiq- Despite the fact that 3D structural information on the MDM2 uitin ligase activity in a high through-put natural products screen RING domain is now available [63,64,92,93], several aspects of its [86]. Like the deazaflavins, sempervirine was observed to inhibit E3 ligase activity remain unclear, including exact delineation of both MDM2-dependent p53 ubiquitinylation and MDM2 autoubiq- the nucleotide-binding site and the active site itself, as well as the uitinylation. Again, treatment of cancer cells harbouring wild-type nature of the overall catalytic mechanism. p53 with this compound induced stabilisation of p53 and apopto- sis. The structurally unusual [87] plant alkaloid sempervirine has long been known to possess anticancer activities [88] and perhaps 5. Clinical development of MDM2 inhibitors inhibition of MDM2 E3 ligase activity contributes to these. Certain acridine derivatives (refer structure in Fig. 5, where R At the time of compiling the present report we are aware represents cyclic and noncyclic aliphatic systems) have been shown of at least two MDM2 inhibitors that have actually entered the to stabilise p53 protein levels by blocking p53 ubiquitinylation clinic. The first compound is JNJ-26854165 (Ortho Biotech; John- through a mechanism that differs from what occurs following DNA son & Johnson), which is currently being investigated as an oral damage, where p53 stabilisation is the result of its inability to agent in advanced stage or refractory solid tumours in a phase- recognise and to be tagged for destruction by MDM2 due to post- I trial [94]. The chemical structure of this compound, which was translational modification [89]. The acridine derivates induced p53 apparently discovered using a p53 degradation assay, is shown in transcriptional activity and p53-dependent apoptosis in tumour Fig. 7 [94]. It was reported to induce p53 levels in tumour cell cells in vivo but it remains unclear if they inhibit MDM2 directly. lines and to activate p53 transcriptional activity. However, unlike p53–MDM2 protein interaction inhibitors and E3 ligase inhibitors, JNJ-26854165 apparently blocks the association of MDM2 with the 4.3. MDM2-mediated p53 ubiquitinylation versus MDM2 proteasome both in vitro and in cell-based assays. How exactly this autoubiquitinylation leads to p53 induction remains unclear. Another compound currently undergoing clinical evaluation is The question of how MDM2 E3 ligase inhibitors that do R7112 (Hoffmann-La Roche) [95]. Like JNJ-26854165, R7112 is an not apparently distinguish between autoubiquitinylation and p53 oral agent and is being studied in phase-I trials in haematologic ubiquitinylation work at the molecular level remains open. Sev- neoplasms and advanced solid tumours. No detailed information eral mechanisms can be considered. The most obvious is that they appears to be in the public domain but one assumes that R7112 Please cite this article in press as: Dickens MP, et al. Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology (2009), doi:10.1016/j.semcancer.2009.10.003
  7. 7. ARTICLE IN PRESS G Model YSCBI-848; No. of Pages 9 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx 7 Fig. 6. X-ray crystal structure of the MDM2–MDMX RING domain heterodimer complex (constructed from PDB entry 2VJF [92]). (a) The complex is depicted as a secondary structure cartoon with MDM2 in green and MDMX in cyan. The two zinc ions in each RING domain are shown as spheres, and the coordinating residue side chains as lines. (b) One face of the MDM2 surface, together with the C-terminus of MDMX, forms the likely interaction site with the E2 ubiquitin conjugating enzyme (key residues shown with side chain and labelled). (c) Interaction between the C-terminus of MDMX (cyan) and MDM2 (green CPK surface). in so doing have increased our understanding of the p53–MDM2 protein–protein interaction and the effects of inhibiting it. The evidence is now in favour and with a number of these inhibitors entering clinical trials the ultimate proof of concept may be just around the corner. The next stage in the refinement of p53–MDM2 Fig. 7. Chemical structure of JNJ-26854165, a clinical MDM2 inhibitor that has been protein–protein interaction inhibitors will probably concern the reported to block an association of MDM2 with the proteasome. current lack of cross-inhibition of MDMX. The development of MDM2 E3 ubiquitin ligase inhibitors is less is a compound from the nutlin series. Finally, Ascenta [96] have advanced and remains hampered by the biological complexity of an oral MDM2 inhibitor compound known as AT-219 under late the ubiquitinylation process. There is a real need for a better under- preclinical development. Again the exact nature of the compound standing at the molecular level of how exactly MDM2 functions as has not been disclosed but it is likely to be an optimised member an E3 ubiquitin ligase, so that structure-based drug design efforts of the spiro-oxindole series. can be used. It has been pointed out that E3 ligases in general are conceptually very attractive drug targets but that “ligases are today 6. Conclusions where kinases were 10 to 15 years ago” [97]. Directly inhibiting the p53–MDM2 interaction, as a means of Conflict of interest activating p53, is potentially useful in the treatment of cancers still expressing wild-type p53 and continues to be investigated The authors declare that there are no conflicts of interest. intensively. There have been concerns as to how viable this con- cept would be therapeutically: will it be possible to inhibit a Funding source protein–protein interaction with a drug-like molecule? What are the effects of unleashing p53 on healthy cells? Endeavours to Michael P. Dickens’s studies are sponsored by Cancer Research develop small-molecule inhibitors have addressed these issues and UK through the Beatson Institute for Cancer Research, Glasgow, Please cite this article in press as: Dickens MP, et al. Small-molecule inhibitors of MDM2 as new anticancer therapeutics. Seminars in Cancer Biology (2009), doi:10.1016/j.semcancer.2009.10.003
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