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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
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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 http://www.pymol.org). 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.
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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, http://www.eyesopen.com/).
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
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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
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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
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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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YSCBI-848; No. of Pages 9
8 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx
Scotland, UK. Ross Fitzgerald’s studies are sponsored by Cyclacel [26] Fujiwara T, Tanaka N, Kanazawa S, Ohtani S, Saijo Y, Nukiwa T, et al.
Limited, Dundee, Scotland, UK. Peter M. Fischer’s studies in the Multicenter phase I study of repeated intratumoral delivery of adenovi-
ral p53 in patients with advanced non-small-cell lung cancer. J Clin Oncol
cancer research area relevant to the article are sponsored by Cycla- 2006;24:1689–99.
cel Limited, Dundee, Scotland, UK (studentship to coauthor Ross [27] Guo J, Xin H. Chinese gene therapy. Splicing out the West? Science
Fitzgerald) and Cancer Research UK through the Beatson Institute 2006;314:1232–5.
[28] Shimada H, Matsubara H, Shiratori T, Shimizu T, Miyazaki S, Okazumi S, et al.
for Cancer Research, Glasgow, Scotland, UK (studentship to coau- Phase I/II adenoviral p53 gene therapy for chemoradiation resistant advanced
thor Michael Dickens). These sponsors have no involvement in the esophageal squamous cell carcinoma. Cancer Sci 2006;97:554–61.
study design; collection, analysis and interpretation of data; the [29] Wallraven G, Nemunaitis JJ, Maples PB. Compassionate approval process for
experimental gene-based products. J Clin Oncol 2008;26:1899–900.
writing of the manuscript; the decision to submit the manuscript [30] Mendrysa SM, McElwee MK, Michalowski J, O’Leary KA, Young KM, Perry ME.
for publication. Mdm2 Is critical for inhibition of p53 during lymphopoiesis and the response
to ionizing irradiation. Mol Cell Biol 2003;23:462–72.
[31] Mendrysa SM, O’Leary KA, McElwee MK, Michalowski J, Eisenman RN, Powell
DA, et al. Tumor suppression and normal aging in mice with constitutively
References high p53 activity. Genes Dev 2006;20:16–21.
[32] Zhang R, Wang H, Agrawal S. Novel antisense anti-MDM2 mixed-backbone
[1] Cahilly-Snyder L, Yang-Feng T, Francke U, George DL. Molecular analysis and oligonucleotides: proof of principle, in vitro and in vivo activities, and mech-
chromosomal mapping of amplified genes isolated from a transformed mouse anisms. Curr Cancer Drug Targets 2005;5:43–9.
3T3 cell line. Somat Cell Mol Genet 1987;13:235–44. [33] O’Leary KA, Mendrysa SM, Vaccaro A, Perry ME. Mdm2 regulates p53
[2] Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, et al. independently of p19(ARF) in homeostatic tissues. Mol Cell Biol 2004;24:
Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor 186–91.
transactivation domain. Science 1996;274:948–53. [34] Vassilev LT. MDM2 inhibitors for cancer therapy. Trends Mol Med
[3] Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The mdm-2 oncogene 2007;13:23–31.
product forms a complex with the p53 protein and inhibits p53-mediated [35] Bogan AA, Thorn KS. Anatomy of hot spots in protein interfaces. J Mol Biol
transactivation. Cell 1992;69:1237–45. 1998;280:1–9.
[4] Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of [36] Fischer PM. Peptide, peptidomimetic, and small-molecule antagonists of the
p53. Nature 1997;387:296–9. p53–HDM2 protein–protein interaction. Int J Peptide Res Ther 2006;12:3–19.
[5] Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for [37] Hardcastle IR. Inhibitors of the MDM2–p53 interaction as anticancer drugs.
tumor suppressor p53. FEBS Lett 1997;420:25–7. Drugs Fut 2007;32:883–96.
[6] Tao W, Levine AJ. Nucleocytoplasmic shuttling of oncoprotein Hdm2 is [38] Murray JK, Gellman SH. Targeting protein–protein interactions: lessons from
required for Hdm2-mediated degradation of p53. Proc Natl Acad Sci USA p53/MDM2. Biopolymers 2007;88:657–86.
1999;96:3077–80. [39] Bottger V, Bottger A, Howard SF, Picksley SM, Chene P, Garcia-Echeverria C, et
[7] Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm-2 autoregulatory feedback al. Identification of novel mdm2 binding peptides by phage display. Oncogene
loop. Genes Dev 1993;7:1126–32. 1996;13:2141–7.
[8] Barak Y, Juven T, Haffner R, Oren M. mdm2 expression is induced by wild type [40] García-Echeverría C, Chène P, Blommers MJJ, Furet P. Discovery of potent
p53 activity. EMBO J 1993;12:461–8. antagonists of the interaction between human double minute 2 and tumor
[9] Matheu A, Maraver A, Serrano M. The Arf/p53 pathway in cancer and aging. suppressor p53. J Med Chem 2000;43:3205–8.
Cancer Res 2008;68:6031–4. [41] Sakurai K, Schubert C, Kahne D. Crystallographic analysis of an 8-mer
[10] Dai MS, Shi D, Jin Y, Sun XX, Zhang Y, Grossman SR, et al. Regulation of the p53 peptide analogue complexed with MDM2. J Am Chem Soc 2006;128:
MDM2–p53 pathway by ribosomal protein L11 involves a post-ubiquitination 11000–1.
mechanism. J Biol Chem 2006;281:24304–13. [42] Chene P, Fuchs J, Bohn J, García-Echeverría C, Furet P, Fabbro D. A small syn-
[11] Jin A, Itahana K, O’Keefe K, Zhang Y. Inhibition of HDM2 and activation of p53 thetic peptide, which inhibits the p53-hdm2 interaction, stimulates the p53
by ribosomal protein L23. Mol Cell Biol 2004;24:7669–80. pathway in tumour cell lines. J Mol Biol 2000;299:245–53.
[12] Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM. Mdm2 is a RING [43] Shangary S, Wang S. Targeting the MDM2–p53 interaction for cancer therapy.
finger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem Clin Cancer Res 2008;14:5318–24.
2000;275:8945–51. [44] Hu C-Q, Hu Y-Z. Small molecule inhibitors of the p53–MDM2. Curr Med Chem
[13] Meek DW, Knippschild U. Posttranslational modification of MDM2. Mol Can- 2008;15:1720–30.
cer Res 2003;1:1017–26. [45] Domling A. Small molecular weight protein–protein interaction antagonists:
[14] Shvarts A, Steegenga WT, Riteco N, van Laar T, Dekker P, Bazuine M, et an insurmountable challenge? Curr Opin Chem Biol 2008;12:281–91.
al. MDMX: a novel p53-binding protein with some functional properties of [46] Dey A, Verma CS, Lane DP. Updates on p53: modulation of p53 degradation
MDM2. EMBO J 1996;15:5349–57. as a therapeutic approach. Br J Cancer 2008;98:4–8.
[15] Ramos YF, Stad R, Attema J, Peltenburg LT, van der Eb AJ, Jochemsen AG. Aber- [47] Berg T. Small-molecule inhibitors of protein–protein interactions. Curr Opin
rant expression of HDMX proteins in tumor cells correlates with wild-type Drug Discov Dev 2008;11:666–74.
p53. Cancer Res 2001;61:1839–42. [48] Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo acti-
[16] Linares LK, Hengstermann A, Ciechanover A, Muller S, Scheffner M. HdmX vation of the p53 pathway by small-molecule antagonists of MDM2. Science
stimulates Hdm2-mediated ubiquitination and degradation of p53. Proc Natl 2004;303:844–8.
Acad Sci USA 2003;100:12009–14. [49] Koblish HK, Zhao S, Franks CF, Donatelli RR, Tominovich RM, LaFrance LV, et al.
[17] Lane D. How cells choose to die. Nature 2001;414:25–7. Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human
[18] Vousden KH. Activation of the p53 tumor suppressor protein. Biochim Bio- tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo.
phys Acta 2002;14:47–59. Mol Cancer Ther 2006;5:160–9.
[19] Momand J, Jung D, Wilczynski S, Niland J. The MDM2 gene amplification [50] Leonard K, Marugan JJ, Raboisson P, Calvo R, Gushue JM, Koblish HK, et al.
database. Nucleic Acids Res 1998;26:3453–9. Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists with improved
[20] Bartel F, Meye A, Wurl P, Kappler M, Bache M, Lautenschlager C, et al. Ampli- cellular activity. Bioorg Med Chem Lett 2006;16:3463–8.
fication of the MDM2 gene, but not expression of splice variants of MDM2 [51] Marugan JJ, Leonard K, Raboisson P, Gushue JM, Calvo R, Koblish HK, et al.
MRNA, is associated with prognosis in soft tissue sarcoma. Intl J Cancer Enantiomerically pure 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists.
2001;95:168–75. Bioorg Med Chem Lett 2006;16:3115–20.
[21] Boeckler FM, Joerger AC, Jaggi G, Rutherford TJ, Veprintsev DB, Fersht AR. [52] Parks DJ, Lafrance LV, Calvo RR, Milkiewicz KL, Gupta V, Lattanze J, et al. 1,4-
Targeted rescue of a destabilized mutant of p53 by an in silico screened drug. Benzodiazepine-2,5-diones as small molecule antagonists of the HDM2-p53
Proc Natl Acad Sci USA 2008;105:10360–5. interaction: discovery and SAR. Bioorg Med Chem Lett 2005;15:765–70.
[22] Joerger AC, Ang HC, Fersht AR. Structural basis for understanding onco- [53] Parks DJ, LaFrance LV, Calvo RR, Milkiewicz KL, Jose Marugan J, Raboisson
genic p53 mutations and designing rescue drugs. Proc Natl Acad Sci USA P. Enhanced pharmacokinetic properties of 1,4-benzodiazepine-2,5-dione
2006;103:15056–61. antagonists of the HDM2-p53 protein–protein interaction through structure-
[23] Myers MC, Wang J, Iera JA, Bang JK, Hara T, Saito S, et al. A new family of based drug design. Bioorg Med Chem Lett 2006;16:3310–4.
small molecules to probe the reactivation of mutant p53. J Am Chem Soc [54] Ding K, Lu Y, Nikolovska-Coleska Z, Qiu S, Ding Y, Gao W, et al. Structure-
2005;127:6152–3. based design of potent non-peptide MDM2 inhibitors. J Am Chem Soc
[24] Atencio IA, Grace M, Bordens R, Fritz M, Horowitz JA, Hutchins B, et al. Bio- 2005;127:10130–1.
logical activities of a recombinant adenovirus p53 (SCH 58500) administered [55] Ding K, Lu Y, Nikolovska-Coleska Z, Wang G, Qiu S, Shangary S, et al. Structure-
by hepatic arterial infusion in a Phase 1 colorectal cancer trial. Cancer Gene based design of spiro-oxindoles as potent, specific small-molecule inhibitors
Ther 2006;13:169–81. of the MDM2–p53 interaction. J Med Chem 2006;49:3432–5.
[25] Cristofanilli M, Krishnamurthy S, Guerra L, Broglio K, Arun B, Booser DJ, et [56] Deng J, Dayam R, Neamati N. Patented small molecule inhibitors of
al. A nonreplicating adenoviral vector that contains the wild-type p53 trans- p53–MDM2 interaction. Expert Opin Ther Pat 2006;16:165–88.
gene combined with chemotherapy for primary breast cancer: safety, efficacy, [57] Uhrinova S, Uhrin D, Powers H, Watt K, Zheleva D, Fischer P, et al. Structure
and biologic activity of a novel gene-therapy approach. Cancer (Hoboken, NJ, of Free MDM2 N-terminal domain reveals conformational adjustments that
United States) 2006;107:935–44. accompany p53-binding. J Mol Biol 2005;350:587–98.
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
9. ARTICLE IN PRESS
G Model
YSCBI-848; No. of Pages 9
M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx 9
[58] Showalter SA, Bruschweiler-Li L, Johnson E, Zhang F, Bruschweiler R. Quanti- [80] Yang Y, Ludwig RL, Jensen JP, Pierre SA, Medaglia MV, Davydov IV, et al. Small
tative lid dynamics of MDM2 reveals differential ligand binding modes of the molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate
p53-binding cleft. J Am Chem Soc 2008;130:6472–8. p53 in cells. Cancer Cell 2005;7:547–59.
[59] Shangary S, Qin D, McEachern D, Liu M, Miller RS, Qiu S, et al. Temporal acti- [81] Burgstaller P, Hermann T, Huber C, Westhof E, Famulok M. Isoalloxazine
vation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors derivatives promote photocleavage of natural RNAs at G.U base pairs embed-
and leads to complete tumor growth inhibition. Proc Natl Acad Sci USA ded within helices. Nucleic Acids Res 1997;25:4018–27.
2008;105:3933–8. [82] Kawamoto T, Ikeuchi Y, Hiraki J, Eikyu Y, Shimizu K, Tomishima M, et al. Syn-
[60] Hu B, Gilkes DM, Farooqi B, Sebti SM, Chen J. MDMX overexpression prevents thesis and evaluation of nitro 5-deazaflavins as novel bioreductive antitumor
p53 activation by the MDM2 inhibitor nutlin. J Biol Chem 2006;281:33030–5. agents. Bioorg Med Chem Lett 1995;5:2109–14.
[61] Patton JT, Mayo LD, Singhi AD, Gudkov AV, Stark GR, Jackson MW. Levels of [83] Kawamoto T, Ikeuchi Y, Hiraki J, Eikyu Y, Shimizu K, Tomishima M, et al.
HdmX expression dictate the sensitivity of normal and transformed cells to Evaluation of differential hypoxic cytotoxicity and electrochemical studies of
Nutlin-3. Cancer Res 2006;66:3169–76. nitro 5-deazaflavins. Bioorg Med Chem Lett 1995;5:2115–8.
[62] Hu B, Gilkes DM, Chen J. Efficient p53 activation and apoptosis by simultane- [84] Wilson JM, Henderson G, Black F, Sutherland A, Ludwig RL, Vousden KH, et
ous disruption of binding to MDM2 and MDMX. Cancer Res 2007;67:8810–7. al. Synthesis of 5-deazaflavin derivatives and their activation of p53 in cells.
[63] Popowicz GM, Czarna A, Holak TA. Structure of the human Mdmx pro- Bioorg Med Chem 2007;15:77–86.
tein bound to the p53 tumor suppressor transactivation domain. Cell Cycle [85] Kitagaki J, Agama KK, Pommier Y, Yang Y, Weissman AM. Targeting tumor
2008;7:2441–3. cells expressing p53 with a water-soluble inhibitor of Hdm2. Mol Cancer Ther
[64] Popowicz GM, Czarna A, Rothweiler U, Sszwagierczak A, Krajewski M, Weber 2008;7:2445–54.
L, et al. Molecular Basis for the Inhibition of p53 by Mdmx. Cell Cycle [86] Sasiela CA, Stewart DH, Kitagaki J, Safiran YJ, Yang Y, Weissman AM, et al.
2007;6:2386–92. Identification of inhibitors for MDM2 ubiquitin ligase activity from natu-
[65] Macchiarulo A, Giacche N, Carotti A, Baroni M, Cruciani G, Pellicciari R. Tar- ral product extracts by a novel high-throughput electrochemiluminescent
geting the conformational transitions of MDM2 and MDMX: insights into screen. J Biomol Screen 2008;13:229–37.
dissimilarities and similarities of p53 recognition. J Chem Inf Model 2008, [87] Woodward RB, Witkop B. The structure of sempervirine. J Am Chem Soc
ePub ahead of print (doi:10.1021/ci800146m). 1949;71:379.
[66] McCoy MA, Gesell JJ, Senior MM, Wyss DF. Flexible lid to the p53-binding [88] Beljanski M, Beljanski MS. Three alkaloids as selective destroyers of
domain of human Mdm2: implications for p53 regulation. Proc Natl Acad Sci cancer cells in mice. Synergy with classic anticancer drugs. Oncology
USA 2003;100:1645–8. 1986;43:198–203.
[67] Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell [89] Wang W, Ho WC, Dicker DT, MacKinnon C, Winkler JD, Marmorstein R, et al.
life. EMBO J 1998;17:7151–60. Acridine derivatives activate p53 and induce tumor cell death through Bax.
[68] Fang S, Weissman AM. A field guide to ubiquitinylation. Cell Mol Life Sci Cancer Biol Ther 2005;4:893–8.
2004;61:1546–61. [90] Poyurovsky MV, Priest C, Kentsis A, Borden KL, Pan ZQ, Pavletich N, et al. The
[69] Joazeiro CA, Weissman AM. RING finger proteins: mediators of ubiquitin lig- Mdm2 RING domain C-terminus is required for supramolecular assembly and
ase activity. Cell 2000;102:549–52. ubiquitin ligase activity. EMBO J 2007;26:90–101.
[70] Grossman SR, Deato ME, Brignone C, Chan HM, Kung AL, Tagami H, et al. [91] Uldrijan S, Pannekoek WJ, Vousden KH. An essential function of the extreme
Polyubiquitination of p53 by a ubiquitin ligase activity of p300. Science C-terminus of MDM2 can be provided by MDMX. EMBO J 2007;26:102–12.
2003;300:342–4. [92] Linke K, Mace PD, Smith CA, Vaux DL, Silke J, Day CL. Structure of the
[71] Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W. Mono- versus polyubiqui- MDM2/MDMX RING domain heterodimer reveals dimerization is required
tination: differential control of p53 fate by Mdm2. Science 2003;302:1972–5. for their ubiquitinylation in trans. Cell Death Differ 2008;15:841–8.
[72] Lai Z, Ferry KV, Diamond MA, Wee KE, Kim YB, Ma J, et al. Human mdm2 [93] Kostic M, Matt T, Martinez-Yamout MA, Dyson HJ, Wright PE. Solution struc-
mediates multiple mono-ubiquitination of p53 by a mechanism requiring ture of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53. J
enzyme isomerization. J Biol Chem 2001;276:31357–67. Mol Biol 2006;363:433–50.
[73] Leng RP, Lin Y, Ma W, Wu H, Lemmers B, Chung S, et al. Pirh2, a p53-induced [94] Arts J, Page M, Valckx A, Blattner C, Kulikov R, Floren W, et al. JNJ-26854165—a
ubiquitin-protein ligase, promotes p53 degradation. Cell 2003;112:779–91. novel hdm2 antagonist in clinical development showing broad-spectrum pre-
[74] Dornan D, Wertz I, Shimizu H, Arnott D, Frantz GD, Dowd P, et al. The ubiquitin clinical antitumor activity against solid malignancies. Proc Am Assoc Cancer
ligase COP1 is a critical negative regulator of p53. Nature 2004;429:86–92. Res 2008;49. Abs. 1592.
[75] Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF-BP1/Mule is a critical mediator [95] Roche Pharma Pipeline; 2008. http://www.roche.com/pipeline.htm.
of the ARF tumor suppressor. Cell 2005;121:1071–83. [96] Ascenta’s Pipeline; 2008. http://www.ascenta.com/.
[76] Lai Z, Yang T, Kim YB, Sielecki TM, Diamond MA, Strack P, et al. Differenti- [97] Garber K. Missing the target: ubiquitin ligase drugs stall. J Natl Cancer Inst
ation of Hdm2-mediated p53 ubiquitination and Hdm2 autoubiquitination 2005;97:166–7.
activity by small molecular weight inhibitors. Proc Natl Acad Sci USA [98] Fry DC, Emerson SD, Palme S, Vu BT, Liu C-M, Podlaski F. NMR structure of
2002;99:14734–9. a complex between MDM2 and a small molecule inhibitor. J Biomol NMR
[77] Ma J, Martin JD, Zhang H, Auger KR, Ho TF, Kirkpatrick RB, et al. A second p53 2004;30:163–73.
binding site in the central domain of Mdm2 is essential for p53 ubiquitination. [99] Grasberger BL, Lu T, Schubert C, Parks DJ, Carver TE, Koblish HK, et al. Discov-
Biochemistry 2006;45:9238–45. ery and cocrystal structure of benzodiazepinedione HDM2 antagonists that
[78] Murray MF, Jurewicz AJ, Martin JD, Ho TF, Zhang H, Johanson KO, et al. A activate p53 in cells. J Med Chem 2005;48:909–12.
high-throughput screen measuring ubiquitination of p53 by human mdm2. J [100] Fasan R, Dias RL, Moehle K, Zerbe O, Obrecht D, Mittl PR, et al. Structure-
Biomol Screen 2007;12:1050–8. activity studies in a family of beta-hairpin protein epitope mimetic
[79] Davydov IV, Woods D, Safiran YJ, Oberoi P, Fearnhead HO, Fang S, et al. Assay inhibitors of the p53-HDM2 protein–protein interaction. ChemBioChem
for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2. 2006;7:515–26.
J Biomol Screen 2004;9:695–703.
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