phd lit review

G

Y

R

S

MS

a

KCDMMNPEPNBS

1

iooiimidltl

lMtsltt[

1d

ARTICLE IN PRESSModel

SCBI-848; No. of Pages 9

Seminars in Cancer Biology xxx (2009) xxx–xxx

Contents lists available at ScienceDirect

Seminars in Cancer Biology

journa l homepage: www.e lsev ier .com/ locate /semcancer

eview

mall-molecule inhibitors of MDM2 as new anticancer therapeutics

ichael P. Dickens, Ross Fitzgerald, Peter M. Fischer ∗

chool of Pharmacy & Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK

r t i c l e i n f o

eywords:ancer therapyrug discovery and developmentDM2 inhibitorDMXongenotoxic p53 activationrotein–protein interaction3 ubiquitin ligase inhibitor

a b s t r a c t

It has long been known that traditional anticancer radio- and chemotherapies in part work through director indirect activation of the p53 tumour suppressor pathway. However, many of these strategies are non-selective and genotoxic. The emerging understanding of the pathways that regulate p53 has led to thenotion that it should be possible to activate the p53 pathway in ways that are inherently nongenotoxic.Important targets for pharmacological interference in this respect are MDM2 and MDMX, key negativeregulators of p53. Genetic and pharmacologic studies suggest that blocking the physical interaction ofthese proteins with p53, or inhibiting the catalytic role of MDM2 in tagging p53 for proteasomal degra-

roteasomal degradationutlinenzodiazepinedionepiro-oxindole

dation, both of which lead to an increase in the transcriptional activity of p53, may indeed be an efficientand safe way to eradicate tumour cells that retain wild-type p53. Here we review the rationale for suchstrategies, as well as the current state in the discovery and development of drugs that reactivate p53by inhibiting its inhibitors MDM2 and MDMX. The first compounds that have been shown in modelsystems to be able selectively to kill cancer cells in this way are now entering clinical trials and thepromise of MDM2 inhibitors as a new therapeutic anticancer modality should therefore become clear in

e.
the not-too-distant futur
. Introduction

The p53 tumour suppressor protein is a transcription factor thats activated in response to cellular stress. Depending on the severityf the threat to genome integrity, p53 then imposes cell cycle arrestr apoptosis. Because p53 has strong growth-suppressive activity,t must be tightly regulated to allow normal cells to function. Thiss achieved to a large extent by a protein known as murine double

inutes-2 (MDM2), so called because its gene was first discoveredn DNA associated with paired acentric chromatin bodies, termedouble minutes, in spontaneously transformed mouse 3T3 fibrob-

asts [1]. The corresponding human protein is sometimes referredo as HDM2 but here we shall use the abbreviation MDM2 regard-ess of species.

MDM2 regulates p53 in at least three different ways, i.e. at theevels of p53 function, protein stability, and subcellular location.

DM2 forms a protein–protein interaction with the N-terminalranscription activation domain of p53, thus blocking p53 tran-criptional activity [2,3]. Furthermore, MDM2 is the E3 ubiquitin

Please cite this article in press as: Dickens MP, et al. Small-molecule inhiBiology (2009), doi:10.1016/j.semcancer.2009.10.003

igase that promotes ubiquitin-dependent proteasomal degrada-ion of p53 [4,5]. Finally, MDM2 causes nuclear export of p53 intohe cytoplasm of the cell, moving p53 away from its site of action6].

∗ Corresponding author. Tel.: +44 0115 846 6242; fax: +44 0115 951 3412.E-mail address: [email protected] (P.M. Fischer).

044-579X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2009.10.003

© 2009 Elsevier Ltd. All rights reserved.

While MDM2 controls the protein levels of p53, it is itself undertranscriptional control of p53 and the two are thus linked in a tightautoregulatory feedback loop [7,8]. Depending on the nature ofgenotoxic or nongenotoxic stress a cell may experience, the nega-tive regulation of p53 by MDM2 is interrupted in several differentways. Most importantly, the functions of MDM2 in p53 suppressionare inhibited upon association with the ARF protein, an alterna-tive transcript of the INK4a/ARF tumour suppressor locus, which isinduced upon oncogenic stress (reviewed in [9]). Similarly, MDM2is inhibited upon ribosomal stress by the ribosomal proteins L5, L11,and L23 [10,11]. Furthermore, MDM2 is regulated through post-translational modifications, including autoubiquitinylation [12],sumoylation, and multi-site phosphorylations by a range of kinases,especially the DNA damage-induced kinases (reviewed in [13]).

MDMX (also known as MDM4) is a nonredundant homologueof MDM2 that also regulates p53 [14] and is overexpressed inmany cancers [15]. Unlike MDM2, however, MDMX expression isnot regulated by p53 and MDMX is thus not part of the negativefeedback loop with p53. MDMX also lacks intrinsic ubiquitin ligaseactivity but is itself a target for MDM2 ubiquitinylation. It formsheterodimers with MDM2, which enhances the ability of MDM2 toinduce p53 degradation [16]. MDMX binds p53 at the same site and

bitors of MDM2 as new anticancer therapeutics. Seminars in Cancer

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 ofevading apoptosis, either by disabling p53 directly through muta-tion or deletion, or indirectly by alterations of various components

dx.doi.org/10.1016/j.semcancer.2009.10.003

http://www.sciencedirect.com/science/journal/1044579X

http://www.elsevier.com/locate/semcancer

mailto:[email protected]


ING

Y

2 n Canc

ori(as

nabcstp

pptTtrsa

2

veSag[ae[apvt

oatfttpMgo

3

abddcpdidc

ARTICLEModel


M.P. Dickens et al. / Seminars i

f the pathways that regulate p53 [17]. About half of all cancersetain wild-type p53 [18] and in these the normal regulation of p53s sometimes disrupted through direct overexpression of MDM2in ca. 7% of cancers [19]). MDM2 overexpression due to genemplification is especially frequent (ca. 30%) in human osteogenicarcomas and soft tissue sarcomas [20].

Because of the central role of p53 in tumour suppression,ongenotoxic therapeutic strategies that activate p53 in one way ornother are highly desirable. Depending on p53 status this shoulde able to be achieved in various ways. For example, proof-of-oncept studies have shown that mutant p53 might be able to betabilised or otherwise reactivated pharmacologically [21–23]. Inumours that retain a functional p53 pathway, on the other hand,reventing p53 degradation is an attractive option.

There are many potential therapeutic targets within the p53athway, downstream of the stress response, which offer theossibilities of nongenotoxic p53 activation and bypassing the par-icular defects that could render an upstream target ineffective.he MDM2–p53 regulatory system is one such target. Modula-ion of this system with small molecules is a very active area ofesearch. Here we review current progress in the development ofmall molecules that inhibit the MDM2–p53 protein–protein inter-ction or the ubiquitin ligase activity of MDM2.

. Target rationale and therapeutic window

The key question for any therapeutic strategy that aims to acti-ate the p53 response is whether of not this will result in a selectiveffect on tumour cells as opposed to the cells of healthy tissues.uch specificity of p53 to kill tumour cells, but not normal cells,ppears to underlie the safety of p53 gene therapy, which hasained approval in China and is now being developed elsewhere24–29]. It has been shown that mice with a hypomorphic MDM2llele produce only about 30% of the normal level of MDM2 andxhibit increased transcriptional and functional activation of p5330]. The effects of p53 under these circumstances are not lethals one might expect, although the animals are small and show53-dependent apoptosis of lymphoid cells. Nevertheless they areiable, do not age prematurely, and are resistant to tumour forma-ion [31].

Similarly, in vivo suppression of MDM2 using antisenseligonucleotides has been demonstrated to result in therapeuticntitumour effects without overt toxicity (reviewed in [32]). Fromhese and other results [33] it is clear that the p53 pathway dif-ers significantly in normal and p53 wild-type cancer cells andhat the latter are selectively sensitive to increases in p53 effec-or functions. This notion is enhanced by the results of extensiveharmacological studies, especially those using the nutlin pioneerDM2 inhibitors (discussed in more detail below), which also sug-

est that cancer cells are more susceptible to proapoptotic effectsf p53 than noncancerous cells (reviewed in [34]).

. The p53–MDM2 interaction

Prior to elucidation of the structure of the p53–MDM2 inter-ction it was thought that protein–protein interactions could note effectively inhibited with membrane-permeable and otherwiserug-like small molecules because of the extensive size and poorefinition of protein interfaces. The X-ray crystal structure of aomplex between the N-terminal domain of MDM2 and a 12mer


eptide encompassing residues 16–27 of the p53 transactivationomain showed that the bulk of the p53–MDM2 interaction in fact

nvolved just three lipophilic residues of p53, buried in a well-efined hydrophobic surface cleft in MDM2, of a size that couldlearly be fully occupied by a small molecule [2]. This observation

PRESSer Biology xxx (2009) xxx–xxx

has helped to reshape our perception of protein–protein interac-tions as drug targets, since in many cases these contain so-calledhot spots, where the binding energy of protein–protein interactionsis concentrated [35].

3.1. Early work with peptide antagonists of the p53–MDM2interaction

A detailed discussion of peptide and peptidomimeticapproaches to modulate the p53–MDM2 interaction has beenprovided elsewhere [36–38] and we shall only summarise inoutline some of the early peptide optimisation studies that definedthe pharmacophore model which provided the platform forsubsequent development of nonpeptide inhibitors.

Initially, screening of phage-displayed peptide libraries led tothe discovery of a 12mer peptide MPRFMDYWEGLN with 28-foldpotency increase compared to the corresponding p53 sequence16QETFSDLWKLLF27 [39]. Interestingly, only the three key inter-acting residues (bold type in preceding sequences) were conservedbetween these peptides and the basic pharmacophore feature ofall potent ligands is indeed three suitably oriented hydrophobicgroups. Next, artificial amino acids were used to explore conforma-tional features. This led to the development of a highly optimised8mer peptide that inhibited the p53–MDM2 PPI with low nanomo-lar potency, which represented a >1700-fold increase in affinitycompared with the 12mer p53 peptide [40].

Incorporation of a chloro group at the indole C6 position of thekey Trp residue showed that better occupancy of the binding sitecompared to the cognate ligand could be achieved with substantialpotency gains. The effect of introducing helix-stabilising residuesshowed the importance of a rigid scaffold in presenting the keyresidues in a way that results in optimal shape complementaritywith the binding site. Again this feature was subsequently recapit-ulated with nonpeptidic inhibitors. The increases in affinity broughtabout by inclusion of charged nonnative residues indicated that fur-ther polar contacts not present in the native system could be madeoutside of the main binding cleft.

A complex crystal structure of this high-affinity 8mer peptidebound to MDM2 was solved recently [41] and shows that the pep-tide does indeed bind in the expected manner (Fig. 1). The structuralfeatures of this peptide have been inherited by subsequent small-molecule inhibitors, the best of which are those that mimic thepeptide most closely. The optimised peptide also provided pharma-cological target validation, since it was somewhat permeable andthus able to reach its target MDM2 in intact cells. It was observed toinduce apoptosis selectively in MDM2-overexpressing cancer cellsvia nongenotoxic p53 activation [42].

3.2. Small-molecule p53–MDM2 antagonists

Because of their central role as pioneers for protein–proteininteraction drug target modulators in general, inhibitors of thep53–MDM2 interaction have been reviewed extensively. We donot intend to duplicate these efforts here but direct the interestedreader to some of the most recent reviews [43–47]. One of thesegives an up-to-date summary, covers some 20 distinct classes ofsmall-molecule p53–MDM2 inhibitors, and assesses critically towhat extent these have been validated, i.e. which can be regarded asgenuine p53–MDM2 inhibitors and which operate to block MDM2functions by different mechanisms [45].

Of the small-molecule inhibitor series described to date, three


are of particular importance. The nutlins [48], the benzodi-azepinediones [49–53], and the spiro-oxindoles [54,55] (importantrepresentative members from these series are shown in Fig. 2) allbind MDM2 with low nanomolar affinity and induce cancer cellapoptosis in a p53-dependent manner. Typically these compounds


ARTICLE ING Model

YSCBI-848; No. of Pages 9

M.P. Dickens et al. / Seminars in Canc

Fig. 1. MDM2-binding mode of an optimised p53-derived peptide. Residues ofthe p53 peptide (grey CPK sticks) are labelled (Ac3C, cyclopropylglycine; 6-Cl-Trp,6-chloro-tryptophan; Pmp, phosphonomethylphenylalanine; Aib, aminoisobutyricacid). MDM2 is shown as a green CPK surface. Constructed from PDB entry 2GV2[41]. This and subsequent illustrations showing 3D structures were prepared withto

asIfafr

bhtW(ifiahcf

It has recently transpired that because of the nonredundant but

Fp

he PyMOL programme (DeLano, W.L. The PyMOL Molecular Graphics System (2002)n the World Wide Web http://www.pymol.org).

re only active in cells that express wild-type p53, and p53 tran-criptional products can be observed to be upregulated as a result.mportantly, the presence of posttranslationally unmodified p53ollowing treatment of cells with these compounds shows that theyct in a nongenotoxic manner. Furthermore, optimised analoguesrom these compound series have all been shown to cause tumouregression in xenograft models.

Although chemically distinct, all adhere to the basic hydropho-ic three-pronged pharmacophore model. Each uses a unique, rigid,eterocyclic scaffold to project three lipophilic groups into thehree subpockets in the binding site that are occupied by the F19,

23 and L26 residue side chains in the case of p53 as the ligandFig. 3). As these interactions are predominantly hydrophobic, alter-ng the size of the lipophilic binding groups in order optimally toll the site in a shape-complimentary manner greatly increases


ffinity. Potent members of all three inhibitor chemotypes bearalide-substituted aromatic groups positioned to maximise theseontacts. Indeed the nutlins rely entirely on hydrophobic contactsor their affinity. Several compounds contain chlorophenyl sub-

ig. 2. Chemical structures of potent small-molecule p53–MDM2 interaction inhibitor53-binding cleft of MDM2 are shown in green, red, and purple, respectively. Compare Fi

PRESSer Biology xxx (2009) xxx–xxx 3

stituents that closely mimic the 6-Cl-Trp modification discussedabove in the context of peptide inhibitors.

Because the p53-binding cleft of MDM2 is highly hydrophobic,minimal nonpeptide inhibitors are by necessity very lipophilic andthus lack aqueous solubility. Throughout their development, all ofthe inhibitors have thus evolved to include solvent-exposed polargroups that aid solubility. In the benzodiazepinediones, additionof such solubilising groups improved cellular potency, but at theexpense of some binding affinity. In the case of the spiro-oxindoles,however, addition of a solubilising group to the core structureresulted in increased affinity as well as cellular activity, and led tothe development of the most potent inhibitors. No detailed medic-inal chemistry has been disclosed about the nutlins but the threecompounds presented in the original report [48] vary mostly in thesolubilising group and a review of the MDM2 inhibitor patent lit-erature suggests considerable dependence of biological activity onthe nature of this group [56]. Whether solubilising groups just act asproperty-improving appendages or become an additional pharma-cophore feature depends largely on their attachment point. In thecase of MI-219 the solubilising chain makes additional hydropho-bic contacts outside the main binding cleft and the polar groups arethought to mimic contacts made by the Pmp or Glu residues of theoptimised p53 peptide (Fig. 1). The binding modes of nutlins andbenzodiazepinediones show that their solubilising groups projectdifferently and cannot make similar interactions (Fig. 3).

The fact that all proteins and their binding sites are flexible isalso evident from structural studies with MDM2. When no ligandis bound, the p53-binding cleft of MDM2 exists in a closed confor-mation, which opens upon binding to p53 [57]. A flexible lid coversthe cleft in the unbound state and is displaced upon p53 peptidebinding but not upon binding small molecules [58]. Comparison ofthe MDM2 conformations in the various bound structures shownin Fig. 3 clearly demonstrates that the small-molecule inhibitorsbind to MDM2 so that it adopts a similar conformation as it doesupon binding p53. The partially closed form is evident in the caseof the benzodiazepinedione inhibitor complex, which is the onlysmall-molecule inhibitor complex where the lid region is presentin the MDM2 construct employed.

3.3. Inhibition of the p53–MDMX interaction


overlapping functions of MDM2 and MDMX in the regulation ofp53, and because of the comparatively frequent amplification ofMDMX in cancer cells, an ideal nongenotoxic p53 activator shouldinhibit both MDM2 and MDMX. However, it has been shown that

compounds. The groups that interact with the F19, W23, and L26 subsites of theg. 1. Solubilising groups are indicated in blue.


http://www.pymol.org/

ARTICLE IN PRESSG Model


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( e ligat 1 [48( to anO

t2ftbasis

FaMoYpTa

purple), and the spiro-oxindole MI-219 (blue) with MDM2 are shown in b–e. (f): Thhe same colour schemes as in a–e. Constructed from PDB entries 1YCR [2] (a), 1RVe) was obtained through docking of a multiconformer database of the compound inpenEye Scientific Software, http://www.eyesopen.com/).

he small-molecule MDM2 inhibitors nutlin-3 and MI-219 have60- and >10,000-fold lower affinity, respectively, for MDMX thanor MDM2 [59]. While MDMX overexpression can prevent p53 reac-ivation by nutlin-3 [60,61], an indiscriminate peptide inhibitor haseen shown to activate p53 in cells that overexpress both MDM2


nd MDMX and to induce (expressed intracellularly in thioredoxin-caffolded form from an adenoviral construct) tumour regressionn corresponding xenograft models [62]. Up until the present, nopecific inhibitors of MDMX have been reported.

ig. 4. Structural comparison of MDM2 and MDMX. (a) The coordinates of X-ray crystal snd MDMX (green; PDB entry 3DAB [63]) were aligned and are shown as secondary strDMX, presumably due to the unique presence of the conformationally constrained 95PSP

rientation of the Y99 (MDMX) side chain (labelled green CPK sticks) compared with the99 (MDM2) positions are observed in other MDM2 complex structures such as 1RV1, 1resence of the larger M53 (MDMX) residue compared to L54 (MDM2), the altered positiohe complex (1RV1) between nutlin-2 (grey CPK sticks) and MDM2 (grey surface with L5

ligned and one of the bromophenyl groups of the nutlin-2 ligand from the former can be

nd–MDM2 complexes were aligned and the ligands are shown superimposed using] (b), 1TTV [98] (c), and 1T4E [99] (d). The predicted binding mode of MI-219 [59]MDM2 model derived from 1YCR (using the programmes OMEGA2 and FRED from

Recently, a crystal structure of a complex between MDMX and ap53 peptide was solved, which shows why MDM2 inhibitors havesuch low affinity for MDMX [63,64] (Fig. 4). Mainly this is due to thefact that the L26 subsite in the p53-binding cleft is slightly smaller inMDMX than in MDM2. The Y99 (MDMX) residue is oriented differ-


ently to the corresponding Y100 (MDM2) and a larger M55 (MDMX)replaces L54 (MDM2). Although the two proteins have very similarsecondary and tertiary structure, the presence of a unique and con-formationally constrained 95PSP97 sequence in MDMX at the start

tructure complexes with bound p53 peptides of MDM2 (cyan; PDB entry 1YCR [2])ucture diagrams. The �2′ helix in MDMX adopts a different orientation to that in97 sequence (shown as unlabelled stick model) in MDMX. This results in a differentcorresponding Y100 (MDM2) side chain (cyan CPK sticks from 1YCR). IntermediateT4E, 1T4F, 2AXI and 2GV2 (magenta CPK sticks) [41,48,99,100]. Together with then of Y99 (MDMX) results in occlusion of part of the p53-binding site in MDMX. (b)4 and Y100 in cyan CPK). (c) The MDM2 (1RV1) and MDMX (3DAB) structures wereobserved to clash with the altered M53,Y99 (MDMX) region.


http://www.eyesopen.com/

ING

Y

n Canc

ofbtcbaMbat

ttfMWba

4

4

e(atcsaCifusoadE

tstmoar

udbpuHtuc

4

2d

ARTICLEModel



f the �2′ helix, which supports the Y99 residue, results in a dif-erent orientation of this helix compared to MDM2. In MDMX thisrings Y99 into close proximity of the larger M55, resulting in par-ial obstruction of the p53-binding cleft. A recent computationalomparison of MDM2 and MDMX also suggests that differencesetween the proteins may affect how they recognise p53, as wells small-molecule inhibitors [65]. As the flexible N-terminal lid ofDM2 is known to play a role in molecular recognition [58], it has

een suggested that the significant differences in the lids of MDM2nd MDMX may differentially influence ligand recognition and inurn selectivity [66].

The structure-activity relationships of MDM2 inhibitors showhat increasing the size of the lipophilic binding groups so thathey fill the binding site better greatly increases potency. There-ore optimised MDM2 inhibitors are probably too bulky to bind

DMX, whereas p53 and peptides derived from it can bind both.hile the affinity for MDMX of only very few MDM2 inhibitors has

een reported, it appears that the more potent and optimised theyre for MDM2, the more selective they are for MDM2 over MDMX.

. MDM2 as an E3 ubiquitin ligase

.1. Background

Protein ubiquitinylation involves three ATP-dependentnzymes in a sequential reaction. A ubiquitin activating enzymeE1) forms a thioester bond between its active site cysteine residuend the C-terminal glycine of ubiquitin. Activated ubiquitin ishen transferred from the E1-ubiquitin complex to a ubiquitinonjugating enzyme (E2) by transthioesterification. In the finaltep a ubiquitin protein ligase (E3) binds the E2-ubiquitin complexnd aids in the formation of an isopeptide linkage between the-terminus of ubiquitin and the �-amino group of a lysine residue

n the target protein, or to a ubiquitin already attached [67]. Onceour or more ubiquitins are linked through lysine 48 (K48) ofbiquitin, the modified protein is recognised by the 26S protea-ome, and is degraded. Specificity of the ubiquitinylation processccurs mostly at the level of the E3 enzymes, of which over 1000re known in the human body, whereas there are only around 30ifferent E2 enzymes, and a single E1 (two isoforms referred to as1a and E1b) [68].

MDM2 belongs to the family of E3 ubiquitin ligases that con-ain a RING (really interesting new gene) domain [69]. These aretructurally defined by cross-branched active site histidine and cys-eine residues bound to two zinc ions. Although MDM2 can catalyse

ultiple monoubiquitinylation and polyubiquitinylation reactionsn p53, it remains unclear to what extent MDM2 or other E3 lig-ses (such as p300) are responsible for the p53 polyubiquitinylationequired for efficient degradation in vivo [70–72].

Apart from MDM2, three other proteins can act as E3 ubiq-itin ligases for p53. PIRH2 (p53-induced protein with RING-H2omain), like MDM2, is linked with p53 in an autoregulatory feed-ack loop that controls p53 function [73]. COP1 (constitutivelyhotomorphogenic 1) acts independently of MDM2 as a RING E3biquitin ligase [74] and ARF-BP1 (ARF-binding protein) acts as aECT (homologous to E6-AP carboxyl terminus) E3 ubiquitin ligase

owards p53 [75]. The roles of these E3 ubiquitin ligases in p53 reg-lation are not well understood and there is no evidence that theyan replace MDM2 in the regulation of p53 stability [34].


.2. MDM2 E3 ligase inhibitors

The first report on MDM2 E3 ligase inhibitors dates back to002 and concerns the arylsulfonamide, bisarylurea, and acylimi-azolone compounds shown in Fig. 5, which were discovered in an


MDM2-mediated p53 ubiquitinylation screen of a chemical library[76]. It was shown that all three compounds behaved as simplereversible inhibitors of MDM2 in vitro, that they bound to MDM2in a mutually exclusive manner, and that inhibition was noncom-petitive with respect to both E2 and p53 substrates. Furthermore,the compounds were selective insofar as they did not inhibit E3ligases other than MDM2, and, surprisingly, did not inhibit MDM2autoubiquitinylation.

It is known that while the isolated MDM2 RING domain thatincludes the extreme C-terminus of MDM2 retains E3 ligase activ-ity, ubiquitinylation of p53 by MDM2 also requires the N-terminaldomain, where the main p53 recruitment site resides, as well as thecentral acidic domain, which contains a secondary p53-binding site[77]. One could therefore imagine that the above compounds mightprevent p53 ubiquitinylation not at the level of the MDM2 catalyticactivity but by preventing p53 binding. A lack of effects of the com-pounds on the physical interaction between MDM2 and p53 wasdemonstrated, however, suggesting that the mode of inhibitionmay be allosteric, perhaps by blocking a structural rearrangementof MDM2 necessary for p53 ubiquitinylation but not for MDM2autoubiquitinylation [72].

Regardless of the mechanism of MDM2 inhibition, the selectivitytowards p53 ubiquitinylation as opposed to MDM2 autoubiquitiny-lation by the arylsulfonamide, bisarylurea, and acylimidazolonecompounds in Fig. 5 would be desirable from a therapeutic view-point, since inhibition of both activities might lead to accumulationof MDM2, which in turn would be expected to limit inhibition ofp53 ubiquitinylation and subsequent degradation. However, no cel-lular or in vivo activity data were presented for these compounds,and apparently there has not been any follow-up since the originalreport [76].

The only other p53-selective MDM2 E3 ligase inhibitor inthe public domain concerns a compound (of undisclosed struc-ture) that was also identified in a high through-put chemicallibrary (>600,000 compounds) screen using an MDM2-mediatedp53 ubiquitinylation assay, as well as an MDM2 autoubiquitiny-lation counter screen [78]. It was observed that although most ofthe numerous screening hits identified showed similar activity inthe p53 and autoubiquitinylation assays, a few chemotypes dis-played some selectivity. The most selective compound inhibitedp53 ubiquitinylation with an IC50 value of 8 �M but was inactive atconcentrations up to 100 �M in the autoubiquitinylation assay.

A family of closely related 7-nitro-5-deazaflavin compoundscalled HLI98 (deazaflavins 1–3 in Fig. 5) have been identified asinhibitors of MDM2 E3 ubiquitin ligase activity by high through-put screening of MDM2 autoubiquitinylation [79]. Using cell-basedassays, the lead compound HLI98C was demonstrated to inhibitselectively p53 ubiquitinylation, to increase MDM2 and p53 pro-tein levels, to reactivate p53 function, and to induce p53-dependentapoptosis in cancer cells. However, the HLI98 compounds appear tohave low potency and to promote at least some p53-independentcellular toxicity. Nevertheless, these compounds succeed in show-ing proof of principle that small molecules can inhibit MDM2 E3ubiquitin ligases and may have potential for use in cancer therapy[80].

A potential problem with the HLI98 compounds results fromthe high redox potential of 5-deazaflavins. The nitro group is sus-ceptible to one-electron reduction leading to the generation ofthe nitro anion radical. The planar heteroaromatic system of theHLI98 compounds can intercalate with DNA [81] and the pres-ence of the reactive radical can then result in cytotoxicity through


DNA damage [82,83]. More recent work investigated the abilityof 5-deazaflavin analogues to stabilise and activate p53. Resultsshow that the nitro group present in HLI98 compounds is in factnot essential for 5-deazaflavins to reactive p53. Thus 6-chloro-5-deazaflavin compounds such as deazaflavin 4 in Fig. 5 were found




6 M.P. Dickens et al. / Seminars in Cancer Biology xxx (2009) xxx–xxx

olecul

t

(s1tuipc

u[bupsli

rttdrttc

4a

nue

Fig. 5. Chemical structures of small-m

o induce elevation of p53 levels to a similar extent as HLI98 [84].Very recently, a more soluble and potent deazaflavin

deazaflavin 5 (HLI393) in Fig. 5) was discovered, which pos-esses a 5-dimethylaminopropylamino side chain but lacks the0-aryl group of HLI98 compounds [85]. HLI393 was determinedo be highly water-soluble and to inhibit MDM2-mediated p53biquitinylation with low micromolar cellular potency, resulting

n increased MDM2 and p53 protein levels, leading to selective53-dependent apoptosis in a variety of different cancer cell linesontaining wild-type p53.

Sempervirine was discovered as an inhibitor of MDM2 E3 ubiq-itin ligase activity in a high through-put natural products screen86]. Like the deazaflavins, sempervirine was observed to inhibitoth MDM2-dependent p53 ubiquitinylation and MDM2 autoubiq-itinylation. Again, treatment of cancer cells harbouring wild-type53 with this compound induced stabilisation of p53 and apopto-is. The structurally unusual [87] plant alkaloid sempervirine hasong been known to possess anticancer activities [88] and perhapsnhibition of MDM2 E3 ligase activity contributes to these.

Certain acridine derivatives (refer structure in Fig. 5, where Represents cyclic and noncyclic aliphatic systems) have been showno stabilise p53 protein levels by blocking p53 ubiquitinylationhrough a mechanism that differs from what occurs following DNAamage, where p53 stabilisation is the result of its inability toecognise and to be tagged for destruction by MDM2 due to post-ranslational modification [89]. The acridine derivates induced p53ranscriptional activity and p53-dependent apoptosis in tumourells in vivo but it remains unclear if they inhibit MDM2 directly.

.3. MDM2-mediated p53 ubiquitinylation versus MDM2utoubiquitinylation


The question of how MDM2 E3 ligase inhibitors that doot apparently distinguish between autoubiquitinylation and p53biquitinylation work at the molecular level remains open. Sev-ral mechanisms can be considered. The most obvious is that they

e MDM2 E3 ubiquitin ligase inhibitors.

might block ATP binding or interfere with the E3 catalytic site insome other way. The C-terminal tail of MDM2 was shown to becritical for MDM2 E3 ligase activity [90,91] and a recent X-ray crys-tal structure of an MDM2–MDMX RING domain heterodimer [92]shows that this tail, by inserting into a groove of the partner protein,forms a composite binding site for the E2-ubiquitin complex (Fig. 6).Since this interaction is apparently required for MDM2 E3 ligaseactivity both in trans and in cis (perhaps by a similar tail insertionintramolecularly), it is possible that nonselective inhibitors targetthe E2-binding site or the tail-binding groove directly.

Despite the fact that 3D structural information on the MDM2RING domain is now available [63,64,92,93], several aspects of itsE3 ligase activity remain unclear, including exact delineation ofthe nucleotide-binding site and the active site itself, as well as thenature of the overall catalytic mechanism.

5. Clinical development of MDM2 inhibitors

At the time of compiling the present report we are awareof at least two MDM2 inhibitors that have actually entered theclinic. The first compound is JNJ-26854165 (Ortho Biotech; John-son & Johnson), which is currently being investigated as an oralagent in advanced stage or refractory solid tumours in a phase-I trial [94]. The chemical structure of this compound, which wasapparently discovered using a p53 degradation assay, is shown inFig. 7 [94]. It was reported to induce p53 levels in tumour celllines and to activate p53 transcriptional activity. However, unlikep53–MDM2 protein interaction inhibitors and E3 ligase inhibitors,JNJ-26854165 apparently blocks the association of MDM2 with theproteasome both in vitro and in cell-based assays. How exactly thisleads to p53 induction remains unclear.


Another compound currently undergoing clinical evaluation isR7112 (Hoffmann-La Roche) [95]. Like JNJ-26854165, R7112 is anoral agent and is being studied in phase-I trials in haematologicneoplasms and advanced solid tumours. No detailed informationappears to be in the public domain but one assumes that R7112




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 (structure cartoon with MDM2 in green and MDMX in cyan. The two zinc ions in each RIN(b) One face of the MDM2 surface, together with the C-terminus of MDMX, forms the likewith side chain and labelled). (c) Interaction between the C-terminus of MDMX (cyan) an

Fr

iapho

6

asicptd

ig. 7. Chemical structure of JNJ-26854165, a clinical MDM2 inhibitor that has beeneported to block an association of MDM2 with the proteasome.

s a compound from the nutlin series. Finally, Ascenta [96] haven oral MDM2 inhibitor compound known as AT-219 under latereclinical development. Again the exact nature of the compoundas not been disclosed but it is likely to be an optimised memberf the spiro-oxindole series.

. Conclusions

Directly inhibiting the p53–MDM2 interaction, as a means ofctivating p53, is potentially useful in the treatment of cancerstill expressing wild-type p53 and continues to be investigated


ntensively. There have been concerns as to how viable this con-ept would be therapeutically: will it be possible to inhibit arotein–protein interaction with a drug-like molecule? What arehe effects of unleashing p53 on healthy cells? Endeavours toevelop small-molecule inhibitors have addressed these issues and

constructed from PDB entry 2VJF [92]). (a) The complex is depicted as a secondaryG domain are shown as spheres, and the coordinating residue side chains as lines.ly interaction site with the E2 ubiquitin conjugating enzyme (key residues shownd MDM2 (green CPK surface).

in so doing have increased our understanding of the p53–MDM2protein–protein interaction and the effects of inhibiting it. Theevidence is now in favour and with a number of these inhibitorsentering clinical trials the ultimate proof of concept may be justaround the corner. The next stage in the refinement of p53–MDM2protein–protein interaction inhibitors will probably concern thecurrent lack of cross-inhibition of MDMX.

The development of MDM2 E3 ubiquitin ligase inhibitors is lessadvanced and remains hampered by the biological complexity ofthe ubiquitinylation process. There is a real need for a better under-standing at the molecular level of how exactly MDM2 functions asan E3 ubiquitin ligase, so that structure-based drug design effortscan be used. It has been pointed out that E3 ligases in general areconceptually very attractive drug targets but that “ligases are todaywhere kinases were 10 to 15 years ago” [97].

Conflict of interest

The authors declare that there are no conflicts of interest.


Funding source

Michael P. Dickens’s studies are sponsored by Cancer ResearchUK through the Beatson Institute for Cancer Research, Glasgow,


ING

Y

8 n Canc

SLccFftswf

R

ARTICLEModel



cotland, UK. Ross Fitzgerald’s studies are sponsored by Cyclacelimited, Dundee, Scotland, UK. Peter M. Fischer’s studies in theancer research area relevant to the article are sponsored by Cycla-el Limited, Dundee, Scotland, UK (studentship to coauthor Rossitzgerald) and Cancer Research UK through the Beatson Instituteor Cancer Research, Glasgow, Scotland, UK (studentship to coau-hor Michael Dickens). These sponsors have no involvement in thetudy design; collection, analysis and interpretation of data; theriting of the manuscript; the decision to submit the manuscript

or publication.

eferences

[1] Cahilly-Snyder L, Yang-Feng T, Francke U, George DL. Molecular analysis andchromosomal mapping of amplified genes isolated from a transformed mouse3T3 cell line. Somat Cell Mol Genet 1987;13:235–44.

[2] Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, et al.Structure of the MDM2 oncoprotein bound to the p53 tumor suppressortransactivation domain. Science 1996;274:948–53.

[3] Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The mdm-2 oncogeneproduct forms a complex with the p53 protein and inhibits p53-mediatedtransactivation. Cell 1992;69:1237–45.

[4] Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation ofp53. Nature 1997;387:296–9.

[5] Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 fortumor suppressor p53. FEBS Lett 1997;420:25–7.

[6] Tao W, Levine AJ. Nucleocytoplasmic shuttling of oncoprotein Hdm2 isrequired for Hdm2-mediated degradation of p53. Proc Natl Acad Sci USA1999;96:3077–80.

[7] Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm-2 autoregulatory feedbackloop. Genes Dev 1993;7:1126–32.

[8] Barak Y, Juven T, Haffner R, Oren M. mdm2 expression is induced by wild typep53 activity. EMBO J 1993;12:461–8.

[9] Matheu A, Maraver A, Serrano M. The Arf/p53 pathway in cancer and aging.Cancer Res 2008;68:6031–4.

[10] Dai MS, Shi D, Jin Y, Sun XX, Zhang Y, Grossman SR, et al. Regulation of theMDM2–p53 pathway by ribosomal protein L11 involves a post-ubiquitinationmechanism. J Biol Chem 2006;281:24304–13.

[11] Jin A, Itahana K, O’Keefe K, Zhang Y. Inhibition of HDM2 and activation of p53by ribosomal protein L23. Mol Cell Biol 2004;24:7669–80.

[12] Fang S, Jensen JP, Ludwig RL, Vousden KH, Weissman AM. Mdm2 is a RINGfinger-dependent ubiquitin protein ligase for itself and p53. J Biol Chem2000;275:8945–51.

[13] Meek DW, Knippschild U. Posttranslational modification of MDM2. Mol Can-cer Res 2003;1:1017–26.

[14] Shvarts A, Steegenga WT, Riteco N, van Laar T, Dekker P, Bazuine M, etal. MDMX: a novel p53-binding protein with some functional properties ofMDM2. EMBO J 1996;15:5349–57.

[15] Ramos YF, Stad R, Attema J, Peltenburg LT, van der Eb AJ, Jochemsen AG. Aber-rant expression of HDMX proteins in tumor cells correlates with wild-typep53. Cancer Res 2001;61:1839–42.

[16] Linares LK, Hengstermann A, Ciechanover A, Muller S, Scheffner M. HdmXstimulates Hdm2-mediated ubiquitination and degradation of p53. Proc NatlAcad Sci USA 2003;100:12009–14.

[17] Lane D. How cells choose to die. Nature 2001;414:25–7.[18] Vousden KH. Activation of the p53 tumor suppressor protein. Biochim Bio-

phys Acta 2002;14:47–59.[19] Momand J, Jung D, Wilczynski S, Niland J. The MDM2 gene amplification

database. Nucleic Acids Res 1998;26:3453–9.[20] Bartel F, Meye A, Wurl P, Kappler M, Bache M, Lautenschlager C, et al. Ampli-

fication of the MDM2 gene, but not expression of splice variants of MDM2MRNA, is associated with prognosis in soft tissue sarcoma. Intl J Cancer2001;95:168–75.

[21] Boeckler FM, Joerger AC, Jaggi G, Rutherford TJ, Veprintsev DB, Fersht AR.Targeted rescue of a destabilized mutant of p53 by an in silico screened drug.Proc Natl Acad Sci USA 2008;105:10360–5.

[22] Joerger AC, Ang HC, Fersht AR. Structural basis for understanding onco-genic p53 mutations and designing rescue drugs. Proc Natl Acad Sci USA2006;103:15056–61.

[23] Myers MC, Wang J, Iera JA, Bang JK, Hara T, Saito S, et al. A new family ofsmall molecules to probe the reactivation of mutant p53. J Am Chem Soc2005;127:6152–3.

[24] Atencio IA, Grace M, Bordens R, Fritz M, Horowitz JA, Hutchins B, et al. Bio-logical activities of a recombinant adenovirus p53 (SCH 58500) administeredby hepatic arterial infusion in a Phase 1 colorectal cancer trial. Cancer Gene


Ther 2006;13:169–81.[25] Cristofanilli M, Krishnamurthy S, Guerra L, Broglio K, Arun B, Booser DJ, et

al. A nonreplicating adenoviral vector that contains the wild-type p53 trans-gene combined with chemotherapy for primary breast cancer: safety, efficacy,and biologic activity of a novel gene-therapy approach. Cancer (Hoboken, NJ,United States) 2006;107:935–44.

PRESSer Biology xxx (2009) xxx–xxx

[26] Fujiwara T, Tanaka N, Kanazawa S, Ohtani S, Saijo Y, Nukiwa T, et al.Multicenter phase I study of repeated intratumoral delivery of adenovi-ral p53 in patients with advanced non-small-cell lung cancer. J Clin Oncol2006;24:1689–99.

[27] Guo J, Xin H. Chinese gene therapy. Splicing out the West? Science2006;314:1232–5.

[28] Shimada H, Matsubara H, Shiratori T, Shimizu T, Miyazaki S, Okazumi S, et al.Phase I/II adenoviral p53 gene therapy for chemoradiation resistant advancedesophageal squamous cell carcinoma. Cancer Sci 2006;97:554–61.

[29] Wallraven G, Nemunaitis JJ, Maples PB. Compassionate approval process forexperimental gene-based products. J Clin Oncol 2008;26:1899–900.

[30] Mendrysa SM, McElwee MK, Michalowski J, O’Leary KA, Young KM, Perry ME.Mdm2 Is critical for inhibition of p53 during lymphopoiesis and the responseto ionizing irradiation. Mol Cell Biol 2003;23:462–72.

[31] Mendrysa SM, O’Leary KA, McElwee MK, Michalowski J, Eisenman RN, PowellDA, et al. Tumor suppression and normal aging in mice with constitutivelyhigh p53 activity. Genes Dev 2006;20:16–21.

[32] Zhang R, Wang H, Agrawal S. Novel antisense anti-MDM2 mixed-backboneoligonucleotides: proof of principle, in vitro and in vivo activities, and mech-anisms. Curr Cancer Drug Targets 2005;5:43–9.

[33] O’Leary KA, Mendrysa SM, Vaccaro A, Perry ME. Mdm2 regulates p53independently of p19(ARF) in homeostatic tissues. Mol Cell Biol 2004;24:186–91.

[34] Vassilev LT. MDM2 inhibitors for cancer therapy. Trends Mol Med2007;13:23–31.

[35] Bogan AA, Thorn KS. Anatomy of hot spots in protein interfaces. J Mol Biol1998;280:1–9.

[36] Fischer PM. Peptide, peptidomimetic, and small-molecule antagonists of thep53–HDM2 protein–protein interaction. Int J Peptide Res Ther 2006;12:3–19.

[37] Hardcastle IR. Inhibitors of the MDM2–p53 interaction as anticancer drugs.Drugs Fut 2007;32:883–96.

[38] Murray JK, Gellman SH. Targeting protein–protein interactions: lessons fromp53/MDM2. Biopolymers 2007;88:657–86.

[39] Bottger V, Bottger A, Howard SF, Picksley SM, Chene P, Garcia-Echeverria C, etal. Identification of novel mdm2 binding peptides by phage display. Oncogene1996;13:2141–7.

[40] García-Echeverría C, Chène P, Blommers MJJ, Furet P. Discovery of potentantagonists of the interaction between human double minute 2 and tumorsuppressor p53. J Med Chem 2000;43:3205–8.

[41] Sakurai K, Schubert C, Kahne D. Crystallographic analysis of an 8-merp53 peptide analogue complexed with MDM2. J Am Chem Soc 2006;128:11000–1.

[42] Chene P, Fuchs J, Bohn J, García-Echeverría C, Furet P, Fabbro D. A small syn-thetic peptide, which inhibits the p53-hdm2 interaction, stimulates the p53pathway in tumour cell lines. J Mol Biol 2000;299:245–53.

[43] Shangary S, Wang S. Targeting the MDM2–p53 interaction for cancer therapy.Clin Cancer Res 2008;14:5318–24.

[44] Hu C-Q, Hu Y-Z. Small molecule inhibitors of the p53–MDM2. Curr Med Chem2008;15:1720–30.

[45] Domling A. Small molecular weight protein–protein interaction antagonists:an insurmountable challenge? Curr Opin Chem Biol 2008;12:281–91.

[46] Dey A, Verma CS, Lane DP. Updates on p53: modulation of p53 degradationas a therapeutic approach. Br J Cancer 2008;98:4–8.

[47] Berg T. Small-molecule inhibitors of protein–protein interactions. Curr OpinDrug Discov Dev 2008;11:666–74.

[48] Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo acti-vation of the p53 pathway by small-molecule antagonists of MDM2. Science2004;303:844–8.

[49] Koblish HK, Zhao S, Franks CF, Donatelli RR, Tominovich RM, LaFrance LV, et al.Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress humantumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo.Mol Cancer Ther 2006;5:160–9.

[50] Leonard K, Marugan JJ, Raboisson P, Calvo R, Gushue JM, Koblish HK, et al.Novel 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists with improvedcellular activity. Bioorg Med Chem Lett 2006;16:3463–8.

[51] Marugan JJ, Leonard K, Raboisson P, Gushue JM, Calvo R, Koblish HK, et al.Enantiomerically pure 1,4-benzodiazepine-2,5-diones as Hdm2 antagonists.Bioorg Med Chem Lett 2006;16:3115–20.

[52] Parks DJ, Lafrance LV, Calvo RR, Milkiewicz KL, Gupta V, Lattanze J, et al. 1,4-Benzodiazepine-2,5-diones as small molecule antagonists of the HDM2-p53interaction: discovery and SAR. Bioorg Med Chem Lett 2005;15:765–70.

[53] Parks DJ, LaFrance LV, Calvo RR, Milkiewicz KL, Jose Marugan J, RaboissonP. Enhanced pharmacokinetic properties of 1,4-benzodiazepine-2,5-dioneantagonists of the HDM2-p53 protein–protein interaction through structure-based drug design. Bioorg Med Chem Lett 2006;16:3310–4.

[54] Ding K, Lu Y, Nikolovska-Coleska Z, Qiu S, Ding Y, Gao W, et al. Structure-based design of potent non-peptide MDM2 inhibitors. J Am Chem Soc2005;127:10130–1.

[55] Ding K, Lu Y, Nikolovska-Coleska Z, Wang G, Qiu S, Shangary S, et al. Structure-based design of spiro-oxindoles as potent, specific small-molecule inhibitors


of the MDM2–p53 interaction. J Med Chem 2006;49:3432–5.[56] Deng J, Dayam R, Neamati N. Patented small molecule inhibitors of

p53–MDM2 interaction. Expert Opin Ther Pat 2006;16:165–88.[57] Uhrinova S, Uhrin D, Powers H, Watt K, Zheleva D, Fischer P, et al. Structure

of Free MDM2 N-terminal domain reveals conformational adjustments thataccompany p53-binding. J Mol Biol 2005;350:587–98.


ING

Y

n Canc

ery and cocrystal structure of benzodiazepinedione HDM2 antagonists that

ARTICLEModel



[58] Showalter SA, Bruschweiler-Li L, Johnson E, Zhang F, Bruschweiler R. Quanti-tative lid dynamics of MDM2 reveals differential ligand binding modes of thep53-binding cleft. J Am Chem Soc 2008;130:6472–8.

[59] Shangary S, Qin D, McEachern D, Liu M, Miller RS, Qiu S, et al. Temporal acti-vation of p53 by a specific MDM2 inhibitor is selectively toxic to tumorsand leads to complete tumor growth inhibition. Proc Natl Acad Sci USA2008;105:3933–8.

[60] Hu B, Gilkes DM, Farooqi B, Sebti SM, Chen J. MDMX overexpression preventsp53 activation by the MDM2 inhibitor nutlin. J Biol Chem 2006;281:33030–5.

[61] Patton JT, Mayo LD, Singhi AD, Gudkov AV, Stark GR, Jackson MW. Levels ofHdmX expression dictate the sensitivity of normal and transformed cells toNutlin-3. Cancer Res 2006;66:3169–76.

[62] Hu B, Gilkes DM, Chen J. Efficient p53 activation and apoptosis by simultane-ous disruption of binding to MDM2 and MDMX. Cancer Res 2007;67:8810–7.

[63] Popowicz GM, Czarna A, Holak TA. Structure of the human Mdmx pro-tein bound to the p53 tumor suppressor transactivation domain. Cell Cycle2008;7:2441–3.

[64] Popowicz GM, Czarna A, Rothweiler U, Sszwagierczak A, Krajewski M, WeberL, et al. Molecular Basis for the Inhibition of p53 by Mdmx. Cell Cycle2007;6:2386–92.

[65] Macchiarulo A, Giacche N, Carotti A, Baroni M, Cruciani G, Pellicciari R. Tar-geting the conformational transitions of MDM2 and MDMX: insights intodissimilarities and similarities of p53 recognition. J Chem Inf Model 2008,ePub ahead of print (doi:10.1021/ci800146m).

[66] McCoy MA, Gesell JJ, Senior MM, Wyss DF. Flexible lid to the p53-bindingdomain of human Mdm2: implications for p53 regulation. Proc Natl Acad SciUSA 2003;100:1645–8.

[67] Ciechanover A. The ubiquitin-proteasome pathway: on protein death and celllife. EMBO J 1998;17:7151–60.

[68] Fang S, Weissman AM. A field guide to ubiquitinylation. Cell Mol Life Sci2004;61:1546–61.

[69] Joazeiro CA, Weissman AM. RING finger proteins: mediators of ubiquitin lig-ase activity. Cell 2000;102:549–52.

[70] Grossman SR, Deato ME, Brignone C, Chan HM, Kung AL, Tagami H, et al.Polyubiquitination of p53 by a ubiquitin ligase activity of p300. Science2003;300:342–4.

[71] Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W. Mono- versus polyubiqui-tination: differential control of p53 fate by Mdm2. Science 2003;302:1972–5.

[72] Lai Z, Ferry KV, Diamond MA, Wee KE, Kim YB, Ma J, et al. Human mdm2mediates multiple mono-ubiquitination of p53 by a mechanism requiringenzyme isomerization. J Biol Chem 2001;276:31357–67.

[73] Leng RP, Lin Y, Ma W, Wu H, Lemmers B, Chung S, et al. Pirh2, a p53-inducedubiquitin-protein ligase, promotes p53 degradation. Cell 2003;112:779–91.

[74] Dornan D, Wertz I, Shimizu H, Arnott D, Frantz GD, Dowd P, et al. The ubiquitinligase COP1 is a critical negative regulator of p53. Nature 2004;429:86–92.

[75] Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF-BP1/Mule is a critical mediatorof the ARF tumor suppressor. Cell 2005;121:1071–83.

[76] Lai Z, Yang T, Kim YB, Sielecki TM, Diamond MA, Strack P, et al. Differenti-ation of Hdm2-mediated p53 ubiquitination and Hdm2 autoubiquitinationactivity by small molecular weight inhibitors. Proc Natl Acad Sci USA2002;99:14734–9.

[77] Ma J, Martin JD, Zhang H, Auger KR, Ho TF, Kirkpatrick RB, et al. A second p53binding site in the central domain of Mdm2 is essential for p53 ubiquitination.Biochemistry 2006;45:9238–45.


[78] Murray MF, Jurewicz AJ, Martin JD, Ho TF, Zhang H, Johanson KO, et al. Ahigh-throughput screen measuring ubiquitination of p53 by human mdm2. JBiomol Screen 2007;12:1050–8.

[79] Davydov IV, Woods D, Safiran YJ, Oberoi P, Fearnhead HO, Fang S, et al. Assayfor ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2.J Biomol Screen 2004;9:695–703.


[80] Yang Y, Ludwig RL, Jensen JP, Pierre SA, Medaglia MV, Davydov IV, et al. Smallmolecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activatep53 in cells. Cancer Cell 2005;7:547–59.

[81] Burgstaller P, Hermann T, Huber C, Westhof E, Famulok M. Isoalloxazinederivatives promote photocleavage of natural RNAs at G.U base pairs embed-ded within helices. Nucleic Acids Res 1997;25:4018–27.

[82] Kawamoto T, Ikeuchi Y, Hiraki J, Eikyu Y, Shimizu K, Tomishima M, et al. Syn-thesis and evaluation of nitro 5-deazaflavins as novel bioreductive antitumoragents. Bioorg Med Chem Lett 1995;5:2109–14.

[83] Kawamoto T, Ikeuchi Y, Hiraki J, Eikyu Y, Shimizu K, Tomishima M, et al.Evaluation of differential hypoxic cytotoxicity and electrochemical studies ofnitro 5-deazaflavins. Bioorg Med Chem Lett 1995;5:2115–8.

[84] Wilson JM, Henderson G, Black F, Sutherland A, Ludwig RL, Vousden KH, etal. Synthesis of 5-deazaflavin derivatives and their activation of p53 in cells.Bioorg Med Chem 2007;15:77–86.

[85] Kitagaki J, Agama KK, Pommier Y, Yang Y, Weissman AM. Targeting tumorcells expressing p53 with a water-soluble inhibitor of Hdm2. Mol Cancer Ther2008;7:2445–54.

[86] Sasiela CA, Stewart DH, Kitagaki J, Safiran YJ, Yang Y, Weissman AM, et al.Identification of inhibitors for MDM2 ubiquitin ligase activity from natu-ral product extracts by a novel high-throughput electrochemiluminescentscreen. J Biomol Screen 2008;13:229–37.

[87] Woodward RB, Witkop B. The structure of sempervirine. J Am Chem Soc1949;71:379.

[88] Beljanski M, Beljanski MS. Three alkaloids as selective destroyers ofcancer cells in mice. Synergy with classic anticancer drugs. Oncology1986;43:198–203.

[89] Wang W, Ho WC, Dicker DT, MacKinnon C, Winkler JD, Marmorstein R, et al.Acridine derivatives activate p53 and induce tumor cell death through Bax.Cancer Biol Ther 2005;4:893–8.

[90] Poyurovsky MV, Priest C, Kentsis A, Borden KL, Pan ZQ, Pavletich N, et al. TheMdm2 RING domain C-terminus is required for supramolecular assembly andubiquitin ligase activity. EMBO J 2007;26:90–101.

[91] Uldrijan S, Pannekoek WJ, Vousden KH. An essential function of the extremeC-terminus of MDM2 can be provided by MDMX. EMBO J 2007;26:102–12.

[92] Linke K, Mace PD, Smith CA, Vaux DL, Silke J, Day CL. Structure of theMDM2/MDMX RING domain heterodimer reveals dimerization is requiredfor their ubiquitinylation in trans. Cell Death Differ 2008;15:841–8.

[93] Kostic M, Matt T, Martinez-Yamout MA, Dyson HJ, Wright PE. Solution struc-ture of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53. JMol Biol 2006;363:433–50.

[94] Arts J, Page M, Valckx A, Blattner C, Kulikov R, Floren W, et al. JNJ-26854165—anovel hdm2 antagonist in clinical development showing broad-spectrum pre-clinical antitumor activity against solid malignancies. Proc Am Assoc CancerRes 2008;49. Abs. 1592.

[95] Roche Pharma Pipeline; 2008. http://www.roche.com/pipeline.htm.[96] Ascenta’s Pipeline; 2008. http://www.ascenta.com/.[97] Garber K. Missing the target: ubiquitin ligase drugs stall. J Natl Cancer Inst

2005;97:166–7.[98] Fry DC, Emerson SD, Palme S, Vu BT, Liu C-M, Podlaski F. NMR structure of

a complex between MDM2 and a small molecule inhibitor. J Biomol NMR2004;30:163–73.

[99] Grasberger BL, Lu T, Schubert C, Parks DJ, Carver TE, Koblish HK, et al. Discov-


activate p53 in cells. J Med Chem 2005;48:909–12.[100] Fasan R, Dias RL, Moehle K, Zerbe O, Obrecht D, Mittl PR, et al. Structure-

activity studies in a family of beta-hairpin protein epitope mimeticinhibitors of the p53-HDM2 protein–protein interaction. ChemBioChem2006;7:515–26.


http://dx.doi.org/10.1021/ci800146m

http://www.roche.com/pipeline.htm

http://www.ascenta.com/

phd lit review

Business

egulates p53

p53 suppressionare

protein levels of p53

p53 tranactivity

negative regulation

nuclear export of p53

nonredundant homologueof

human protein