Download INHIBITING THE p53–MDM2 INTERACTION: AN IMPORTANT

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INHIBITING THE p53–MDM2
INTERACTION: AN IMPORTANT
TARGET FOR CANCER THERAPY
Patrick Chène
p53 is an attractive therapeutic target in oncology because its tumour-suppressor activity can
be stimulated to eradicate tumour cells. Inhibiting the p53–MDM2 interaction is a promising
approach for activating p53, because this association is well characterized at the structural and
biological levels. MDM2 inhibits p53 transcriptional activity, favours its nuclear export and
stimulates its degradation, so inhibiting the p53–MDM2 interaction with synthetic molecules
should lead to p53-mediated cell-cycle arrest or apoptosis in p53-positive stressed cells.
Novartis, K125 443,
CH-4002 Basel,
Switzerland.
e-mail:
[email protected]
doi:10.1038/nrc991
102
The p53 tumour suppressor is present at a low concentration in normal cells. Stress, such as hypoxia or DNA damage, causes p53 to accumulate in the nucleus, where it is
active1. Depending on the cellular stress and cell type, the
activation of p53 can lead to various responses (FIG. 1). For
example, DNA damage might result in growth arrest to
allow for repair of the damage, or apoptosis — both of
these responses aim to prevent damaged cells from proliferating and passing mutations on to the next generation2.
p53 acts as a transcription factor, and is able to activate
many genes to induce these specific functions. As cells
that lack functional p53 are unable to respond appropriately to stress, they can accumulate mutations that favour
the development of cancer. The influence of p53 loss in
cancer has been shown in vivo by inactivating the Trp53
gene (which encodes p53) in mice 3. These mice develop
normally, but are tumour prone. By 6 months of age,
74% of the animals have developed tumours, and, within
10 months, all of them are dead. In humans, about 50%
of tumours are thought to possess a mutated form of
TP53 (REF. 4) and, in some tumour types, these mutations
are associated with poor prognosis and treatment failure5.
For these reasons, many pharmaceutical companies and
academic laboratories are engaged in drug discovery
activities to target tumours with defective p53 (REFS 6,7).
In many other tumours, however, p53 is present
in its wild-type form, which offers the possibility of
stimulating its tumour-suppressor activity to eradicate
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the tumours. The involvement of p53 in effective cancer
treatment is shown by the fact that radiotherapy induces
various proteins that recognize damage and transmit
this information to p53, which, in turn, induces cell
death8. A new approach consists of stimulating p53 by
inhibiting its interaction with MDM2. MDM2 and p53
are part of an auto-regulatory feedback loop9,10 (FIG. 2).
MDM2 is transcriptionally activated by p53 (REF. 11) and
MDM2, in turn, inhibits p53 activity in several ways.
MDM2 binds to the p53 transactivation domain12 and
thereby inhibits p53-mediated transactivation13. MDM2
also contains a signal sequence that is similar to the
nuclear export signal of various viral proteins14 and,
after binding to p53, it induces its nuclear export15. As
p53 is a transcription factor, it needs to be in the nucleus
to be able to access the DNA; its transport to the cytoplasm by MDM2 prevents this. Finally, MDM2 is a
ubiquitin ligase16, so is able to target p53 for degradation
by the proteasome17,18. In normal cellular conditions,
p53 is constantly degraded by MDM2, and is therefore
present at low levels.
The importance of MDM2 in the control of p53 activity is demonstrated with Mdm2 gene-knockout mice19,20.
Their embryos die very early during gestation, but additional deletion of Trp53 rescues them from death. This
indicates that, during development, MDM2’s ability to
control p53 is essential. It has also been shown that
MDM2 overexpression blocks p53-mediated cell-cycle
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Summary
• The tumour suppressor p53 induces cell death by apoptosis in response to various
stress conditions, such as oncogene activation or DNA damage.
• The loss of p53 tumour-suppressor activity — either by mutation/deletion of the TP53
gene or by inhibition of the p53 protein — favours the development of cancer.
• The MDM2 protein is a negative regulator of p53. After binding to p53, it inhibits its
transcriptional activity, favours its nuclear export and stimulates its degradation.
• The overexpression of MDM2 in various tumours inhibits p53, therefore favouring
uncontrolled cell proliferation.
• The inhibition of the p53–MDM2 interaction is an attractive strategy to activate p53mediated apoptosis in tumours with overexpressed MDM2, but wild-type p53.
• Several low-molecular-weight compounds and peptides that inhibit the p53–MDM2
interaction have been obtained. The peptidic inhibitors show an antiproliferative effect
in tumour cells overexpressing MDM2.
Cellular stress
DNA damage
Activated oncogenes
Hypoxia
Ribonucleotide depletion
Telomere erosion
Cytoplasm
p53
Properties of the p53–MDM2 complex
Cellular responses
Apoptosis
Cell-cycle arrest
DNA repair
Differentiation
Senescence
p53 targets
Nucleus
Figure 1 | The p53-mediated response. p53 exists in nonstressed cells at a very low concentration. Under stress
conditions, the p53 protein accumulates in the cell, binds in its
tetrameric form to p53-response elements and induces the
transcription of various genes that are involved in cell-cycle
control, apoptosis, DNA repair, differentiation and senescence.
The loss of p53 tumour-suppressor activity by
mutation/deletion of TP53 or inhibition of p53 allows the
proliferation of the cells that are damaged under the stress
conditions. This uncontrolled proliferation can lead to tumour
development.
NUCLEAR MAGNETIC
RESONANCE
(NMR). A technique that uses
the magnetic properties of
certain atomic nuclei (such as
1
H, 13C and 15N) to determine
the structure of the proteins.
X-RAY CRYSTALLOGRAPHY
A technique that uses the
diffraction of the X-rays to
determine the structure of the
proteins.
samples shows that MDM2 is amplified in 7% of these
tissues30. The highest frequency of MDM2 amplification
is observed in soft-tissue tumours, osteosarcomas and
oesophageal carcinomas. Furthermore, many reports
describe the overexpression of MDM2 in different types
of tumour 31–33. The presence of high levels of MDM2 in
these tumours might be an important element for their
survival, because it decreases their ability to activate p53.
The design of compounds that prevent the interaction between p53 and MDM2 is therefore an attractive strategy for activating p53 tumour-suppressor
activity in tumours. Two different approaches have so
far been described. The first one, which consists of
designing MDM2 antisense oligodeoxynucleotides,
has already been reviewed34 and will not be covered
here. The second one consists of synthesizing lowmolecular-weight compounds that, after binding at
the interface between the p53 and the MDM2 proteins, prevent their association. So, what progress has
been made in this area of research?
arrest and apoptosis21. The activation of p53 tumoursuppressor activity therefore depends on its association with MDM2. Several pathways activate p53 via
the control of its interaction with MDM2. For example, DNA damage induces the phosphorylation of
different p53 residues (Ser15, Thr18 or Ser20), which
prevents them from binding to MDM2 (REFS 22–27).
Alternatively, the activation of oncogenes such as
c-MYC or RAS prevents MDM2-mediated degradation of p53 via expression of ARF, which — after binding
to MDM2 — abolishes MDM2-mediated degradation
of p53 (REFS 28,29). It is important to note that ARF does
not bind at the p53–MDM2 interface and, therefore, is
not a competitive inhibitor.
Overexpression of the MDM2 protein should have
negative consequences for the cell, because it diminishes its ability to activate the p53 pathway under stress
conditions. The analysis of more than 3,000 tumour
NATURE REVIEWS | C ANCER
The regions involved in the interaction between p53
and MDM2 were first identified in a yeast two-hybrid
screen35 and in immunoprecipitation experiments12.
The MDM2-binding domain on p53 was localized at
the amino terminus, from residues 1–41 (REF. 35) or
1–52 (REF. 12); the p53-binding domain on MDM2 was
also localized at the amino terminus, from residues
1–118 (REF. 35) or 19–102 (REF. 12). Site-directed experiments have subsequently shown the importance of a
few key p53 residues — Leu14, Phe19, Leu22 and
Trp23 (REF. 36) — and a minimal MDM2-binding site
on the p53 protein was mapped with p53-derived
peptides from residues 18–23 (REF. 37). The strength of
the interaction between p53 peptides and MDM2
fragments has been determined with several methods22,25,38,39. The experimental values of Kd (apparent
dissociation constant) range from 60 to 700 nM
depending on the length of the p53-derived peptides.
NUCLEAR MAGNETIC RESONANCE (NMR) studies of p53derived peptides show that they are loosely folded in
solution40–42. These measurements, together with the
X-ray data (see below), indicate that the peptide
probably adopts a helical conformation upon binding
to MDM2. The regions that correspond to residues
13–119 of Xenopus laevis MDM2 and residues
17–125 of human MDM2 have been crystallized in
complex with a peptide that corresponds to amino
acids 15–29 of p53 (p53 15–29) and their structures
have been determined by X-RAY CRYSTALLOGRAPHY39.
p5315–29 binds into a large cleft that is present at the
surface of MDM2 (FIG. 3a). The residues 19–25 form an
α-helix and residues 17, 18 and 26–29 take a more
extended conformation. Thr18 is particularly important for the stability of the helix39 and the regulation of
the p53–MDM2 interaction by phosphorylation22,23.
A detailed structural analysis of the interface
between p53 and MDM2 reveals many factors that must
be considered when aiming to inhibit this interaction.
Only one of the two partners (MDM2) has a structurally
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DNA damage
p53
Ubiquitylation
Cytoplasm
Inhibitors of
the p53–MDM2
interaction
Induces p53
degradation
MDM2
Favours nuclear
export
Proteasome
Activated
oncogenes
?
Nucleus
p53 targets
ARF
Inhibitors of
MDM2
Tumour-suppressor
activity
Blocks
transactivation
p53-independent
activities
MDM2
Figure 2 | Regulation of p53 by MDM2. p53 and MDM2 form an auto-regulatory feedback loop. p53 stimulates the expression of
MDM2; MDM2 inhibits p53 activity because it blocks its transcriptional activity, favours its nuclear export and stimulates its
degradation. Different cellular signals, such as DNA-damage or oncogene activation, induce p53 activation. DNA damage favours
p53 phosphorylation, preventing its association with MDM2. Activated oncogenes activate the ARF protein, which prevents the
MDM2-mediated degradation of p53. Similarly, inhibitors of the p53–MDM2 interaction should activate p53 tumour-suppressor
activity in tumour cells that express wild-type p53. These compounds, because they bind to MDM2, could also affect the p53independent activities of MDM2.
well-defined binding site. The inhibitors should therefore aim to mimic the other partner (p53). One of the
two interfaces (p53) is formed by only one segment of
contiguous amino acids, allowing the design of peptidic
inhibitors (p53 mimics). Three residues — Phe19, Trp23
and Leu26 (FIG. 3b) — contribute to a large extent to the
interaction and consequently to the binding energy of
the p53 peptides43. The inhibitors of the p53–MDM2
interaction will have to contain mimics of these amino
acids. There are only three hydrogen bonds connecting
p53 to MDM2, and at least the most buried one (via p53
Trp23) will have to be preserved to ensure sufficient
affinity of the inhibitors. The interface between both
proteins is rather small (the calculated accessible surface
area44 that is buried at the interface on MDM225–109 and
p5317–29 is about 660 Å2 and 809 Å2, respectively), indicating that it might be possible to design relatively small
inhibitors. This is important because molecules with
molecular weights that are higher than 500 Da usually
have a lower oral bioavailability. The interface is twisted
(the planarity 44 of the MDM2 interface is 3.1). The less
flat the interface between two partners, the greater the
tendency of one of the two partners to be buried (here,
p53). The burial of an inhibitor is usually linked to its
partial desolvation, which leads to a favourable entropic
contribution in the binding energy. The p53–MDM2
interface is hydrophobic (70% of the atoms at the interface are non-polar), and therefore inhibitors of the
p53–MDM2 interaction will have to contain lipophilic
groups. The presence of lipophilic groups usually
improves the binding energy because of the favourable
contribution of entropy. However, highly lipophilic
inhibitors will show a decrease in bioavailability.
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p53 and MDM2 family members
Recently, two TP53-related genes (TP63 and TP73) and
one MDM2-related gene (MDMX) have been
identified45–47. Several splice variants of TP63, TP73 and
MDM2 are expressed in the cell48–50 — for example,
more than 40 different splice variants of MDM2 have
been identified — which further increases the number
of p53 and MDM2 family members. These proteins
share many properties with p53 or MDM2, but they
also have distinct cellular functions50,51. Several lines of
evidence indicate that p53 is more involved in tumour
suppression, whereas p73 and p63 are more important
during development and differentiation52. This is particularly well exemplified with the different phenotypes
that are observed with the Trp53-, Trp63- and Trp73knockout mice53.
p53 has an overall primary sequence similarity of
69% and 62% with p73 and p63, respectively, and
MDM2 has an overall primary sequence similarity of
37% with MDMX. This homology indicates that p53
and MDM2 might associate with other family members. Indeed, MDMX has been shown to bind to p53
(REF. 47) and, not surprisingly, inhibitors of the
p53–MDM2 interaction also disrupt the p53–MDMX
interaction54. However, because small structural differences exist between MDM2 and MDMX, it might be
possible in the future to identify compounds that more
specifically inhibit one of these two interactions. p73
and p63 also associate with MDM2 (REFS 55,56), as would
be expected from their strong homology with p53. p73derived peptides have a similar affinity for MDM2 as do
p53-derived peptides22,57. p73 and p63 also interact with
MDMX47,56. Altogether, the available data indicate that
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a Side
negative contribution of entropy in the binding
energy. To minimize this effect, the non-natural amino
acids α-amino isobutyric acid (AIB) and 1-amino-cyclopropanecarboxylic acid (AC3C) were introduced in the
peptides to enhance their pre-organization in solution.
These modifications led to more potent peptides61,62 (4
and 5, TABLE 1). Circular dichroism and NMR measurements confirm an increased pre-organization of peptides 4 and 5 in solution61,62. However, because both
peptides are still flexible in solution, their cyclization
could further enhance their pre-organization and,
therefore, their potency. Two complementary strategies
have been adopted to further improve the affinity
(enthalpic contribution) of peptide 5. The first one
was to replace the residue corresponding to p53 Tyr22
with a phosphonomethylphenylalanine (PMP) to favour
the formation of a salt bridge with MDM2 (REF. 62).
The second one was to substitute the residue corresponding to p53 Trp23 for 6-chloro-tryptophan
(6CLTRP), to fill a small hydrophobic cavity left unoccupied by wild-type p53 in the MDM2 cleft62. The
replacement of the residue corresponding to p53 Tyr22
led to an ~7-fold increase in potency (6, TABLE 1) and
the second strategy led to a further 60-fold increase in
potency (7, TABLE 1). Altogether, blocking the conformation and enhancing the affinity of peptide 3, leads
to an increase of potency of about 1,780-fold.
Top
N
N
C
C
b
Phe19
Trp23
Leu26
PHAGE DISPLAY
A technology that is used for
displaying a protein (or peptide)
on the surface of a
bacteriophage, which contains
the gene(s) that encodes the
displayed protein(s), thereby
physically linking the genotype
and phenotype.
IC50
The concentration of an
inhibitor that is required to
inhibit 50% of the p53–MDM2
interaction.
AIB
(α-amino isobutyric acid). A
non-natural amino acid that is
used to favour helical
conformations in peptides.
AC3C
(1-aminocyclopropanecarboxylic acid). A
non-natural amino acid that is
used to stabilize 310-helix
conformations in peptides.
PMP
(Phosphonomethylphenylalanine).
A tyrosine substituted at its
hydroxyl group by a
phosphonomethyl moiety.
Figure 3 | Structure of the p53–MDM2 complex. a | The
surface of MDM225–109 is in white and the backbone of p5317–29
is in green. Two different views of the complex are presented,
and the amino (N) and carboxyl (C) termini of the p53 peptide
are indicated. b | The p5317–29 backbone is in grey and the side
chains of Phe19, Trp23 and Leu26 are represented. The
surface of MDM225–109 is in yellow.
inhibitors of the p53–MDM2 interaction might also
affect the other p53 and MDM2 family members. For
example, it has been shown that MDM2 and MDMX
inactivate the transcriptional activity and the apoptotic
function of two splice variants of p73, p73α and p73β58.
Therefore, inhibitors of the p53–MDM2 interaction
might also activate the p73-dependent pathway.
Inhibitors of the p53–MDM2 interaction
Peptide inhibitors. Early experiments have shown that
p53-derived peptides inhibit the p53–MDM2 interaction35 and that the minimal MDM2 binding site on the
p53 molecule can be reduced to p5318–23 (REF. 37). This
indicates that peptides could be used as starting points
in the design of inhibitors of the p53–MDM2 interaction. An initial effort to obtain more potent peptides
was to display different peptide libraries on PHAGES59.
This approach allowed the identification of a 12-mer
peptide (2, TABLE 1) that is 29 times more potent in vitro
than the corresponding p53 wild-type peptide (1, TABLE
1)60. Truncations of peptide 2 show that its size can be
reduced to eight residues (3, TABLE 1), keeping an IC
value in the micromolar range60.
The introduction of non-natural amino acids can
further enhance binding because of two factors —
they can help to organize the structural conformation
of the peptide in solution (entropy) before binding
and can alter chemical characteristics that directly
affect binding (enthalpy). The p53 peptides take a helical structure when bound to MDM2, but adopt several
conformations in solution40,41, probably leading to a
50
6CLTRP
(6-chloro-tryptophan).
Tryptophan with a chlorine at
position-6 (corresponds to Cη2).
ELISA
(Enzyme-linked
immunosorbent assay). A solidphase immunoassay that detects
the interaction between proteins
and specific antibodies.
NATURE REVIEWS | C ANCER
Non-peptidic or natural inhibitors. Little has been
reported on non-peptidic or natural inhibitors of the
p53–MDM2 interaction. Majeux et al. have developed
a computational approach for the evaluation of electrostatic desolvation energies of receptor and ligand
following binding 63. They docked into MDM2, using
computing tools, a library of rigid fragments representing building blocks that are frequently found in
drugs. This analysis indicates that 1,4-benzodiazepine2-one (8, TABLE 1) can mimic Phe19 and Trp23 and
that C-3 substituted derivatives of this compound
could also mimic Leu26.
The screening of microbial extracts that were generated from the fermentation of a diverse collection of
microorganisms led to the identification of the fungal
metabolite, chlorofusin64 (9, TABLE 1). Chlorofusin
inhibits the p53–MDM2 interaction with an IC50 of
4.6 µM in an enzyme-linked immunosorbent assay
(ELISA). The binding mode of chlorofusin is not known
and the published data do not show whether this natural compound binds at the interface between p53
and MDM2.
Recently, Zao and collaborators have reported the
synthesis of polycyclic compounds65. These molecules
are built on the same building block, which is used as a
scaffold for substitutions that mimic Phe19 and Trp23
(10, TABLE 1). One of these compounds induces p53
accumulation/activation and apoptosis in cell lines that
express wild-type p53.
Finally, it has been shown that chalcone derivatives
inhibit the p53–MDM2 interaction66 (11, TABLE 1). On
ELISA and gel-shift assays, these compounds showed
an ability to inhibit the p53–MDM2 interaction. The
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Table 1 | Inhibitors of the p53–MDM2 interaction
Compound Structure
IC50 (µM) Type of
References
compound
1
Ac-Gln-Glu-Thr-Phe19-Ser-Asp-Leu-Trp23-Lys-Leu-Leu26-Pro-NH2 8.7
Wild-type p53
2
Ac-Met-Pro-Arg-Phe19-Met-Asp-Tyr-Trp23-Glu-Gly-Leu26-Asn-NH2 0.3
Phage-derived
peptide
62
3
Ac-Phe19-Met-Asp-Tyr-Trp23-Glu-Gly-Leu26-Asn-NH2
8.9
Truncated phagederived peptide
62
4
Ac-Glu-Thr-Phe19-Aib-Asp-Aib-Trp23-Lys-Aib-Leu26-Aib-Glu-NH2
5.2
Constrained
wild-type
peptide
61
5
Ac-Phe19-Met-Aib-Tyr-Trp23-Glu-Ac3c-Leu26-Asn-NH2
2.2
Constrained
peptide 3
62
6
Ac-Phe19-Met-Aib-Pmp-Trp23-Glu-Ac3c-Leu26-Asn-NH2
0.3
Peptide 5 with
a PMP
at position 22
62
7
Ac-Phe19-Met-Aib-Pmp-6ClTrp23-Glu-Ac3c-Leu26-Asn-NH2
0.005
Peptide 6 with
a 6ClTrp
at position 22
62
ND
1,4 benzodiazepine2-1
63
4.6
Chlorofusin
64
ND
Polycyclic compound
65
117
Chalcone derivative
66
8
N
NH
O
9
OH
O
H
N
O
O
O
NH
O
H2N
O
O
HN
OH
O
HN
Cl
O
HN
H
N
O
O
N
N
H
HN
H2N
O
O
O
O
O
N
H
HO
O
10
OH
N
H
O
11
O
O
Cl
Cl
O
O
OH
Compounds 1 to 7 are peptides. Compounds 8, 10 and 11 are synthetic molecules. Compound 9 is a natural compound. Aib, α-amino
isobutyric acid; Ac3c, 1-amino-cyclopropanecarboxylic acid; PMP, phosphonomethylphenylalanine; 6ClTrp, 6-chloro-tryptophan.
binding mode of these compounds was determined by
NMR spectroscopy. They bind to the p53 binding site on
the MDM2 protein. However, they do not fully occupy
the cleft and only interact with the region that is involved
in the interaction with Trp23, which might explain their
low potency (between 50 µM and 250 µM in ELISA).
peptides to the leader sequence of the antennapedia
protein (referred to below as tagged peptides) 69.
Finally, it has been shown that peptide 7 can be used
directly in cellular experiments70,71. However, data are
not yet available on the biological properties of the
non-peptidic inhibitors.
Biological properties of the inhibitors
Induction of p53 activity. The experimental results essentially agree with what would be expected, given our current knowledge of the p53–MDM2 interaction. Once
transfected in MDM2-overexpressing osteosarcoma cells
(SA1), the GST inhibitors bind to MDM2, preventing its
interaction with p53 as shown in immunoprecipitation
experiments68. This induces a redistribution of cellular
p53 — so that it is found predominantly in the nucleus68
— and its accumulation67,68,70,71. This would be a predicted response following administration of inhibitors of
the p53–MDM2 interaction, as these molecules should
Various strategies have been used to study the biological effect of inhibitors of the p53–MDM2 interaction
in cellular systems. Böttger and collaborators have
inserted peptide 2 or the corresponding wild-type
peptide into a loop region of the Escherichia coli
thioredoxin protein (referred to below as TIP)67.
Wasylyk and collaborators have fused the same peptide
(or its dimer variant) to the glutathione S-transferase
protein (referred to below as GST)68. Kanovsky and
collaborators have coupled wild-type p53-derived
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prevent MDM2-mediated degradation of p53 (REFS
17,18). However it has not been shown directly that
these inhibitors actually block MDM2-mediated
ubiquitylation of p53.
This nuclear accumulation of p53 also induces its
transcriptional activation — transfection or microinjection of TIP inhibitors in various tumour cell lines
induces the activation of a p53-responsive reporter
gene67. Similar results are obtained with GST
inhibitors68. Peptide 7 and GST inhibitors also stimulate
the transcription of the endogenous MDM2 and
CDKN1A genes, and expression of the corresponding
proteins (MDM2 and WAF1, also known as p21 and
CIP1)68,70,71. By contrast, peptide 7 does not induce the
expression of WAF1 in the p53-null Saos-2 osteosarcoma cells, or in p53-mutant-expressing SK-BR-3
cells71. Similarly, GST inhibitors have no effect in head
and neck carcinoma HSC2 cells that lack p53 (REF. 68).
FACS
(Fluorescence-activated cell
sorting). A technique that is used
in flow cytometry to detect cells
that are labelled with fluorescent
dyes.
TUNEL
(TdT-mediated dUTP-X nickend labelling). A method that is
used to measure DNA strand
breaks during apoptosis.
E6 PROTEIN
A viral oncoprotein that is
derived from certain human
papillomavirus types that are
associated with increased risk of
cervical cancer. E6 binds to and
targets p53 for ubiquitinmediated degradation.
Inhibition of cell proliferation. Inhibitors of the
p53–MDM2 interaction also affect the proliferation of
tumour cell lines. TIP inhibitors induce a decrease in the
number of S-phase bromodeoxyuridine-positive T22
cells, indicating a cell-cycle arrest 67. In SA1 cells, the GST
inhibitors decrease colony formation in a colony-forming assay, induce an increase in the number of cells with
sub-G1 and G0/G1 contents of DNA, as measured by
FACS, and, finally, stimulate cell death from apoptosis, as
determined in a TUNEL assay 68. The effect of these
inhibitors is abolished in the presence of the human
papillomavirus 16 (HPV16) E6 PROTEIN, showing that they
stimulate the p53-dependent pathway. Peptide 7 induces
a cell-cycle arrest in the HCT116 colon carcinoma cell
line, which contains low amounts of MDM2, whereas it
induces apoptosis in SA1 and JAR (choriocarcinoma)
cells, which have a higher MDM2 concentration70. In
SA1 cells, peptide 7 induces apoptosis via the release of
cytochrome c from the mitochondria, and the activation
of caspase-9 and caspase-3. The absence of apoptosis in
HCT116 cells is not the consequence of an alteration in
the p53-dependent apoptotic pathway, as ectopic expression of p53 stimulates apoptosis in HCT116 cells with
deleted CDKN1A (REF. 72). This shows that, in some cell
lines, inhibition of the p53–MDM2 interaction is not
sufficient for the induction of apoptosis.
Toxicity to normal tissues? Blaydes and WynfordThomas reported that inhibition of the p53–MDM2
interaction prevents the proliferation of normal human
fibroblasts, raising the possibility that inhibitors of the
p53–MDM2 interaction could be toxic for healthy tissues73. Preliminary evidence shows that inhibitors of
the p53–MDM2 interaction are more toxic for tumour
cells than for normal cells70. The effect of peptide 7 in
two non-tumour cell lines — NHDF710 (normal
human dermal fibroblasts) and HMEC2595 (human
mammary epithelial cells) — was compared in a proliferation assay with its effect in SA1 cells. The peptide,
which activates p53 in all three cell lines, has a greater
effect on the proliferation of SA1 cells than on that of
the two non-tumour cell lines.
NATURE REVIEWS | C ANCER
The results obtained with the tagged peptides are
different69. These peptides are toxic to various tumour
cell lines that express wild-type p53, but not to normal cells. However, they are also toxic to tumour cells
that lack p53. This, together with the fact that no p53dependent apoptotic markers have been identified
after treatment with the tagged peptides, indicates
that they induce cell death via a p53-independent
mechanism. As the three tagged peptides — p5312–20
(this peptide does not contain Trp23 and Leu26), p5317–26
and p5312–26 — are equally cytotoxic, but a p53-unrelated
tagged peptide is not, the authors explain the observed
cytotoxicity by the fact that the peptides contain the
same common sequence: Glu-Thr-Phe-Ser.
Conclusions and perspectives
The growth or the death of cells depends to a large
extent on the fine-tuning of the p53–MDM2 interaction. As MDM2 controls p53 activity at the post-translational level, inhibition of the p53–MDM2 interaction
allows an immediate p53-mediated response.
Compounds that prevent this interaction might be
useful as new anticancer agents that activate the p53
pathway in tumours.
The design of inhibitors of protein–protein interaction is, today, a challenge in medicinal chemistry74.
The feasibility of such an approach depends on the
structure of the protein–protein interface. A flat, large
and polar interface is more difficult to target than a
twisted, small and apolar interface. The p53–MDM2
interface corresponds in many aspects to the second
type of interface. The p53-binding site on MDM2 is a
deep, rather small and hydrophobic cleft. The
p53–MDM2 interface therefore contains several
structural features that are favourable to the design of
inhibitors. It has been shown that it is possible to
obtain very potent peptidic inhibitors, but these
molecules are by no means drugs. It is now very
important to identify potent low-molecular-weight
compounds that block this interaction. Some pharmaceutical companies have run random screens with
their compound libraries, but the results are, so far,
disappointing: only weak inhibitors have been identified. This can be explained by a variety of factors. The
correct compounds are not present in the screened
libraries because the pharmaceutical companies,
which have mainly targeted enzymes in the past, do
not have libraries with the structural diversity that is
required to target protein–protein interactions.
Another factor is the high complementarity of the
p53–MDM2 interface. It has been shown that an
optimal ratio of receptor volume to ligand volume
exists for good molecular recognition75. It might be
difficult to find molecules that fill the Phe19, Trp23
and Leu26 sub-pockets with an optimal ratio by random screening. De novo design might therefore be
the best way to generate high-affinity compounds. In
such an approach, a first sub-pocket (for example,
Trp23) will be filled with the correct pharmacophore
mimic and, using rigid spacers (for entropic reasons),
the two other sub-pockets will be filled in a stepwise
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REVIEWS
manner. This approach, which is obviously more
time-consuming in chemistry, might ultimately be
the most productive.
On the biological side, it has been shown that inhibition of the p53–MDM2 interaction with synthetic
inhibitors such as peptides leads to activation of p53.
However, all of the data available today come from
in vitro experiments. It is therefore of utmost importance to show that inhibitors of the p53–MDM2 interaction have an antiproliferative effect in vivo.
Unfortunately, the potency and/or the physico-chemical properties of the inhibitors that are available at present do not allow us to carry out such experiments.
Several other points also remain to be examined.
Inhibitors of the p53–MDM2 interaction will probably
affect other p53 and MDM2-family members. It is
therefore important to determine the biological consequences of the interaction between the inhibitors and
these proteins. This might be a promising area for drug
discovery, as p53 is deleted or mutated in many
tumours. Another aspect is that the inhibitors of the
p53–MDM2 interactions could also interfere with
other MDM2-dependent pathways. It has been shown
that MDM2 does more than just keep p53 under control. For example, it interacts with various cellular proteins, such as TAFII250 (REF. 76), Numb77 and MTBP78.
The influence of the inhibitors of the p53–MDM2
interaction on these interactions has not been studied
and because the inhibitors bind to MDM2, they could
also affect some of the p53-independent functions of
MDM2. Are inhibitors of the p53–MDM2 interaction
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Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: CDKN1A | Mdm2 | MDM2 | MDMX | MYC | p53 | RAS |
TP63 | TP73 | Trp53 | Trp63 | Trp73
FURTHER INFORMATION
MDM2 database: http://www.infosci.coh.org/mdm2
p53 home page: http://p53.curie.fr
Structure of p53–MDM2:
http://www.rcsb.org/pdb/cgi/explore.cgi?pid=13806103734896
1&pdbId=1YCR
Access to this interactive links box is free online.
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