Targeting the MDM2–p53 Protein–Protein
Interaction for New Cancer Therapy: Progress
and Challenges
Shaomeng Wang, Yujun Zhao, Angelo Aguilar, Denzil Bernard, and Chao-Yie Yang
University of Michigan Comprehensive Cancer Center and Departments of Internal Medicine,
Pharmacology, and Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109
Correspondence: shaomeng@ umich.edu
MDM2 is a primary cellular inhibitor of p53. It inhibits p53 function by multiple mecha-
nisms, each of which, however, is mediated by their direct interaction. It has been proposed
that small-molecule inhibitors designed to block the MDM2–p53 interaction may be effec-
tive in the treatment of human cancer retaining wild-type p53 by reactivating the p53 tumor
suppressor function. Through nearly two decades of intense efforts, a number of structurally
distinct, highly potent, nonpeptide, small-molecule inhibitors of the MDM2–p53 interac-
tion (MDM2 inhibitors) have been successfully designed and developed, and at least seven
such compounds have now been advanced into human clinical trials as new anticancer
drugs. This review offers a perspective on the design and development of MDM2 small-
molecule inhibitors and discusses early clinical data for some of the MDM2 small-molecule
inhibitors and future challenges for the successful clinical development of MDM2 inhibitors
for cancer treatment.
MDM2: A PRIMARY CELLULAR
INHIBITOR OF p53
B
y controlling expression of a large number
of genes, the transcription factor p53 plays a
vital role in the regulation of many cellular pro-
cesses, including cell-cycle progression, apopto-
sis, senescence, DNA repair, and metabolism,
and functions as a powerful tumor suppressor
(Vousden and Lu 2002; Toledo and Wahl 2006;
Stiewe 2007; Brown et al. 2009; Wade et al.
2013). Mice lacking the p53 protein develop
normally but are prone to the development of
a variety of tumors (Kemp et al. 1993). Perhaps
unsurprisingly, TP53, the gene encoding p53, is
mutated or deleted in 50% of human cancers,
rendering p53 nonfunctional as a tumor sup-
pressor (Feki and Irminger-Finger 2004).
Because of the critical role of p53 in regula-
tion of many cellular processes, the level and
the activity of p53 are tightly controlled. The
murine double minute 2 (MDM2) oncogene
is a primary cellular regulator and inhibitor
of p53. The role of MDM2 as a primary nega-
tive endogenous regulator of p53 is unambigu-
ously established by the fact that MDM2-null
is embryonically lethal in mice, and they can
only be rescued by concurrent deletion of the
TP53 gene (Jones et al. 1995; Montes de Oca
et al. 1995).
Editors: Guillermina Lozano and Arnold J. Levine
Additional Perspectives on The p53 Protein available at www.perspectivesinmedicine.org
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MDM2 and p53 regulate each other mutu-
ally through the autoregulatory feedback loop
shown in Figure 1 (Wu et al. 1993; Freedman
et al. 1999). In cells containing wild-type p53,
on activation by a variety of stimuli, p53 tran-
scribes the MDM2 gene, leading to an increase
of MDM2 mRNA and protein. The MDM2 pro-
tein, in turn, binds to the p53 protein directly
through their amino termini and inhibits p53
function through three major mechanisms: (1)
On binding, MDM2 directly ubiquitinates p53
through its E3 liga se activity, promoting protea-
somal degradation of p53; (2) the interaction
of MDM2 with p53 blocks the binding of p53
to its targeted DNA, rendering p53 ineffective as
a transcription factor; and (3) MDM2 promotes
export of p53 out of the cell nucleus, making
p53 inaccessible to its targeted DNA and further
reducing its transcriptional ability (Wu et al.
1993; Freedman et al. 1999; Juven-Gershon
and Oren 1999). Through these three inhibitory
mechanisms, MDM2 functions as an effective
p53 antagonist in cells with wild-type p53.
Consistent with its role as an efficient in-
hibitor of the p53 tumor suppressor func-
tion, MDM2, when overexpressed, is oncogenic
(Ganguli et al. 2000; see Oliner et al. 2016). In
human tumors, overexpression of the MDM2
protein can be caused by gene amplification.
The MDM2 gene is amplified in an average of
7% of human cancers based on an analysis of
28 different types of cancers involving 4000
human tumor samples (Momand et al. 1998),
but a higher frequency of MDM2 gene ampli-
fication occurs in certain types of tumors,
including well-differentiated liposarcomas
(.80%), soft tissue tumors (20%), osteosarco-
mas (16%), and esophageal carcinomas (13%)
(Weaver et al. 2008, 2009). In further support of
its powerful inhibitory role of the p53 tumor
suppressor function, MDM2 gene amplification
and TP53 gene mutation are largely mutually
exclusive in human cancers (Oliner et al. 1992;
Shvarts et al. 1996; Wasylishen and Lozano
2016). In addition to MDM2 gene amplifica-
tion, MDM2 overexpression can be the result
of a variet y of other mechanism s, such as sin-
gle-nucleotide polymorphism, enhanced tran-
scription, or increased translation (Capoulade
et al. 1998; Momand et al. 2000; Bond et al.
2004, 2005). Pathologically, MDM2 overexpres-
sion has been correlated with poor clinical prog-
nosis and poor response to current cancer ther-
apies (Capoulade et al. 1998; Momand et al.
2000; Bond et al. 2004, 2005).
BLOCKING THE MDM2–p53 INTERACTION
AND REACTIVATING p53 AS A NEW CANCER
THERAPEUTIC STRATEGY
Because MDM2 functions as a primar y in-
hibitor of the p53 tumor suppressor func-
tion, agents that target MDM2 can reactivate
wild-type p53. MDM2 inhibits p53 through
several mechanisms that are dependent on its
direct interaction with p53. Hence, peptides or
nonpeptide small molecules designed to block
the MDM2–p53 protein–protein interaction
!"!#
$%&
$%&
$%&
p53
p53
p53
MDM2
Nuclear export
Inhibition
of transcriptional
activity
Poly-ubiquitination
and proteasomal degradation
Ubiquitination
Ubiquitin
Figure 1. Autoregulatory loop of p53 and MDM2. Activation of p53 transcribes MDM2 mRNA and increases
MDM2 protein, which in turn inhibits p53 activity by three mechanisms.
S. Wang et al.
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can lead to an increase of p53 protein and tran-
scriptional activation of p53. By harnessing the
powerful tumor suppressor function of p53,
such compounds may have a therapeutic poten-
tial for the treatment of human cancer retaining
wild-type p53.
Biochemical studies have mapped the
MDM2– p53 protein–protein interaction to
the first 120 amino-terminal amino acid res-
idues of MDM2 and the first 30 amino-terminal
residues of p53 (Capoulade et al. 1998; Mo-
mand et al. 2000). The determination of a
high-resolution cocrystal structure of MDM2
complexed with residues 15– 29 of a p53 pep-
tide (Fig. 2A) (Kussie et al. 1996) in 1996 pro-
vided the atomic details of their interaction and
suggested the feasibility of the design of non-
peptide, drug-like, small-molecule inhibitors
capable of blocking the MDM2–p53 interac-
tion. Specifically, the cocrystal structure shows
that the p53 peptide adopts an a-helical con-
formation and interacts with MDM2 primarily
through three hydrophobic residues, Phe19,
Trp23, and Leu26, which cluster together and
bind into a well-defined hydrophobic pocket in
MDM2. Although natural p53 peptides have
only micromolar binding affinities to MDM2,
peptides designed using unnatural amino acids
can achieve low nanomolar binding affinities
(Garcı
´
a-Echeverrı
´
a et al. 2000), further sup-
porting the feasibility of designing high-affinity,
nonpeptide, small-molecule inhibitors to block
the MDM2 – p53 interaction.
NUTLINS: THE FIRST POTENT AND SPECIFIC
SMALL-MOLECULE INHIBITORS OF THE
MDM2–p53 INTERACTION
Despite intense research efforts in academic
laboratories and pharmaceutical companies,
design of highly potent, specific, nonpeptide
small-molecule inhibitors of the MDM2 –p53
interaction with a well-defined mechanism of
action has proven to be much more difficult
than originally anticipated. The breakthrough
came in 2004 with the discover y of the nutlins
by Vassilev et al. (2004) from Hoffmann-La
Roche. Among the initial nutlins reported, nut-
lin-3a (Fig. 3) binds to MDM2 with an IC
50
(half-maximal inhibitory concentration) value
of 90 n
M and shows a cellular mechanism of
action consistent with targeting the MDM2 –
p53 interaction. Nutlin-3a effectively activates
wild-type p53 in cancer cells, potently inhibits
cell growth in cancer cell lines retaining wild-
typ e p53 in a dose-dependent manner, and
shows . 10-fold selectivity over cancer cell lines
harboring p53 mutation or with p53 deletion.
Activation of p53 by nutlin-3a leads to tran-
scription of p53-regulated genes, including
Phe19
Phe19
Trp23
Trp23
Leu26 Leu26
Leu22
Leu22
p53
p53
MDM2
Nutlin-2
MDM2
AB
Figure 2. Cocrystal structures. (A) Cocrystal structure of MDM2 (surface rendering) in complex with p53 (stick
model). p53 protein uses primarily three key residues (Phe19, Trp23, and Leu26) to interact with a well-defined,
surface hydrophobic pocket in MDM2. (B) Superposition of the cocrystal structures of nutlin-2/MDM2
complex and p53/MDM2 complex (PDBIDs: 1YCR and 4HG7). Nutlin-2 is shown by yellow sticks and the
three key p53-binding residues are shown by green sticks with the MDM2 protein shown in the surface
rendering.
MDM2–p53 Protein–Protein Interactions
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MDM2 and the cell-cycle regulator p21. Inter-
estingly, although nutlin-3a activates p53 in
nontumorigenic NIH-3T3 cells and inhibits
cell proliferation, it fails to kill the cells. Im-
pressively, nutlin-3a is orally bioavailable. At
high doses (100 and 200 mg/kg) and a twice
daily dosing schedule, oral administration of
nutlin-3a effectively inhibits tumor growth in
the xenograft model of the human osteosarco-
ma SJSA-1 cell line containing an amplified
MDM2 gene, while showing no signs of toxicity
to mice. The cocrystal structure of nutlin-2,
a close analog of nutlin-3a, in a complex with
MDM2 clearly shows that the nutlins mimic
the three key p53-binding residues (Phe19,
Trp23, and Leu26) (Fig. 2B) (Vassilev et al.
2004). The preclinical data obtained using the
nutlins show that highly potent and selective
nonpeptide small-molecule inhibitors of the
MDM2–p53 interaction may have a therapeutic
potential for the treatment of human cancers
retaining wild-type p53. The discovery of the
nutlins has also inspired other research groups
to design new MDM2 inhibitor s with higher
CI
CI O
O
O
S
N
N
EtO
N
N
Nutlin-3a RG-7112 (RO5045337)
CI
CI
O
O
O
O
N
NH
N
N
CI
CI
CI
CI
CI
CI
O
O
O
S
O
Me
CO
2
H
N
O
MI-77301 (SAR405838) MI-888 AMG-232
OH
OH
O
O
F
F
N
H
CO
2
H
OMe
NH
NH
O
N
N
N
NO
O
O
O
Cl
Cl
RG7388 (RO5503781) NVP-CGM097
Cl
CN
F
F
N
H
NH
NH
N
H
N
H
Figure 3. Chemical structures of representative MDM2 inhibitors.
S. Wang et al.
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potency and selectivity and better pharmacoki-
netics.
RG7112 (RO5045337): THE FIRST MDM2
INHIBITOR ADVANCED INTO HUMAN
CLINICAL TRIALS
Further optimization of nutlin-3a by scientists
from Hoffmann-La Roche to improve its
binding affinity to MDM2, cellular potency,
pharmacokinetics, and chemical stability ulti-
mately resulted in the discovery of RG7112
(RO5045337) (Fig. 3), the first MDM2 inhibitor
to be advanced into human clinical trials
(Vu et al. 2013; Siu et al. 2014). RG7112 has a
binding affinity to MDM2 (IC
50
¼ 18 nM),
which is better than that of nutlin-3. It effectively
inhibits cell growth in cancer cell lines with wild-
type p53 (IC
50
¼ 0.18–2.2 mM) and is several
times more potent than nutlin-3. RG7112 shows
good selectivity over cancer cell lines with a p53
mutation (IC
50
¼ 5.7– 20.3 mM). It effectively
activates w ild-type p53 in vitro and in v ivo
and shows good oral pharmacokinetic proper-
ties in mice. In two xenograft models of SJSA1
and MHM osteosarcoma cell lines with MDM2
gene amplification and overexpression of
MDM2 protein, RG7122 dose-dependently in-
hibits tumor growth and is capable of achieving
partial tumor regression with oral administra-
tion, without signs of toxicity in mice.
OTHER CLASSE S OF HIGHLY
POTENT AND SELECTIVE NONPEPTIDE,
SMALL-MOLECULE INHIBITORS
OF THE MDM2–p53 INTERACTION
Inspired by the discovery of the nutlins, several
classes of potent and selective nonpeptide
small-molecule inhibitors of the MDM2 –p53
interaction have been designed and developed
using different strategies. Some of them have
achieved much higher affinities to MDM2
and better antitumor activity than nutlin-3 or
RG7112 in animal models of human cancer.
Using a computational structure-based
design strategy, our laboratory has designed spi-
ro-oxindoles as a new class of nonpeptide
small-molecule inhibitors (Ding et al. 2005).
Extensive optimization has resulted in the dis-
covery of MI-77301 (Fig. 3) (Wang et al. 2014)
and MI-888 (Zhao et al. 2013a,b), which bind to
MDM2 with K
i
values of 0.88 and 0.44 nM,re-
spectively, and are 50 times more potent than
nutlin-3 in the same binding assay. MI-77301
and MI-888 show .10,000-fold selectivity over
MDMX, a protein closely homologous with
MDM2. In human cancer cell lines, both MI-
77301 and MI-888 effectively activate wild-type
p53 at concentrations of 30–100 n
M. Consistent
with their higher binding affinities to MDM2
and higher potencies in activation of p53, these
compounds are more than 10 times more po-
tent than nutlin-3 in inhibition of cell growth in
cancer cell lines retaining wild-type p53, and
they show .100- fold cellular selectivity over
cancer cell lines harboring mutated p53 or
with p53 deletion. In the SJSA-1 xenograft
model, both compounds, orally administered
daily, are capabl e of achieving complete and
long-lasting tumor regression without signs of
toxicity. A single, oral dose of MI-77301 is ca-
pable of achieving complete tumor regression of
the SJSA-1 tumors in mice ( Wang et al. 2014).
The cocrystal structure of MI-77301 in a com-
plex with human MDM2 protein (Wang et al.
2014) shows that consistent with its design, MI-
77301 mimics all three of the key p53-binding
residues and further is involved in additional
hydrophobic and hydrogen-bonding interac-
tions. A substituted phenyl group in MI-77301,
for example, has a p-p stacking interaction with
the His96 residue of MDM2. MI-77301 also
induces refolding of the unstructured residues
10– 25 of the MDM2 amino-terminal region,
making these residues a part of the binding
pocket and further enhancing the MDM2 –
MI-77301 binding affinity by 25 times.
AMG-232 (Fig. 3), which contains a piper-
idin-2-one scaffold, was discovered by Amgen
scientists through structure-based design and
extensive optimization (Rew et al. 2012; Sun
et al. 2014). AMG-232 has a K
d
value of
0.045 n
M with MDM2 and is probably the
most potent MDM2 inhibitor reported to date
(Sun et al. 2014). It inhibits cell proliferation
with IC
50
values of 9.1 nM and 10 nM in the
SJSA-1 and HCT-116 cell lines, respectively,
MDM2–p53 Protein–Protein Interactions
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