Michael A. Dyer
Aart G. Jochemsen, R. Kiplin Guy and
Samantha A. Cicero, Brenda A. Schulman,
Catherine A. Regni, Donald Bashford,
Zhu, Nicholas Mills, David C. Smithson,
FangyiLeggy A. Arnold, Antonio M. Ferreira,
Damon Reed, Ying Shen, Anang A. Shelat,
First Small Molecule Inhibitor of MDMX
Identification and Characterization of the
Cell Biology:
doi: 10.1074/jbc.M109.056747 originally published online January 15, 2010
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Identification and Characterization of the First Small
Molecule Inhibitor of MDMX
*
□
S
Received for publication, August 17, 2009, and in revised form, January 8, 2010 Published, JBC Papers in Press, January 15, 2010, DOI 10.1074/jbc.M109.056747
Damon Reed
‡1
, Ying Shen
‡1
, Anang A. Shelat
§
, Leggy A. Arnold
§
, Antonio M. Ferreira
¶
, Fangyi Zhu
§
, Nicholas Mills
§
,
David C. Smithson
§
, Catherine A. Regni
¶
, Donald Bashford
¶
, Samantha A. Cicero
‡
, Brenda A. Schulman
¶储
,
Aart G. Jochemsen**, R. Kiplin Guy
§
, and Michael A. Dyer
‡储2
From the Departments of
‡
Developmental Neurobiology,
§
Chemical Biology and Therapeutics, and
¶
Structural Biology and
储
Howard Hughes Medical Institute, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 and the **Department of
Molecular and Cellular Biology, Leiden University Medical Center, 2333 ZA Leiden, Netherlands
The p53 pathway is disrupted in virtually every human tumor.
In ⬃50% of human cancers, the p53 gene is mutated, and in the
remaining cancers, the pathway is dysregulated by genetic
lesions in other genes that modulate the p53 pathway. One com-
mon mechanism for inactivation of the p53 pathway in tumors
that express wild-type p53 is increased expression of MDM2 or
MDMX. MDM2 and MDMX bind p53 and inhibit its function by
distinct nonredundant mechanisms. Small molecule inhibitors
and small peptides have been developed that bind MDM2 in the
p53-binding pocket and displace the p53 protein, leading to
p53-mediated cell cycle exit and apoptosis. To date, peptide
inhibitors of MDMX have been developed, but no small mole-
cule inhibitors have been reported. We have developed bio-
chemical and cell-based assays for high throughput screening of
chemical libraries to identify MDMX inhibitors and identified
the first MDMX inhibitor SJ-172550. This compound binds
reversibly to MDMX and effectively kills retinoblastoma cells in
which the expression of MDMX is amplified. The effect of
SJ-172550 is additive when combined with an MDM2 inhibitor.
Results from a series of biochemical and structural modeling
studies suggest that SJ-172550 binds the p53-binding pocket of
MDMX, thereby displacing p53. This lead compound is a useful
chemical scaffold for further optimization of MDMX inhibitors
that may eventually be used to treat pediatric cancers and vari-
ous adult tumors that overexpress MDMX or have similar
genetic lesions. When combined with selective MDM2 inhibi-
tors, SJ-172550 may also be useful for treating tumors that
express wild-type p53.
Tumorigenesis is a multistep process that involves dysregu-
lation of several pathways that are crucial for cell growth and
survival (1). The p53 pathway regulates cell survival in response
to cellular stress (e.g. DNA damage) or oncogenic stress (e.g. Rb
pathway dysregulation) (2, 3) and is suppressed in virtually
every human cancer by genetic lesions in the p53 gene or other
components of the pathway (4). Approximately half of all can-
cers express wild-type p53, and considerable research over the
past decade has focused on inducing p53-mediated cell death in
these tumors (4, 5). Most efforts to date have focused on inhib-
iting MDM2, a negative regulator of p53 (6–14).
Another key regulator of the p53 pathway is a protein related
to MDM2 called MDMX (15–17). MDM2 and MDMX share
homology in their p53-binding domains, but MDMX is
believed to regulate p53 through distinct mechanisms. Specifi-
cally, MDM2 primarily regulates p53 stability and subcellular
localization, whereas MDMX may directly regulate p53 tran-
scription (17–21). MDMX is genetically amplified in 19% of
breast carcinomas, 19% of colon carcinomas, 18% of lung car-
cinomas, and a smaller percentage of gliomas (17). One of the
best characterized tumors with an MDMX amplification is ret-
inoblastoma. Approximately 65% of human retinoblastomas
have increased MDMX copy number, which correlates with
increased MDMX mRNA and protein (22). Previous studies
have demonstrated that the MDMX amplification suppresses
p53-mediated cell death in Rb pathway-deficient retinoblasts
(22).
A general consensus is emerging that to efficiently induce a
p53 response in tumor cells that express wild-type p53, it may
be necessary to inactivate both MDM2 and MDMX (18, 23, 24).
To date, no screens to identify small molecule inhibitors of
MDMX have been reported, and MDM2 inhibitors probably do
not bind as efficiently to MDMX because of structural differ-
ences in the p53-binding pockets of the two proteins (25–27).
Consistent with this theory, nutlin-3a binds MDMX with at
least a 40-fold weaker equilibrium binding constant than for
MDM2 (22).
Therefore, to identify small molecules that bind MDMX and
prevent its interaction with p53, we developed biochemical and
cell-based assays suitable for high throughput screening (HTS)
3
of chemical libraries. Using this approach, we have identified
the first MDMX inhibitor, SJ-172550, and demonstrated that it
*This work was supported, in whole or in part, by National Institutes of Health
grants from NCI, Cancer Center Support. This work was also supported by
Howard Hughes Medical Institute, American Cancer Society, Research to
Prevent Blindness, Pearle Vision Foundation, International Retinal
Research Foundation, Pew Charitable Trust, and American Lebanese Syr-
ian Associated Charities (to M. A. D.).
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental “Materials and Methods,” Figs. 1–9, Table 1, and additional
references.
1
Both authors contributed equally to this work.
2
To whom correspondence should be addressed: MS323, St. Jude Children’s
Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105. Tel.: 901-
595-2257; Fax: 901-595-3143; E-mail: michael.dyer@stjude.org.
3
The abbreviations used are: HTS, high throughput screening; FP, fluores-
cence polarization; FITC, fluorescein isothiocyanate; MALDI, matrix-as-
sisted laser desorption ionization; IR, ionizing radiation; GST, glutathione
S-transferase; shRNA, small hairpin RNA.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10786 –10796, April 2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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can efficiently kill MDMX-amplified retinoblastoma cells.
SJ-172550 functions in an additive manner with the MDM2
inhibitor nutlin-3a, thereby confirming the importance of tar-
geting both of these negative regulators of p53 in cancer cells.
This validated MDMX inhibitor provides a valuable lead com-
pound and chemical scaffold for further chemical modification
to develop a high affinity MDMX inhibitor with good bioavail-
ability, pharmacokinetics, and pharmacodynamics.
EXPERIMENTAL PROCEDURES
Plasmid Constructs and Protein Production—The p53-bind-
ing domain of mouse and human MDMX (amino acids 1–185)
and human MDM2 (amino acids 1–188) were amplified by PCR
and cloned into the pGEX-4T1 plasmid. Recombinant GST
fusion proteins were prepared in BL21 (DE3) Escherichia coli
cells. The lysates were cleared by spinning at 100,000 ⫻g,
and the supernatant was loaded onto a 5-ml GSTrap Fast-
Flow column (GE Healthcare). Subsequent purification
included a Mono Q column and an S200 gel filtration col-
umn. Peak fractions were combined and dialyzed against
phosphate-buffered saline (pH 7.6) containing 2 mMphenyl-
methylsulfonyl fluoride.
Fluorescence Polarization Assays—Fluorescence polarization
(FP) assays were conducted in assay buffer containing 10 mM
Tris (pH 8.0), 42.5 mMNaCl, and 0.0125% Tween 20. The wild-
type p53 peptide (amino acids 15–29) was GSGSSQETF-
SDLWKLLPEN, and the mutant AAA-p53 peptide was
GSGSSQETFADLAKLAPEN. The FP assays were carried out
using 2.5 nMFITC peptide (or 15 nMTexas Red) and 1
M
GST-MDMX or GST-MDM2. For MDM2-p53 or MDMX-p53
inhibitor assays, small molecules were preincubated with the
recombinant protein for 30 min. The labeled peptide was then
added and incubated for 45 min. FP assays were conducted in
384-well black microplates (Corning Glass). The FP FITC
assays were analyzed using an EnVision multilabel plate reader
with a 480-nm excitation filter, a 535-nm static and polarized
filter, and an FP FITC dichroic mirror. The unlabeled compet-
itor peptide and nutlin-3 were used as positive controls, and the
alanine-substituted p53 peptide (AAA-p53) was used as a neg-
ative control. To minimize the possibility of false-positives
caused by endogenous fluorescence from the compounds in the
library, we also developed an FP assay with the Texas Red fluo-
rophore. This assay was conducted as described above, except it
required a 555-nm excitation filter, a 632-nm static and polar-
ized filter, and a Texas Red FP dichroic mirror.
Chemical Library and High Throughput Screening—The
screening library consisted of 295,848 unique compounds from
commercial sources (ChemDiv, ChemBridge, and Life Chemi-
cals) arrayed individually at 10 mMin DMSO in 384-well
polypropylene plates. The purity of compounds was reported
by the vendor as ⱖ90%. HTS was carried out on a system devel-
oped by high resolution engineering with integrated plate incu-
bators (Liconic). Plates were transferred from instrument to
instrument by a Stau¨bli T60 robot arm. Assay materials were
dispensed in bulk by using Matrix Wellmates (Matrix Technol-
ogies). Compound plates were centrifuged in a Vspin plate cen-
trifuge (Velocity11). All compound transfers were accom-
plished by using a 384-well pin tool with 10-nl hydrophobic
surface-coated pins (V & P Scientific). These pins allowed for
the delivery of 25 nl to achieve a final compound concentration
of 10
M. The fluorescent signal was measured using an EnVision
multilabel plate reader.
RESULTS
Characterization of an MDMX-p53 Binding Assay for High
Throughput Screening—To identify MDMX inhibitors by HTS
of chemical libraries, we developed an FP assay (28) to detect
the binding of the p53 peptide to GST-MDMX-(1–185) in 384-
well plates. This assay is based on the retention of polarization
during fluorescence spectroscopy of the p53 peptide conju-
gated to a fluorophore such as FITC (Fig. 1A). FP is inversely
proportional to the rotational diffusion of the fluorophore, and
for our experiments, it was measured using an FP spectrometer.
The polarization of free p53-FITC peptide (2.5 nM) was 100 mP,
and with increasing concentrations of purified GST-MDMX-
(1–185) protein (supplemental Fig. 1, A–D), the polarization
peaked (100%) at 280 mP (Fig. 1B). Similar data were obtained
using GST-MDM2-(1–188) (Fig. 1Band supplemental
Fig. 1, A–D). From these curves, the EC
50
for p53 peptide-
MDMX was 0.36
Mand that for p53 peptide-MDM2 was 0.23
M(Fig. 1B). Biacore experiments provided similar binding
constants for GST-MDMX-(1–185) (K
d
⫽1.05
M) and GST-
MDM2-(1–188) (K
d
⫽1.03
M)(supplemental Fig. 1E). Iso-
thermal titration calorimetry measurements using a purified
minimal p53-binding domain of MDMX-(23–111) confirmed
these binding constants (supplemental Fig. 1, F–H).
To test the specificity of our FP assay for MDMX/MDM2
binding to p53, we performed a competition experiment with
unlabeled p53 peptide and a version of the AAA-p53 peptide
that is defective for binding to MDM2/MDMX (29). The pro-
tein concentration in this assay was 1
M, and the p53-FITC
peptide concentration was 2.5 nM. The EC
50
values from the
competition experiments were 0.42 and 0.30
Mfor GST-
MDMX-(1–185) and GST-MDM2-(1–188), respectively (Fig.
1C), whereas AAA-p53 showed no evidence of competition at
any concentration tested (0.5–200
M) (Fig. 1C).
To test whether our FP assay was suitable for identifying
small molecule inhibitors of MDMX-p53 binding, we per-
formed a dose-response experiment using nutlin-3a at con-
centrations ranging from 0.5 nMto 300
M(Fig. 1D). Nut-
lin-3a was originally identified as an MDM2 inhibitor (6), but
it also binds to MDMX, albeit with a much weaker K
d
value
(22). The protein concentration was held constant at 1
M,
and the peptide concentration was 2.5 nMfor each concen-
tration of nutlin-3a tested. The EC
50
value for binding of
nutlin-3a to MDM2 was 0.28
Mand that to MDMX was 20.1
M(Fig. 1D).
High Throughput Screening of a Chemical Library to Identify
Novel MDMX Inhibitors—To identify novel MDMX inhibitors,
we performed an HTS of the St. Jude chemical library (295,848
unique compounds) by using the GST-MDMX-(1–185)/p53-
FITC peptide FP assay (Fig. 1E). A total of 356,352 wells (com-
pounds and controls) were screened over the course of 13 days
by using 928 plates (chemical structures and screening data are
available for download at the following url: www.stjuderesearch.
org/guy/data/mdmx). We selected the mouse MDMX (MdmX)
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protein for the HTS, because expression of the recombinant
protein in E. coli was more efficient than human MDMX.
Compounds were screened at a final concentration of 10
M.
The scatterplot of activities demonstrates clear separation
between the positive and negative controls (Fig. 1E). The
average z-prime for the assays was positive but low (⬍0.4)
because of variance in the control distributions (supple-
mental Fig. 2D). However, the reference peptide EC
50
value was
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stable and within experiment error, and no major plate artifacts
were detected (supplemental Fig. 2).
To reduce the probability of selecting false-positive com-
pounds from the primary screen, we performed a receiver oper-
ating characteristic analysis using different activity thresholds
for compound selection. This analysis led us to select the 70%
activity threshold to obtain 3,596 compounds for subsequent
validation. To eliminate compounds with intrinsic fluorescence
emission spectra that overlapped with FITC, we performed a
secondary FP assay using a Texas Red-conjugated p53 peptide.
This analysis was performed on all 3,596 compounds that met
the 70% activity cutoff (10
M) in triplicate. The top 1,000 com-
pounds were then selected from the Texas Red FP assay based
on their percentage inhibition. An additional 152 structural
analogs of these 1000 compounds were included (total 1,152) to
provide sufficient coverage of chemical scaffolds to begin to
FIGURE 1. Biochemical assays for high throughput screening to identify MDMX inhibitors. A, schematic of the FP assay used to identify MDMX inhibitors.
The protein used for the screen consisted of residues 1–185 of MDMX fused to GST. The peptide (orange) was conjugated to FITC (green) for the primary screen
and to Texas Red for secondary assays. B, plot of the percentage of bound p53-FITC peptide (at a fixed concentration) with increasing concentrations of MDMX
(squares) or MDM2 (triangles). C, plot of the percentage of p53-FITC peptide associated with indicated proteins in the presence of increasing concentrations of
unlabeled wild-type p53 peptide or unlabeled alanine-substituted p53 peptide (AAA-p53) as a negative control. D, plot of the percentage of p53-FITC peptide
associated with the indicated proteins in the presence of increasing concentrations of nutlin-3a. B–D, each data point is the mean ⫾S.D. of triplicate
experiments. E, scatterplot of HTS of a chemical library for MdmX inhibitors. The blue data points indicate compounds that were selected for further analysis,
and the black data points are compounds that did not exhibit activity in the HTS. DMSO was used as a negative control (red), and the unlabeled p53 peptide
(green) was used as a positive control. The density plot illustrates the clear separation of the positive and negative control samples across the entire screen. Each
day of screening is separated by a yellow line.
FIGURE 2. Identification of diverse chemotypes with candidate MDMX inhibitors. A, work flow schematic of the primary HTS, secondary analysis, and
dose-response and cell-based assays. Numbers of compounds that were selected for each round of analysis are indicated. B, histogram of the distribution of
MDMX activities for the 1,152 compounds in this study. Gray bars represent the number of compounds within the indicated range of activity from the FITC-FP
primary screen; black bars represent the number of those compounds that were confirmed as true positives via dose response. Similarly, the light and dark blue
bars represent the distributions from the Texas Red FP retest screen. The Texas Red FP assay is better than the FITC FP assay at discriminating true-positives from
false-positives. C, distribution of EC
50
values for MdmX, MDMX, and MDM2 for all 1,152 compounds calculated from the dose response in triplicate using the
Texas Red FP assay. D–J, visual representation of seven chemotype clusters. The black triangles are Murcko scaffolds, and screened compounds are represented
as nodes that are connected to their parent scaffold by gray lines. Nodes are colored by potency against MDMX (blue ⫽high and gray ⫽low) and sized
according to selective cytotoxicity for retinoblastoma cells versus BJ cells (large ⫽selective for retinoblastoma). Large dark blue circles have low binding
constants for MDMX and selective cytotoxicity for retinoblastoma cells. Eleven compounds were selected from these chemotype clusters for further
characterization.
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establish structure-activity relationships for our candidate
MDMX inhibitors (Fig. 2A).
Analysis of Active Compounds from MDMX Inhibitor High
Throughput Screening—To further characterize the 1,152
active compounds, we measured the binding constant of each
compound to MdmX (mouse), MDMX (human), and MDM2
(human) by performing a dose-response assay in triplicate (Fig.
2, A–C). Compounds that showed activity ⱖ70% in the primary
FITC MdmX assay displayed a wider range of activities in the
Texas Red retest assay (Fig. 2B, compare gray bars to light blue
bars). Moreover, the median retest activity was a better predic-
tor of validated compounds (compounds with a well behaved
dose response) than the primary screen single point activity.
Therefore, the to further characterize the 1,152 active com-
pounds, we measured the binding constant of each compound
to MdmX (mouse), MDMX (human), and MDM2 (human) by
performing a dose-response assay in triplicate (Fig. 2, A–C). FP
assay demonstrated better discriminatory power than the FITC
FP assay.
To complement these biochemical studies, we carried out a
cell-based assay to further characterize the activity of the 1,152
compounds on retinoblastoma cells that have an MDMX
amplification (Weri1) or a cell line that is p53-deficient
(SJmRbl-8) (22). CellTiter-Glo (Promega) assay was used to
measure intracellular ATP levels as an indicator of viability.
SJmRbl-8 cells and Weri1 cells showed a linear relationship
between luminescence and cell number (supplemental
Fig. 3, Aand B). As a positive control for cytotoxicity, we used
vincristine, which is a microtubule inhibitor that disrupts chro-
mosome segregation during mitosis and kills both cell lines
with similar LC
50
values (supplemental Fig. 3C). As a positive
control for p53-selective cytotoxicity, we used nutlin-3a, which
selectively kills Weri1 cells with an MDMX amplification and is
less cytotoxic against p53-deficient SJmRbl-8 cells (supple-
mental Fig. 3D). We also used BJ cells, an hTERT-immortalized
human foreskin fibroblast cell line, as an additional control to
estimate the general cytotoxicity of compounds in our lead
compound collection. We carried out a dose-response cytotox-
icity assay for each cell line on all 1,152 compounds in triplicate.
Overall, the assay performed well in this HTS format, and we
identified a subset of compounds from our active compounds
set with significant selective cytotoxicity against the retinoblas-
toma cells (supplemental Fig. 3, Eand F).
To integrate and visualize the dose-response data from the
biochemical assays of MDMX and MDM2 and the cell-based
data and chemical scaffolds represented in the 1,152 com-
pounds, we overlaid biochemical and cell-based data onto a
network graph constructed to represent related families of che-
motypes within the lead compound set (supplemental Fig. 4).
This approach allowed us to quickly identify scaffolds with the
desired profiles showing strong binding to MDMX and cyto-
toxicity against an MDMX-amplified retinoblastoma cell line.
On the basis of these data, we selected 11 representative com-
pounds from clusters 1, 4, 5, 7, 8, 11, and 54 for further analysis
(Fig. 2, D–J,supplemental Fig. 4, and supplemental Table 1).
Several clusters that looked promising based on the aforemen-
tioned criteria were eliminated from further analysis because of
the presence of potentially problematic chemical functional-
ities such as unsubstituted quinones, maleimides, Michael
acceptors, and thioesters; potential redox activity; or other
medicinal chemistry liabilities (supplemental Fig. 4).
SJ-134433 and SJ-044557 Covalently Modify the MDMX
Protein—Among the 11 compounds selected for further analy-
sis, SJ-134433 and SJ-044557 had excellent profiles (sup-
plemental Fig. 6) with good binding constants for MDMX,
some selectivity for MDMX over MDM2, and efficient killing of
retinoblastoma cells with selectivity for the Weri1 line
(supplemental Table 1). To begin characterizing these com-
pounds, we performed an isothermal denaturation assay and a
redox assay on SJ-134433, SJ-044557, and the other nine com-
pounds (supplemental Fig. 5 and supplemental Table 1). The
rationale underlying the isothermal denaturation assay is that
binding of a small molecule to the p53-binding pocket of
MDMX may stabilize the protein, and in the presence of the
SYPRO orange hydrophobic dye, the temperature for denatur-
ation and dye binding would shift (30, 31). Indeed, GST-
MDMX-(1–185) showed a melting point shift in the presence
of p53 peptide from 46.9 ⫾0.6 °C for native GST-MDMX-(1–
185) protein to 50.8 ⫾0.6 °C for GST-MDMX-(1–185) protein
bound to the p53 peptide (supplemental Fig. 5, Aand B). How-
ever, neither SJ-044557 nor SJ-134433 exhibited a thermal
shift; in fact, they appeared to destabilize the protein
(supplemental Fig. 5).
To explore the possibility that these two compounds exhib-
ited redox activity, we performed an assay to detect compounds
capable of reducing resazurin to resorufin, a redox couple rele-
vant to oxygen tension in mammalian cells (32). DMSO was
used as a negative control, and a 1,6-dimethylpyrimido[5,4-
e][1,2,4]triazine-5,7(1H,6H)-dione-containing compound was
used as the positive control. SJ-044557 and SJ-134433 showed
some redox activity (supplemental Table 1). Next, we explored
the stability of the compounds in our FP buffer to determine
whether they were unstable and if any of the degradation prod-
ucts were reactive species that covalently modified the MDMX
protein and blocked p53 binding. Both compounds were unstable
after 24 h, and SJ-134433 degraded after2hinFPbuffer
(supplemental Fig. 6, Band E). To directly determine whether the
purified MDMX-(23–111) protein was covalently modified by
FIGURE 3. SJ-172550 reversibly binds MDMX. A, heat map of normalized activity for the 11 compounds selected for follow-up characterization. Dark blue is
more favorable for each measurement, and the compounds are listed in order of binding constant for MDMX from best (top) to worst (bottom). These data
suggested that with subsequent biochemical analyses, SJ-134433 and SJ-044557 were less suitable for follow up, and SJ-172550 was preferred. High perform-
ance liquid chromatography of compound SJ-172550 showed that it is stable for 24 h in FP buffer (Band C), and MALDI mass spectrometry showed that it does
not covalently modify the MDMX-(23–111) protein (D). The MDMX protein was incubated with SJ-172550 for2hinFPbuffer and then dialyzed away in a large
excess of dialysis buffer. The ability of the dialyzed protein to bind the p53-FITC peptide was then measured using the FP assay (red line). E, EC
50
value for
binding to p53 peptide after removal of SJ-172550 was indistinguishable from that of the untreated protein. A similar experiment with nutlin-3a also
demonstrated that it binds reversibly to MDMX. F, SJ-044557 and SJ-134433, which covalently modified MDMX, did not reversibly bind MDMX (red lines). A.U.,
absorbance units.
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either compound, we performed high resolution mass spectrom-
etry following incubation with SJ-044557 or SJ-134433. Both
showed a shift in their mass consistent with covalent modifications
(supplemental Fig. 6, Hand I). Although these inhibitors might
prove useful as tools for interrogating MDMX function, they are
not suitable for further development and were thus abandoned.
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SJ-172550 Is Stable and Reversibly Binds MDMX to Inhibit
p53 Binding—A more detailed analysis of the 11 selected com-
pounds revealed that SJ-172550 had an excellent chemical pro-
file, with respect to chemical stability, thermal stability, redox
potential, and solubility. Unlike SJ-134433 and SJ-044557,
SJ-172550 was stable in solution (Fig. 3, Band C) and did not mod-
ify the MDMX protein by covalent binding in our FP assay buffer
(Fig. 3D). Moreover, the compound exhibited strong thermal sta-
bilization (supplemental Fig. 5) and had undetectable redox activ-
ity (Fig. 3Aand supplemental Table 1). To confirm that SJ-172550
binds MDMX reversibly, we incubated the compound with
MDMX for2hinFPbuffer and then removed the compound by
dialysis. As a positive control, we used the p53 peptide and nutlin-
3a. Our results showed that SJ-172550 bound MDMX reversibly
(Fig. 3E), unlike SJ-044557 or SJ-172550 (Fig. 3F).
SJ-172550 Inhibits MDMX-p53 Binding in Cultured Cells—
To test whether SJ-172550 and nutlin-3a have additive or
synergistic effects on retinoblastoma cells, we performed an
isobologram experiment with these two compounds. The data
suggested that nutlin-3a and SJ-172550 act in an additive man-
ner to kill MDMX-amplified human retinoblastoma cells (Fig.
4A). Next, we exposed Weri1 and RB355 retinoblastoma cells
and ML-1 leukemia cells (with wild-type p53) to SJ-172550 (20
M) for 20 h and analyzed the p53 and activated caspase-3 levels
by immunofluorescence to study the mechanism of cell death.
As positive controls, we exposed Weri1 cells to nutlin-3a (5
M)
for 20 h or 5 gray ionizing radiation (IR). DMSO was used as the
negative control. As expected, the Weri1 cells exposed to nut-
lin-3a or IR showed a robust accumulation of p53 (Fig. 4, Band
D). In contrast, the cells exposed to SJ-172550 did not exhibit
the same level of accumulation of p53 (Fig. 4, Band D). This is
consistent with the role of MDMX in regulating transcriptional
activation of p53-responsive promoters but not p53 protein levels.
Apoptosis was robustly induced after exposure to SJ-172550
(Fig. 4, Cand E), and cells exited the cell cycle (Fig. 4F). Real
time RT-PCR and immunoblotting analysis of these cells
revealed that there was also an induction of p53 target genes,
but it was not as robust as that observed with nutlin-3a or IR
(Fig. 4, Gand H). More importantly, the cell death mediated by
SJ-172550 was p53-dependent (Fig. 4, I–K). In addition,
HCT116 cells were sensitive to SJ-172550, but p53-deficient
HCT-116 cells were not (supplemental Fig. 8). SJSA-X cells
expressing high levels of MDMX were also sensitive to
SJ-172550 (supplemental Fig. 8).
To determine whether SJ-172550 disrupts the MDMX-p53
interaction of cells in culture, we performed co-immunoprecipita-
tion experiments in the presence of the compound. Reciprocal
co-immunoprecipitation experiments with antibodies against
MDMX and p53 in C33A (human cervical carcinoma) cells dem-
onstrated partial inhibition of MDMX-p53 binding in cells
(supplemental Fig. 7). Similar data were obtained using human
embryonic retina cells and Weri1 retinoblastoma cells (supple-
mental Fig. 7). Together, these results suggest that the MDMX-
p53 interaction was at least partially inhibited by SJ-172550,
despite its relatively low cell permeability (see supple-
mental Table 1).
Computational Model of SJ-172550 Binding to MDMX—X-
ray crystallographic studies have provided high resolution
structures of MDM2 and MDMX bound to p53 (Fig. 5 and
supplemental Fig. 9) (26, 27, 33, 34). We overlaid these two
structures and determined that the C
␣
root mean square
deviation was 3.9 Å, suggesting that although the overall fold
was well conserved, the tertiary structures of MDM2 or
MDMX bound to p53 are significantly different. This can be
more readily visualized using a space-filling representation
of the overlaid structures. In particular, the structure of the
p53-binding pocket of MDMX was smaller than that of
MDM2 (supplemental Fig. 9B). When nutlin-3a was bound
to MDM2, the tertiary structure of the pocket underwent a
small change (C
␣
root mean square deviation ⫽0.82 Å)
(supplemental Fig. 9C). When the structure of MDM2 bound
to nutlin-3a was overlaid with that of MDMX bound to p53,
the smaller binding pocket of MDMX may explain the lower
affinity binding of nutlin-3a to MDMX, as compared with
that of MDM2 (supplemental Fig. 9, Dand E).
We used both AutoDock 4.2 (35) and the fastdock algorithm in
Scigress Explorer version 7.7 to model the binding of nutlin-3a to
MDM2 and found excellent agreement between the computa-
tional model and the native conformation reported in previous
co-crystallization studies (supplemental Fig. 9F) (6). Using the
same computational approach, the binding of SJ-172550 to the
p53-binding pocket of MDMX was modeled, yielding two impor-
tant structures (Fig. 5, Band D, and supplemental Fig. 9G).
Together, these results provide a plausible mechanism of action
for SJ-172550. SJ-172550 may occlude the p53-binding pocket of
MDMX, thereby inhibiting p53 binding. To test this directly, we
generated a series of seven mutants in the MDMX-binding pocket
(Fig. 5, Cand D,supplemental Fig. 9, Fand G, and supplemen-
tal material) and purified the protein for binding studies (Fig. 5,
E–G,supplemental Fig. 9, Hand Iand supplemental informa-
tion). Some of the mutants (e.g. H54F) were predicted to displace
SJ-172550 without affecting peptide binding, and other mutants
(e.g. M53L) were predicted to make the binding pocket of MDMX
more like MDM2 and thereby reduce binding of SJ-172550 and
FIGURE 4. SJ-172550 disrupts the MDMX-p53 interaction in cells maintained in culture. A, isobologram shows the additive inhibition of Weri1 cell growth when
the combination of SJ-172550 and nutlin-3a was used to treat cells. Multiple ratios were tested, and the plot shows that the LC
50
value (dashed line) was additive but
not synergistic (shaded area) or adverse (area above the dashed line). The error bars represent standard deviation from two independent experiments. Band C,
immunostaining of compound-treated Weri1 cells. The top panels are merged images of differential interference contrast (DIC) and 4⬘,6-diamidino-2-phenylindole
(DAPI) staining, and the lower panels show p53 levels (B) or caspase-3 activation (C). Nutlin-3a and IR were used as positive controls, and DMSO was used as a negative
control. D–F, quantification of the percentage of immunopositive cells shown in Band Cand bromodeoxyuridine (BrdU). G, real time PCR quantification of p53 target
genes p21,MDM2, and E2F1 activated by drug or IR treatments. MDMX levels were also tested. H, immunoblot and quantification for p21 protein levels activated by
drug or IR treatment. Gy, gray. Iand J, histogram of the proportion of p53 or activated caspase-3 immunopositive cells following treatment with 5 gray IR, nutlin-3a or
SJ-172550. In parallel samples, p53 was knocked down using a p53 shRNA, and MDMX was knocked down using an MDMX shRNA (22), or the samples were treated
with a control (scrambled) shRNA. Each bar represents the mean ⫾S.D. scoring in triplicate of 100 cells for each condition and each shRNA. K, representative images
of Weri1 retinoblastoma cells following treatment with SJ-172550 when they lacked p53 (p53 shRNA) or MDMX (MDMX shRNA) as compared with a control shRNA.
Arrows indicate activated caspase-3 immunopositive cells in the red (Cy3) channel. shRNA, small hairpin RNA. Scale bars, 10
M.
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increase binding of nutlin-3a without affecting peptide binding.
These data provide additional validation for our proposed mech-
anism of SJ-172550 inhibition of the MDMX-p53 interaction.
DISCUSSION
Although several small molecule inhibitors of MDM2 have
been identified (6, 14), this is the first report to identify a small
molecule inhibitor of MDMX. There is growing evidence that
MDM2 and MDMX inhibit p53 through distinct mechanisms
and that simultaneous inhibition of these two proteins in tumor
cells that express wild-type p53 may be more effective at killing
the cells than the inhibition of MDM2 alone. Our results com-
plement important previous studies on high affinity peptide
inhibitors of MDMX and MDM2 (9, 34). MDMX inhibitors
FIGURE 5. Aand B, space-filling model of the overlaid MDM2-nutlin-3a (teal) with MDMX-p53 (pink) showing SJ-172550 bound to the p53-binding pocket
of MDM2/MDMX in the secondary docking pose. C, position of p53 peptide based on the MDM2/peptide crystal structure. D, primary docking pose of
SJ-172550 to MDMX superimposed on the crystal structure of p53 peptide bound to MDM2. Cand D, the gray is the solvent-excluded surface of MDM2
(Protein Data Bank code 2Z5T), and the blue is the surface for MDMX. Green residues represent single mutations, and residues shown in red formed a
quadruple mutant. Mutations designed to displace SJ-172550 based on the models of the most energetically favorable docking poses (Fig. 5, Band D)
are Q58D, M61F, Y66I, and Q71D. A second series of residues were changed to make the MDMX-binding pocket more like MDM2 to determine whether
SJ-172550 was binding in the p53-binding pocket. These mutations include M53L, H54F, and a quadruple mutant (QUAD) with P95H/S96R/P97K/R103Y.
From top to bottom, the green residues are Met-53, His-54, Gln-58, Met-61, Tyr-66, and Gln-71 with the red residues being Arg-103, Pro-97, Ser-96, and
Pro-95. E, plot of direct binding of each MDMX protein to Texas Red-labeled p53 peptide exactly as described for the HTS. Each point is the mean ⫾S.D.
of triplicate assays. The EC
50
values for each protein are indicated. F, competition experiments with each MDMX mutant and increasing concentrations
of SJ-172550. Each data point is the mean ⫾S.D. of triplicate assays. The proteins that did not show direct binding of p53 peptide could not be analyzed
in this competition experiment. G, summary of the EC
50
values for direct binding of p53 peptide and competition by nutlin-3a and SJ-172550. The shaded
column (M53L) is one of the mutants that was predicted to make MDMX more like MDM2 and increase nutlin-3a binding while inhibiting SJ-172550
binding without affecting peptide binding. n.d., not determined; WT, wild type.
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alone (peptide or small molecule) may be useful for treating
tumors such as retinoblastoma that show increased MDMX
expression (22). In addition, they may be effective when com-
bined with MDM2 inhibitors to induce a robust p53 response in
cancer cells that express wild-type p53 (36).
We have developed and optimized a biochemical assay for
HTS of MDMX inhibitors. We screened a diverse chemical
library and identified compound SJ-172550 as the first small
molecule inhibitor of MDMX with a low micromolar binding
constant. SJ-172550 reduced p53 binding in vitro and had little
or no redox activity. It did not covalently modify MDMX but
rather thermostabilized the protein and reversibly bound it,
which was consistent with our modeling of SJ-172550 binding
to the p53-binding pocket of MDMX. When retinoblastoma
cells expressing wild-type p53 and high levels of MDMX were
exposed to SJ-172550 in vitro, they showed evidence of p53-
mediated cytotoxicity. More importantly, the death was p53-
dependent because an shRNA to p53 prevented SJ-172550-me-
diated cell death (Fig. 4, Jand K). Although these data point to a
p53-dependent cell death mechanism, they do not rule out the
possibility of off-target binding of SJ-172550. Indeed, many lead
compounds and drugs show off-target effects.
In combination with the MDM2 inhibitor nutlin-3a,
SJ-172550 showed additive cytotoxicity in cells that expressed
wild-type p53. Thus, we propose that SJ-172550 binds the p53-
binding pocket of MDMX, thereby freeing p53 to induce apo-
ptosis. This compound represents a bona fide lead molecule
and, together with the other promising lead scaffolds from this
study, can now be used for further medicinal chemical analyses,
including optimization of affinity, specificity, and cell perme-
ability and assessment of pharmacokinetics and toxicity.
It has been shown that MDM2 and MDMX can form a het-
erodimer through their Ring domains, and this may regulate
MDM2-mediated degradation of p53 (37–39). We found that
the overall p53 protein levels are not dramatically altered fol-
lowing exposure of cultured cells to SJ-172550. The identifica-
tion of the first small molecule inhibitor will allow researchers
to probe this mechanism further by comparing the effect of
MDMX protein loss to inhibition of MDMX-p53 binding on
p53 stability.
One of our key findings from the analysis of biochemical
and cell-based assay data was that the compounds that had
the best binding constants for MDMX were not necessarily
the ones that were most suitable for follow-up. For example,
SJ-134433 had a very good binding constant for MDMX, but
further analysis showed it was unstable in FP buffer, did not
thermostabilize MDMX, had significant redox activity, and
covalently modified the protein. These data clearly empha-
size the importance of performing comprehensive charac-
terization of active compounds to rule out nonspecific
mechanisms of action that make compounds unsuitable for
further development. They also emphasize the need to select
candidates for further work based on a balance of chemical
and biological properties, rather than purely on potency or
biochemical mechanism of action.
We have not yet fully validated the mechanism of action of
SJ-172550, but it seems probable based upon our modeling and
data that it binds the p53-binding pocket of MDMX and frees
p53 to activate its target genes leading to cell cycle exit and
apoptosis. Consistent with this model, we observed moderate
p53 pathway activation in MDMX-amplified retinoblastoma
cells and partial disruption of the MDMX-p53 interaction in
cell lysates from retinoblastoma cells and other cell lines. We do
not believe that the mechanism of action is through p53 protein
stabilization based on immunoblotting and single cell immuno-
staining. A structural alignment of the binding pockets of
MDMX and MDM2 was produced using the backbone atoms
and provides some clues about where SJ-172550 may bind
MDMX to induce p53 pathway activation. Mutations gener-
ated in the p53-binding pocket of MDMX provided additional
support for this proposed mechanism. Additional x-ray crystal-
lography and other structural studies are required to defini-
tively show that SJ-172550 binds to the p53-binding pocket of
MDMX. Nonetheless, this is the first small molecule MDMX
inhibitor that has been identified with a low micromolar bind-
ing constant. It is important to note that SJ-172550 also binds
MDM2, although less effectively. Our compound is clearly less
effective against MDM2 than nutlin-3a (6) or other MDM2
inhibitors (14), and it is difficult to predict whether further
refinement of MDMX binding will similarly improve MDM2
binding or lead to a high affinity MDMX-selective inhibitor.
Acknowledgments—We thank Taosheng Chen, Fu-Yue Zeng, Wenwei
Lin, Jimmy Cui, and David Bouck for assistance with HTS at the St.
Jude High Throughput Screening Center. We thank Aaron Kosinski
and David Miller for assistance with protein preparation. We thank
Erin H. Seeley, Jamie L. Allen, and Richard M. Caprioli (all of the
Mass Spectrometry Research Center, Vanderbilt Medical Center,
Nashville, TN) for assistance with high resolution MALDI mass spec-
trometry analyses. We also thank Brett Waddell for assistance with
Biacore analysis and Angie McArthur for editing the manuscript.
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