1,4,5-Trisubstituted Imidazole-Based p53-MDM2/MDMX Antagonists with Aliphatic Linkers for Conjugation with Biological Carriers

Article abstract of DOI:10.1021/acs.jmedchem.7b00104

The tumor suppressor protein p53, the guardian of the genome , is inactivated in nearly all cancer types by mutations in the TP53 gene or by overexpression of its negative regulators, oncoproteins MDM2/MDMX. Recovery of p53 function by disrupting the p53-MDM2/MDMX interaction using small-molecule antagonists could provide an efficient nongenotoxic anticancer therapy. Here we present the syntheses, activities, and crystal structures of the p53-MDM2/MDMX inhibitors based on the 1,4,5-trisubstituted imidazole scaffold which are appended with aliphatic linkers that enable coupling to bioactive carriers. The compounds have favorable properties at both biochemical and cellular levels. The most effective compound 19 is a tight binder of MDM2 and activates p53 in cancer cells that express the wild-type p53, leading to cell cycle arrest and growth inhibition. Crystal structures reveal that compound 19 induces MDM2 dimerization via the aliphatic linker. This unique dimerization-binding mode opens new prospects for the optimization of the p53-MDM2/MDMX inhibitors and conjugation with bioactive carriers.

Full text of DOI:10.1021/acs.jmedchem.7b00104

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Article
1,4,5-Trisubstituted Imidazole-Based p53-MDM2/MDMX Antagonists
with Aliphatic Linkers for Conjugation with Biological Carriers
Aleksandra Twarda-Clapa, Sylwia Krzanik, Katarzyna Kubica, Katarzyna Guzik, Beata
Labuzek, Constantinos G. Neochoritis, Kareem Khoury, Kaja Kowalska, Miroslawa
Czub, Grzegorz Dubin, Alexander Dömling, Lukasz Skalniak, and Tad A Holak
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 May 2017
Downloaded from http://pubs.acs.org on May 9, 2017
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1,4,5-Trisubstituted Imidazole-Based p53-
MDM2/MDMX Antagonists with Aliphatic
Linkers for Conjugation with Biological Carriers
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Aleksandra Twarda-Clapa,†,# Sylwia Krzanik,†, Katarzyna Kubica,‡, Katarzyna Guzik,‡
Beata Labuzek,‡ Constantinos G. Neochoritis,§ Kareem Khoury,§ Kaja Kowalska,∥ Miroslawa


Czub,‡ Grzegorz Dubin,†, Alexander Dömling,§ Lukasz Skalniak,*,‡ and Tad A. Holak*,‡, ,

∥
† Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30-387 Cracow, Poland
‡ Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Cracow, Poland
§ Department of Drug Design, University of Groningen, Antonius Deusinglaan 1, 9700 AD
Groningen, The Netherlands
∥ Max Plank Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
 Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387
Cracow, Poland
Keywords: p53, MDM2, MDMX, protein-protein interaction, inhibitor, dimerization, cancer
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ABSTRACT
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The tumor suppressor protein p53, the “guardian of the genome”, is inactivated in nearly all
cancer types by mutations in the TP53 gene or by overexpression of its negative regulators,
oncoproteins MDM2/MDMX. Recovery of p53 function by disrupting the p53-
MDM2/MDMX interaction using small-molecule antagonists could provide an efficient non-
genotoxic anticancer therapy. Here we present the syntheses, activities and crystal structures
of the p53-MDM2/MDMX inhibitors based on the 1,4,5-trisubstituted imidazole scaffold
which are appended with aliphatic linkers that enable coupling to bioactive carriers. The
compounds have favorable properties at both biochemical and cellular levels. The most
effective compound 19 is a tight binder of MDM2 and activates p53 in cancer cells that
express the wild type p53, leading to cell cycle arrest and growth inhibition. Crystal structures
reveal that compound 19 induces MDM2 dimerization via the aliphatic linker. This unique
dimerization-binding mode opens new prospects for the optimization of the p53-
MDM2/MDMX inhibitors and conjugation with bioactive carriers.
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INTRODUCTION
The tumor suppressor p53 is inactivated in the majority of human cancers, which is
accomplished either by mutations in the TP53 gene or by overexpression of p53 negative
regulators, primarily MDM2 and/or MDMX.1-4 Restoration of the function of p53 has been
suggested as a promising strategy against cancer.1,2,5 Mutations in the p53-encoding gene are
found in about half of all human cancers and may be combated by gene therapy.6 In the
remaining 50% of cancer cases, in which wild type p53 (p53wt) is present, restoration of its
activity by disrupting the interaction with MDM2/X proteins could be achieved by the use of
small-molecule antagonists of the MDM2/MDMX-p53 interaction. It has been shown that
such antagonists induce p53 downstream proteins, including p21, which leads to the induction
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of the G0/G1 and G2/M cell cycle arrests and additionally induction of apoptosis in some
cancer cell types.7-10 Such an approach promises a broad range non-genotoxic therapy.1,11
Several classes of small-molecule MDM2 antagonists entered clinical trials.3,12-15 However,
there are many challenges before entering clinical practice, mainly concerning
pharmacological properties, and a constant progress is needed to improve the potency of the
compounds and provide precise targeting of cancer cells.16-19
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The interaction of the N-terminal domain of MDM2 and MDMX with the N-terminal
transactivation domain of p53 is mediated by three key side chains of p53 α-helix: Phe19,
Trp23 and Leu26, which occupy hydrophobic subpockets on the MDM2 or MDMX
surface.20,21 These interactions form a three finger pharmacophore, which is present in the
most of the currently available small-molecule MDM2 inhibitors.22,23 Apart from this
canonical binding mode, several new approaches were recently proposed,24,25 for instance
utilizing the transient states of MDM2 for the design of the small molecules occupying yet
another, fourth subpocket within MDM2 (inhibitor YH300, 3-(2-(tert-butylamino)-1-(N-(4-
(4-chlorobenzyloxy)benzyl)formamido)-2-oxoethyl)-6-chloro-1H-indole-2-carboxylic acid).26
A promising class of the p53-based stapled peptides have been recently proposed as another
novel group of antagonists.27,28
Previously, we reported a novel compound based on an imidazole scaffold (6-chloro-3-(1-
(4-chlorobenzyl)-4-phenyl-1H-imidazol-5-yl)-1H-indole-2-carboxylic acid, WK23, named
thereafter 1.1) and capable of disrupting the MDM2-p53 interaction in vitro.29 In the present
study, we characterize compound 1 (Table 1), a derivative of 1.1. Compared to 1.1, the
derivatives of 1 have stronger binding to MDM2 and the improved p53-activating and
growth-inhibitory activities in cell-based assays. We also report a series of modifications of 1
with aliphatic linkers at the carboxylic group that is attached to the indole ring system
(Table 1). This further improves biological properties of the derivatives of 1. Our structure-
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based analysis suggests the potential use of such aliphatic groups for conjugation of the
compounds with biological carriers to improve solubility, stability and precise targeting to
malignant neoplasms.
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RESULTS AND DISCUSSION
Optimization of Compound 1. In a previous study we identified 1 as one of the most
potent derivatives of 1.1 in disrupting the p53/MDM2 interaction in in vitro biochemical
assays.29 Here we evaluate the activity of 1 in the cells and optimize its p53-activation,
growth-inhibitory and cell-cycle arrest properties by decorating the 1 scaffold (Table 1).
Analyzing the co-crystal structure of 1.1 with MDM2, we identified position R2 as a
potential site for compound expansion. The substituents at this position should point away
from the MDM2 molecule allowing for attachment of solubility/permeability enhancing
moieties or linkers for conjugation with targeting biological molecules, without significantly
affecting the affinity. Such assumptions were tested in compounds 13-19.
1 was obtained in the three-component (3CR) van Leusen reaction (Figure 1).30 Position R2
was modified to obtain both amide (2-13) and ester (14-19) derivatives using classical
chemistry (SI Schemes S1-S2, SI Tables S3-S4). Amide derivatives included two kinds of
substituents, polar groups expected to improve the pharmacokinetic properties of 1 and
aliphatic moieties tested to evaluate if additional binding at the surface of MDM2 may be
obtained. Ester derivatives included aliphatic linkers containing terminal reactive group
envisioned for future conjugation with biological carriers/targeting molecules. The binding of
selected derivatives was initially tested by NMR titration31,32 and affinities (Ki) of all
evaluated derivatives were tested by fluorescence polarization (FP) assay (Table 1, plots
presented in SI Table S5).
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Different modifications at R2 position had different effects on the affinity towards MDM2,
demonstrating that even though this substituent predictably should point away from the
protein molecule, certain moieties were still poorly accepted. Modification with 4-
morpholinopiperidinyl (8), 2-morpholinoethyl (14) or 6-succinyloxyhexyl (16) showed no
negative effect on affinity. The binding of 3, 4, 6, 18, and 19 was slightly negatively affected,
but the effect was negligible. The effect of other moieties was more pronounced with the
affinities increasing above two digit nanomolar range (e.g. 2, 5, 7, 9, and 13). Bulky
substituents were not accepted at all at R2 position (10-12). Both tested modification
chemistries (amide vs ester) were compatible with MDM2 binding (e.g. 8 and 14), although
particular substituents favored certain chemistries as demonstrated by an order of magnitude
better affinity of 14 compared to 13. Since the modifications were introduced primarily with
the aim of improving the effect of compounds on cells, all the derivatives which affinity was
not significantly negatively affected were further evaluated in cellular assay as described
below.
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Potency of tested compounds towards MDMX followed the trend established for MDM2,
although the affinities towards the former protein were around three orders of magnitude
lower than those towards the latter as previously observed for 1.1. 1 was the most potent
MDMX binder with affinity (Ki) of 3.15 µM. Clearly, despite significant homology of MDM2
and MDMX, subtle differences in their p53 binding pockets33 preclude optimization of 1
scaffold for concurrent binding to both those proteins.
Bista et al. have demonstrated that extending the Leu26-mimicking substituent within the
three finger pharmacophore in the context of a 6-chloroindole scaffold opens an inducible
noncanonical pocket by constraining Tyr100 in a position beneficial for binding larger
substituents. Such four pocket binding mode was associated with improvement of affinity.26
Here we tested if similar advantageous effect may be obtained within 1 scaffold. To this end
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we introduced elongated benzyl amines in position R1. These modifications (21-24) resulted
in significant reduction of affinity towards MDM2 demonstrating that 1 scaffold is not suited
for exploring the noncanonical pocket observed by Bista et al.26 Interestingly, in this
particular group of compounds the drop of affinity towards MDM2 was not associated with
significant change in potency against MDMX, again demonstrating differences in the p53
binding pockets of those two proteins.
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In parallel we evaluated the affinity of ethyl ester precursors (SI Table S2) of the 20-24
series, but these compounds (20a-24a) have entirely lost their affinity both towards MDM2
and MDMX. This is relatively surprising given that the R2 position is modified with much
larger substituents in 13-19, but this phenomenon was not further evaluated in systematic
manner.
Derivatives of Compound 1 Induce p53-target Genes and Arrest the Cell Cycle. The
on target effect of our 1,4,5-trisubstituted imidazoles in a biological context of a living cell
was evaluated by assessing the expression level of p53, MDM2 and p21 proteins, which are
the products of well-established p53 target genes. All compounds that had Ki values in the FP
assay lower than 2 µM were tested. The second generation MDM2 antagonist RG7388
(Roche;
4-{[(2R,3S,4R,5S)-3-(3-Chloro-2-fluoro-phenyl)-4-(4-chloro-2-fluorophenyl)-4-
acid,
cyano-5-(2,2-dimethyl-propyl)-pyrrolidine-2-carbonyl]-amino}-3-methoxybenzoic
named thereafter 2.1)34 was used as a positive control. 2.1 induced expression of all of the
three proteins already 7 h after the treatment (Figures 2A,B). The densitometry analysis
revealed that the level of p21 expression was increased even stronger after 24 h of the
treatment (Figure 2C). Out of the tested compounds, 1, 13, 14, 15, 16, and 19 induced
accumulations of p53, MDM2 and p21, suggesting the activation of the p53 pathway (Figures
2A,B, SI Figure S1). From these compounds, only for 13, 14 and 19 the induction of p21
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expression was further increased at 24 h of the treatment as observed for 2.1, possibly
reflecting improved stability, retention or activity of the compounds in the cells (Figure 2C).
In parallel, the off target effects/general toxicity were assessed by comparing the influence
of tested agents on the viability of p53wt U-2 OS and p53-null Saos-2 cells. Accordingly, the
compounds most active in the western blot analysis induced growth inhibition of U-2 OS
(p53wt) cells at lower concentrations than the Saos-2 (p53-/-) cells suggesting on target effect
(Table 2). Compound 15 revealed high toxicity towards Saos-2 cells and was removed from
further analysis. As a control, nutlin-3 was used in the MTT assay, confirming its selectivity
towards the p53wt cells (Table 2). The remaining tested compounds were disqualified due to
their weak impact on the growth of the U-2 OS cell line (IC50 values above 30 M, e.g. 3, 18
or 20) or high toxicity towards the Saos-2 cells (selectivity lower than 1.5, e.g. 2, 6 or 15).
Accumulation of p21 upon activation of p53 is expected to trigger cell cycle arrest in the
p53wt cells. The effect of the five most potent compounds on the cell cycle progression was
tested in U-2 OS cells. Treatment with the 5 µM concentration of 13, 14 or 19 resulted in a
markedly decreased S-phase and increased G1 fraction (Figure 3). At higher concentrations
(10 µM) 14 and 19 resulted in nearly complete depletion of the S-phase compartment, while
for 1, 16 and 13 the increase of the G1 fraction, in the expense of S phase, was clearly
observed (Figure 3). The observed extent of cell cycle arrest was comparable to that obtained
using 1 µM 2.1, a compound currently undergoing clinical trials.
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Clearly, the selected compounds show the potency in blocking the progression of the cell
cycle in a p53-dependent manner, and addition of the 2-morpholinoethyl or, especially, 6-
succinyloxyhexyl substituent at position R2 is profitable for the biological activity of
compound 1. In conclusion, compound 19 presents the best biological properties, as
demonstrated by strong and sustained activation of p53-dependent expression of p21, the
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highest selectivity in the MTT assay and the strongest induction of cell cycle arrest of p53wt
cells.
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Crystal Structure of MDM2-19 Complex Reveals Compound Induced Dimerization.
In order to better understand the structural basis of the interactions of evaluated compounds at
the binding pocket of MDM2, we determined the crystal structure of 19 in complex with the
p53 binding domain of MDM2(18-125) (construct of MDM2 comprising residues 18-125). 19
was selected as the most efficient, cell-available, p53-activating derivative. At the same time
the compound is potentially directly suitable for further conjugation with biological carriers
for its reactive group at a long aliphatic linker.
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The crystals diffracted to the 1.85 Å resolution and contained four protein molecules in the
asymmetric unit. Each protein contained a single inhibitor molecule, all of which were well
defined by their respective electron densities (SI Figure S2). No additional inhibitor molecules
were found within the structure. In each case the inhibitor locates at the p53 binding pocket
and occupies all the subpockets (Phe19, Trp23 and Leu26) of a canonical three-finger
pharmacophore (Figure 4). The interaction of 19 with MDM2 is defined primarily by
hydrophobic contacts. The 6-chloroindole substituent inserts into tryptophan pocket and
assumes an orientation almost identical to the indole side chain in the native p53-MDM2
complex while the chlorine atom potentiates the interaction at the bottom of the binding
pocket. Further comparable to p53 interaction, the nitrogen within 6-chloroindole participates
in a hydrogen bond with the carbonyl oxygen of Leu54. As such, the overall arrangement of
6-chloroindole moiety follows a classical binding observed in a large number of MDM2
inhibitors of different classes and repeating the indole interaction involved in p53 binding.16
4-chlorobezyl substituent fills the Leu26 pocket in an orientation in which the chlorine atom
points towards Tyr100. Phe19 pocket accommodates 4-fluorophenyl ring directed at Tyr67.
The imidazole scaffold of 19 further contributes to the interaction by forming hydrophobic
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contacts with Val93. As such, the binding of the core of the compound and aromatic
substituents is almost identical to that previously described for 1.1. This is not surprising since
1.1 is a precursor of 19 (SI Figure S3).
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Most interesting are the interactions of the aliphatic linker unique to 19. Comparable
affinities of 1 and 19 did not portend any pronounced protein – linker interactions. In fact, in
the crystal structure the linker protrudes outside the binging pocket. Only the disposition of
Gln72 and His73 sidechains differs from that observed in 1.1 containing structure. This
adjustment is necessary to accommodate the linker (SI Figure S3). Remarkably, however, the
protruding linker immediately contacts a second inhibitor molecule located within the
adjacent MDM2 molecule. The interaction is reciprocal as the linker of the inhibitor molecule
within the pocket of the adjacent MDM2 in turn contacts the inhibitor molecule within the
first MDM2. As such, the two MDM2 molecules form a dimer build around a dimer of two
inhibitor molecules (Figures 5A,B). The primary contacts between the inhibitor molecules
involve hydrogen bonds between the imidazole ring nitrogens and the nitrogens within the
aliphatic linker (Figure 5C). The overall dimer is further stabilized by reciprocal intra-MDM2
hydrogen bonds formed by Gln59 and Thr63 (Figure 5D). Moreover, the oxygen from the
carboxylic group terminating the aliphatic linker of each 19 molecule forms a water-mediated
contact with the side chains (Gln72, His96) of MDM2 (Figures 5A,C).
To test if the observed 19-mediated MDM2(18-125) dimerization was unrelated to the lack
of the N-terminal part of the MDM2 domain in the evaluated construct we have solved the
crystal structure of 19 in complex with a different construct of MDM2(1-125). Moreover,
these crystals were obtained in different conditions and belonged to a different space group
providing a partial control of the influence of solvent conditions and crystal packing. Despite
these differences the dimerization mode within both structures is virtually identical,
suggesting that dimer formation is compound induced rather than an effect of particular
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experimental conditions. The MDM2 molecules within the dimers seen in the two crystal
forms align with RMSD of 0.198 Å for corresponding Cα atoms. The inhibitor dimers and
interactions between the MDM2 molecules are also identical in both structures. The only
difference is in amino acids 11-17 which were not present in the first construct, but are
present and defined by electron density in the second structure. This fragment closes the
binding pocket from both sides of the dimer (SI Figure S4), but has no significant effect on
the mode of dimerization. Residues 1-10 were not defined by electron density.
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Small molecule induced dimerization of MDM2 has already been previously reported by
Graves et al. and by Surmiak et al., respectively for RO-2443 (Roche; (Z)-5-((6-chloro-7-
methyl-1H-indol-3-yl)methylene)-3-(3,4-difluorobenzyl)imidazolidine-2,4-dione,
named
thereafter 3.1)35 and 3-pyrrolin-2-ones/2-furanones.36 In all instances the dimer is mediated by
direct interaction of the inhibitor moieties facing outside of the binding pocket of MDM2.
However, when the structures of 19, 3.1 and 3-pyrrolin-2-ones/2-furanones induced dimers
are compared by aligning one of the MDM2 molecules within the dimer, the positions of the
partner molecules do not significantly overlap demonstrating different overall dimer
arrangement (SI Figures S5-S6). The interactions between the inhibitor molecules are also
significantly different. In 19, it is the amide within the linker of one inhibitor molecule that
forms a hydrogen bond with the nitrogen of the imidazole ring of adjacent inhibitor and vice
versa (Figure 5). In 3.1, aromatic stacking between the indolyl-hydantoin groups of adjacent
inhibitors is primarily involved (SI Figure S5), whereas in 3-pyrrolin-2-ones/2-furanones it is
a water mediated interaction between the inhibitor molecules and a halogen bond formed by
the inhibitor and adjacent protein molecule (SI Figure S6).
Overall, although target protein dimerization through the interaction of exposed moieties of
inhibitors is common for 19, 3.1 and 3-pyrrolin-2-ones/2-furanones, the detailed molecular
mechanisms and the overall geometry of the resulting dimers are largely dissimilar.
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19 Induces Dimerization of MDM2 in Solution and Dissociates p53-MDM2 Complex.
The arrangement of the molecules in the crystal does not necessarily have to reflect the real
biological assembly. Therefore, we evaluated if dimerization of MDM2 is induced by 19 in
solution. The retention time characterizing MDM2 in analytical gel filtration decreased in the
presence of 19 indicating formation of higher molecular weight species, presumably dimers
(based on column calibration with globular protein standard). 19-induced shifts in the elution
volume were observed for all MDM2 constructs evaluated in this study (1-118, 1-125, 18-
125; Figure 6A; SI Figure S7), so the observed dimerization is not induced by artificially
exposing certain MDM2 surface.
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Increase in the molecular mass of functional assembly results in increased relaxation time
in NMR manifesting in signal broadening in the recorded spectra. To confirm 19-induced
dimerization of MDM2, the protein was titrated with the tested compound and the line width
1
of well separated aliphatic signals in the region form -0.5 ppm to 1.0 ppm of 1D H NMR
spectra was monitored. Line width changes were not observed upon titration with 2.1, a
reference compound known not to affect the monomeric state of MDM2 in solution. Titration
with 19 resulted in marked broadening of peaks indicating the dimerization (Figure 6B, SI
Figure S7). Similar results were obtained regardless the MDM2 construct used (1-118, 18-125
and 1-125). These results further support the 19-induced MDM2 dimerization in solution.
Next we asked if 19 is able to dissociate an existing p53-MDM2 complex. To test this, we
employed a binding competition experiment, known as the 1D AIDA-NMR (Antagonist-
Induced Dissociation Assay).37 The method relies on monitoring the line width changes
associated with the changes in the molecular weight upon complex formation. The well
separated, sharp signal originating from Trp23 of p53 transactivation domain broadens to the
level of noise upon binding to MDM2. Displacement of p53 peptide from MDM2 complex by
1
an MDM2 inhibitor results in the recovery of the specific signal in 1D H NMR spectrum.
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Upon titration of p53-MDM2 complex with 19, recovery of the specific Trp23 signal was
observed, demonstrating that the compound effectively dissociates the p53-MDM2 interaction
(Figure 6C), liberating p53 from the inhibition by its negative regulator.
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CONCLUSIONS
Derivatization of the previously reported 1.1, first into compound 1 and later by modification
of the carboxyl moiety of the chloroindole scaffold, has led to a number of compounds with
affinities comparable to the starting structure, but characterized by significantly improved
activity in cell based assays. The best obtained compounds activated p53 in cancer cells to the
level comparable to the golden standard–nutlin-3a, that is induced the cell cycle arrest,
increased the expression of proapoptotic p21 and had a markedly increased cytotoxicity
towards p53 positive cells. Crystal structure of an example compound (19) revealed a
classical three finger pharmacophore binding mode identical to that of 1.1, but at the same
time an unexpected, inhibitor induced dimerization of the target protein. The dimerization was
confirmed in solution. Such unusual binding mode provides entirely new repertoire of
optimization possibilities. Further, the long aliphatic linkers, containing functional groups,
allow for future attachment of presented series of compounds to biological carriers (e.g.
hyaluronic acid)38 for improved pharmacodynamics and better delivery.
EXPERIMENTAL SECTION
Synthesis. Compounds 1 and 20-24 were obtained in the 3CR van Leusen reaction. 1 was
further modified at position R2 to obtain amide (2-13) and ester (14-19) derivatives using
classical chemistry. The purity of all final compounds - determined by the chromatographic
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UPLC or elemental analysis - was 95% or higher. For details of synthesis and analysis, see
the SI.
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Protein Expression and Purification. Variants of the N-terminal domain of human
MDM2 (1-118; 1-125 and 18-125) were cloned into the pET-20 (Novagen) and expressed in
E. coli strain BL21-CodonPlus(DE3)-RIL as described previously.39 In brief, cells were
cultured at 37°C. Protein expression was induced with 1 mM IPTG at OD600 of 0.8 and
cultured for additional 5 h at 37°C. Cells were collected by centrifugation and lysed by
sonication. Inclusion bodies were collected by centrifugation, washed with PBS containing
0.05% Triton-X100 and subsequently solubilized in 6 M guanidine hydrochloride in 100 mM
Tris-HCl, pH 8.0, containing 1 mM EDTA and 10 mM β-mercaptoethanol. The protein was
dialyzed against 4 M guanidine hydrochloride pH 3.5 supplemented with 10 mM β-
mercaptoethanol. Following, the protein was refolded by dropwise addition into 10 mM Tris-
HCl, pH 7.0, containing 1 mM EDTA and 10 mM β-mercaptoethanol and slow mixing
overnight at 4°C. Ammonium sulfate was added to the final concentration of 1.5 M and the
refolded protein was recovered on Butyl Sepharose 4 Fast Flow (GE Healthcare). The protein
was eluted using 100 mM Tris-HCl pH 7.2 containing 5 mM β-mercaptoethanol and further
purified by gel filtration on HiLoad 16/600 S75 (GE Healthcare) in 5 mM Tris-HCl pH 8.0
containing 50 mM NaCl and 5 mM β-mercaptoethanol (crystallization buffer) or in 50 mM
phosphate buffer pH 7.4 containing 150 mM NaCl and 5 mM DTT (FP/NMR buffer).
N-terminal domain of human MDMX (1-134) was cloned into pET-46Ek/LIC vector
(Novagen). The vector was transformed into E. coli strain BL21-CodonPlus(DE3)-RIL cells.
The cells were cultured at 37°C and protein expression was induced with 0.5 mM IPTG at
OD600 nm of 0.6. The recombinant protein expression was carried for 12 h at 20°C. The
protein was purified in native conditions by metal affinity chromatography. The preparation
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was polished by gel filtration on HiLoad 16/600 S75 (GE Healthcare) in 50 mM phosphate
buffer pH 7.4 containing 150 mM NaCl and 5 mM DTT (FP/NMR buffer).
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Fluorescence Polarization Assay. The assay was performed in 50 mM NaCl, 10 mM Tris
pH 8.0, 1 mM EDTA containing 5% DMSO. To determine the optimal concentration of the
protein for the competition binding assay, the effective concentration of MDM2 (1-118) and
MDMX (1-134) was each time ascertained by determining the apparent Kd towards 5’FAM-
LTFEHYWAQLTS (P2; 10 nM). Competition assay was performed by contacting serial
dilutions of tested compounds with 10 nM P2 at protein concentration yielding f0=0.8.40
Fluorescence polarization was determined at 485 nm excitation and 535 nm emission 15 min
after mixing all assay components. All tests were performed using Corning black 96-well
NBS assay plates at room temperature.
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Cell Lines and Treatment with Compounds. U-2 OS (p53wt), HCT 116 (p53wt) and
Saos-2 (p53-/-) cells were cultured as monolayers in McCoy's 5A Medium containing L-
glutamine (Lonza) and supplemented with 10% fetal bovine serum (Biowest) at 37°C and 5%
CO2 in a humidified atmosphere. All compounds were prepared as 50 mM stock solutions in
DMSO. The cells were exposed to the inhibitors for indicated time periods. In each
experiment, the final concentration of DMSO was kept constant and below detectable effect
level.
Western Blot Analysis. U-2 OS cells and HCT 116 cells (120 000 cells per well) were
seeded on 12-well transparent plates (Falcon). The inhibitors were added 24 h following
seeding and the cells were cultured for additional 7 or 24 h. Cells were lysed with RIPA
buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma). The protein lysate was
clarified by centrifugation and analyzed on 12% SDS polyacrylamide gel (Bio-Rad). The
proteins were transferred to PVDF membrane (Merck Millipore). The membranes were
blocked for 1 h in 5% BSA (Bioshop) in TTBS (100 mM Tris-HCl pH 8.0 containing 0.05%
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Tween-20 and 150 mM NaCl). Blots were probed with following antibodies: anti-MDM2
(Thermo Fisher Scientific, cat. no. 700555), p21 (Cell Signaling, cat. no. 2947), both at
1:2000 dilution, anti-p53 (Santa Cruz Biotechnology, cat. no. sc-6243) at 1:200 dilution or
anti-GAPDH (Cell Signaling, cat. no. 2118) at 1:3000 dilution. Anti-rabbit antibody
conjugated with horseradish peroxidase (Cell Signaling, cat. no. 7074) at 1:3000 dilution was
used to detect the primary antibodies. The blots were developed using ECL Western Blotting
KIT (Bio-Rad) and visualized using ChemiDoc System (Bio-Rad). Densitometric analysis
was performed using ImgeLab software (Bio-Rad).
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Cell Viability Assay. U-2 OS cells (500 cells per well) and Saos-2 cells (1500 cells per
well) were seeded on 96-well transparent plates (Falcon). Cells were treated with inhibitors
24 h following the seeding and cultured for additional 5 days. Thiazolyl blue tetrazolium
bromide (MTT) was added at the final concentration of 500 ng mL-1 and the cells were
incubated for 1 h at 37°C. The medium was removed and formazan crystals were dissolved in
isopropanol containing 40 mM HCl. Absorbance was determined at 570 nm relative to
650 nm reference. Data was normalized to DMSO-treated control. Data was analyzed using
Origin software (OriginLab). IC50 values were calculated by fitting of ‘Dose Resp’ curves to
experimental datasets.
Cell Cycle Analysis. U-2 OS cells were seeded at 300 000 per 60∙15 mm cell culture dish
(Eppendorf), treated with tested inhibitors for 24 h, trypsinized and fixed with 70% ethanol.
The cell concentration was adjusted to 1 x 106 cells/sample and DNA was labeled with PI-
RNAse solution (PBS, 5 g mL-1 propidium iodide, 0.01 mg mL-1 RNase A) for 30 min at
25°C. The cells analyzed for cell cycle DNA content using LSRFortessa cell analyzer (BD
Bioscences).
Crystal Structure Determination. Human MDM2 (residues 18-125 and 1-125) was
prepared in 5 mM Tris-HCl pH 8.0 containing 50 mM NaCl and 5 mM β-mercaptoethanol.
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Molar excess (3×) of 19 was added and the complex was concentrated to 20 mg mL-1.
Screening for crystallization conditions was performed using sitting drop vapor diffusion
setup and commercially available buffer sets (Qiagen, Hampton Research). Crystals of the
MDM2(18-125)-19 complex appeared after few days at 4°C in 0.1 M Na HEPES pH 7.5
containing 10% isopropanol and 20% PEG 4000. The MDM2(1-125)-19 complex crystallized
at 4°C in 0.1 M HEPES pH 7.5 containing 0.2 M NaCl and 25% PEG 3350. The data
collection and refinement description and statistics (SI Table S6) are summarized in the SI.
Analytical Gel Filtration. MDM2(18-125, 1-118 and 1-125) at 1 mg mL-1 was contacted
with 3× molar excess of tested inhibitor for 30 min and the distribution on different molecular
weight species was analyzed by gel filtration in the crystallization buffer using S75 10/300
GL column (GE Healthcare).
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NMR Experiments. All NMR spectra were acquired at 300 K using a Bruker Avance 600
15
MHz spectrometer. Uniform N isotope labelling was achieved by expression of the protein
in the M9 minimal medium containing 15NH4Cl as the sole nitrogen source. MDM2(1-
118)41,42 was prepared in 50 mM phosphate buffer pH 7.4 containing 150 mM NaCl and
5 mM DTT. 10% (v/v) of D2O was added to the samples to provide lock signal. Water
suppression was carried out using the WATERGATE sequence.43 Stock solutions of
inhibitors used for titration were prepared in d6-DMSO. The spectra were processed with
1
TopSpin 3.2 software. H-15N heteronuclear correlations were obtained using the fast HSQC
45
pulse sequence.44 Assignment of the amide groups of MDM2 was obtained after
The AIDA experiments were performed according to 37.
.
ASSOCIATED CONTENT
Supporting Information
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Details on chemical synthesis of compounds, X-ray, FP and NMR may be found in the
Supporting Information. This material as well as Molecular Formula Strings are available free
of charge via the Internet at http://pubs.acs.org.
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Accession Codes
The final models and structure factors for MDM2-19 complexes were deposited into Protein
Data Bank with the following accession numbers: 5J7G (MDM2 18-125) and 5J7F (MDM2
1-125).
AUTHOR INFORMATION
Corresponding Author
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*
T.
A.
Holak:
holak@biochem.mpg.de,
+48 126632287;
L.
Skalniak:
lukasz.skalniak@uj.edu.pl, +48 126632041.
Author Contributions
 These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research has been supported (to TAH) by the Maestro grant UMO-2012/06/A/ST5/00224
from the National Science Centre, Poland, by the Project operated within the Foundation for
Polish Science TEAM Program, co-financed by the EU European Regional Development
Fund and the Marie Curie FP7-Reintegration-Grant within the 7th European Community
Framework Programme and (to GD) UMO-2011/01/D/NZ1/01169 from the National Science
Centre. ATC was supported by Preludium grant UMO-2015/17/N/NZ1/00025, KK - by
Preludium grant UMO-2015/17/N/ST5/01942, LS
-
by Sonata grant UMO-
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2016/21/D/NZ7/00596 and GD - by Sonata bis grant UMO-2012/07/E/NZ1/01907 from the
National Science Centre, Poland. AD was supported by the NIH (1R21GM087617,
1R01GM097082, and 1P41GM094055). The research was carried out with the equipment
purchased thanks to the financial support of the European Union structural funds (contract no.
POIG.02.01.00-12-064/08 and POIG.02.01.00-12-167/08) and European Regional
Development Fund in the framework of the Polish Innovation Economy Operational Program
(contract no. POIG.02.01.00-12-023/08). This project has received funding from the European
Union’s Framework Programme for Research and Innovation Horizon 2020 (2014-2020)
under the Marie Skłodowska-Curie Grant Agreement No. 675555, Accelerated Early staGe
drug discovery (AEGIS). SK received the financial support from the Faculty of Biochemistry,
Biophysics and Biotechnology of Jagiellonian University, a partner of the Leading National
Research Center (KNOW) supported by the Ministry of Science and Higher Education.
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ABBREVIATIONS USED
3CR,
3-Component Reaction; AIDA, Antagonist-Induced Dissociation Assay; FP,
Fluorescence Polarization; GAPDH, Glyceraldehyde-3-Phosphate Dehydrogenase; MTT, 3-
(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; MDM2, Murine Double
Minute-2; MDMX, Murine Double Minute-X; PVDF, Polyvinylidene Fluoride; RMSD, Root
Mean Square Deviation.
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Engh, R. A.; Holak, T. A. Chalcone derivatives antagonize interactions between the human
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oncoprotein MDM2 and p53. Biochemistry 2001, 40, 336–344.
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TABLES, FIGURES AND FIGURE LEGENDS
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Table 1. Inhibitory activities of compound 1 and its derivatives against the p53-
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MDM2/MDMX interaction using the FP assay.
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Ki M2
[µM]
Ki MX
[µM]
Ki M2
[µM]
Ki MX
[µM]
Code
1*
R2
Code
13
R2
0.006
0.225
0.023
0.038
2.31
3.15
n.a.
14.7
29.6
n.a.
n.a.
n.a.
0.121
0.017
0.059
0.015
0.098
0.054
0.058
n.a.#
40.2
n.a.
2
3
4
5
6
7
14
15
16
17
18
19
9.75
n.a.
0.038
28.0
12.4
26.3
Code
20a±
20°
R1
Ki M2
Ki MX
6.02
n.a.
0.008
35.3
8
0.021
n.a.
4.91
n.a.
21a
21
0.656
n.a.
27.4
n.a.
n.a.
n.a.
9
0.060
n.a.
4.40
n.a.
22a
22
10
11
12
0.433
n.a.
4.71
n.a.
23a
23
n.a.
0.322
n.a.
6.76
n.a.
24a
n.a.
0.157
4.17
24
*compounds 1-19 have 4-chlorophenyl group in R1 position; compounds 20-24 have hydroxyl group in R2
°
position; ±a stands for ethyl ester (20a-24a) of the respective acidic forms of 20-24; # n.a. stands for not active.
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Table 2. Selectivity of the tested compounds towards the p53 positive cells. IC50 values were
obtained from the MTT assay. Selectivity is calculated as a ratio of IC50 values towards the
p53 negative (Saos-2) and positive (U-2 OS) cells.
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U-2 OS
Saos-2
Code
Selectivity
IC50 [µM]
IC50 [µM]
11.08 ± 1.83 38.56 ± 3.61
3.48
3.20
2.23
1.95
1.61
7.59
19
17.42 ± 3.49 55.82 ±13.49
1
16
5.71 ± 1.39
3.67 ± 0.28
7.27 ± 0.63
2.66 ± 0.39
12.73 ± 2.66
7.15 ± 1.33
11.69 ± 1.30
20.16 ± 4.71
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14
Nutlin-3
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Figure 1. The Van Leusen reaction used for the synthesis of the series scaffold. The
substituents are varied by changing the amine and isocyanide components (for details see the
SI).
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Figure 2. Accumulation of products of the p53 gene and p53-regulated genes upon treatment
of cells with the selected compounds. A,B. The U-2 OS cells and HCT 116 cells (both p53wt)
were treated with 5, 10 or 20 M of the indicated compounds for 7 or 24 h. Expression levels
of MDM2 and p21 were assessed by western blot analysis. The GAPDH level served as a
protein loading control. C. Densitometry analysis of the results shown in A and B. Values
were normalized to the GAPDH level and to the DMSO-treated control.
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