The hydrophobically-tagged MDM2-p53 interaction inhibitor Nutlin-3a-HT is more potent against tumor cells than Nutlin-3a

Article abstract of DOI:10.1039/c9cc07795b

We present the first application of hydrophobic tagging to a non-covalent inhibitor of protein-protein interactions. Nutlin-3a-HT, created by fusing the hydrophobic tag HyT13 to the MDM2-p53 interaction inhibitor Nutlin-3a, prevented cellular accumulation of MDM2 upon p53 reactivation, and had a stronger effect on cell viability and the induction of apoptosis than Nutlin-3a.

Full text of DOI:10.1039/c9cc07795b

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The hydrophobically-tagged MDM2–p53
interaction inhibitor Nutlin-3a-HT is more
potent against tumor cells than Nutlin-3a†
Cite this: Chem. Commun., 2019,
55, 14351
Received 4th October 2019,
Accepted 4th November 2019
Florian Nietzold,‡ Stefan Rubner‡ and Thorsten Berg
*
DOI: 10.1039/c9cc07795b
rsc.li/chemcomm
We present the first application of hydrophobic tagging to a non- androgen receptor,10 dihydrofolate reductase,7b the E3 ligase
covalent inhibitor of protein–protein interactions. Nutlin-3a-HT, cereblon,11 the DNA binding protein TDP-43,12 and the tau
created by fusing the hydrophobic tag HyT13 to the MDM2–p53 protein.13
interaction inhibitor Nutlin-3a, prevented cellular accumulation of
We recently presented the first application of hydrophobic
MDM2 upon p53 reactivation, and had a stronger effect on cell tagging to an existing inhibitor of a protein–protein interaction
viability and the induction of apoptosis than Nutlin-3a.
domain by fusing Poloxin-2,15 an irreversible inhibitor of the
protein–protein interaction domain of the serine/threonine
Loss of function of the tumor suppressor p53 by mutation or kinase Plk1, with two different hydrophobic adamantane-based
deletion is found in a large proportion of human tumors.1 In tags,16 which led to proteasome-dependent degradation of the
tumor cells bearing wild-type p53, the major mechanism of p53 target protein.9a,10,16a In theory, irreversibly-binding bioactive
inhibition is via overexpression of the E3 ubiquitin ligase molecules are preferable for the hydrophobic tagging approach,
MDM2, which inactivates p53 by binding to its transactivation because their use results in a maximal display time of the
domain, promoting nuclear export, and inducing its proteaso- hydrophobic tag on the protein surface. However, the vast
mal degradation.2 Overexpression of MDM2 can be caused by
gene amplification, which is found in 7% of human cancers,
with the highest frequency of gene amplification observed in
soft tissue tumors (20%).3 Inhibitors of the MDM2–p53 inter-
action relieve inhibition of p53 by binding to MDM2, leading to
increased p53 concentrations and the induction of apoptosis in
tumor cells.4 The Nutlin series of cis-imidazolines, including
Nutlin-3a (1, Fig. 1A), bind to MDM2 in a reversible manner, and
were the first potent small-molecule modulators of the MDM2–p53
interaction.5 Currently, a number of MDM2–p53 interaction inhi-
bitors are being tested in clinical trials.6
Hydrophobic tagging of small molecules and peptides has been
shown to be a powerful approach by which to degrade target
proteins. Upon binding of a bioactive molecule to its binding
partner, the hydrophobic tag is presented on the protein surface,
mimicking a partially unfolded protein.7 This is thought to activate
the cell’s quality control machinery, and after a failed refolding
attempt by chaperones, the protein is degraded by the proteasome.8
Applications of this concept include the pseudokinase Her3,9 the
Institute of Organic Chemistry, Leipzig University, Johannisallee 29, 04103 Leipzig,
Germany. E-mail: tberg@uni-leipzig.de; Fax: +49 341 9736599
Fig. 1 (A) Structure of Nutlin-3a (1). (B) Co-crystal structure of 1 bound to
MDM2 (PDB ID: 4J3E).14 (C) Structures of HyT13 7a and Nutlin-3a-HT (2).
(D) Building blocks for the synthesis of 2.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc07795b
‡ These authors contributed equally.
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majority of bioactive molecules designed in chemical biology
and medicinal chemistry bind non-covalently to their protein
targets. We aimed to explore the feasibility of expanding the
application of hydrophobic tagging to include reversibly bind-
ing small-molecule inhibitors of protein–protein interactions.
The mode of binding of Nutlin-3a to MDM2 has been
determined by X-ray crystallography.14 The two chlorophenyl
moieties and the isopropoxy group of Nutlin-3a insert deeply
into the hydrophobic pocket of MDM2 (Fig. 1B). In contrast, the
piperazinone moiety is located at the rim of the hydrophobic
pocket, with the amide nitrogen pointing towards the solvent.
On the basis of this structure, we designed the fusion molecule
2 dubbed Nutlin-3a-HT, in which a hydrophobic tag based on
HyT13 7a is connected to the piperazinone moiety of 1 via the
amide nitrogen (Fig. 1C). Binding of 2 to MDM2 should result
in the presentation of the hydrophobic tag on the surface of
MDM2, without significantly interfering with protein binding.
Synthesis of 2 was achieved from the imidazoline precursor 3,
the piperazinone 4, and the hydrophobic tag amine 5 (Fig. 1D).
Compound 3 was obtained in a variation of the protocol for
Nutlin-3a synthesis developed by Johnston and co-workers17 in
diastereomerically pure form and an enantiomeric excess (e.e.)
of 99% (Fig. S1–S3, ESI†). The piperazinone building block 4 was
synthesized in a 4-step procedure from piperazin-2-one by N-Boc
protection, N-alkylation with 6-bromohexanoic acid, esterifica-
tion and N-Boc deprotection in 53% yield (Fig. 2A). N-Alkylation
of N-Boc-protected piperazin-2-one with bromohexanoic acid ethyl
ester was not successful. The hydrophobic amine 5 is generated
via Staudinger reaction from the azide 6 (Fig. 2B), which itself
is accessible in 5 synthetic steps.16 N-Acetylation of 5 afforded
the control compound 7, which lacks the MDM2-recognition
elements of 2.
Coupling of the piperazinone 4 to 3 afforded the diamide 8,
which was cyclized to the cis-imidazoline 9 using the Hendrick-
son reagent18 (Fig. 2C). Ester hydrolysis to 10 and coupling to
the hydrophobic tag amine 5 provided Nutlin-3a-HT (2) in a
procedure that involved a total of 23 synthetic steps based on
commercially available starting materials.
Fig. 2 Synthesis of (A) piperazinone 4, (B) hydrophobic tag amine 5 and
control compound 7, and (C) Nutlin-3a-HT (2).
In a fluorescence polarization (FP)-based assay, Nutlin-3a-HT
(2) displaced a fluorophore-labeled, p53-derived peptide from
MDM2 with low micromolar activity (IC50 = 2.83 Æ 0.40 mM,
Ki = 1.24 Æ 0.18 mM)19 (Fig. 3). Although this represents an 8-fold
lower activity against MDM2 compared to Nutlin-3a (1) (IC50
=
0.35 Æ 0.03 mM, Ki = 0.15 Æ 0.01 mM),19 previous studies have also
observed a modest loss in activity upon conjugation of a bioactive
molecule to a hydrophobic tag.10,16b Compound 7 did not inhibit
the MDM2–p53 interaction, indicating its suitability for use as
negative control compound in cell-based assays.
Exposure of HCT-116 (human colon carcinoma) cells, which
carry wild-type p53, to Nutlin-3a (1) for 24 h induced a dose-
dependent increase in the protein levels of MDM2, p53, and the
cell cycle inhibitor p21 (Fig. 4A). These observations are con-
sistent with the literature and are expected for a MDM2–p53
interaction inhibitor, since both MDM2 and p21 are transcrip-
tionally activated by p53. Nevertheless, the observation that the
levels of MDM2 are increased to a higher extent than the levels
Fig. 3 Activities of Nutlin-3a (1), Nutlin-3a-HT (2) and control compound
7 against the MDM2–p53 interaction in competitive fluorescence polar-
ization assays.
of p53 and p21 (Fig. 4B) by 1 represents an inherent drawback
of occupancy-based MDM2–p53 interaction inhibitors, which is
likely to limit their clinical efficacy.
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after the degradation of a particular MDM2 molecule has been
decided by the cell’s quality control machinery, leading to con-
comitant degradation of p53. This model would also explain the
slightly reduced protein levels of the p53 transcriptional target p21
in the presence of 2 (Fig. 4A). In a recent study on MDM2-targeting
PROTACs based on the MDM2–p53 interaction inhibitor MI-1061
and cereblon ligands, a similar effect was observed for some of the
PROTACS.21
Consistent with the results of the Western blots, HCT-116
cells treated with 2 showed a higher degree of apoptosis than
cells treated with 1 did (39 Æ 7% apoptotic cells for 30 mM 2 vs.
15 Æ 1% for 30 mM 1, Fig. 5A and Fig. S5, ESI†). The Western
blot data (Fig. 4) and the apoptosis assay data (Fig. 5A) are also
consistent with the results of cell viability assays obtained after
the same time of compound exposure (24 h), which show a
dose-dependent reduction of cell viability for either compound,
with stronger effects observed for 2 (Fig. 5B). The efficacy of
both 1 and 2 increases with longer exposure times of 48 h and 120 h.
After 48 h of exposure, 2 had an IC50 value of 10.5 Æ 2.5 mM, whereas
1 only exhibited a 41% reduction of cell viability at 30 mM (Fig. S6,
ESI†). After 120 h, 2 exhibited an IC50 value of 0.83 Æ 0.17 mM, which
Fig. 4 (A) Effect of Nutlin-3a (1), Nutlin-3a-HT (2), and control compound
7 on the protein levels of MDM2, p53, and p21 (n = 3). (B and C) Fold
increase of the protein levels of MDM2, p53, and p21 compared to the
untreated DMSO control in the presence of (B) compound 1 and (C)
compound 2. Mean values and standard deviations are shown. *p o 0.05,
**p o 0.01 (t-test, two-tailed, paired).
In contrast, exposure of HCT-116 cells to hydrophobically- is 2.4-fold lower compared to 1 (IC50 = 2.00 Æ 0.09 mM, Fig. 5C).
tagged Nutlin-3a-HT (2) for 24 h resulted in only a minimal Because of the high activity of both 1 and 2 after 120 h, both
increase of MDM2 levels, whilst inducing a much stronger and compounds had a similar effect on cell viability at the highest
dose-dependent increase in the levels of p53 and its transcrip- concentrations tested (5 mM and 10 mM). The negative control
tional target p21 (Fig. 4A and Fig. S4, ESI†). The concentrations compound 7 did not affect the apoptotic rate or cell viability of
of p53 and p21 in cells treated with 2 are raised to a higher HCT-116 cells under any of the conditions tested, excluding
extent than the concentrations of MDM2 (Fig. 4C). This sug- non-specific effects caused by the hydrophobic tag as the
gests that the degradation-inducing mechanism of action of reason for the superior activity of 2. Together with the known
2 is superior to the occupancy-based mechanism of action of 1. MDM2-selective effects of 1,5 combined cell-based data are
The control compound 7 did not alter the protein levels of any
of the three proteins (Fig. 4A), excluding any non-specific effect
of the adamantyl group.
Release of MDM2-induced p53 inhibition by treatment with the
untagged Nutlin-3a (1) leads to a significant increase in the levels of
both MDM2 and p21 (Fig. 4 and Fig. S4, ESI†), with 30 mM 1
inducing a 42-fold increase in MDM2 and a 14-fold increase in p21.
In contrast, 30 mM of the hydrophobically-tagged Nutlin-3a-HT (2)
increases p21 levels significantly more strongly than MDM2 levels
(8.7-fold versus 2.4-fold) (Fig. 4 and Fig. S4, ESI†). This observation,
in combination with the known degradation-targeting properties of
the hydrophobic tag HyT13,7a,16a supports the idea that binding
and degradation of MDM2 is the mechanism of action of 2. The
much weaker induction of MDM2 by 2 as compared to 1 cannot be
attributed to the reduced affinity for MDM2 seen in FP assays
(Fig. 3), because this would not explain the increased p53 and p21
levels in the presence of 2 (Fig. 4A).
We note that the levels of p53 in cells treated with 2 are
generally lower than in cells treated with 1 (Fig. 4A). We hypothe-
size that proteasomal degradation of MDM2 by 2 may not be
limited to MDM2 itself, but may also affect, to a lesser extent,
proteins bound to the MDM2 molecules marked for proteasomal
degradation. Consistent with this model, proteasomal degradation
Fig. 5 (A) Rate of apoptosis in HeLa cells treated with 1, 2, and control
compound 7 for 24 h as analyzed by flow cytometry (n = 4). (B and C) Cell
viability assays of HCT-116 cells after (B) 24 h, and (C) 120 h exposure to 1,
2, and 7 (n = 3). Cell viability in the presence of DMSO control was defined
as 100%. Mean values and standard deviations are shown. *p o 0.05,
**p o 0.01, ***p o 0.001 (t-test, two-tailed, paired).
of a non-ubiquitinated protein when bound to an ubiquitinated
protein has been reported.20 It is conceivable that an exchange of 2
for p53 as the binding partner of MDM2 may occur in the window
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the mechanism of action of Nutlin-3a-HT (2).
In this study, we report the synthesis and evaluation of Nutlin-3a-
HT (2) as the first hydrophobically-tagged small-molecule inhibitor of
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This work was generously supported by the Deutsche For-
schungsgemeinschaft (BE 4572/3-1), as well as the European
Union and the Free State of Saxony, European Regional
Development Fund. We thank the Core Unit Fluorescence
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(Kathrin Jager and Andreas Losche) for support with the flow
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Conflicts of interest
There are no conflicts of interest to declare.
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