Discovery of Novel Isatin-Based p53 Inducers

Article abstract of DOI:10.1021/acsmedchemlett.5b00011

A series of isatin Schiff base derivatives were identified during in silico screening of the small molecule library for novel activators of p53. The compounds selected based on molecular docking results were further validated by a high-content screening a

Full text of DOI:10.1021/acsmedchemlett.5b00011

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Letter
DISCOVERY OF NOVEL ISATIN-BASED P53 INDUCERS
Pavel Davidovich, Vasilisa Aksenova, Varvara Petrova, Dmitri Tentler, Daria Orlova, Sergey Smirnov, Vladislav
Gurzhiy, Andrei L Okorokov, Alexander Garabadzhiu, Gerry Melino, Nick Barlev, and Viacheslav Tribulovich
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 06 Jul 2015
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DISCOVERY OF NOVEL ISATIN-BASED P53 INDUCERS
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*P. Davidovich , V. Aksenova , V. Petrova , D. Tentler , D. Orlova , S. Smirnov , V. Gurzhiy4, A. L. Okorokov5, A.
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Garabadzhiu1, G. Melino1,3, N. Barlev1,2, and V. Tribulovich1
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1St. Petersburg Technological Institute, 26, Moskovskii Av., St. Petersburg, 190013, Russia.
2Institute of Cytology, 4, Tikhoretsky Av., St. Petersburg, 194064, Russia
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3MRC Toxicology Unit, University of Leicester, Lancaster Rd., Leicester LE1 9HN, United Kingdom.
4St. Petersburg State University, Universitetsky Av. 26, St. Petersburg, 198504, Russia.
5Wolfson Institute for Biomedical Research, University College London, Gower St., London WC1E 6BT, United
Kingdom.
ABSTRACT
A series of isatin Schiff base derivatives were identified during in silico
screening of the small molecule library for novel activators of p53. The
compounds selected based on molecular docking results were further
validated by a high-content screening assay using U2OS human
osteosarcoma cells with an integrated EGFP-expressing p53-dependent
reporter. The hit compounds activated and stabilized p53, as shown by
Western blotting, at higher rates than the well-known positive control
Nutlin-3. Thus, the p53-activating compounds identified by this approach
represent useful molecular probes for various cancer studies.
KEYWORDS: in silico, isatin Mannich and Schiff bases, p53 activators, in vivo assay, protein-protein interactions,
MDM2
There are several pharmacological methods to
Introduction
stimulate p53 antitumor activity: a) small molecules
The p53 protein functions primarily as
a
(SM) that cause genotoxic stress (for example,
doxorubicin)7; b) re-activators of mutant p53, which
revert the p53 molecule to the wild-type conformation9;
and c) SM PPI inhibitors that block the ubiquitin-
mediated proteasomal degradation of p538. The latter
“class” of molecules seems to be the most promising as
it generally exhibits lower toxicity levels.
sequence-specific transcription factor1, and it drives the
expression of a large number of genes2 in response to
various stimuli3. The current view is that p53, in addition
to its crucial role in anticancer responses4, also plays a
role in inflammation5. The attenuation of p53 activity
(commonly caused by point mutations or overexpression
of its negative regulatory proteins) leads to uncontrolled
cell proliferation.
As mentioned above, p53 is subject to ubiquitin-
dependent degradation, and the principal p53-specific E3
ubiquitin ligase is mouse double minute (MDM2)
protein. Perhaps not surprisingly, MDM2 is a major
pharmacological target. Importantly, unlike acetylation10
or methylation, MDM2-dependent ubiquitination of p53
on several lysine residues in its C-terminus significantly
destabilizes the protein10. In recent years, MDM2 has
been recognized as the critical protein to target for p53
stabilization11.
P53 has a wide network of protein-protein
interactions6 (PPI), and some of the interacting proteins
are key players in the regulation of tumor growth7.
However, among the wide variety of p53 PPIs, only
several interacting proteins are directly responsible for
p53 activity and stability. Therefore, blunting these
critical p53-related PPIs has been shown to be a
successful strategy for cancer treatment8.
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The p53 protein can be structurally divided into We have virtually screened the in-house library of
four domains: the N-terminal region that includes two
transactivation domains (TADs), the proline-rich region,
the central core DNA-binding domain, and the C-
terminal tetramerization and regulatory domain12.
38,000 compounds available at the St. Petersburg
Technological Institute. Analysis of the affinity data
along with visual inspection allowed us to identify a list
of potential MDM2-p53 PPI IMSBDs inhibitors.
Because the identified IMSBDs can exist in two
isomeric states28, they were treated as separate E- and Z-
isomers. Some of the IMSBDs were poorly soluble,
whereas others had low inhibitory activity and high
cytotoxicity levels (only the N-substituted benzamide
isatin Schiff base derivatives showed significant p53
activation levels (Table 1) accompanied by low
toxicity).
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The p53 TADs are the main interface for
interaction with the regulatory protein MDM2. The α-
helical part of TAD1 (key residues Phe19, Trp23 and
Leu26) within the p53 protein interacts with MDM2 by
binding to its N-terminal hydrophobic pocket. The p53-
binding part of MDM2 is rigid, and its solved structure
is widely used in molecular docking studies13.
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Most of the MDM2 inhibitors mimic the structure
of the TAD1 α-helix14 and hence form the same
intermolecular contacts with the hydrophobic MDM2
pocket as the original p53. An alternative concept of
stapled peptides was shown to be a successful strategy in
attempts to inhibit MDM215. Nutlin-3 is the most studied
SM inhibitor of the MDM2-p53 interaction; it has been
examined through in vitro and in vivo16 experiments.
Several NMR17 and X-ray18 studies have specified the
binding pose and details of its interactions with MDM2.
Nutlin-3 was able to reactivate p53 in a non-genotoxic
manner in clinical trials19. To date, many Nutlin
derivatives with higher affinities for MDM2 have been
developed. Isoindolinones are derivatives of one of the
promising “class” of inhibitors showing in vivo and in
vitro activity20. Benzylideneindoline-2-ones have
recently been presented as MDM2-p53 PPI inhibitors
that exhibit in cellulo activity21. More detailed
information about PPI inhibitors is presented in the
recently published review22.
The identified compounds represent a combination
of Mannich29 and Schiff bases that are known to have a
tendency for hydrolysis. Therefore, we separately
analyzed two different possible hydrolysis reactions
(Figure S7) of IMSBD4 using UV-vis spectrometry. As
seen in Figure S6, incubation of the isatin Mannich base
for 12 hrs at pH 7.5 and 37 ºC led to its almost complete
hydrolysis. In contrast, the isatin Schiff base exhibited a
much lower rate of hydrolysis. Thus, we predicted that
even though the selected compounds undergo
hydrolysis, they still exert their biological functions as
non-N-substituted Schiff bases.
Importantly, we found that the compounds bind
with BSA, which is abundantly present in serum. The
interaction of compounds with both BSA and MDM2
might also prevent these compounds from undergoing
hydrolysis. For instance, Kimble-Hill et al.24 reported
successful crystallization of the non-hydrolyzed Mannich
base
(
N-[(1-piperazinyl)methyl]-isatin)
with
the
The isatin (1H-indole-2,3-dione) scaffold
(benzylideneindoline-2-one analog) is widely used in
drug discovery studies23. N-substituted isatin piperazine
derivatives have been reported as selective inhibitors for
aldehyde dehydrogenases in in vitro assays24, and the
crystal structures of protein-ligand complexes have
validated the mechanism of their action. Piperidine
analogs show inhibitory activity against another class of
metabolic proteins, carboxylesterases25. N-alkylated
isatins have recently been described as caspase-3 activity
inhibitors26. Isatin-benzothiazole Schiff base derivatives
have shown anti-cancer activity in breast tumor cell lines27.
Here, we report the discovery of isatin Mannich
and Schiff base derivatives (IMSBDs) that activate the
p53 transcription factor. These compounds were
identified by in silico screening (Table S2), and their
activities were validated in cellulo using a human bone
osteosarcoma epithelial U2OS cell line, which stably
expressed a p53-driven enhanced green fluorescent
protein (EGFP) reporter.
ALDH3A1 protein using the same pH and buffer system
in the crystallization assay as we used in our hydrolysis
studies. Thus, the larger peak shifts in the UV-vis spectra
in the culture media than in the buffer (Figure S6) can be
explained by the effect of BSA binding.
We analyzed the ability of both ISBD isomers to
dock to the p53-binding pocket of MDM2. The (E)-
ISBDs docked to the p53-binding pocket with lower ꢀG
than the Z-form by approximately 1 kcal/mol. As seen in
Figure 2, the (E)-ISBD2 isomer forms mainly
hydrophobic interactions with the MDM2 p53-binding
site (Leu pocket, π-π stacking interaction with His96 and
Tyr100). The calculated affinity of (S,E)-ISBD1 to the
p53-binding pocket of MDM2 is one-fold lower in Ki
units than the corresponding value for ISBD2. We
propose that the identified ISBDs can act as a p53 α-
helix mimetic (Figure 1), whereby the phenyl ring of
ISBD2 overlays the Phe19 residue and the isatin core
overlays the hydrophobic Leu26 of the p53 peptide.
Furthermore, the methylated aliphatic carbon in (S,E)-
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ISBD1 alters the conformation of the ligand, causing the
phenyl ring to overlay the Trp23 residue of the -helix.
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episomal plasmid that contains the EGFP coding
α
sequence under the control of five repeated p53 response
elements taken from the promoter region of the p21
gene, which is the bona fide target of p53 transcriptional
activity.
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Upon treatment with activating SMs (e.g.,
MDM2-p53 PPI inhibitors), cellular p53 is stabilized by
the acetylation modification30. Subsequently, p53 binds
the response elements and triggers transcription of the
EGFP gene, whose product emits fluorescence.
Therefore, the efficacy of p53-activating SMs was
measured indirectly as the number of EGFP-positive
cells.
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Figure 1. Superimposed representation of the docked
poses of ISBD2 (blue) and ISBD1 (rose) with the p53
As shown in Figure 3, the presence of EGFP-
positive cells (green) demonstrates that IMSBD4
activates p53-dependent transcription of the EGFP
reporter.
TAD1 α-helix (cyan).
One of the major limitations of p53-activating
ISBDs is the presence of E-to-Z interconversion, which
lowers the overall binding affinity to MDM2. As
previously reported28, ISBDs crystallize preferentially in
the (E)-conformation but convert to the (Z)-form when
dissolved. The topology of the (Z)-form differs
substantially from that of the (E)-conformer (Figure S10).
We performed 2D NMR experiments on
IMSBD4 to obtain the relationship of E to Z solution
concentrations. The spectral data (supporting
information) confirm the molar (E-to-Z) ratio to be 8:1
Figure 3. A representative image of the U2OS-pLV cell
line response to treatment with Nutlin-3 (2.5
and IMSBD4 (2.5 M) (B)
ꢁM) (A)
ꢁ
in polar DMSO (
ε = 46.8) and in less polar chloroform
(
ε
= 4.7). We suggest that the ratio between these
Quantitatively, the potency of compounds was
estimated as the ratio between the percentage of EGFP-
positive cells after treatment with IMSBDs (2.5 µM, 48
hrs) and the effect of a well-characterized non-genotoxic
MDM2 inhibitor, Nutlin-3. N-Mannich bases of ISBDs
substituted with N-(benzyl)benzamide demonstrated
higher levels of p53 activation than the control Nutlin-3
inhibitor (Table 1). EGFP activation and cytotoxicity
(Hoechst stain) values in p53+ and p53- cells treated
with various IMSBDs are shown as ratios between the
effects of compounds and the effects mediated by
Nutlin-3 treatment, which were arbitrarily set as 1.
isomers remains approximately the same when dissolved
in culture medium for in vivo studies. We also confirmed
the presence of the E-configuration in the solid state by
solving the crystal structure of IMSBD4 (Table S1, Figure
S2).
To validate our in silico predictions, we decided
to study the effect of SMs on p53 stabilization and
activation. To this end, we developed a cell-based
reporter system using the p53-positive U2OS cell line
(Figure 2).
Table 1. EGFP activation
and cytotoxicity after
treatment with IMSBDs
Number of cells
EGFP
Total
p53+ p53–
IMSBDs
R1, R2,
IMSBDs/
Nutlin
Figure 2. A schematic representation of the test-system
Bn-piperazine, Me
Bn-piperidine, H
Bn-piperazine, H
Ph-piperazine, H
2-F-Ph-piperazine, H
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8.70
0.04 0.27
0.14 0.32
0.10 0.19
0.22 0.40
0.11 0.24
containing five p53 response elements
4.90
4.20
3.90
3.60
Unlike in vitro models, this approach is less prone
to false-positive signals due to the lower background of
off-target effects. The resulting cell line features an
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the level of MDM2 in U2OS-pLV cells. This
discrepancy also indicates that IMSBDs may stabilize
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methylbenzylamine, H
3.10
0.14 0.41
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As seen in Table 1, different IMSBDs activate
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p53 by a different mechanism(s) from the one exerted by
Nutlin-3. For instance, a close ISBD analog, 4-
hydroxybenzylideneindolin-2-one, has been reported as
a micromolar inhibitor of Polo-like kinase 4, which is
indirectly involved in the regulation of p5332. However,
the exact molecular mechanism of p53 stabilization
requires additional investigation.
p53 in U2OS-pLV cells (as measured by the number of
cells “with EGFP”) at various levels but are consistently
more effective than Nutlin-3. We also measured the
cytotoxic effect of SMs by counting the number of cells
that survived after the treatment. The cytotoxicity of
IMSBDs was clearly and consistently higher in p53+
cells versus p53-cells (Table 1, compare the last two
rows), which indicates that these effects are p53
dependent. The fact that IMSBD cytotoxicity was higher
than that of Nutlin-3 suggests that these compounds may
have additional targets and/or activate the apoptotic p53
pathway, which is mechanistically different from the
pathway activated by Nutlin-3 in these cells.
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Conclusions
Using in silico and in vivo studies, we have
identified several isatin-based derivatives that display
strong p53 stimulatory effects. Based on in silico
modeling, we assume that these novel pharmacological
activators of p53 may disrupt the PPI between p53 and
MDM2. However, further studies are required to
establish the precise molecular mechanism of this
phenomenon.
The difference in activity between IMSBD2-
IMSBD6 is not significant (compared to IMSBD1),
which supports the hypothesis that the common Schiff
base is the active part of IMSBDs and that the variable
Mannich base has no significant effect on the activity of
SMs. Slight differences in the activity can be explained
by the differences in physical properties (e.g., logP and
PSA) of IMSBDs, whereas the binding poses of ISBD
and IMSBD to MDM2 are generally similar (Figure S9).
To directly assess the effect of IMSBDs on the
stability of the p53 protein, we performed Western
blotting (WB), which allows measurement of the
intracellular levels of the p53 protein. The representative
WB data using IMSBD4 are shown in Figure 4.
Treatment of U2OS-pLV cells with IMSBD4 for 12 hrs
resulted in p53 stabilization comparable to the effect of
Nutlin-3 but was less pronounced than Doxorubicin
(DOXO).
AUTHOR INFORMATION
Corresponding Authors
*(P.D.) E-mail: davidovich.pavel@technolog.edu.ru
Present Address
∥NIH, Bethesda, Maryland 20892, United States
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
Funding
The work was supported by the Ministry of Education and
Science of Russia (contracts 11.G34.31.0069, 14.B25.31.0013
and 14.132.21.1334) and by the BBSRC (BB/E019862/1).
ASSOCIATED CONTENT
Supporting information
Synthetic
procedures,
analytical
and
computational data, and biological assay details can be
found in the supporting information. This information is
available free of charge via the Internet at
http://pubs.acs.org
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DISCOVERY OF NOVEL ISATIN-BASED P53 INDUCERS
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*P. Davidovich1, V. Aksenova1,2∥, V. Petrova1,3, D. Tentler1,2, D. Orlova1, S. Smirnov4, V. Gurzhiy4, A. L.
Okorokov5, A. Garabadzhiu1, G. Melino1,3, N. Barlev1,2, and V. Tribulovich1
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Graphical Abstract
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Superimposition representation of the docked poses of ISBD2 (blue) and ISBD1 (rose) with p53 TAD1 αꢀ
helix (cyan).
123x108mm (300 x 300 DPI)
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A schematic representation of the test-system that contained five p53 response elements
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A representative image of U2OS-pLV cell line response on the treatment with Nutlin-3 (2.5 µM) (A)
88x66mm (300 x 300 DPI)
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A representative image of U2OS-pLV cell line response on the treatment with IMSBD4 (2.5 µM) (B)
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WB analysis of the p53 protein levels after the treatment with compounds
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