Photoactivation of MDM2 Inhibitors: Controlling Protein-Protein Interaction with Light

Article abstract of DOI:10.1021/jacs.8b04870

Selectivity remains a major challenge in anticancer therapy, which potentially can be overcome by local activation of a cytotoxic drug. Such triggered activation can be obtained through modification of a drug with a photoremovable protecting group (PPG), and subsequent irradiation in the chosen place and time. Herein, the design, synthesis and biological evaluation is described of a photoactivatable MDM2 inhibitor, PPG-idasanutlin, which exerts no functional effect on cellular outgrowth, but allows for the selective, noninvasive activation of antitumor properties upon irradiation visible light, demonstrating activation with micrometer, single cell precision. The generality of this method has been demonstrated by growth inhibition of multiple cancer cell lines showing p53 stabilization and subsequent growth inhibition effects upon irradiation. Light activation to regulate protein-protein interactions between MDM2 and p53 offers exciting opportunities to control a multitude of biological processes and has the potential to circumvent common selectivity issues in antitumor drug development.

Full text of DOI:10.1021/jacs.8b04870

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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Photoactivation of MDM2 Inhibitors: Controlling Protein−Protein
Interaction with Light
Mickel J. Hansen,†,∥,‡Femke M. Feringa,‡,∥ Piermichele Kobauri,† Wiktor Szymanski,†,§
,†
́
Rene H. Medema,* and Ben L. Feringa*
†Centre for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The
Netherlands
‡Oncode Institute, Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands
§Department of Radiology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The
Netherlands
S
* Supporting Information
ABSTRACT: Selectivity remains a major challenge in
anticancer therapy, which potentially can be overcome by
local activation of a cytotoxic drug. Such triggered
activation can be obtained through modification of a
drug with a photoremovable protecting group (PPG), and
subsequent irradiation in the chosen place and time.
Herein, the design, synthesis and biological evaluation is
described of a photoactivatable MDM2 inhibitor, PPG-
idasanutlin, which exerts no functional effect on cellular
outgrowth, but allows for the selective, noninvasive
activation of antitumor properties upon irradiation visible
light, demonstrating activation with micrometer, single
cell precision. The generality of this method has been
demonstrated by growth inhibition of multiple cancer cell
lines showing p53 stabilization and subsequent growth
inhibition effects upon irradiation. Light activation to
regulate protein−protein interactions between MDM2
and p53 offers exciting opportunities to control a
multitude of biological processes and has the potential
to circumvent common selectivity issues in antitumor
drug development.
Figure 1. A schematic representation of the principles behind
phototriggered p53 stabilization. Caged inhibitor (PPG-idasanutlin) is
not able to inhibit the MDM2−p53 protein−protein interaction,
which results in p53 ubiquitylation and degradation. Irradiation with
ancer is one of the major causes of death in the
Cdeveloped world. Long-standing drawbacks of cancer 400 nm light releases the active inhibitor idasanutlin which prevents
MDM2−p53 binding and as a consequence increases the p53 level,
chemotherapy are its inherent toxicity and associated adverse
effects. To fight these selectivity issues, targeting pathways that
are exclusively needed for cancer cell survival have been
explored.1−4 One way of controlling these cellular pathways is
by interfering with cancer cell-specific protein−protein
interactions (PPIs). Interestingly, by controlling PPIs, remote
control of a specific protein can be achieved, which opens up
new targeting strategies in anticancer treatment (see Figure 1).
The best known tumor suppressor protein, p53, is heavily
involved in PPIs and plays an important role in cell-cycle
control, apoptosis, DNA repair and cellular stress responses.5,6
Activation of p53 by various types of stress can drive cellular
senescence, which is an irreversible cell-cycle arrest, to prevent
potential transformation of the damaged cell. Utilizing its role
in apoptosis and senescence, reactivation of the p53 signaling
leading to senescence or cell death.
major concern in p53 reactivating therapies is its effect on
normal cells, since upregulation of p53 protein expression by
itself is sufficient to induce senescence or apoptosis in all
cycling cells.9,10 Therefore, the selective activation of the p53
pathway in cancerous tissue is a key challenge, as it would
greatly increase the potential success for therapeutical
application.
One of the main repressors of p53 activity is the MDM2-
protein. MDM2 interacts with p53 to promote its ubiquity-
lation, making it a target for degradation by the proteasome
Received: May 9, 2018
pathway remains a preeminent target for cancer treatment.7,8
A
© XXXX American Chemical Society
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Figure 2. Strategy toward photocleavable nutlin derivatives. (a) Idasanutlin, a potent MDM2 inhibitor allowing the stabilization of p53 levels in
tumor cells. (b) Molecular docking showcases the possible interaction with Lys90 as a potential site to alter the activity (PDB: 4JRG).29 (c)
Irradiation of PPG-idasanutlin led to the formation of idasanutlin and PPG(6) as the sole products. (d) Absorption spectra of PPG-idasanutlin,
idasanutlin and PPG(6) in buffer.30 (e) UV−vis spectra of PPG-idasanutlin upon exposure to 400 nm light showing a clean photochemical
conversion (isosbestic point at 350 nm) to the desired products, see SI for detailed UPLC−MS studies.
(Figure 1).11−13 The regulatory PPI between p53 and MDM2
makes the latter an interesting target in anticancer drug
development. Recently, a class of MDM2 inhibitors (nutlins)
have been developed allowing the selective activation of the
tumor suppressing p53 pathway (Figure 1; structure of
idasanutlin shown).14−16 Nutlins bind to the p53-binding site
of MDM2, inhibiting proteolytic breakdown of p53, resulting
in the stabilization of p53 which arrests rapid cell division and
can induce senescence.9
To ultimately increase the selectivity of such MDM2
inhibitors and to utilize them as a research tool to investigate
MDM2−p53 interactions, photopharmacological strategies17,18
can be applied in which a drug is modified with a
photoswitch,17,18 or photoremovable protecting group.19−21
Masking of a functional group in a pharmacophore with a
photoremovable protecting group allows its selective light-
triggered activation. Ideally, the photoprotected drug is
inactive, while after photodeprotection the active drug is
liberated, taking advantage of the noninvasive nature of
light.22,23
Herein, we describe the design, synthesis and biological
evaluation of a photoactivatable MDM2 inhibitor PPG-
idasanutlin. The principle of phototriggered p53 stabilization
is shown in Figure 1. The caged inhibitor (PPG-idasanutlin) is
not capable of blocking the MDM2−p53 protein−protein
interaction, resulting in p53 degradation. Photochemical
release of idasanutlin prevents MDM2−p53 binding, triggering
senescence or cell death. The system described herein allows,
for the first time, the selective light-activation of tumor-
arresting p53 in living cells.
Our molecular design was based on a recently developed
MDM2 inhibitor, idasanutlin (see Figure 2a), which showed
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Figure 3. Functional p53 induction upon λ = 400 nm irradiation in PPG-idasanutlin treated cells. (a) RPE-1 cells were treated with indicated
compounds (all 10 μM final) and fixed 4 h after 5 min (∓ 400 nm) irradiation.34 (b) Quantification of the mean p53 intensity per nucleus in cells
treated as in (a).35 (c) Representative Western blot showing p53 protein levels in cells 4 h after addition of DMSO or PPG-idasanutlin and
irradiation for indicated time periods. Hsp90 is used as a loading control. (d) Selective outgrowth disadvantage in RPE-1 cells 6 days after PPG-
idasanutlin treatment +400 nm irradiation for 5 min. (e) Representative Western blot showing p53 protein levels in three cell lines (U2OS, RKO,
BJhTert) 4 h after indicated treatments. (f) Selective outgrowth inhibition in indicated cell lines 6 days after PPG-idasanutlin treatment +400 nm
irradiation for 5 min.36
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Figure 4. Spatiotemporal control of PPG-idasanutlin. (a) Schematic representation of microwell setup for laser irradiation of individual RPE-1 cells
to activate PPG-idasanutlin. Laser target area (represented by red circle) for single pulse (0.1 s irradiation at 5 μm interspaced position) indicated
with scale. Individual irradiated cells followed by measuring nuclear p53-venus levels (fluorescence) every 15 min for 3 h after laser irradiation.
Approximately 200 cells in each microwell. (b) Percentage of cells that divide within 8 h after indicated treatments.38 (c,d) p53-venus fluorescent
signal in individual RPE-1 cells tracked over time after indicated treatments as represented in (a).39
high potency, moderate selectivity and good bioavailabil-
ity.7,14,16 From limited SAR studies,29 it can be concluded that
the m-methoxybenzoic acid group plays a potential role in
binding affinity, cellular potency/stability and pharmacokinetic
properties (see Figure 2a, marked red). Synthetic modification
of the m-methoxybenzoic acid potentially renders the nutlin
derivative inactive. The possibility to alter the activity of
idasanutlin by masking of this functional group was further
established by docking studies suggesting that a potential
interaction with Lys90 is prevented in the protected compound
(Figure 2b and SI for computational details).
Encouraged by these preliminary docking studies, we
designed a photoactivatable idasanutlin (PPG-idasanutlin)
which would potentially show a difference in activity between
the protected and photodeprotected forms. We selected the
coumarin scaffold as the PPG of choice, which is known to
allow for a fast deprotection with biocompatible visible light (λ
> 400 nm) without the generation of toxic side products. The
hydroxymethylcoumarin was preferred over the normal
hydroxycoumarin because of its improved hydrolytic stability
and increased rate of photocleavage.21,24,25
at pH = 7.0, photodeprotection with λ = 400 nm light was
observed, showing solely the formation of idasanutlin and
hydroxycoumarin. The rate of photocleavage proved to be
high, allowing the major photorelease of idasanutlin within 5
min of irradiation, with a 0.1% quantum yield.22 Moreover, no
significant spontaneous hydrolysis of PPG-idasanutlin for >24
h in buffer at room temperature was observed. This allows the
application of PPG-idasanutlin under physiological assay
conditions using short irradiation times with biocompatible
visible (>400 nm) light.
Next, the biological activity of PPG-idasanutlin was
investigated. Initial studies aimed at confirming a difference
in p53 activation upon λ = 400 nm light exposure after
addition of the protected idasanutlin derivative (PPG-
idasanutlin). Nontransformed, p53-proficient retinal pigment
epithelial cells (RPE-1) were treated with DMSO (control),
nutlin-3, idasanutlin or PPG-idasanutlin followed by ∓
irradiation with 400 nm light (Figure 3). Immunofluorescent
staining revealed a significant increase in nuclear p53 protein
levels in cells 4 h after addition of nutlin-3 or idasanutlin,
regardless of the irradiation with 400 nm light. Importantly,
treatment with PPG-idasanutlin only resulted in a significant
increase in p53 protein level when these cells were irradiated
with 400 nm light (photorelease of idasanutlin, see Figure
3a,b).31 To examine the level of control over the dose−
Following the synthesis of the desired PPG-idasanutlin (see
SI for details),21,26−28 we investigated its photochemical
behavior under physiological conditions. From UV−vis
spectroscopy and UPLC−MS measurements in aqueous buffer
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response of idasanutlin (employing PPG-idasanutlin), p53
protein levels in RPE-1 cells were determined by immunostain-
ing after both increasing duration of 400 nm light irradiation
and varying doses of PPG-idasanutlin (Figure 3c and Figure
S16). The clear dose−response dependent accumulation of
p53 protein shows the highly effective light controllable dose
responsiveness of the biological effect using PPG-idasanutlin
(Figure 3c).
Subsequently, the functional ability to photocontrol growth
of rapidly dividing cells was investigated. Colony outgrowth of
RPE-1 cells treated with PPG-idasanutlin was selectively
blocked after irradiation with 400 nm, while irradiation did
not perturb outgrowth of DMSO treated cells (Figure 3d).32
This showcases the use of 400 nm light in living systems as a
valid approach to photocontrol biological function. It should
be emphasized that in the outgrowth experiment seen in Figure
3d, treatment of cells with PPG-idasanutlin without 400 nm
irradiation did not show any growth inhibition, confirming the
lack of inherent activity of the protected idasanutlin. In other
words PPG-idasanutlin has no functional effect on p53
stabilization nor compromises cellular outgrowth.33
To verify whether (re)activation of p53 by our light
controllable PPG-idasanutlin is more generally applicable and
not dependent on the nontransformed RPE-1 cells used in
these experiments, additional nontransformed (BJ-hTert) and
tumor (RKO (colon carcinoma), U2OS (osteosarcoma)) cell
lines were included for follow-up analysis. Selective stabiliza-
tion of p53, after treatment with PPG-idasanutlin and light
irradiation, was observed in all cell lines tested (Figure 3e).
The light-controlled p53 activation invariably led to a dramatic
reduction in cellular outgrowth (Figure 3f) proving the
possibility to control tumor cellular growth using PPG-
idasanutlin and light.
To demonstrate the spatiotemporal control of the designed
system, we sought to investigate the selective enhancement of
p53 levels in individual RPE-1 cells within a cell population
using light irradiation.37 Using RPE-1 cells that stably
expressed a venus-tagged version of p53 (p53-venus), p53
protein accumulation could be tracked (see SI for details) with
high time-resolution in individual cells by live-cell microscopy.
RPE p53-venus cells were grown in 620 μm wide microwells
and a 405 nm laser was used to irradiate individual cells in the
colony with a single 0.1 s pulse at 5 μm inter spaced positions
to acquire micrometer precision (Figure 4a). To determine the
functionality of the high spatiotemporal control obtained in
this setup, cell cycle progression was monitored in single cells
following laser activation of PPG-idasanutlin (photorelease of
idasanutlin). Functional p53 activation will halt cell division,
causing fewer cells to pass through mitosis.9 Indeed, the
percentage of cells that divide within 8 h after the indicated
treatment strongly drops in cells that were irradiated after
treatment with PPG-idasanutlin (Figure 4b). This shows that a
specific cellular fate can be induced at single-cell resolution by
laser irradiation as presented in Figure 4a. Quantification of the
nuclear p53-venus signal at 15 min intervals in single cells
treated with PPG-idasanutlin revealed the selective stabiliza-
tion of p53 protein following irradiation with the 405 nm laser
(Figure 4c). A significantly lesser extent of p53 stabilization
was detectable in neighboring cells that were not irradiated by
the 405 nm laser (Figure 4c). The limited stabilization of the
nonirradiated neighboring cells is most likely explained by
diffusion of activated PPG-idasanutlin (idasanutlin) within the
excess of liquid cell culture medium in this 2D cell culture
setup. In contrast, p53 stabilization was completely absent in
nonirradiated cells from adjacent wells at micrometer distance,
where diffusion could not take place. In addition, p53 levels did
not increase due to laser-induced damage to the cells, since
p53 levels were unaltered in cells following an identical
irradiation protocol in absence of PPG-idasanutlin (Figure 4d).
Together these results show the selective activation of PPG-
idasanutlin resulting in the release of idasanutlin, using an
extremely short (0.1 s) pulse of 405 nm laser irradiation at
micrometer, single-cell resolution, which offers promising
opportunities for future studies using PPG-idasanutlin in 3D
settings like (tumor) tissue.
In summary, the PPG-idasanutlin reported herein allows the
photocontrol of protein−protein interactions and their func-
tional outcome. Stabilization of p53 and consequent cell
growth arrest could be obtained by MDM2 inhibition upon
photoactivation with biocompatible 400 nm light. Excitingly,
spatiotemporal control was achieved with microsecond
irradiation at micrometer, single-cell resolution. This con-
stitutes, to the best of our knowledge, the first system that
externally and indirectly controls p53 levels with light. Next to
a promising concept toward selective anticancer therapy, the
designed system can also function as a molecular tool to
investigate MDM2−p53 interactions as well as selectively
interfere with the numerous cellular processes regulated by
p53.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b04870.
Materials and methods, synthetic schemes, experimental
procedures, photochemical data, NMR spectra and
HRMS of new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*r.medema@nki.nl
*b.l.feringa@rug.nl
ORCID
Wiktor Szymanski: 0000-0002-9754-9248
Ben L. Feringa: 0000-0003-0588-8435
Author Contributions
∥M.J.H. and F.M.F. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by The Netherlands
Organization for Scientific Research (NWO−CW, Top grant
to B.L.F. and NWO VIDI grant no. 723.014.001 for W.S.), the
Dutch Cancer Foundation (KWF; NKI 2014−6787), the
Royal Netherlands Academy of Arts and Sciences (KNAW),
the Ministry of Education, Culture and Science (Gravitation
programme 024.001.035), and the European Research Council
(Advanced Investigator Grant no. 694345 to B.L.F.). We thank
all Medema, Rowland, Jacobs and Feringa lab members for
helpful discussions on this study. We kindly thank Dr. Galit
Lahav for sharing the RPE-1 p53-venus cell line.
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Rev. 2015, 44, 3358−3377.
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