Multifunctional Compounds for Activation of the p53-Y220C Mutant in Cancer

Article abstract of DOI:10.1002/chem.201802677

The p53 protein plays a major role in cancer prevention, and over 50 % of cancer diagnoses can be attributed to p53 malfunction. The common p53 mutation Y220C causes local protein unfolding, aggregation, and can result in a loss of Zn in the DNA-binding domain. Structural analysis has shown that this mutant creates a surface site that can be stabilized using small molecules, and herein a multifunctional approach to restore function to p53-Y220C is reported. A series of compounds has been designed that contain iodinated phenols aimed for interaction and stabilization of the p53-Y220C surface cavity, and Zn-binding fragments for metallochaperone activity. Their Zn-binding affinity was characterized using spectroscopic methods and demonstrate the ability of compounds L4 and L5 to increase intracellular levels of Zn²⁺ in a p53-Y220C-mutant cell line. The in vitro cytotoxicity of our compounds was initially screened by the National Cancer Institute (NCI-60), followed by testing in three stomach cancer cell lines with varying p53 status’, including AGS (WTp53), MKN1 (V143A), and NUGC3 (Y220C). Our most promising ligand, L5, is nearly 3-fold more cytotoxic than cisplatin in a large number of cell lines. The impressive cytotoxicity of L5 is further maintained in a NUGC3 3D spheroid model. L5 also induces Y220C-specific apoptosis in a cleaved caspase-3 assay, reduces levels of unfolded mutant p53, and recovers p53 transcriptional function in the NUGC3 cell line. These results show that these multifunctional scaffolds have the potential to restore wild-type function in mutant p53-Y220C.

Full text of DOI:10.1002/chem.201802677

A Journal of
Accepted Article
Title: Multifunctional Compounds for Activation of the p53 Y220C
Mutant in Cancer
Authors: Jessica J Miller, Christophe Orvain, Shireen Jozi, Ryan M
Clarke, Jason R. Smith, Anais Blanchet, Christian Gaiddon,
Jeffrey J Warren, and Tim Storr
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To be cited as: Chem. Eur. J. 10.1002/chem.201802677
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Multifunctional Compounds for Activation of the p53 Y220C
Mutant in Cancer
Jessica J. Miller,[a] Christophe Orvain,[b] Shireen Jozi,[a] Ryan M. Clarke,[a] Jason R. Smith,[a] Anais
Blanchet,[b] Christian Gaiddon,*[b] Jeffrey J. Warren,*[a] and Tim Storr*[a]
Abstract: The p53 protein plays a major role in cancer prevention,
and over 50% of cancer diagnoses can be attributed to p53
malfunction. The common p53 mutation Y220C causes local protein
unfolding, aggregation, and can result in a loss of Zn in the DNA-
binding domain. Structural analysis has shown that this mutant
creates a surface site that can be stabilized using small molecules,
and we report herein a multifunctional approach to restore function
to p53-Y220C. We designed a series of compounds containing
iodinated phenols aimed for interaction and stabilization of the p53-
restoration of function to mutant p53 is an acknowledged
chemotherapeutic target.[7] Herein, we describe the synthesis,
characterization, and in vitro testing of a new series of bifunctional
ligands designed to restore function in mutant p53, specifically the
Y220C point mutation.
The majority of p53 mutations are point mutations localized to the
protein’s core DNA-binding (p53C) domain,[6] and a number of small
molecules have been developed in an effort to reactivate mutant
p53.[8] One such example is APR-246 (Scheme 1a), which has
shown positive results in a Phase I/II clinical trial.[9] APR-246 is a
pro-drug and is activated to a Michael acceptor that binds covalently
to cysteine residues of mutant p53, resulting in protein
reactivation.[10] Due to the non-specific nature of this process, APR-
246 also binds other proteins, and modification of Thioredoxin
reductase 1 leads to increased reactive oxygen species (ROS)
production, an additional cytotoxic mechanism.[11]
Y220C
surface
cavity,
and
Zn-binding
fragments
for
metallochaperone activity. We characterized their Zn-binding affinity
using spectroscopic methods and demonstrate the ability of
compounds L4 and L5 to increase intracellular levels of Zn2+ in a
p53-Y220C-mutant cell line. The in vitro cytotoxicity of our
compounds was initially screened by the National Cancer Institute
(NCI-60), followed by testing in three stomach cancer cell lines with
varying p53 status’, including AGS (WTp53), MKN1 (V143A), and
NUGC3 (Y220C). Our most promising ligand, L5, is nearly 3-fold
more cytotoxic than cisplatin in a large number of cell lines. The
impressive cytotoxicity of L5 is further maintained in a NUGC3 3D
spheroid model. L5 also induces Y220C-specific apoptosis in a
cleaved caspase-3 assay, reduces levels of unfolded mutant p53,
and recovers p53 transcriptional function in the NUGC3 cell line.
These results show that our multifunctional scaffolds have the
potential to restore wild-type function in mutant p53-Y220C.
Introduction
The p53 protein, referred to as the “guardian of the human
genome,”[1] is a tetrameric transcription factor that regulates the
expression of target genes to induce antiproliferative cellular
responses.[2] Among those genes are those that initiate apoptosis,
DNA repair, and cell cycle arrest of damaged cells.[3] However, in
over 50% of cancer diagnoses, p53 does not carry out its essential
function.[4] The most common alterations to p53 are point mutations
that affect tertiary structure or alter the protein’s ability to bind
DNA.[5] The net result is that cells bearing malfunctioning p53 are
susceptible to enhanced proliferation and survival.[6] There is
significant therapeutic potential for p53, and pharmacological
Scheme 1. Chemical structures of (a) APR-246 (PRIMA-1MET) (b)
NSC319726 (ZMC1) (c) PhiKan083 and (d) iodinated phenol small molecule
shown to bind the p53 Y220C mutant pocket.
The p53C domain contains a single Zn2+ ion that is required for
proper protein folding and function.[12] Mutations to p53C can disrupt
protein stability and/or cause loss of Zn.[13] Consequently, the
discovery of small molecule Zn chaperones aimed to restore wild-
type function in mutant p53 has generated much attention.[8c, 14] For
example, the thiosemicarbazone ligand ZMC1 (Scheme 1b) induces
conformational “wild-type-like” change in the common Zn-binding
p53 mutation (R175H), and restores p53 transactivation function.[14]
Further studies indicate that subtle tuning of the Zn-binding affinity of
the metallochaperones is critical for p53 reactivation,[15] as it
functions by repopulating Zn-deficient p53C with Zn2+.[16] More
broadly, targeted metal ion chelation and redistribution has been
shown as a promising anti-cancer strategy,[17] and a number of
recent studies have highlighted both the novelty and complexity of
this approach.[8c, 18]
[a]
[b]
J.J. Miller, S. Jozi, R.M Clarke, J.S Smith, Prof. J.J. Warren, Prof. T.
Storr , Department of Chemistry, Simon Fraser University, 8888
University Drive, Burnaby (Canada)
tim_storr@sfu.ca, j_warren@sfu.ca
C. Orvain, C. Gaiddon, Inserm UMR_S 1113, Molecular
Mechanisms of Stress Response and Pathologies, Université de
Strasbourg, Strasbourg, France. gaiddon@unistra.fr
Supporting information for this article is given via a link at the end of
the document.
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Mutations to the p53C domain can result in structural destabilization
such that the protein unfolds at or below physiological
temperatures.[3a] The Y220C point mutation is
a
common
destabilizing p53 mutation and contributes to about 75,000 new
cancer cases each year.[8b] It results in a cavity at the surface of the
protein, which decreases the melting point of the protein by ~2 °C
and contributes to unfolding and ultimately aggregation.[3a] Small
molecules that bind to the Y220C cavity were developed using in
silico and in vitro screening, and the carbazole-based molecule
PhiKan083 (Scheme 1c) was identified to raise the melting
temperature (Tm) and slow the rate of thermal denaturation of p53-
Y220C.[19] More recently, derivatives of halogenated phenols have
been reported by Boeckler and co-workers as a class of molecules
that bind to the p53-Y220C cavity (Scheme 1d).[8b] It is hypothesized
that halogen-bonding interactions with amino acids inside the cavity
plays an important role in binding. The halogenated compounds
modestly increase the melting temperature of a p53-Y220C model
protein and slow the rate of thermally induced protein
unfolding/aggregation.[8b] These results demonstrate that an
iodinated-phenol core can be a starting point for envisioning new
molecular designs. In addition to protein unfolding, p53 Y220C is
Scheme 2. Chemical Structure of ligands L1-L5.
20]
prone to the loss of Zn2+ in the DNA-binding domain,[12-13,
compatibility, respectively. L4 and L5 include additional metal-
binding groups to promote 1:1 Zn2+ to ligand complex formation.
presenting a new opportunity for drug design
Multifunctional drugs, namely agents with more than one therapeutic
mechanism, have gained increasing acceptance in recent years.[21]
Multifunctional drugs offer potential advantages over their
monofunctional counterparts, namely the potential to produce
additive or synergistic effects by acting on multiple targets or
designing one component to enhance druggable characteristics of
the therapeutic molecule such as blood-brain-barrier penetration or
tissue specificity.[22] Such characteristics have been successful at
enhancing drug efficacy and lowering toxic side effects, providing a
Molecular Docking
To investigate the potential binding mode of L1-L5 with the mutation-
induced cavity in p53-Y220C we employed molecular docking of the
[8b]
ligands with available X-ray data
Our modelling results predict
that the iodinated phenol moiety in L1-L5 orients in the p53-Y220C
cavity (Figure 1, S12-14) in a similar fashion to a known p53-Y220C
ligand that incorporates the same diiodophenol pharmacophore.[8b] In
addition, the DPA metal-binding unit in L4 and L5 does not
significantly alter the interaction, with our lead compound L5 (vide
infra) overlapping with X-ray data (PDBID: 4AGQ) for a known p53-
Y220C binding ligand (Fig. S11).
new avenue in drug discovery from a “one-drug-one-target” to a
23]
“one-drug-multiple-target” strategy.[21d,
Herein, we report a new
series of bifunctional ligands designed to restore wild-type activity in
p53-Y220C by serving as structural stabilizers and as Zn-
chaperones. Our new series of compounds feature different binding
groups that tune Zn2+ affinities and promote interactions with p53-
Y220C.
Zinc Binding Properties.
Previously reported models for Zn-binding in p53 describe two
possible ligation sites, the native binding site (Kd1) and non-native
(Kd2) sites.[26] The native Zn2+ Kd1 for WTp53 is estimated to be on
Results and Discussion
Ligand Design and Synthesis
the order of 10-12 M.[16, 27] This value derives from the low intracellular
levels of free zinc,[16,
and the fact that under physiological
27]
A series of ligands (L1-L5) that are designed to restore wild-type
function in p53-Y220C were synthesized and characterized (Scheme
2). The ligand series was designed with two motifs in mind: (1) a
p53-Y220C binding diiodophenol core,[8b] and (2) Zn-binding groups
to promote metallochaperone activity.[8c, 16, 24] Metal-binding groups
were installed at the 2-position, following rationales for related p53-
binding molecules.[8b] Complexes of L1 with a number of different
metal ions have been investigated for their anti-cancer activity,
however, the neutral metal complexes have low aqueous
solubility.[25] The carboxylic acid (L2) and polyethylene glycol (L3)
groups were added to promote water solubility and biological
conditions, WTp53 is predominantly in the holo (zinc-bound) form.[28]
Metallochaperones designed to rescue zinc-binding in p53 mutants
should therefore have Zn2+ affinities that are less than that of the
native site (Kd1), yet higher than that of non-native sites (Kd2),
estimated to be about 10-6 M for WTp53.[8c] The p53-Y220C mutant
is prone to the loss of Zn2+ due to local unfolding and increased
aggregation, so the exact value of Kd1 is unknown. Assuming that
the p53 Y220C mutant should have a slightly weaker Zn affinity than
WTp53, we designed Zn-metallochaperones for p53 Y220C where
the Zn affinity is in between Kd2 and Kd1 (10-9 < Kd chelator < 10-12).
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are reported in Table 1. The calculated pM values for L1-L3
demonstrate a high Zn2+-affinity at physiological pH, however, exhibit
limited biological activity (vide infra). The calculated pZn values for
the 1:1 complexes L4 and L5 are comparable (7.9 and 8.4
respectively), and match well with reported Zn-affinities for a ligand
series containing the same metal-binding fragment.[29] These values
are to afford approximate dissociation constants (Kd) in the low
nanomolar range, an affinity appropriate for functioning as Zn
metallochaperones for p53-Y220C.
Table 1. Stability constants (log K) of the Zn complexes of L1-L5 and
calculated pM values[a] (errors are for the last digit).
Log K
pZn[a]
(pH 7.4)
ZnL
ZnLH
ZnLH2
ZnL2
10.34(5)
Figure 1. A low energy pose of L5 in the mutation-induced cavity for p53-
Y220C (PDBID: 4AGQ)[8b]. The halogen bonding interaction (ILeu145) is
shown with a yellow line. The hydrogen bond network between the conserved
water, D228, and V147 is shown in red. The Van der Waals surface of the
protein is shown in gray. Atom colours: carbons represented in gray in the
protein and teal for the ligand. Oxygen is shown in red, nitrogen in blue, and
sulfur in yellow. Polar hydrogens are shown in white. Non-polar hydrogens are
omitted for clarity.
L1
L2
L3
L4
L5
9.2
10.1
9.4
14.63(1)
15.439(6)
13.59(1)
14.72(1)
14.63(3)
6.327(5)
5.572(4)
6.427(5)
7.802(8)
8.98(2)
-
-
-
-
10.65(4)
10.04(3)
7.9
-
-
8.4
2.89(3)
Spectrophotometric (UV-visible) pH titrations were carried out to
characterize ligand speciation and Zn-affinity for L1-L5. These
studies show that, at biological pH (7.4), both 1:1 and 2:1 ligand:Zn2+
complexes are present for L1, L2, and L3 (Figure 2).[25] Verani and
co- workers proposed that the 1:1 Zn:L1 species is the biologically
active form.[25] Our speciation results are in accord with this
observation, predicting that [ZnL1]+ is the major constituent in
solution at pH 7.4. In contrast, speciation diagrams of ligands L4 and
L5 are described by a model with only 1:1 ligand to metal species
present (Figure 2). This result is consistent with the presence of an
additional Zn-binding N-(2-pyridylmethyl) moiety in these ligands.[29]
Complete data sets, models, and simulations are given in the
Supporting Information.
[a] pZn was calculated using pZn = (–log[Zn2+ free), where Zn2+ is determined
]
from the Hyss model.[31] [L1-L3] = 6.25 µM, [L4-L5] = [Zn2+] = 12.5 µM, 25 °C,
I = 150 mM NaCl.
Zn complexes of L4 and L5 were also isolated and characterized
using 1H NMR, MS, and X-ray crystallography (for ZnL4Cl, Figure 3),
and are in accord with the 1:1 binding of L4 and L5 to Zn2+ modelled
above for variable pH titrations. Complete crystallographic
information is in the Supporting Information (Table S2).
Figure 3. Figure 3. ORTEP of ZnL4Cl (50 % probability) using POV-Ray,
excluding hydrogen atoms and solvent. Selected interatomic distances [Å]:
Zn(1)-N(1-3): 2.093-2.280; Zn(1)-O(1): 1.954; Zn(1)-Cl(1): 2.286.
Figure 2. (left) Simulated species distribution plot of Zn2+ + L1. (right)
Simulated speciation plot of Zn2+ + L5. Speciation diagrams made using
HySS2009.
Increasing intracellular levels of Zn2+ in the p53 Y220C cell line
NUGC3.
Analysis of the speciation diagrams for each ligand provides the Zn2+
affinity of each ligand at physiological pH. The concentration of free
Zn2+ present in solution at a given pH, referred to as pM (pZn = –
log[Znunchelated]), is a direct estimate of the metal-ligand affinity when
all species in solution are considered.[30] Calculated values for pZn
We investigated whether L4 and L5 could serve as Zn-
metallochaperones and increase intracellular levels of Zn2+ in the
stomach cancer p53-Y220C cell line NUGC3. NUGC3 cells were
incubated with the fluorescent Zn2+ sensitive probe FZ3-AM
(1µM),[32] followed by incubation with L4 or L5 (15 µM), 50 µM ZnCl2
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Figure 4. Treatment of NUGC3 (p53 Y220C) with L4 and L5 increases
intracellular Zn2+. (a) Imaging of intracellular Zn2+ levels in complete serum-
free media. NUGC3 cells were incubated with 1 µM FZ3-AM[24] for 20 minutes
at 37°C, followed by incubation with indicated treatment (ZnCl2 = 50 µM, L4 =
L5 = 15 µM, 50 µM PYR) for 2 hours. 150 µM TPEN was added following
incubation with ZnCl2 and L5. Cells were imaged using a Nikon ApoTome
microscope and fluorescence-quantified using ImageJ. All images were taken
at indicated magnification. (B) Fluorescence intensity of FZ3-AM at 488 nm
demonstrating relative Zn2+ levels. Blue line indicates mean values, while
black error bars demonstrate the 95% confidence interval.
* indicates
statistical differences from control with p < 0.0001, • indicates statistical
differences from control with p < 0.01, and Δ indicates statistical difference
from L5 + ZnCl2 with p < 0.0001. No statistical differences are observed
between the control and L5 + ZnCl2 + TPEN.
and to a lesser extent, L4, to serve as Zn-metallochaperones in
NUGC3 cells containing the p53-Y220C mutation.
In Vitro cytotoxicity assays.
L1-L5 and the corresponding zinc complexes of L4 and L5 (ZnL4Cl
and ZnL5Cl) were submitted to the National Cancer Institute’s NCI-
60 program for in vitro screening against a panel of 60 human
cancer cell lines. After initial 1-dose screening (10 µM), L1-L3 were
rejected from further testing due to their insufficient cytotoxicity
(Table S3). The inactivity of these 2:1 Zn2+-binding ligands could be
due to different factors, including limited cell uptake of the ligands,
and/or limited solubility of neutral complexes formed from available
Zn2+ found in cell culture media.[33] These ligands were not subjected
to further testing and will not be discussed further. The activity of the
1:1 Zn2+ binding ligands, L4 and L5, was significantly greater and the
aggregate results from their 5-dose testing are displayed in Table 2.
L4 and L5 were found to have a broad range of cytostatic (GI50, 0.4 -
2.2 µM) and cytotoxic (LC50, 4.6 – 93.8 µM) activity. L5 shows the
most promising results, exhibiting high cytostatic activity in
combination with a cytotoxic activity that is almost three times more
potent than that of cisplatin. This combination of both cytostatic and
cytotoxic activity can offer major advatages in the treatment of
cancers.[34]
and subsequent imaging. Pyrithione was used as a positive control
for Zn uptake. Both L4 and L5 increased intracellular levels of Zn2+
in NUGC3 cells, as indicated by increased intracellular fluorescence
(Figure 4). However, due to the similar Zn Kd values of FZ3-AM and
L4/L5 (Zn2+ Kd = ~ 15 nM for FZ3-AM[32], 13 nM for L4 and 4 nM for
L5) Zn-binding to the fluorophore in this experiment is likely
restricted, and thus total Zn uptake is underestimated. L5 increased
intracellular Zn2+ levels more than 4-fold compared to the untreated
control, and a two-fold increase over treatment is observed with L4
(Figure 4). Addition of the strong membrane-permeable Zn-chelator,
N-N-N’-N’-tetrakis-(2-picolyl)-ethylenediamine (TPEN, Kd = 26 fM)[33]
following treatment with L5 results in
a significant loss of
fluorescence, indicating the ability of L5 to deliver intracellular Zn2+
to stronger Zn chelates. These results demonstrate the ability of L5
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Table 2. Mean GI50 and LC50 values for L4, L5, and cisplatin from the NCI-
60 screen
Ligand
NSC Number[a]
GI50[b] (µM)
LC50[b] (µM)
L4
788646
788647
119875
2.2
1.5
1.5
70.3
15.0
44.0
L5
Cisplatin
[a] NSC number is the compounds internal ID number at the National
Cancer Institute. [b] GI50 values correspond to the dose that inhibits 50% of
cell growth compared to non-treated controls, while LC50 indicates the
concentration required to kill 50% of treated cells.[35]
Interestingly, their corresponding Zn complexes (ZnL4Cl) and
ZnL5Cl) exhibited lower biological activity at the initial test
concentration of 10 µM (Table S3), and did not meet the necessary
threshold for 5-dose testing in the NCI-60 panel. However, further
investigation of the Zn complexes using stomach cancer cell lines
AGS (WTp53) and NUGC3 (p53-Y220C) showed that their
cytotoxicity increases at higher concentrations (Figures S37-40),
with ZnL4Cl exhibiting increased cytotoxicity in comparison to L4 at
concentrations > 10 µM. Conversely, L5 is more cytotoxic than
ZnL5Cl in both AGS and NUGC3 cell lines at all concentrations
studied.
A heat map of the 5-dose NCI-60 screen for L4 and L5 is shown in
Figure 5, and summarizes the patterns of in vitro cytotoxicity (GI50
and LC50) from low activity (blue) to high activity (red). The most
striking result is the level of cytotoxicity displayed by L5 across most
cell lines. However, L4 and L5 have little cytotoxic effect on
leukemia cell lines (> 100 µM in all cases). Chemoresistance in
leukemia cancers are common, especially in multiple myeloma
(RPMI-8226).[36] In contrast, L4 and L5 are highly cytotoxic on the
melanoma cell line SK-MEL-5, with GI50 values 2.6 and 1.5 µM and
LC50 values of 13.0 and 5.2 µM, respectively. Note that the NCI-60
panel does not presently contain p53-Y220C mutant cell lines,
though many other p53 mutants are included. In addition, several
highly lethal cancer types are not represented in the NCI-60 screen
(i.e. gastric cancer, pancreatic cancer).
Figure 5. Heat map showing the in vitro cytotoxicity (left: log10 GI50; right: log10
LC50) of L4 and L5 in the NCI-60 screen. Blue indicates low cytotoxicity (100
µM) and red indicates high cytotoxicity (0.01 µM).
cisplatin and oxaliplatin. The compounds however, do not display
higher in vitro cytotoxicity in NUGC3 p53-Y220C expressing cells
compared to the AGS wild-type p53 cell line at the 24-hour timepoint.
MKN1 is insensitive to L4 (IC50 51.6 µM), however, the low IC50
value for L5 (1.2 µM) highlights its increased cytotoxicity in
comparison to the other ligands in the series.
Table 3. In vitro cytotoxicity (IC50 values)[a] data for stomach cancer cell lines
AGS, MKN1, and NUGC3
For these two reasons, we further investigated the biological activity
of our compounds in gastric cancer cells. Gastric cancer is the
second leading cause of cancer-related deaths worldwide, and its
lethality is particularly high as indicated by a 5-year survival rate of
around 30%.[37] In addition, there are gastric cancer cell lines with
different p53 status’, including p53- Y220C. We tested the in vitro
cytotoxicity of our ligand series on three human gastric cancer cell
lines: AGS, which contains wild-type p53, MKN1, with the V142A
point mutation, and NUGC3, which has the p53-Y220C mutant. The
data are reported in Table 3 as IC50 values. The results are in
agreement with the data obtained at the NCI-60 screen; L1-L3
displayed minimal cytotoxic activity (Table S3), whereas L4 and L5
were highly cytotoxic on two stomach cancer cell lines tested, AGS
and NUGC3, showing a significant improvement upon IC50 values of
Ligand
AGS (WTp53)
MKN1 (p53V143A
)
NUGC3 (p53Y220C
)
L4
2.7±0.2
1.6±0.06
25.0±2.4
7.4±0.9
51.6±3.6
1.2±0.05
4.3±0.35
6.9±0.06
2.7±0.1
L5
1.7±0.02
20.0±1.7
50.0±2.7
Cisplatin
Oxaliplatin
[a] IC50 is the concentration needed for 50% reduction of survival based on
survival curves.[38]
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To further characterize the anticancer potential of our lead cytotoxic
compound L5, we assessed its cytotoxic activity in non-adherent 3D
aggregate cultures of NUGC3 cells. The use of 3D cell cultures is
becoming increasingly important in drug discovery due to their ability
to more accurately represent physiological conditions, including cell
signalling processes (cell- to cell and cell-extracellular matrix
involved in cell proliferation).[39] Therefore, the use of 3D cell cultures
can better predict lead compounds for in vivo testing before entering
clinical trials.[40] 3D spheroids of NUGC3 cells were treated with IC75
concentrations obtained from 2D cultures of L5, and compared to
treatment with oxaliplatin, one of the leading drugs for gastric cancer
treatment.[41] Even at the markedly lower dose of L5 administered
compared to oxaliplatin (19.5 µM vs 250 µM at IC75), L5 remained
more cytotoxic (Figure 6).
Figure 7. AGS cells (a) and NUGC3 cells (b) were treated for 48 hours with
the IC50 and IC75 concentrations of indicated compound. Proteins were
extracted, and 20 µg were separated on SDS PAGE. Cleaved caspase-3
(Caspase 3*), p53, and actin were then detected by Western blot analysis.
concentration) is notably increased in NUGC3 (p53-Y220C) cells in
comparison to AGS (WTp53) cells under the same conditions. This
suggests that the apoptotic effect of L5 is potentiated in the Y220C
mutant cell line (vide infra).
Figure 6. NUGC3 cells (300 cells/well; 96 wells/plate) were grown for 4 days
and then treated for 3 days at IC50 and IC75 concentrations as indicated. 3D
cultures were observed by microscopy to estimate clone size (left) and cell
survival was assayed using rezasurin (right). * indicates statistical differences
from non-treated control with p < 0.001 as established by Anova followed by
Tukey test.
To further explore the mechanism by which L5 imparts biological
activity of p53-Y220C, we investigated whether L5 could bind
directly to the p53-Y220C mutant protein using surface plasmon
resonance (SPR) (Figure S41a). Unfortunately, binding at low
micromolar concentrations was not observed, and limited solubility of
L5 beyond 400 µM prevents detection of higher micromolar affinity
binding (Figure S41B), a range relevant to a compound with the
same 3,5-diiodophenol pharmacophore (Kd = 225 µM by ITC and
184±23 µM by NMR).[8b] Immunoprecipitation experiments using the
conformation-specfic antibody Pab 240 (recognizing unfolded p53),
however, showed that treatment with L5 reduced the levels of of
unfolded p53, indicating that compound treatment leads to a change
in conformation of mutant p53 protein (Figure S42).
Apoptotic effects of L4 and L5 in human gastric cancer cell
lines.
To investigate the molecular bases for the cytotoxicity of compounds
L4 and L5, and probe whether this mechanism is p53-dependent,
we examined whether L4 and L5 could cleave caspase-3, an
indicator of apoptosis,[42] in both AGS and NUGC3 cell lines. We also
analysed the levels of p53 expression in the cells. AGS and NUGC3
cells were treated with the indicated compounds at both IC50 and
IC75 concentrations for 48 hours, and then cleavage of caspase-3
and p53 protein levels were assessed by Western blot. As expected,
oxaliplatin induces an increase in p53 protein levels and low levels of
cleaved caspase-3 in AGS cells are present (Figure 7).[43] Treatment
with L5 results in the presence of low caspase-3 levels comparable
to oxaliplatin, again highlighting its increased cytotoxicity compared
to L4, which does not induce any changes in either caspase-3
cleavage or p53 expression levels. In contrast to oxaliplatin, however,
the increase in cleaved caspase for L5 is not coupled with an
induction of p53 protein level in the AGS (WTp53) cell line. In the
NUGC3 cell line, oxaliplatin induces an increase in cleaved caspase-
3 at both IC50 and IC75 concentrations. In addition, oligomeric forms
of p53 are observed with molecular weights corresponding to dimers
upon treatment with IC75 concentrations of oxaliplatin, which could
indicate p53 activation.[44] Interestingly, the level of cleaved caspase-
3 upon treatment with L5 at concentrations as low as 19.5 µM (IC75
Upregulation of p53 transcriptional targets.
Based on the high in vitro cytotoxicity of L5 and its increased activity
in p53-Y220C expressing cells in the cleaved caspase assay
mentioned above, we further investigated the role of p53 in the
biological activity of this lead compound. We measured the
expression level of p53 and several p53 target genes that are
involved in either cell cycle arrest (p21)[3a] or apoptosis (NOXA,
7b]
PUMA)[3a,
in p53-Y220C expressing and p53-silenced NUGC3
cells. p53 expression was silenced using siRNA (Figure 8). NUGC3
cells were treated for 24 hours at the IC50 concentration and the
expression level of p53 target genes were measured by RT-PCR.
Upon transfection of sip53, expression levels for NOXA and p21
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increase under non-treated (NT) controls compared to NT under
siCT conditions. This is likely because mutant p53 can bind and
inactivate p63 and p73 proteins,[45] both of which also induce cell
cycle arrest and apoptosis by regulating p53 target genes such as
NOXA and p21.[46] Therefore, removing mutant p53 with sip53
restores function in p63 and p73, causing their expression levels to
increase. In the presence of p53-Y220C (siCT), expression levels of
all three target genes was significantly higher when treated with L5
compared to non-treated controls (NT), and are of similar levels
induced by oxaliplatin, despite the lower concentration of L5 (IC50 L5
= 1.7 µM vs 50 µM for oxaliplatin) administered (Figure 8). The
increase in genes PUMA and NOXA are indicative of an activation in
apoptosis by L5, which correlates to the results obtained in Figure 7.
Upon treatment with sip53, expression levels of PUMA and NOXA
decrease in the case of L5, however, increase when treated with
oxaliplatin. This is likely a result of the ability of oxaliplatin to induce
apoptosis via PUMA in a p53-independent manner.[47]
Strikingly, treatment of NUGC3 cells with IC50 values of L5 resulted
in a 4-fold increase in p21 expression compared to the non-treated
control. These results are obtained without increasing p53
expression levels, unlike oxaliplatin, suggesting the restoration of
wild-type function in existing p53-Y220C. Furthermore, the
diminished experession of p53-Y220C upon treatment with siRNA
significantly reduced the impact of L5 on the mRNA level of the p53
target genes. These results indicate restoration of transcriptional
activity to the p53-Y220C mutant, and that at least part of the
biological activity of L5 is due to restoration of wild-type p53 function.
Summary
The pharmacological reactivation of p53 is a key target in cancer
research, and there is significant promise in the development of
small molecules to restore wild-type function to specific p53 mutants.
In this work we have designed a series of multifunctional molecules
to reactivate the common p53-Y220C mutant. We show that
compounds L4 and L5 exhibit Zn metallochaperone activity in the
Y220C mutant NUGC3 cell line. Characterization of their in vitro
cytotoxicity in the NCI-60 screen and on stomach cancer cell lines
AGS, MKN1, and NUGC3 identified lead compound L5, which
displayed increased cytotoxicity compared to cisplatin and oxaliplatin.
Additionally, L5 remained cytotoxic on 3D cell cultures, an important
characteristic as these systems more directly mimic physiological
conditions. Further investigation into the mechanism of action shows
that L5 induces apoptosis in the NUGC3 cell line via caspase-3, but
not in AGS cells under the same conditions. This cytotoxicity is
achieved at 19.5 µM, a value 10-fold larger for oxaliplatin (250 µM,
IC75). Similar reports for Y220C-dependent induction of apoptosis
using small molecules have been reported at higher concentrations,
48]
albeit on shorter time scales.[8b,
L5 also restores p53
transcriptional activity in the p53-Y220C mutant NUGC3 cell line.
Upregulation of p53 target genes PUMA, NOXA, and p21 is
observed, an effect that is decreased upon knockdown of p53.
These results are obtained in the absence of increased p53
expression, suggesting restoration of wild-type function.[45c]
Remarkably, our results for L5 are obtained at low doses of 1.7 µM
49]
(L5 IC50), demonstrating the potent activity of this scaffold.[48a,
Although binding to recombinant p53-Y220C was not observed at
such low concentrations via SPR, further structural modifications can
be made to increase protein affinity and ensure further target
selectivity.
Given the high levels of in vitro cytotoxicity of L5 on both p53-Y220C
and WTp53 cell lines, it likely that the activity of L5 is due to both
p53-dependent and p53-independent pathways. Indeed, recent
studies on p53 activating scaffolds show that increased ROS
generation is an important component of the observed cytotoxicity, in
addition to p53 activation.[8c, 49] In the case of metal-binding agents,
Figure 8. NUGC3 cells were transfected with control siRNA (siCT) or siRNA
directed against p53 (sip53) and then treated for 24 hours with IC50
concentrations of indicated compounds. Top: Proteins were extracted, and 20
µg were separated on SDS PAGE. p53 and actin were then detected by
Western blotting. Bottom: Total RNAs were extracted and RT-qPCR
performed to measure the expression of P53, P21, PUMA and NOXA. Bars
represent means of triplicates with error bars. * indicates statistical differences
from NT (siCT) with p<0.001, Δ indicates statistical differences from NT (siCT)
with p<0.01, • indicates statistical differences from L5 (siCT) with p<0.001.
ROS-associated toxicity could be due to the in situ formation of
18a]
redox-active Cu complexes.[8c,
We plan to investigate cellular
levels of ROS species upon L5 treatment both in wild-type and p53-
Y220C cell lines moving forward. Overall, our novel series of
bifunctional scaffolds have the potential to restore wild-type function
in the p53-Y220C mutant and L5 is a promising scaffold for future
structure-activity relationship studies to increase affinity for p53-
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10.1002/chem.201802677
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Y220C and improve selectivity. In combination with major
technological advancements in gene sequencing capability and a
shift towards personalized medicine, the development of small
molecules capable of mutant-specific p53 reactivation holds
significant promise.
Michael Smith Career Investigator Award (T.S), and NSERC
Discovery Grant (RGPIN05559) and the SFU William and
Isabella Steel Fund (J.J.W). J.J.M thanks NSERC and SFU for
postgraduate fellowships. R.M.C. acknowledges NSERC for a
postgraduate fellowship. Work at the Universitꢀ de Strasbourg is
supported by the CNRS, Inserm, ARC, Ligue contre le cancer.
Keywords: p53 • drug design • metallochaperone • protein
stability • apoptosis
Acknowledgements
The authors thank the National Cancer Institute for NCI-60
screening, and acknowledge the assistance of David Weber in
visualization of the NCI-60 heat map. Prof. Selivanova and
group are thanked for helpful discussions. Work at Simon
Fraser University is supported by a Natural Sciences and
Engineering Research Council (NSERC) Discovery Grant and a
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