Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization

Article abstract of DOI:10.1021/jacs.9b11232

The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.

Full text of DOI:10.1021/jacs.9b11232

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Article
Quinazoline Ligands Induce Cancer Cell Death through Selective
STAT3 Inhibition and G‑Quadruplex Stabilization
§
§
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Jan Jamroskovic, Mara Doimo, Karam Chand, Ikenna Obi, Rajendra Kumar, Kristoffer Brannstrom,
̈
Mattias Hedenstrom, Rabindra Nath Das, Almaz Akhunzianov, Marco Deiana, Kazutoshi Kasho,
̈
Sebastian Sulis Sato, Parham L. Pourbozorgi, James E. Mason, Paolo Medini, Daniel Ohlund,
Sjoerd Wanrooij,* Erik Chorell,* and Nasim Sabouri*
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ABSTRACT: The signal transducer and activator of transcription 3
(STAT3) protein is a master regulator of most key hallmarks and
enablers of cancer, including cell proliferation and the response to
DNA damage. G-Quadruplex (G4) structures are four-stranded
noncanonical DNA structures enriched at telomeres and oncogenes’
promoters. In cancer cells, stabilization of G4 DNAs leads to
replication stress and DNA damage accumulation and is therefore
considered a promising target for oncotherapy. Here, we designed and
synthesized novel quinazoline-based compounds that simultaneously
and selectively affect these two well-recognized cancer targets, G4
DNA structures and the STAT3 protein. Using a combination of in
vitro assays, NMR, and molecular dynamics simulations, we show that
these small, uncharged compounds not only bind to the STAT3
protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of
STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a
G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an
increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is
the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also
simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.
therefore have great potential for synergism with compounds
that cause DNA damage. Two recognized cancer targets along
this line that have lately gained a lot of attention are G-
quadruplex (G4) DNA structures and the STAT3 protein. G4
DNA structures are four-stranded secondary DNA structures
that play important roles in regulating gene expression. In the
human genome, it is estimated that G4 structures can form at
over 700 000 positions.3 G4 structures are over-represented in
oncogenes and regulatory genes, and under-represented in
housekeeping and tumor suppressor genes,4,5 and therefore
suggested to be promising chemotherapeutic targets. This is
further supported by the high occurrence of G4 structures in
the telomeres and by their ability to inhibit telomerase action
and obstruct DNA replication and repair, which leads to
activation of the DNA damage response pathway resulting in
INTRODUCTION
■
Drug resistance presents a major challenge in cancer therapy.
The combination of two or more therapeutic agents with
different targets is therefore used with the aim to improve the
therapeutic effect and reduce the development of drug
resistance. Likewise, a single molecule active on two distinct
cancer targets should result in similar therapeutic benefits and
also reduce the risk of drug−drug interactions. However, this
strategy is rare, likely because it is difficult to develop such
dual-target compounds.
A well-known strategy to combat cancer is to cause DNA
damage. This is detrimental to the majority of cancer cells
because of their dysfunctional DNA repair mechanisms,
resulting in apoptosis. For instance, breast cancer cells that
are BRCA1/BRCA2 deficient, and therefore defective in
repairing their DNA through homologous recombination, are
treated in clinics with DNA-damaging agents, such as cis-platin
and poly(ADP-ribose) polymerase (PARP) inhibitors.1 How-
ever, many cancer cells circumvent this by blocking
programmed cell death and become resistant to treatment.2
The use of compounds that target antiapoptotic pathways
Received: October 18, 2019
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Scheme 1. Scheme of Compounds Synthesis
apoptosis.6,7 Furthermore, cancer cells possess more G4 DNA
structures compared to noncancerous cells,8 and clinical trials
have been conducted with the G4-stabilizing compound CX-
5461 for treatment of BRCA1/2-deficient tumors9 as well as
compound CX-3543 for treatment of carcinoid and neuro-
endocrine tumors.10
RESULT AND DISCUSSION
■
4f and 8g Selectively Bind and Stabilize G4
Structures in Vitro. We previously screened >30 000
compounds for their ability to bind to three different G4
structures, and we identified quinazoline-based compound 5b
as having the ability to both bind and stabilize G4 structures.16
Furthermore, we reported16 that a compound with structural
resemblance to 5b had been identified in a screen for
compounds that selectively inhibit phosphorylation of
STAT3 at tyrosine 705 (pSTAT3).17 Intrigued by this
potential for dual targeting, we designed and synthesized a
library of 47 analogues (Scheme 1). Initially, we determined
the G4 stabilization effect of these analogues by measuring the
progression of Taq DNA polymerase on DNA templates
carrying G4 structures with different topologies and one non-
G4 control DNA template,16,18 and we identified several
selective G4-stabilizing compounds and structure−function
relationships (Figures S1−S3). For our subsequent experi-
ments, we selected compounds 4f and 8g (Scheme 1) because
these were the most effective in stabilizing the different G4
topologies without affecting the non-G4 DNA (Figure S3).
Dose-dependent studies of these two compounds showed an
up to 10-fold improvement in G4-associated inhibition of
DNA polymerization compared to the original hit 5b,
suggesting that these two compounds efficiently stabilize G4
structures (Figures 1a, 1b, and S4−S9a). The topologies of the
G4 structures had slightly different impacts on the stabilization
ability of the compounds, and 5b and 8g had stronger
preferences for parallel DNA structures than a hybrid DNA
structure. 4f stabilized the ribosomal parallel and hybrid DNA
structures equally well and showed very strong preference for
the well-characterized parallel c-MYC Pu24T G4 structure
(Figures 1a, 1b, and S4−S9a). By surface plasmon resonance
(SPR) and microscale thermophoresis (MST) the dissociation
constant (KD) of 4f for the c-MYC Pu24T G4 DNA structure
was estimated to be ∼180 nM (Figure 1c−f). Moreover, the
compounds were selective for G4 DNA over single-stranded
DNA (ssDNA) because the affinity of 4f and 8g measured by
The Janus kinase/signal transducer and activator of
transcription (JAK/STAT) signaling pathway plays important
roles in cell growth and survival. Activation of the members of
the STAT family of proteins through phosphorylation is thus
tightly regulated, and loss of this control correlates with
pathological conditions. In particular, uncontrolled/constitu-
tive active STAT3 is frequently detected in several cancer
types,11,12 and STAT3 is therefore considered to be a
promising cancer drug target.13 Unphosphorylated and inactive
STAT3 exists in a monomeric state and localizes mainly in the
cytoplasm. When STAT3 is phosphorylated, it dimerizes and
translocates into the nucleus where it promotes transcription of
target genes, of which many are oncogenes.14 Subsequently,
downstream pathways act in cancer cell survival, proliferation,
invasion, and metastasis.2 Thus, inhibition of STAT3
phosphorylation blocks its activation and represents one of
the main strategies in STAT3-related drug development.15
Here, we synthesized 47 quinazoline analogues and analyzed
them with biochemical and biophysical methods, molecular
modeling, microscopy, and cell experiments. These studies
reveal the mechanism by which the quinazolines selectively
stabilize G4 DNA structures in cells. Additionally, we show
that the same lead compounds also block phosphorylation of
the STAT3 protein without affecting STAT1. Treatment of
human cells with the compounds increased DNA damage and
induced apoptosis. Importantly, treated breast cancer-derived
cells showed reduced viability compared to noncancerous cells
from breast tissue. Thus, we present quinazoline compounds
that selectively bind to two independent chemotherapeutic
targets, which represent a novel chemotherapeutic strategy.
B
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intercalative binding mode. To investigate these potential
binding modes, we next performed molecular dynamics (MD)
simulations based on the NMR results. When 4f was modeled
on the top of the first G-tetrad, it mostly interacted with G-4
and G-8 in the first G-tetrad but also with G-17, although no
chemical shift changes were observed for G-17 in the NMR
experiments (Figure 2c−e). When the compound was
intercalated between the first and the second G-tetrad it also
generated a stable structure where 4f mostly interacted with G-
4 and G-8, leaving G-17 largely unaffected in accordance with
the NMR results (Figure 2c, 2f, and 2g). The MD-predicted
affinity for the top-binding mode was weaker than that for the
intercalating conformation (Table S1). The intercalative
binding mode would explain 4f’s high affinity despite being
neutral and having a low molecular weight, which is rare for
compounds that target large and flat binding surfaces such as
end stacking with G4 structures. Nevertheless, the intercalative
binding mode is unusual for G4-stabilizing compounds, and
even though our data match better for this binding mode, the
most commonly described end-stacking binding mode cannot
be excluded without further structural elucidation.
We also performed MD simulations with 8g, which
suggested different types of end-stacking binding modes for
this compound (Figure S11b and Table S2). In fact, modeling
of 8g in the intercalative binding mode did not give good
results and prohibited subsequent MD simulations (data not
shown).
HeLa Cells Are Sensitive to 4f and 8g Resulting in
Replication Stress, DNA Damage, and Apoptosis. Next,
we investigated whether the compounds are able to enter into
cultured human cells. We first took advantage of the
fluorescence properties of 8g (Figure S12a) and performed
in vivo live imaging of HeLa cells by 2-photon excitation
microscopy and confocal laser scanning microscopy (CLSM).
We detected the accumulation of 8g in the nucleolar G4-rich
regions already at 10 min after the start of the treatment
(Figure S12b−d). Detection of nucleolar localization is typical
for many fluorescent G4 ligands.20−24 Moreover, we confirmed
4f and 8g uptake by Caco-2 cell permeability experiments
(Table S3). Together these data show that both 4f and 8g are
able to enter into human cells.
Figure 1. Compounds 4f and 8g selectively stabilize G4 structures in
vitro. Dose response analyses of the Taq-polymerase stop assay with
(a) 4f and (b) 8g with the different G4 templates (hybrid telomeric
G4, magenta; parallel ribosomal G4, blue; parallel c-MYC Pu24T G4,
green; antiparallel cdc13+ promoter DNA, red) and non-G4 DNA
(black) templates used in the primary Taq-polymerase assay screens
(Figures S4, S5, and S8). Numbers represent the estimated IC50
values. Binding of 4f to the c-MYC Pu24T G4 DNA structure was
measured by SPR showing the (c) sensorgrams and (d) dose response
curve as well as by MST analysis showing the (e) binding curves and
(f) dose response curve. IC50 and KD values show mean of three
measurements SD.
Because human cancer cell lines, HeLa cells in particular,
have increased amounts of G4 DNA structures compared to
noncancerous cells like human primary fibroblasts (HPFs),25
we compared the effect of 5b, 4f, and 8g on these two cell
types. Increasing concentrations of all three compounds were
toxic to both HPFs and HeLa cells (4f and 8g were both more
toxic compared to 5b) (Figure 3a and 3b). Notably, while the
dose response of 5b was the same in the two cell types, 4f was
slightly more toxic to HeLa cells and 8g had a significantly
stronger effect on HeLa cells compared to HPFs (at 2.5−7.5
μM) (Figure S13a−c). The largest difference in cell survival
was observed when HPFs (90.4% viable cells) and HeLa cells
(8.6% viable cells) were treated with 2.5 μM 8g (Figure S13c),
showing that HeLa cells are about 10-fold more sensitive to 8g
than HPFs.
One explanation for the observed cell viability effects might
be perturbed DNA replication.9 To examine the effect of 8g on
DNA replication, we performed DNA fiber analysis in HeLa
cells (Figure 3c and 3d). The mean DNA replication tract
length was significantly shorter in 8g-treated cells compared to
mock-treated cells (p = 5.4 × 10−19) (Figure 3e), suggesting
that 8g affects the DNA replication speed. DNA replication
SPR and/or fluorescence titrations for the ssDNA control
oligonucleotide was negligible (Figures S9 and S10).
To analyze the compounds’ binding interactions with G4
DNA structures, we performed nuclear magnetic resonance
(NMR) studies with the c-MYC Pu24T G4 DNA structure by
monitoring chemical shift changes of the imino protons of
guanines in the G4 structure.19 In agreement with the SPR,
MST, and/or fluorescence titration results, the NMR data
showed that both 4f and 8g bound to the c-MYC Pu24T G4
structure (Figures 2a, 2b, and S11a). However, the chemical
shift changes could not be quantified for 8g because line
broadening of the imino peaks was observed instead of a new
set of peaks (Figure S11a), which suggests multiple binding
modes or fast on−off rates. By mapping the peak shift changes
induced by 4f to the c-MYC Pu24T G4 NMR structure, we
found that 4f strongly affected two of the guanines on one side
of the top G-tetrad (G-4 and G-8) (Figure 2a−c) and guanine
G-5 in the second G-tetrad located below G-4 and G-8 (Figure
2c). The strong effect on one side of the top G-tetrad could
potentially be explained by binding interactions with the 5′
DNA sequence flanking the G4 or by a more atypical
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1
Figure 2. Binding modes of 4f to the c-MYC Pu24T G4 structure. (a) Imino region of the H NMR spectrum of c-MYC Pu24T in the absence
(bottom) and presence of 0.5 equiv of 4f (middle) and 1 equiv of 4f (top). New set of well-defined peaks appeared upon addition of 4f, originating
from the 4f:DNA complex. At a 1:2 ratio of 4f to DNA, both free and bound forms of the imino peaks were observed, which were used to assign the
bound form. Peaks marked with asterisks originated from the DNA in complex with 4f. Sizes of the induced chemical shift changes could be
determined by observing exchange peaks in a 2D NOESY spectrum (b) of 0.5 equiv of 4f binding to c-MYC Pu24T G4 DNA. Off-diagonal peaks
represent cross-peaks from the exchange between free and bound form of DNA. (c) Cartoon showing the structure of c-MYC Pu24T G4 DNA and
interactions of the different guanines in the presence of 4f based on the NMR data in a and b. (d) Top and (e) side views of an end-stacking
binding model compared to (f) top and (g) side views of an intercalative binding model from two of the largest MD clusters of the c-MYC Pu24T
G4 DNA-4f complex. Color coding is the same in c−g. Observed chemical shift changes in the NMR data are shown in red (large shifts), yellow
(moderate shifts), and blue (no/small shifts).
tract length was not determined in 4f-treated cells. Decreased
DNA replication speed could potentially be a sign of DNA
damage accumulation, which can be detected by phosphory-
lated histone H2A.X (γH2A.X) protein levels.26
other hand, high radiation doses lead to phosphorylation of
ATM and consequently DNA repair activation and thus have a
less pronounced effect on cell survival.28 We confirmed a
similar mechanism for 8g by measuring apoptosis with
increasing compound concentrations. At 5 μM, the proportion
of apoptotic cells was about 2-fold higher (23.1%) compared
to cells treated with either 10 or 20 μM 8g (10.7% and 13%,
respectively) (Figures 4c and S14a). This finding explains the
inverted cell viability dose response with 8g (Figure 3a).
4f Treatment Is Toxic to Triple-Negative Breast
Cancer Tumor Cells. On the basis of our primer extension
assays and SPR analysis, we observed efficient stabilization of
telomeric G4 DNA by 4f (Figures 1a, S3b, S5a, and S9d).
Furthermore, HeLa cells treated with 4f had ∼4-fold increase
in the formation of internuclear chromatin bridges (ICBs)
(Figure 4d and 4e), a hallmark of telomere instability.29 Similar
to 4f, the G4-stabilizing compounds, CX-5461 and CX-3543,
also induce replication defects, DNA damage, and telomere
instability, all important properties for DNA-targeting cancer
drugs.9,10 In fact, the highly aggressive triple-negative breast
cancer cell lines, MDA-MB-231 and MDA-MB-436, are among
the most sensitive breast cancer cell lines toward CX-5461.9
Furthermore, MDA-MB-436 cells have a BRCA1 mutation that
results in loss of nuclear BRCA1 protein expression.30 We
treated these cell lines with 4f to examine if MDA-MB-231 and
MDA-MB-436 are also more sensitive to 4f than healthy
We treated HeLa cells with increasing concentrations of 5b,
4f, and 8g based on the effects detected in the cell viability
assay (Figure 3a), and we found an increased γH2A.X signal
compared to the mock-treated cells (Figure 4a and 4b). We
also analyzed the upstream phosphorylation of ATM serine/
threonine kinase (ATM), the major kinase involved in the
phosphorylation of H2A.X.27 The individual presence of all
three compounds increased ATM phosphorylation levels,
confirming that they all induce a DNA damage checkpoint
response (Figure 4a and 4b). Surprisingly, 8g showed a
differential dose response for ATM and H2A.X phosphor-
ylation (Figure 4a and 4b). At 5 μM 8g, the γH2A.X levels
were increased but the ATM phosphorylation levels were
unchanged, whereas at higher compound concentrations both
ATM and H2A.X were phosphorylated (Figure 4a and 4b).
These data suggest that cells treated with 5 μM 8g undergo
ATM-independent phosphorylation of H2A.X, while at higher
concentrations the compound causes increased γH2A.X
through the ATM-dependent pathway. It has been reported
that in cells treated with ionizing radiation a lower radiation
dose leads to a strong decrease in the cell survival rate as a
result of ATM-independent H2A.X phosphorylation. On the
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number of BG4 foci could already be detected after 1 h of
treatment with 20 μM 8g (Figure S14b). In contrast, cells
treated with 5 μM 8g, a concentration that caused
phosphorylation of H2A.X without ATM activation, did not
show an increase in the number of BG4-positive foci/cell,
suggesting that the ATM-independent phosphorylation of
H2A.X is not dependent on G4 stabilization (Figure S14b).
Together, these data support the hypothesis that 8g and 4f are
able to stabilize G4 DNA structures in cells and that this
induces replication stress and DNA damage and thus reduces
cell viability. However, 8g affected the viability more strongly
compared to 4f, and this cannot be explained only by G4 DNA
stabilization because 4f is at least as effective as 8g in stabilizing
G4 DNA structures both in vitro and in human cells (Figures
1a, 1b, 5, and S3a and b).
Phosphorylation of STAT3 Is Inhibited by 4f and 8g
by Direct Binding. To examine if the reduced viability is due
to unsuccessful STAT3 activation, we tested if our compounds
affect pSTAT3 levels in human cells. We treated HeLa cells
with 4f, 5b, or 8g, which indeed resulted in a reduction of the
pSTAT3 protein levels for all three compounds (Figure 6a). In
4f-treated cells, pSTAT3 reduction occurred at 50 μM (Figure
6a and 6b), a concentration at which G4 stabilization was also
increased (Figure 5c). In contrast, in 8g-treated cells pSTAT3
was inhibited already at 5 μM (Figure 6a and 6b), a
concentration at which we did not detect any increase in the
number of BG4-positive foci (Figure S14b). Together these
data suggest that 4f and 8g act on both G4 structures and
pSTAT3, although 8g-dependent pSTAT3 inhibition occurs at
lower concentrations than the G4 structure stabilization,
resulting in the activation of two different processes that
ultimately lead to cell death (Figure 4c). At the highest
concentration tested for each of the compounds, total STAT3
levels were also affected (Figure 6a). However, the reduction
of pSTAT3 occurred at lower compound concentrations, and a
dose-dependent reduction of the pSTAT3/STAT3 ratio was
observed (Figure 6b), indicating that the reduced pSTAT3
levels were not dependent on the total STAT3 protein levels.
Because STAT3 levels are positively autoregulated,31 STAT3
downregulation might represent a consequence of pSTAT3
inhibition. In agreement, we confirmed a direct interaction
between 4f and 8g with STAT3 protein by SPR analysis (4f KD
= 45 μM, 8g KD = 15.5 μM) (Figures 6c−e and S15), showing
that 4f and 8g bind to the STAT3 protein and might therefore
directly interfere with STAT3 phosphorylation.
STAT1, another member of the STAT family of proteins
that have antiproliferative and pro-apoptotic functions,32
shares around 50% amino acid sequence homology with
STAT3.33 Importantly, none of the compounds tested here
affected the total or phosphorylated levels of STAT1 (Figure
6a and 6b), indicating that the compounds selectively inhibit
STAT3 over STAT1. Therefore, 4f and 8g not only stabilize
G4 structures but also selectively inhibit the STAT3-mediated
pathway, which is an important pathway in cancer therapeutics.
8g Localizes into the Nucleus in S. pombe Cells and
Perturbs Replication Fork Progression. Although the
JAK/STAT signaling pathway is essential for multicellular
organisms, it is not present in unicellular organisms such as the
fission yeast Schizosaccharomyces pombe.34,35 However, the
positions of many G4 structures are conserved between S.
pombe and multicellular organisms,36,37 and unresolved G4
structures result in fork pausing and DNA damage,37,38
indicating that G4 structures also form in S. pombe. Therefore,
Figure 3. HeLa cells are sensitive to the novel compounds resulting in
replication stress. Cell viability assay of (a) HeLa and (b) HPFs
treated for 48 h with 5b, 4f, or 8g at the indicated concentrations.
Data represent the mean SD, n ≥ 3. (c) Schematic of the DNA fiber
analysis. (d) Representative images of replication tracts with different
lengths. Intact DNA fibers displaying iodo-deoxyuridine (IdU) labels
(red) flanked by chloro-deoxyuridine (CldU) labels (green). (e)
Quantification of the fiber length (kb) in treated (8g) versus mock
cells (−). Data represent populations of individual DNA fibers for
each condition of the final experiment (63 for control and 52 for
treatment). Mean 2SD is indicated. Welch-corrected two-sample t
tests of ln-transformed data were used, and p value is indicated.
epithelial cell lines derived from benign proliferative breast
tissue, MCF-10a. Indeed at 7 μM, a concentration that was not
toxic for the control breast cell lines, we found reduced
viability of both the MDA-MB-231 and the MDA-MB-436
tumor cell lines, 60% and 55%, respectively (Figure 4f). In
addition, and similar to CX-5461, both cell lines were more
sensitive to 4f compared to the noninvasive and less aggressive
breast cancer cell line MCF-7, which is BRCA1+/+ and does
not contain known mutations in DNA damage repair genes
(Figure 4f).
These data demonstrate that triple-negative breast cancer
cell lines are more sensitive to 4f treatment than cell lines
derived from control breast tissue, suggesting that 4f, similar to
CX5461, which is in clinical trial phase I,9 may be a good drug
candidate in treating triple-negative breast cancer and target
tumor cells that are deficient in DNA damage repair pathways.
Similar to 4f, the breast cancer cells MCF-7, MDA-MB-231,
and MDA-MB-436 are more sensitive to 8g compared to the
cell line derived from benign proliferative breast tissue, MCF-
10a (Figure S13d).
Treatment with 4f and 8g Results in Increased BG4
Foci in HeLa Cells. To determine if 4f and 8g stabilize G4
DNA structures in human cell culture, we used the anti-G4
DNA antibody BG425 and performed immunofluorescence
microscopy to visualize and quantify G4 DNA structures in
HeLa cells. At the compound concentrations that resulted in a
DNA damage response (Figure 4a and 4b), we found that the
number of BG4 foci per cell nucleus increased significantly in
the treated cells compared to mock-treated cells (4f p = 5.52 ×
10−13 and 8g p = 1.06 × 10−6) (Figure 5). The increased
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Figure 4. Compounds 4f and 8g induce DNA damage response and genome instability in HeLa cells. (a) Immunoblot analysis of soluble (for
pATM, ATM, and PCNA) and chromatin-bound (for γH2A.X and H2AX) protein fractions extracted from HeLa cells treated for 12 h with 5b, 4f,
or 8g at the indicated concentrations. PCNA was used as loading control of the soluble protein fractions. (b) Quantification of the immunoblot
analysis in a. Error bars represent the mean absolute error (n = 2) for 5b and 4f and the mean SD (n = 3) for 8g. (c) Number of apoptotic cells
(annexin V-positive cells) measured by flow cytometry. HeLa cells were treated for 12 h with 8g at the indicated concentrations and stained with
propidium iodide and annexin V. Data represent the mean SD (n = 3). Analysis of the data was performed using two-sample t tests with assumed
equal variance, and p values are indicated. (d) Representative images of HeLa cells treated for 12 h with 50 μM 4f (right) or DMSO (left). Cell
nuclei were stained with DAPI upon cell fixation. Black arrows indicate ICBs. Scale bars indicate 40 μm. (e) Fold change in the number of ICBs in
treated versus untreated cells. At least 350 cells from six technical replicates were counted for each treatment, and data represent the mean SD of
three independent experiments. Analysis of the data was performed using the two-sample t test. (f) Viability of different cell lines. Cells were treated
for 48 h with 7 μM 4f. Data represent the mean SD (n = 3). Analysis of the data was performed using the two-sample t test.
to confirm that the effects of 4f and 8g on HeLa cells are a
consequence of G4 stabilization and not merely a result of the
pSTAT3 inhibition, we tested the effect of these compounds in
S. pombe.
arrested cells that were unable to progress through the cell
cycle (Figure 7d).
To more directly examine the effect of 8g on DNA
replication, we performed DNA fiber analysis (Figure 7e). The
mean DNA replication tract length was significantly shorter in
8g-treated cells compared to mock-treated cells (p = 1.6 ×
10−7) (Figure 7e), suggesting that 8g affects DNA replication
progression in S. pombe in a STAT3-independent manner.
For this study, we employed an S. pombe mutant strain in
which the multidrug-resistant response pathway has been
partly deleted, as wild-type S. pombe cells are multidrug
resistant due to very efficient drug efflux pumps.39,40 We found
that 8g localized into the nucleus (Figures 7a and S16a) and
affected the growth of asynchronous S. pombe cells (Figure
7b). 4f did not affect cell growth, probably due to a limited cell
uptake in S. pombe (Figure 7b).39 Cells treated with 8−12 μM
8g showed a 4-fold decrease in the number of doublings and
altered cell morphology compared to mock-treated S. pombe
cells (Figure 7b and 7c). To determine if the reduced cell
growth was due to slower S-phase, we examined whether 8g
affects the cell cycle progression of synchronized S. pombe cells
(Figure S16b). Synchronized cells released from the G2 phase
treated with 5 μM 8g showed both delayed (∼100 min after
release instead of ∼80 min) and prolonged (120 min instead of
80 min) S-phase compared to mock-treated cells (Figure 7d).
Increasing the concentration of 8g to 10 μM resulted in G2-
CONCLUSIONS
■
We show that the quinazoline compounds can selectively
stabilize G4 structures both in vitro and in human cell culture.
In addition, the quinazoline lead compounds also selectively
inhibit the STAT3-mediated pathway by binding to the
STAT3 protein (Figure S17) without affecting the STAT1
protein. We show that our compounds induce replication
stress, telomere and genome instability, and apoptosis and
might therefore be beneficial for use in cancer therapy. In fact,
we detected reduced viability of aggressive breast cancer cells
compared to cells from healthy breast tissue treated with the
lead compound.
F
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Figure 6. Phosphorylation of STAT3 is inhibited by direct binding of
4f and 8g to the STAT3 protein. (a) Total cell lysate from HeLa cells
treated for 12 h at the indicated concentrations of 5b, 4f, or 8g
immunoblotted with the indicated antibodies. Actin was used as the
loading control. (b) Quantification of the immunoblot analysis in a.
Error bars represent the mean absolute error of two independent
experiments. SPR sensorgrams of (c) 4f and (d) 8g binding to
STAT3 protein with corresponding (e) dose response curves
(trifluoroacetic acid salt (TFA) of 8g was used (see Supporting
Information) to increase the solubility of 8g within the concentration
range used in this experiment). Average of three measurements is
shown SD, and full graphs are presented in Figure S15.
Figure 5. Treatment with 4f and 8g results in increased BG4 foci in
HeLa cells. (a) Representative images of HeLa cells stained with the
BG4 antibody after treatment for 12 h. (b and c) Quantification of
BG4-positive cell nuclei. Data represent populations of individual cells
for each condition of the final experiment: (b) DMSO (−) = 133
cells, 8g 20 μM = 111 cells; (c) DMSO (−) = 130 cells, 4f 50 μM =
85 cells). Means
2SD are indicated. Analysis of the data was
performed using Welch-corrected two-sample t tests of ln-transformed
data, and p values are indicated.
Although both stabilization of G4 structures and pSTAT3
inhibition have independently shown promising effects in
inhibiting cancer growth in different model systems,14,41
neither strategy has yet resulted in successful clinical
trials.10,42−44 The ability to affect these two targets with a
single low molecular weight compound represents a chemo-
therapeutic concept with potential benefits such as synergism
and reduced drug resistance and thus may be of high
therapeutic relevance in the clinics.
two-sided Welch-corrected t test in the case of unequal variance.
Unequal variance was determined by the F test. Effect sizes and the
means with asymmetric 2SD were calculated. In the ICB experiment
and flow cytometry, a two-sided Student’s t test with assumed equal
variance was used to determine significant differences. A p value <
0.05 was considered significant. All calculations were performed in
Microsoft Excel and OriginPro 2016 software. Microscopy of BG4
immunostaining of human cells was single blinded using the DAPI
channel for sample acquisition. Fiber analysis and ICB experiments
were not blinded.
Taq DNA Polymerase Assay. All DNA molecules used in the
assay were purchased from Eurofins Genomic (Table S4), and the
experiment was performed as described previously.16 In brief, each
reaction contained 40 nM template DNA incubated with 25 μM
compound, and the control reaction used 5% DMSO in place of the
compound. Each reaction was run for 10 min. The final quantification
was the average value of two independent experiments along with the
absolute error. For the dose response analysis, 40 nM template DNA
was incubated with a 0.06, 0.16, 0.4, 1, 2.6, 6.4, 16, or 40 μM
EXPERIMENTAL SECTION
Compound Synthesis. Detailed procedures for compound
synthesis are described in the Supporting Information.
■
Statistical Analysis. The minimal sample sizes for the microscopy
experiments (BG4 immunostaining of human cells and S. pombe and
fiber analysis) were determined by pilot experiments. Distribution
plots and quantile−quantile plots were used to graphically examine
the normality of the sample distributions. Transformation to natural
logarithms was performed if required. p values were calculated by a
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Figure 7. 8g localizes into the nucleus in S. pombe cells and slows down cell growth during S-phase. (a) Representative image of S. pombe cells
stained by 25 μM 8g and 0.25% DMSO (−). Final pictures are cropped of the full field images shown in Figure S12a. (b) Number of doublings per
12 h of S. pombe cells treated with 8g. Error bars represent SD, n = 3. (c) Representative images of S. pombe cells from b treated with 0.25%
DMSO (−) or 10 μM 8g. Arrows indicate “pear”-like and other cell morphology deformations. (d) FACS analysis of synchronized S. pombe cells
grown in PMG media treated with 5 and 10 μM of 8g. Representative FACS profiles are shown, n = 3. (e) Quantification of the DNA fiber length
(kb) in treated (8g) versus mock cells (−). Data represent populations of individual DNA fibers for each condition of the final experiment (n = 40
per treatment). Mean SD is indicated. Two-sample t tests were used, and p value is indicated.
concentration of the compounds or with 5% DMSO as a control
reaction. The experiments were performed in the same way as in the
primary screening. The final quantification was the average value of
three independent experiments along with the standard deviation.
IC50 values were calculated by fitting the data from each experiment
to the dose response function in the OriginPro 2016 software.
Surface Plasmon Resonance. The SPR experiment with DNA
molecules was performed on a ProteOn XPR (Biorad) at 25 °C. A
final concentration of 5 μM biotin-labeled oligonucleotides (Table
S4) was folded into G4 structures in 10 mM potassium phosphate
buffer, pH = 7, 150 mM KCl at 95 °C for 5 min and cooled down to
room temperature overnight. Folded oligonucleotides were immobi-
lized on a neutravidin-coated NLC sensor chip (Biorad) at a rate of
30 μL/min until maximal response unit (RU) values were reached
(ribosomal G4 DNA, 780 RU; telomeric G4 DNA, 720 RU; c-MYC
Pu24T G4 DNA, 1020 RU; c-kit G4 DNA, 780 RU; ssDNA, 1150
RU). Compounds 4f and 8g were diluted in SPR buffer (10 mM
potassium phosphate buffer, pH = 7, 150 mM KCl, 0.05% Tween 20,
and 5% DMSO) and injected at a flow rate of 50 μL/min for 120 s.
Signal from a reference surface was subtracted, and data were solvent
corrected for DMSO in order to obtain the true RU values. The
apparent dissociation constants (KD) were calculated by fitting the
data to a single-site binding function in the OriginLab 2016 software.
All data were smoothed for visualization purposes only. The SPR
experiment with STAT3 protein was performed on a Biacore T200
(GE Healthcare). A total of 10 ng/μL of his-STAT3 protein
(SignalChem) (diluted in 1× phosphate-buffered saline (PBS),
0.005% Tween 20, and 5% DMSO) was immobilized on the NTA
sensor chip at a flow rate of 5 μL/min until 1500 maximal RU.
Compounds 4f, 8g, and 8g as TFA salt were injected in triplicate at a
flow rate of 50 μL/min for 120 s. Compound 8g showed signs of
aggregation, so 8g as TFA salt with improved solubility was used
instead to get clean kinetics. The signal from a reference surface was
subtracted, and the KD values were calculated by fitting the averaged
data from the sensorgrams to a single-site binding function in
GraphPad Prism 8.0.
Microscale Thermophoresis. c-MYC Pu24T DNA labeled with
CY5 at the 5′ end was folded in 10 mM potassium phosphate and 100
mM KCl (pH 7.4) by heating at 95 °C for 5 min followed by cooling
to room temperature. All experiments were performed in 10 mM
potassium phosphate (pH 7.4), 100 mM KCl, 0.05% Tween 20, and
4% BSA, the DNA concentration was held constant at 25 nM, and the
4f concentration varied from 0.15 nM to 1.25 μM (14 dilution steps).
The samples were loaded into standard MST-grade glass capillaries,
and the MST experiment was performed using a Monolith NT.115
(Nano Temper, Germany) with 40% LED power. Data were analyzed
using the Nano Temper analysis software, and KD was calculated by
fitting the data to the Hill equation in OriginPro 8.0.
Nuclear Magnetic Resonance. The G4 DNA stock solution was
prepared by folding 200 μM c-MYC Pu24T in 10 mM potassium
phosphate buffer (pH = 7.4) and 35 mM KCl by heating to 95 °C and
slowly cooling to room temperature overnight. An effective DNA
concentration of 180 μM was obtained by adding 10% D2O. NMR
samples were prepared in 3 mm NMR tubes by adding 1 equiv of 4f
or 8g to the DNA stock solution. For 4g, an additional sample with
0.5 equiv of compound was also prepared. All spectra were recorded
at 298 K on a Bruker 850 MHz Avance III HD spectrometer equipped
with a 5 mm TCI cryoprobe. Excitation sculpting was used in the 1D
1H experiments, and 256 scans were recorded. The 2D NOESY
experiment was recorded with 32 scans, 256 t1 increments, a
relaxation delay of 1.1 s, and a mixing time of 200 ms. Processing was
performed with zero filling in the indirect dimension and using 90°-
shifted squared sine-bell apodization in both dimensions for the
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NOESY spectrum. Processing was performed in Topspin 3.5 (Bruker
Biospin, Germany). The NMR peaks for c-MYC Pu24T G4 DNA
were assigned according to a previously published structure.19
Molecular Dynamics Simulations. The c-MYC Pu24T solution
structures (PDB ID: 2MGN) were downloaded from the Protein
Data Bank.19 Eight c-MYC Pu24T-4f structures were modeled based
on various 4f binding modes (Figure S18) and four binding modes of
8g (Figure S19) using the Openbabel,45 Avogadro,46 and Chimera47
software packages. Each complex was placed inside the center of a
dodecahedron box, solvated by adding water molecules, and
neutralized by adding an excess of 100 mM KCl using GROMACS
tools.48 The DNA was simulated with the Amber99SB49 force-field
parameters with PARMBSC150 improvements, and the tip3p model51
was used for water molecules. Before assigning GAFF force-field
parameters52 to 4f and 8g, its partial atomic charges were computed
by the RESP method53 using AmberTool54 after geometry
optimization by PM6 and B3LYP/6-31g(d,p) methods in two stages
using the Gaussian package.55 Subsequently, MD simulations were
performed using GROMACS-201648 as previously described.56 The
obtained MD trajectories were combined and clustered on the basis of
principle component analysis using gmx_clusterByFeatures (https://
gmx-clusterbyfeatures.readthedocs.io). The binding energy was
calculated with the MM/PBSA method using the g_mmpbsa
tool.57,58 Standard errors were calculated using the block-averaging
method.59
S. pombe DNA Fiber Analysis. For the DNA fiber analysis, we
used the S. pombe strain (bfr1::hygr pmd1::natr cdc25−22
pfh1+::ura4+-nmt-pfh1-GFP leu1−32::[hENT1 leu1+] his7−366::[hsv-
tk his7+] ade6-M21? his3-D1? telo-his3?).
Cells were grown to 107 cells/mL in the presence of 1.5 μM 8g or
0.015% (v/v) DMSO at 25 °C in liquid EMMII (Formedium) media
for about 12 h. Next, the cultures were diluted to 5 × 106 cells/mL,
and the concentration of 8g was increased to 3 μM before arresting
the cells in G2 phase at 37 °C for 4 h. The cells were released from
G2 phase by shifting the temperature back to 25 °C. A 66 μM final
concentration of bromodeoxyuridine (BrdU) was added 30 min after
release from the G2 phase, and cells were allowed to incorporate
BrdU into their DNA for 35 min. After addition of a stop solution
(250 mM EDTA, pH 8.0, 0.16% sodium azide), cells were harvested
by centrifugation and resuspended in cold 70% ethanol. A 200 U/mL
lyticase from Arthrobacter luteus (Sigma-Aldrich) was used to digest
the cell wall prior to stretching DNA fibers on microscopic slides.
BrdU incorporated into DNA was detected using rat anti BrdU clone
BU1/75 (ICRI) primary antibody (ABD Serotec) and Goat anti Rat
IgG Alexa Fluor 568 secondary antibody (Life technologies), while
anti-DNA antibody single-stranded clone 16−19 primary antibody
(Sigma-Aldrich) and Goat Anti Mouse IgG2a (γ2a) Alexa Fluor 488
secondary antibody (Life technologies) were used to detect ssDNA.
Stained DNA fibers were visualized using an Axio Imager Z1
microscope (Zeiss), and images of untangled DNA fibers were taken
at random from different fields. Only DNA fibers with BrdU label
having intact ssDNA ends or DNA fibers with BrdU label measuring
more than 70 μm were selected for analysis using Zen 2.6 blue edition
(Zeiss) and ImageJ software packages. The experiments were repeated
independently twice with two biological replicates.
Spectrophotometric Measurements. A 10 μM concentration
of 8g was diluted into 100 mM KCl and 10.0 mM TRIS pH = 7.5
with and without the same equivalent of folded c-MYC Pu24T G4
DNA, and UV−vis absorption spectra were recorded by a T90+ UV/
vis spectrometer (PG instruments Ltd.).
Spectrofluorimetric Measurement. Emission and excitation
spectra of 5 μM 8g in 100% DMSO were recorded in a quartz cuvette
with a 1 cm path length on a Jasco Spectrofluorometer FP-6500.
Fluorimetric Titrations. A 2.0 μM concentration of 8g (in 100
mM KCl and 10.0 mM TRIS pH = 7.5, 0.025% DMSO) was titrated
by DNA or RNA oligonucleotides folded in the same buffer. The
isosbestic point, λexc = 305 nm, was used for 8g excitation, and
fluorescence spectra (λem = 315−675 nm) were recorded by a Jasco
FP-6500 spectrofluorometer. DNA/RNA background fluorescence
was subtracted from all data. Peak values at λem = 546 nm were fitted
into hyperbolic binding function in Graphpad Prism 8.0 available at
https://www.graphpad.com/support/faq/fitting-binding-of-
fluorescent-ligands/.
S. pombe Growth and Doubling Time. The S. pombe
(bfr1::hygr pmd1::natr ade6-M210 leu1) strain39 that had genes
deleted in the multidrug-resistance response was the kind gift of the
laboratory of Dr. Tarun Kapoor (Rockefeller University). The cells
were exponentially grown at 30 °C in minimal medium EMMII
(Formedium), and 1 × 106 cells/mL were treated with 8g (1.6, 3.1,
6.3, 8, 10, 12.5, 25, 50, and 100 μM) or 0.25% DMSO for 12 h. The
number of doublings per 12 h was calculated.
Cell Culture and Compound Preparation. HeLa cells
(epitheloid cervix carcinoma, purchased from Sigma-Aldrich) and
HPFs from healthy adults (a kind gift from Leonardo Salviati,
University of Padova, Italy) were cultured at 37 °C in 7% CO2 in
DMEM high glucose medium with Glutamax (Gibco) supplemented
with 1 mM sodium pyruvate, penicillin-streptomycin, and 10% (for
HeLa) or 20% (for HPFs) fetal bovine serum. The MDA-MB-436 cell
line (ATCC No. HTB-130) was obtained from the American Type
Culture Collection (ATCC). The breast cancer cell lines MCF-7
(ATCC No. HTB-22) and MDA-MB-231 (ATCC No. HTB-26) and
the nontumorigenic epithelial cell line MCF 10A (ATCC No. CRL-
10317) were kindly provided by Professor Jenny Persson (Depart-
̊
ment of Molecular Biology, Umea University, Sweden). Cells were
tested to confirm the absence of mycoplasma. Compounds were
dissolved in DMSO to 10 (4f) or 20 mM (8g and 5b), aliquoted, and
stored at −20 °C. Prior to the addition to cells, the compounds were
dissolved in the culture medium at the final concentration required.
Cell Viability. Cell viability was measured using the PrestoBlue
cell viability reagent (Invitrogen) according to the manufacturer’s
recommendations. Briefly, 5000 (for HeLa), 4000 (for HPFs, MDA-
MB-231, MCF-7, and MDA-MB-436), or 3500 (for MCF-10a) cells/
well were seeded in complete medium on 96-well plates the day
before the treatment. Compounds were dissolved in medium at the
indicated concentrations and added to cells. At 48 h after treatment,
10 μL of PrestoBlue was added to each well and the cells were
incubated at 37 °C for three additional hours. Fluorescence
(excitation 560 nm, emission 590 nm, 10 nm bandwidth) was
recorded using a Synergy H4 microplate reader (Biotek).
S. pombe Cell Synchrony and Flow Cytometry Analysis. S.
pombe (bfr1::hygr pmd1::natr, cdc25−22) cells were used, and the
experiment was performed as described previously.38 Cells were
treated with 5 or 10 μM 8g immediately after G2 release. Samples
were taken as described in Figure 5, and analysis was performed on a
Beckman Coulter Cytomics FC500 flow cytometer. The experiment
was repeated at least three times for each condition.
S. pombe Fluorescence Microscopy. Cells were exponentially
grown at 30 °C in minimal medium PMG (Formedium). An amount
of 5 ×106 cells/mL was treated with 8g (final concentration 25 μM)
or 0.25% DMSO (control) for 30 min, washed in PMG medium, and
immobilized on poly-L-lysine-coated glass slides. Localization of 8g
was immediately analyzed by a confocal microscope Leica SP8
FALCON using a HC PL APO 63×/1.40 OIL CS2 objective, hybrid
detector, and Diode 405 nm laser with recorded emission between
520 and 620 nm. Final image was captured with an opened pinhole to
4. To determine nuclear localization of 8g, the background intensity
of the fluorescence signal was decreased in both samples by identically
treating the images with the ImageJ software.60
DNA Fiber Analysis for HeLa Cells. Asynchronous HeLa cells at
70% confluence were seeded at 1 × 105 cells 18 h prior to the 24 h
treatment with 10 μM 8g or 0.1% DMSO (control cells). Cells were
pulse labeled with 25 μM iodo-deoxyuridine (IdU) in fresh medium
containing 10 μM 8g or 0.1% DMSO for 30 min. Subsequently, cells
were incubated for 30 min in fresh medium containing 200 μM
chloro-deoxyuridine (CIdU) and 10 μM 8g or 0.1% DMSO followed
by a 1 h incubation in fresh medium with 200 μM thymidine. Cells
were then harvested and resuspended in cold PBS. DNA fiber
stretching was performed as previously described.61 Briefly, stretched
DNA fibers were immunostained with primary antibodies for IdU
detection, for CIdU detection, and for ssDNA detection along with
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their respective Alexa Fluor antibodies. The antibodies used and their
dilutions are shown in Table S5. Stained DNA fibers were visualized
using an Axio Imager Z1 microscope (Zeiss), and images were
captured randomly from different fields containing untangled fibers.
Only fibers containing IdU labels flanked by CIdU labels with intact
ssDNA ends were selected for analysis using the ZEN 2.3 (Zeiss) and
ImageJ software packages.60 A minimum of 131 individual DNA fibers
were measured for each experimental condition in two independent
experiments. Measurements were made in micrometers and converted
to kilobases using a conversion factor for the length of a labeled track
of 1 μm corresponding to roughly 2 kb.62
63× oil objective (NA 1.40) using identical acquisition settings. Cell
nuclei were focused on the DAPI channel, and BG4-positive foci were
counted in a semiautomatic mode using a customized Cell Profiler
(Broad Institute) pipeline. All images were processed using ImageJ
software.
In Vivo Cell Microscopy. Around 100 000 cells were seeded the
day before treatment on glass-bottomed microwell dishes (MaTek
Corp.). The cells were treated with 50 μM 8g for the indicated time
points, then washed with 1× PBS, and resuspended in complete
DMEM medium without phenol red and supplemented with 25 mM
Hepes. Fluorescence was imaged within 20 min from the end of
compound treatment with a Scientifica 2P galvo microscope equipped
with a Spectra Physics Mai Tai DeepSee Ti:sapphire laser. Acquisition
was made with a 20× water immersion objective designed for 2-
photon applications (Olympus XLUMPLFN 20XW, NA = 1).
Fluorescence was detected with two GaAsP PMTs from Hamamatsu
(emission filter green 525 nm/50, red 585 nm/40). The voltage was
kept at 700 V. For CLSM, HeLa cells were treated with 20 μM 8g for
30 min in DMEM medium. After 30 min, DMEM medium was
replaced by DMEM medium without phenol red and cells were
imaged by the confocal microscope Leica SP8 FALCON using a HC
PL APO 63× /1.40 water CS2 objective and Diode 405 nm laser with
4% power to avoid autofluorescence of cells. Emission was recorded
between 520 and 620 nm by hybrid detector (HyD). Maximum
intensity projection of Z-stack images was used for visualization, and
final images were processed using Fiji (ImageJ) software.
Fluorescence signal in treated and untreated images was enhanced
for visualization purpose only. For quantification, regions of interest
were selected in cell cytoplasm and nucleoli, and the average
fluorescence signal from the selected areas was used.
Protein Extraction and Immunoblotting. For H2A.X and
ATM analysis, HeLa cells were seeded on 10 cm dishes the day before
treatment in order to have 80−90% confluency the day after. Cells
were treated for 12 h at the indicated concentrations. Detergent-
solubilized protein fractions (for ATM/pATM analysis) and nuclear
histone-bound protein fractions (for H2A.X/γH2A.X analysis) were
extracted as previously described.63 For STAT protein analysis, HeLa
cells were seeded on 6-well plates the day before treatment in order to
have 80−90% confluency the day after. Cells were treated for 12 h at
the indicated concentrations and solubilized for 30 min on ice in
RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium
deoxycholate, and 50 mM Tris-HCl (pH 8.0)). After high-speed
centrifugation, the supernatant was collected for further analysis.
Buffers for protein extraction were supplemented with 1× EDTA-free
Halt protease inhibitor cocktail (ThermoFisher Scientific), 1 mM
NaOV4, and 3 mM NaF. Protein amounts were quantified using a
BCA protein assay kit (ThermoScientific). Equal amounts (15 μg) of
protein were separated on 4−20% SDS-TGX (Bio-Rad) gels and
transferred to 0.45 μM nitrocellulose membranes (GE Healthcare Life
Sciences) using a Mini-Protean electrophoresis system (Bio-Rad).
Membranes were blocked in 5% nonfat milk for 2 h. Primary
antibodies were incubated overnight at 4 °C, and horseradish
peroxidase-conjugated-secondary antibodies were incubated 1 h at
room temperature. The antibodies used and their dilutions are shown
in Table S5. All washes and incubations were performed in Tris-
buffered saline with Tween 20. Chemiluminescent detection was
performed using ECL Western blotting substrates (ThermoScientific)
and a ChemiDoc Touch Imaging System (Bio-Rad). Signal
quantification was performed using the ImageQuant TL software
(GE Healthcare Life Sciences).
Apoptosis Assay. HeLa cells (120 000 cells/well) were seeded on
6-well plates the day before the treatment. Cells were treated for 12 h
with 8g at the indicated concentrations, and the number of apoptotic
cells was detected by flow cytometry using the FITC/Annexin V Dead
Cell Apoptosis Kit (Molecular Probes) according to the manufac-
turer’s instructions. Stained cells were measured with a Cytomics
FC500 (Beckman Coulter) equipped with a 488 nm argon laser. A
total of 30 000 cells were collected for each sample. Propidium iodide
emission was detected on the FL4 channel (675 nm), FITC emission
was detected on the FL1 channel (525 nm), and the FL4 channel was
manually compensated over the FL1 channel. Data were analyzed
with the CXP Analysis software (Beckman Coulter). The analysis was
performed on ungated cells, quadrants were determined on the
untreated sample, and the same parameters were used for analyzing all
samples.
ICB Assay. About 60 000 HeLa cells were seeded on 13 mm glass
coverslips the day before treatment. Cells were treated for 12 h with
50 μM 4f, and the ICB assay was performed as previously described.64
Images were processed by ImageJ Software using the gray scale and
invert functions, and cell nuclei and ICBs were counted.
Caco-2 Cell Permeability Assay. Caco-2 cell monolayers
(passage 94−105) were grown on permeable filter supports and
used for the transport study on day 21 after seeding. Prior to the
experiment, a drug solution of 10 μM was prepared and warmed to 37
°C. The Caco-2 filters were washed with prewarmed HBSS prior to
the experiment, and the experiment was started by applying the donor
solution to the apical or basolateral side. The transport experiments
were carried out at pH 7.4 in both the apical and the basolateral
chambers. The experiments were performed at 37 °C and with a
stirring rate of 500 rpm. The receiver compartment was sampled at
15, 30, and 60 min, and at 60 min a final sample was also taken from
the donor chamber in order to calculate the mass balance of the
compound. The samples (100 μL) were transferred to a 96-well plate
containing 100 μL of methanol and warfarin as IS and were sealed
until LC-MS/MS analysis.65
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.9b11232.
BG4 Immunostaining. BG4 immunostaining was performed
using a protocol modified from ref 25. Briefly, 60 000 cells were
seeded on 13 mm glass coverslips the day before treatment. After
treatment with the compounds, the cells were fixed in 2%
paraformaldehyde and permeabilized in 0.1% Triton X-100 at room
temperature. Cells were blocked in 2% nonfat milk followed by
incubation with BG4-FLAG, anti-FLAG, and Alexa Fluor-conjugated
antibodies. Each incubation was for 1 h at 37 °C in a humidified
chamber. The antibodies used and their dilutions are shown in Table
S5. All washes and incubations were performed in 1× PBS buffer. Cell
nuclei were stained with 0.2 μg/mL diamidino-2-phenylindole
(DAPI) solution prior to mounting the coverslips on glass slides
with DAKO mounting medium (Agilent Technologies). Cells were
imaged with a Zeiss AxioImager Z1 equipped with an Apotome and a
Supporting figures and tables; compound synthesis
(PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Sjoerd Wanrooij − Department of Medical Biochemistry and
̊
̊
Biophysics, Umea University, Umea 90736, Sweden;
Email: sjoerd.wanrooij@umu.se
̊
Erik Chorell − Department of Chemistry, Umea University,
̊
Umea 90736, Sweden; orcid.org/0000-0003-2523-1940;
Email: erik.chorell@umu.se
J
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Nasim Sabouri − Department of Medical Biochemistry and
and MIMS Excellence by Choice Postdoctoral Programme (to
M.De.). D.O. was also supported by the Swedish Society of
Medicine (SLS-786661), federal funds through the county
̈
̊
̊
Biophysics, Umea University, Umea 90736, Sweden;
orcid.org/0000-0002-4541-7702; Email: nasim.sabouri@
umu.se
̈
council of Vasterbotten (VLL-643451, VLL-832001), the
Cancer Research Foundation in Northern Sweden (LP 18-
2202), the Swedish Foundation for International Cooperation
in Research and Higher Education (PT2015-6432), and The
Authors
Jan Jamroskovic − Department of Medical Biochemistry and
̊
̊
Biophysics, Umea University, Umea 90736, Sweden;
orcid.org/0000-0001-6871-7663
̈
Sjoberg Foundation. We thank the Kapoor lab at Rockefeller
University for providing the multidrug-resistant S. pombe
strain, Susan Forsburg and Sarah Sabatinos for providing the
parental strains for DNA fiber analysis, Leonardo Salviati
(Padova University, Italy) for providing HPF cells, Jenny
Mara Doimo − Department of Medical Biochemistry and
̊
̊
Biophysics, Umea University, Umea 90736, Sweden
̊
Karam Chand − Department of Chemistry, Umea University,
̊
Umea 90736, Sweden
̊
Persson (Umea University) for providing the breast cell lines,
Ikenna Obi − Department of Medical Biochemistry and
Chiara Frasson (Padova University, Italy) for advice on
̊
̊
Biophysics, Umea University, Umea 90736, Sweden
̊
cytofluorimetry, Igor Iashchishyn (Umea University) and
̊
Rajendra Kumar − Department of Chemistry, Umea University,
Lenka Kuglerova (SLU) for advice on statistical analysis,
̊
Umea 90736, Sweden; orcid.org/0000-0002-7268-9519
̊
Gorazd Stojkovic (Umea University) for help with sequencing
̈
̈
Kristoffer Brannstrom − Department of Medical Biochemistry
and Biophysics, Umea University, Umea 90736, Sweden
Mattias Hedenstrom − Department of Chemistry, Umea
University, Umea 90736, Sweden
Rabindra Nath Das − Department of Chemistry, Umea
University, Umea 90736, Sweden; orcid.org/0000-0001-
6347-2169
gels, and the SciLifeLab Drug Discovery and Development
Platform ADME of the Therapeutics Facility, Department of
Pharmacy, Uppsala University. We also thank Irene Martinez
Carrasco, Naga Venkata Gayathri Vegesna, the Knut and Alice
Wallenberg foundation program “NMR for Life” for NMR
spectroscopy support, and the Biochemical Imaging Center at
̊
̊
̊
̈
̊
̊
̊
̊
Umea University and the National Microscopy Infrastructure
Almaz Akhunzianov − Department of Medical Biochemistry
(VR-RFI 2016-00968) for providing support and assistance in
microscopy. The MD simulations were performed on resources
provided by the Swedish National Infrastructure for Comput-
̊
̊
and Biophysics, Umea University, Umea 90736, Sweden;
Institute of Fundamental Medicine and Biology, Kazan Federal
University, Kazan 420008, Russia
̊
ing (SNIC) at HPC2N Umea, Sweden.
Marco Deiana − Department of Medical Biochemistry and
̊
̊
Biophysics, Umea University, Umea 90736, Sweden;
orcid.org/0000-0002-7815-4494
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.9b11232
Author Contributions
§J.J., M.D., and K.C.: These authors contributed equally.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Knut and Alice Wallenberg
Foundation (to N.S., S.W., and D.O.), the Swedish Society for
̈
Medical Research (to N.S.), the Swedish Research Council (to
̈
̊
D.O., E.C., N.S., and S.W.), the Medical Faculty of Umea
University (to N.S.), the Wenner-Gren Foundation (to S.W.,
E.C., and M.Do.), the Kempe Foundations (to E.C., grant
SMK-1632), the Åke Wiberg Foundation (to E.C.), the
̈
Swedish Cancer Society (to N.S. and D.O.), the HORIZON
2020-MSC Individual fellowship (to M.Do., grant agreement
No 751474), JSPS Overseas Research Fellowships (to K.K),
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