Small molecule-mediated induction of endoplasmic reticulum stress in cancer cells

Article abstract of DOI:10.1039/d1md00095k

The endoplasmic reticulum (ER) is one of the crucial sub-cellular organelles controlling myriads of functions including protein biosynthesis, folding, misfolding and unfolding. As a result, dysregulation of these pathways in the ER is implicated in cancer development and progression. Subsequently, targeting the ER in cancer cells emerged as an interesting unorthodox strategy in next-generation anticancer therapy. However, development of small molecules to selectively target the ER for cancer therapy remained elusive and unexplored. To address this, herein, we have developed a novel small molecule library of sulfonylhydrazide-hydrazones through a short and concise chemical synthetic strategy. We identified a fluorescent small molecule that localized into the endoplasmic reticulum (ER) of HeLa cells, induced ER stress followed by triggering autophagy which was subsequently inhibited by chloroquine (autophagy inhibitor) to initiate apoptosis. This small molecule showed remarkable cancer cell killing efficacy in different cancer cells as mono and combination therapy with chloroquine, thus opening a new direction to illuminate ER-biology towards the development of novel anticancer therapeutics.

Full text of DOI:10.1039/d1md00095k

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RESEARCH ARTICLE
Small molecule-mediated induction of
endoplasmic reticulum stress in cancer cells†
Cite this: DOI: 10.1039/d1md00095k
Shalini Pandey,ab Virender Kumar Sharma,c Ankur Biswas,a
b
Mayurika Lahiri c and Sudipta Basu
*
The endoplasmic reticulum (ER) is one of the crucial sub-cellular organelles controlling myriads of
functions including protein biosynthesis, folding, misfolding and unfolding. As a result, dysregulation of
these pathways in the ER is implicated in cancer development and progression. Subsequently, targeting the
ER in cancer cells emerged as an interesting unorthodox strategy in next-generation anticancer therapy.
However, development of small molecules to selectively target the ER for cancer therapy remained elusive
and unexplored. To address this, herein, we have developed
a novel small molecule library of
sulfonylhydrazide-hydrazones through a short and concise chemical synthetic strategy. We identified a
fluorescent small molecule that localized into the endoplasmic reticulum (ER) of HeLa cells, induced ER
stress followed by triggering autophagy which was subsequently inhibited by chloroquine (autophagy
inhibitor) to initiate apoptosis. This small molecule showed remarkable cancer cell killing efficacy in
different cancer cells as mono and combination therapy with chloroquine, thus opening a new direction to
illuminate ER-biology towards the development of novel anticancer therapeutics.
Received 17th March 2021,
Accepted 15th July 2021
DOI: 10.1039/d1md00095k
rsc.li/medchem
the stress remains unmitigated.14 Very recently, this
deregulated ER homeostasis has been implicated in various
Introduction
The endoplasmic reticulum (ER) is a vital organelle that is
known to govern the synthesis, folding and processing of over
a third of all the cellular proteins.1–4 Several ER associated
chaperones assist in ensuring proper folding and modification
of these proteins before they traffic out of the ER.5–7 Despite
the robustness of this protein folding machinery, the success
rate for optimum protein folding is low owing to the wide
range of cellular disturbances that disrupt its efficiency.8 As a
result, several unfolded and misfolded proteins start
accumulating inside the ER lumen leading to a state known as
ER stress.9,10 In response to this burden of unfolded proteins,
a cytoprotective program known as the unfolded protein
response (UPR) is launched by the cell.11–13 Together, the
three mechanistically different branches of the UPR regulate
the expression of numerous genes that resolve this ER stress
and maintain homeostasis or induce apoptotic signals in case
pathological states and particularly with cancer.15,16 The
baseline activity level of the ER stress response system is
elevated in cancer cells as compared to normal cells. Thus, the
ER stress signalling is involved in tumorigenesis and
development leading it to be the new Achilles heel in the
development of cancer therapeutics.17–19
Over the last couple of decades, small molecules have
emerged as important tools to illuminate myriads of
biological phenomena in cancer cells leading to the
development of novel anti-cancer therapies.20,21 In this
context, few small molecules have been explored that target
various aspects of the unfolded protein response and ER
physiology.22–30 Molecules like salubrinal, GSK2606414, and
sunitinib are well reported to manipulate one or more arms
of ER stress pathways.31 However, due to the scarcity of the
targeting moieties, the development of ER targeted
therapeutics is still in its infancy and therefore the presence
of such ER stress inducers and UPR inhibitors is limited.
Hence, there is a serious need to develop novel small
molecules that can serve as ER stress modulators. Very
recently, a few molecules were demonstrated to accumulate
inside the ER in cancer cells to induce ER stress or as a tool
to understand ER physiology.32–35 For instance, Rocchi et al.
reported the development of HA15 that serves as an ER stress
inducer and overcomes BRAF-inhibitor resistance in
melanoma cells.36 On the other hand, Xiao et al. reported an
a Department of Chemistry, Indian Institute of Science Education and Research
(IISER)-Pune, Homi Bhabha Road, Pashan, Pune, 411008, India.
E-mail: ankur.biswas@students.iiserpune.ac.in
b Discipline of Chemistry, Indian Institute of Technology (IIT) Gandhinagar, Palaj,
Gandhinagar, Gujarat, 382355, India. E-mail: Sudipta.basu@iitgn.ac.in
c Department of Biology, Indian Institute of Science Education and Research
(IISER)-Pune, Homi Bhabha Road, Pashan, Pune, 411008, India
† Electronic supplementary information (ESI) available: 1H NMR, 13C NMR, and
HR-MS spectra, cell viability, fluorescence spectra, western blot quantification,
and cell viability assay. See DOI: 10.1039/d1md00095k
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ER-H2O2 probe to understand the generation of hydrogen Results and discussion
peroxide at the ER during apoptosis.37
A remarkable
Synthesis of the small molecule library
highlight in these small molecules is the presence of
sulfonamide moieties. Furthermore, it was reported that the
sulphonamide moiety facilitates the internalization of small
molecules in the intracellular ER owing to the presence of
sulphonamide receptors on the ER surface.38,39 Moreover,
sulphonamide as well as hydrazide-hydrazone containing
small molecules demonstrated diverse biological activities
making them privileged structures for natural and non-
natural products (Scheme 1a).40–47
Inspired by the scarcity of novel ER stress modulators, we
develop a focussed library of sulfonohydrazide-hydrazone
based small molecules (4′, Scheme 1a) to identify novel ER
stress inducers in cancer cells. To address this, herein, we
The synthesis of sulfonohydrazide-hydrazone molecules is
shown in Scheme 1b. Briefly, commercially available
substituted aromatic sulfonyl chlorides (1′) were reacted with
hydrazine monohydrate at
0 °C to obtain substituted
aromatic sulfonyl hydrazides (2′) in 60–70% yield (Fig. S1,
ESI†). Further, these aromatic sulfonyl hydrazides were
reacted with different aromatic aldehydes (3′) in the presence
of p-toluenesulfonic acid as a catalyst to afford 66 different
sulfonylhydrazide-hydrazones (4′) in 50–80% yield (Fig. S2,
1
ESI†). The final products were characterized by H NMR, 13C
NMR and HR-MS (Fig. S3–S200, ESI†).
To evaluate their cancer cell killing activity, we first
incubated these library members in HeLa cervical cancer
cells at a concentration of 30 μM for 24 hours. The cell
viability was quantified by the MTT assay. Interestingly, only
four compounds (compounds 1, 10, 17 and 55) were found to
induce more than 60% HeLa cell killing even at 30 μM
concentration (Fig. S201, ESI†). After finding out the
potential lead compounds, we evaluated their cancer cell
killing ability in a dose dependent manner. HeLa cells were
incubated with compounds 1, 10, 17 and 55 for 24 h in
different concentrations and the cell viability was evaluated
by the MTT assay. It was observed that at 24 h post
incubation, compounds 1, 10, 17 and 55 induced HeLa cell
killing with IC50 values of 9 μM, 16 μM, 18 μM and 19 μM
respectively (Fig. 1). From this cell viability assay, it was
evident that compound 1 showed the best cell killing efficacy
with the lowest IC50 value. Interestingly, we observed that the
electron donating groups (Cl, Br, dansyl) on the benzene ring
attached to the sulfonyl moiety increased the cytotoxicity
effect, whereas, the electron withdrawing group (NO2)
reduced the cytotoxicity. Moreover, we also observed that the
6-bromoindole moiety also increased the cytotoxicity of the
report
a
short and concise synthesis of
based small
a
library of
molecules
sulfonohydrazide-hydrazone
comprising 66 compounds (Scheme 1b). Upon screening
these compounds in the HeLa cell line, we identified four
molecules as potential ER stress inducers. One of the four
lead molecules was found to be fluorescent in nature and
localized inside the subcellular ER to induce ER stress
through CHOP and IRE-1α pathways (Scheme 1c).
Interestingly, the ER stress induced by the lead molecule
triggered autophagy followed by apoptosis in HeLa cells.
Further inhibition of autophagy by chloroquine (CQ), a
classic inhibitor that decreases the autophagic flux by
inhibiting the autophagosome–lysosome fusion,48 resulted in
improved cell killing in lung and breast cancer cells
including drug resistant-breast cancer cells.
compounds.47 However,
a
detailed structure–activity
relationship (SAR) analysis needs to be performed to
understand the effect of the functional groups in the
sulfonylhydrazide-hydrazone library. Moreover, compound 1
Scheme
1 (a) Structures of the biologically active sulphonamide
natural products and sulfonylhydrazide-hydrazone focused library. (b)
Synthetic scheme of the sulfonylhydrazide-hydrazone library. (c)
Schematic representation of the mechanism of action of compound 1
in cancer cells to induce ER stress.
Fig. 1 Cell viability assay of compounds 1, 10, 17 and 55 in HeLa cells
at 24 h post-incubation in a dose dependent manner. ***p < 0.0001,
the p value is calculated using two-way ANOVA.
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was found to be fluorescent with λmax (emission) at 538 nm
(Fig. S202, ESI†). This inherent fluorescence property of
compound 1 will aid in its cellular tracking into the
endoplasmic reticulum (ER), and hence we chose to carry out
further studies using compound 1.
stress levels lead to an increment in the expression levels of
protooncogene CHOP which is a marker for ER stress induced
apoptosis.51,52 To demonstrate the onset of ER stress we
evaluated the levels of CHOP in HeLa cells by an
immunofluorescence assay. We treated HeLa cells with
compound 1 for 24 h. We then incubated the cells with a
primary antibody specific to CHOP for 4 h followed by
incubation with an Alexa-Fluor 594 (red fluorescence) tagged
secondary antibody. The nuclei of the cells were stained with
DAPI (blue fluorescent). We then visualized the cells with
confocal microscopy. The fluorescence microscopy images
showed that the control cells expressed negligible CHOP which
was evident from no red fluorescence signals (Fig. 3a). However,
the cells treated with compound 1 showed remarkably increased
red fluorescence signals indicating that compound 1 increased
(1.7 fold) the expression of CHOP protein in HeLa cells.
Furthermore, the overlap of red and blue fluorescence to give
purple fluorescence indicated the accumulation of CHOP in the
ER homing
We hypothesized that the presence of the sulphonamide
moiety in compound 1 will enable its localization specifically
inside the subcellular ER.49,50 To validate this hypothesis, we
incubated the HeLa cells with compound 1 (concentration =
5 μM) (green fluorescent) for two different time points of 3 h
and 6 h followed by staining the ER with ER Tracker Red. We
then visualised the cells using confocal laser scanning
microscopy (CLSM). It was observed that compound 1 started
accumulating in the ER within 3 h and the accumulation
increased at 6 h (Fig. 2 and S203, ESI†) which was observed
from the yellow fluorescence resulting from merging of red
and green fluorescence. The ER localization of compound 1
was further quantified from the CLSM images through
Pearson's coefficients of 0.84 and 0.86 and Mander's
coefficients of 0.98 and 0.97 at 3 h and 6 h, respectively
(Table S1, ESI†). These confocal images and quantification
showed that indeed compound 1 homed into the subcellular
ER in HeLa cancer cells over 6 h.
nucleus which is
a
marker of ER stress induction.52
Interestingly, the immunofluorescence staining of CHOP
showed a characteristic punctate structure after treatment with
compound 1 which is the hallmark of ER stress (Fig. S204,
ESI†). We also evaluated the expression of CHOP by gel
electrophoresis. We treated HeLa cells with compound 1 for 24
ER stress
Once internalized inside the ER, compound 1 is expected to
induce ER stress in the HeLa cervical cancer cells. Elevated ER
Fig.
3 (a) Confocal images of HeLa cells after treatment with
compound 1 for 24 h followed by treatment with a CHOP primary
antibody and an Alexa Fluor 594-tagged secondary antibody (red).
Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (b and c) The
western blot images of CHOP, IRE1α, PERK, BiP and caspase-12
proteins as the markers of ER stress in HeLa cells after treatment with
compound 1 for 24 h.
Fig. 2 Confocal microscopy images of HeLa cells at 3 h and 6 h post-
incubation with compound 1 (green fluorescence). The cells were
counter stained with ER-Tracker Red dye. Scale bar = 10 μm.
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h, followed by lysis of the cells. We separated the sub-cellular
proteins to perform western blot analysis. In accordance with
the confocal microscopy study, the cells after treatment with
compound 1 showed a significant increase (1.6 fold) in the
expression of CHOP compared to the non-treated control cells
(Fig. 3b and S205a, ESI†). To further estimate the ER stress, we
evaluated the expression levels of another ER stress marker
IRE1-α.53 We found that the cells treated with compound 1
exhibited an enhanced expression level of IRE1-α as compared
to the control cells (1.8 fold) (Fig. 3b and S205b, ESI†). We also
evaluated the expression of PERK and BiP as ER stress markers
and activation of caspase-12 as an ER stress-mediated apoptosis
marker by gel electrophoresis.10,36,54 We found that compound
1 induced enhanced expression of PERK (1.6 fold), BiP (1.6 fold)
and caspase-12 (1.4 fold) compared to the non-treated control
cells (Fig. 3c and S205c–e, ESI†). These immunofluorescence
assays and gel electrophoresis revealed that compound 1 indeed
induced ER stress in HeLa cells.
The ER is the major site of lipid metabolism as several
enzymes involved in it are compartmentalized in the ER.55 As
a result, excessive ER stress is known to increase lipid
accumulation inside the cell.56 We anticipated that HeLa cells
treated with compound 1 will also show an enhanced lipid
content in them. To validate this hypothesis, we performed
an Oil Red O staining experiment.57,58 We treated HeLa cells
with compound 1 for 24 h followed by staining the cells with
Oil-Red-O dye. Oil Red O is a lipid soluble lysochrome dye
which stains the lipid droplets in cells or frozen tissues.59 We
also stained the nucleus of the cells with blue fluorescent
DAPI. We visualised the Oil-Red-O stained cells by confocal
microscopy. The confocal microscopy images showed that the
control cells contained traces of lipid molecules as expected
it was evident that compound 10 also enhanced the red
fluorescence signal significantly compared to the non-treated
control cells (Fig. S206, ESI†). This confocal microscopy
exhibited that indeed the lipidic content was increased inside
the cells after treatment with compound 1 and compound 10
because of elevated ER stress.
Autophagy induction
ER stress induction acts as a potent trigger for autophagy which
is primarily a cytoprotective secondary response to the excessive
protein build up in the ER.60–62 To investigate if autophagy was
launched as a survival mechanism by the HeLa cells in response
to treatment with compound 1, we evaluated the cellular
expression level of LC3B, a key autophagy marker, by an
immunofluorescence assay.63 We treated HeLa cells with
compound 1 for 24 h followed by fixing the cells with
paraformaldehyde. These fixed cells were then permeabilized
with a buffer containing 0.1% Tween-20 followed by incubation
with a primary antibody specific to LC3B for 4 h. The cells were
then incubated with a secondary antibody tagged with Alexa-
Fluor-488 (green fluorescent). The nuclei of the cells were
stained with blue fluorescent DAPI. The cells were visualised by
confocal microscopy. The green fluorescence signals were hardly
seen in the control cells (Fig. 5a). However, the cells treated with
compound 1 exhibited highly enhanced green fluorescence (5
fold) compared to the control cells. More interestingly, we
observed the formation of autophagosomes (green puncta in
the images) which is regarded as the hallmark of autophagy.64
To confirm that the fluorescence signal of compound 1 does
not interfere with the immunofluorescence assay, we treated
the HeLa cells with non-fluorescent compound 10 for 24 h. The
cells were then fixed, permeabilized and stained with a primary
antibody specific to LC3B and a secondary antibody tagged with
Alexa-Fluor-488. The control cells were treated in a similar
manner without compound 10. The confocal imaging clearly
showed that compound 10 also increased the expression of
LC3B remarkably in the HeLa cells compared to the control
cells (Fig. S207, ESI†).
We also evaluated the expression level of Beclin, another
autophagy marker, by immunofluorescence.65 HeLa cells were
treated with compound 1 for 24 h followed by incubation with a
Beclin specific primary antibody. The cells were then incubated
with a secondary antibody tagged with Alexa-Fluor-594 (red
fluorescent). The nuclei of the cells were stained with DAPI. We
visualised the cells by confocal microscopy and found that an
increased red fluorescence signal was observed in the compound
1 treated cells compared to that in the non-treated control cells (8
fold) (Fig. 5b). The enhanced expression levels of LC3B and Beclin
in the treated cells as evaluated by the immunofluorescence assay
evidently demonstrated that autophagy was induced upon
elevation of ER stress in the HeLa cells.
(Fig. 4). However, compound
1 treatment remarkably
increased the level of the lipids inside the cells, which was
clear from the increased red fluorescence signals from the
Oil-Red-O dye in Fig. 4.
To confirm that the fluorescence signal from compound 1
does not interfere with the Oil-Red-O, we further treated the
HeLa cells with non-fluorescent compound 10 for 24 h and
performed the Oil-Red-O staining. From the confocal imaging
We further validated the expression levels of LC3B and
Beclin by western blot analysis. We treated HeLa cells with
compound 1 for 24 h followed by cell lysis. The cellular proteins
were then subjected to gel electrophoresis. The western blot
Fig. 4 The confocal microscopy images of HeLa cells after incubation
with compound 1 for 24 h followed by staining with Oil Red O dye
(red) and DAPI. Scale bar = 10 μm.
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treated HeLa cells with compound 1 for 24 h followed by
staining the apoptotic and necrotic cells with Annexin V-FITC
and propidium iodide (PI), respectively. The cells were then
analysed by flow cytometry. We found that treatment with
compound 1 resulted in 64.6% cells in the early apoptotic
stage and 17.8% cells in the late apoptotic stage compared to
1.26% and 0.08% in the control cells, respectively (Fig. 6a).
We anticipate that compound 1 was not as effective to induce
apoptosis due to induction of autophagy as a survival
mechanism. We thus hypothesized that the concomitant
inhibition of autophagy will improve the apoptotic outcome
in HeLa cells. To validate this hypothesis, we cotreated HeLa
cells with compound 1 and 50 μM chloroquine, an autophagy
inhibitor, for 24 h and performed a flow cytometry assay.66
We observed that autophagy inhibition indeed improved the
apoptotic outcome with 28.6% and 62% cells in the early and
late apoptotic stages, respectively (Fig. 6a). The flow
cytometry data thus confirmed that compound 1 induced
apoptosis in HeLa cells which further improved upon
inhibition of autophagy with chloroquine.
To further investigate the effect of autophagy inhibition on
the cell killing efficacy of compound 1, we performed a cell
viability assay. We cotreated HeLa cells with compound 1 in a
dose dependent manner along with 50 μM chloroquine for 24
h. The cell viability was measured by the MTT assay. We found
that inhibiting autophagy improved the IC50 value of compound
1 from 9 μM to 4 μM in HeLa cells (Fig. 6b). We also evaluated
the cell killing efficacy of compound 1 on three different cancer
cell lines by the MTT assay. We treated MCF-7 (human breast
cancer), A549 (human lung carcinoma) and MDA-MB-231 (drug
resistant triple negative breast cancer) with compound 1 in a
dose dependent manner for 24 h. We found that compound 1
exhibited IC50 values of 15 μM, 10 μM and 17 μM in MCF-7,
A549 and MDA-MB-231, respectively (Fig. 6b). Furthermore,
inhibition of autophagy by treatment with chloroquine resulted
in enhanced cell killing and improved IC50 values of 10 μM, 5
μM and 13 μM in MCF-7, A549 and MDA-MB-231, respectively
(Fig. 6b). Compound 1 thus efficiently induced cell killing in
Fig. 5 (a and b) Confocal images of HeLa cells after treatment with
compound 1 for 24 h followed by LC3B and Beclin specific primary
antibodies followed by an Alexa Fluor 488 tagged secondary antibody
(green) and an Alexa Fluor 594 tagged secondary antibody (red),
respectively. Scale bar = 10 μm. (c) Western blot images of the LC3-I,
LC3-II and Beclin protein expressions in HeLa cells after treatment with
compound 1 for 24 h.
analysis revealed that treatment with compound 1 resulted in
increased levels of LC3-I (2.6 fold), LC3-II (10 fold) and Beclin (2
fold) indicating the onset of autophagy (Fig. 5c and S208a–c,
ESI†). Moreover, the high LC3-I/LC3-II ratio (3.8 fold) clearly
confirmed the induction of autophagy by compound 1. The
immunofluorescence assay and western blot analysis thus
confirmed that autophagy was triggered upon ER stress
induction on treatment with compound
1 which is in
agreement with our previous reports as well as other
reports.36,49,50
Induction of apoptosis
Fig. 6 (a) Flow cytometry analysis of compound 1 and CQ in HeLa
cells to evaluate apoptosis. (b) Dose dependent cell viability assay of
compound 1 and CQ in HeLa, MCF7, A549 and MDA-MB-231 cells at
24 h post incubation. ***p < 0.001 and **p < 0.01, the p value is
calculated using two-way ANOVA.
Induction of ER stress is expected to induce apoptosis in
HeLa cells. To estimate the apoptosis induced by compound
1 in HeLa cells, we performed a flow cytometry assay. We
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different cancer cell lines which further improved upon
autophagy inhibition.
within 3 h in HeLa cervical cancer cells. The identified small
molecule efficiently induced ER stress mediated apoptosis
and autophagy in HeLa cells. It also induced ER stress
associated apoptosis in lung, breast and drug resistant breast
cancer cell lines. Finally, the treatment in combination with
an autophagy inhibitor, chloroquine, remarkably improved
the cell killing efficacy of the small molecule in the cancer
cell lines. We envision that these small molecules can be
employed in understanding the ER biology and functions in
cancer and pave the way for the development of novel ER
stress inducers thereby improving the cancer therapeutics.
We also evaluated the inhibition of autophagy along with
induction of ER stress with the other lead molecules
(compounds 10, 17 and 55) on HeLa cells. We treated HeLa
cells with CQ in combination with compounds 10, 17 and 55
in a dose dependent manner for 24 h and the cell viability
was measured by the MTT assay. We observed that the
combination treatment of CQ with compounds 10, 17 and 55
induced much improved HeLa cell killing with IC50 = 4.4 μM,
5.5 μM and 16.1 μM respectively (Fig. S209, ESI†).
To be successful in future cancer therapy, compound 1
should selectively induce cancer cell death without collateral
damage to the non-cancerous healthy cells. To evaluate the
effect of compound 1 on healthy cells, we incubated HEK293
human embryonic kidney cells with compound 1 in a dose
dependent manner for 24 h and quantified the cell viability
by the MTT assay. To our surprise, we found that compound
Experimental section
Materials
All the chemicals were purchased from commercial suppliers
unless otherwise noted. MCF-7 cells were obtained from the
European Collection of Authenticated Cell Cultures (ECACC)
(Salisbury, UK). HeLa, HEK-293, MDA-MB-231 and A549 cells
were obtained from the National Centre for Cell Science
(NCCS) (Pune, India).
1
showed only 52.8% cell viability even in 30 μM
concentration with IC50 = 31.7 μM, which was quite high
compared to the IC50 value in different cancer cells (Fig.
S210, ESI†). We further calculated the selectivity index (SI) of
compound 1 in HeLa, MCF7, A549 and MDA-MB-231 cells to
understand its effect on cancer cells compared to the healthy
cells.67 It was found that the SI of compound 1 was 3.52,
2.11, 3.17 and 1.86 in HeLa, MCF7, A549 and MDA-MB-231
cells, respectively, which indicated that compound 1 is more
selective for HeLa and A549 cells, compared to the other
tested cancer cells.
Finally, as a proof of concept, to confirm that ER stress
induction resulted in apoptosis, we treated HeLa cells with
compound 1 and 4-phenyl butyric acid (4-PBA), a chemical
chaperone that alleviates both toxicity and proteomic
alterations induced by an ER stress inducer, by aiding in
protein folding in the ER.68–70 Although 4-PBA is also known
as a potent HDAC inhibitor, it was shown to be non-toxic
even at a higher concentration in HeLa cells.71 We co-treated
HeLa cells with compound 1 along with 15 μM 4-PBA for 24
h and then performed the cell viability assay. We found that
cotreatment with 4-PBA resulted in reduced cell killing (IC50
= 28 μM) in HeLa cells as compared to the treatment with
compound 1 only (Fig. S211, ESI†). From these cell viability
assays, it was evident that compound 1 triggered apoptosis
after induction of autophagy. Furthermore, co-treatment of
compound 1 and an autophagy inhibitor would induce
improved cancer cell killing without inducing significant
damage to the non-cancerous cells.
Synthesis of sulfonohydrazides
The sulfonohydrazides were prepared according to
a
previously reported procedure.72 Briefly, to a solution of
sulfonyl chloride (1 equiv.) in THF at −30 °C, hydrazine
monohydrate (5 equiv.) was added dropwise. The solution
was then allowed to stir for 30 minutes. The progress of the
reaction was monitored through TLC and after the reaction
was over, ethyl acetate was added into the cold reaction
mixture. The reaction mixture was washed multiple times
with ice cold 10% brine solution. The organic layer was
collected and dried over anhydrous Na2SO4 and the solvent
was then evaporated under reduced pressure. The solid
obtained was washed with pentane three times and the
product was stored at 4 °C for further use.
Synthesis of sulfonohydrazide-hydrazones
To a solution of sulfonohydrazide (1 eq.) in ethanol, aldehyde
was added (1 eq.) along with
a catalytic amount of
para-toluene sulfonic acid and the reaction was allowed to
stir at room temperature overnight. The extent of reaction
was monitored by TLC. On completion, the solvent ethanol
was evaporated, and the residue obtained was dissolved in
organic solvents (DCM/ethylacetate). The organic layer was
washed with water, collected and evaporated. The residue
obtained was then purified using column chromatography.
The procedure was utilized to generate a library of 66 small
molecules.
Conclusions
In conclusion, we synthesised a library comprising 66 small
molecules based on sulfonohydrazide-hydrazones through
short and concise synthetic steps. Upon screening, we
identified four molecules as potential ER stress inducers.
Further studies with the dansyl based sulfonohydrazide-
hydrazone (compound 1) revealed its accumulation in the ER
Author contributions
SP, VKS and AB performed all the experiments and collected
the data. SP, ML and SB wrote the manuscript.
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Conflicts of interest
24 Q. Zhang, R. Du, G. R. Reis Monteiro Dos Santos, R.
Yefidoff-Freedman, A. Bohm, J. Halperin, M. Chorev and
B. H. Aktas, Eur. J. Med. Chem., 2020, 187, 111973.
25 H. Bai, T. Chen, J. Ming, H. Sun, P. Cao, D. N. Fusco, R. T.
Chung, M. Chorev, Q. Jin and B. H. Aktas, ChemBioChem,
2013, 14, 1255–1262.
26 S. R. Stockwell, G. Platt, S. E. Barrie, G. Zoumpoulidou, R. H.
te Poele, G. Wynne Aherne, S. C. Wilson, P. Sheldrake, E.
McDonald, M. Venet, C. Soudy, F. Elustondo, L. Rigoreau, J.
Blagg, P. Workman, M. D. Garrett and S. Mittnacht, PLoS
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There are no conflicts to declare.
Acknowledgements
SB acknowledges IIT Gandhinagar internal funding and DST
[SB/NM/NB-1083/2017 (G)] for financial support. SP
acknowledges IISER Pune for a doctoral fellowship. We
sincerely thank Dr. Nirmalya Ballav from IISER Pune for
constructive and helpful scientific discussion and support.
27 M. Gao, L. Duan, J. Luo, L. Zhang, X. Lu, Y. Zhang, Z. Zhang,
Z. Tu, Y. Xu, X. Ren and K. Ding, J. Med. Chem., 2013, 56,
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28 J. Ganz, T. Shacham, M. Kramer, M. Shenkman, H. Eiger, N.
Weinberg, O. Iancovici, S. Roy, L. Simhaev, B. Da'adoosh, H.
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