An Autophagy-Disrupting Small Molecule Promotes Cancer
Cell Death via Caspase Activation
Sang-Hyun Park,[a] Insu Shin,[a] Gun-Hee Kim,[b] Sung-Kyun Ko,[b] and Injae Shin*[a]
A novel autophagy inhibitor, autophazole (Atz), which promoted cancer cell death via caspase activation, is described.
This compound was identified from cell-based high-content
screening of an imidazole library. The results showed that Atz
was internalized into lysosomes of cells where it induced
lysosomal membrane permeabilization (LMP). This process
generated nonfunctional autolysosomes, thereby inhibiting
autophagy. In addition, Atz was found to promote LMPmediated apoptosis. Specifically, LMP induced by Atz caused
release of cathepsins from lysosomes into the cytosol. Cathepsins in the cytosol cleaved Bid to generate tBid, which
subsequently activated Bax to induce mitochondrial outer
membrane permeabilization (MOMP). This event led to cancer
cell death via caspase activation. Overall, the findings suggest
that Atz will serve as a new chemical probe in efforts aimed at
gaining a better understanding of the autophagic process.
Introduction
Autophagy is a self-eating process crucial for cell survival during
nutrient starvation.[1–3] In autophagy, cytoplasmic proteins and
subcellular organelles are delivered to lysosomes for selective
or nonselective degradation. Specifically, cellular components
and organelles are segregated in autophagosomes during
autophagy. A key protein for generation of autophagosomes is
microtubule-associated protein 1 light chain 3 (LC3). LC3-I, a
cytosolic form of LC3, is converted by the cooperative action of
Atg4 and Atg7 to phosphatidylethanolamine-conjugated LC3-II,
which facilitates formation of autophagosomes by anchoring to
the autophagosomal membrane.[4,5] Subsequently, autophagosomes fuse with lysosomes to form autolysosomes in which
engulfed materials are digested to form renewable products by
the action of multiple lysosomal hydrolases.[6] Thus, the functional lysosomes are critical for autophagy, the main role of
which is to serve as an energy source for cell viability under
nutrient-deficient conditions.
Numerous previous studies have shown that the autophagy
level in cancer cells is higher than that in normal cells.[7] This
phenomenon is attributed to the great metabolic and biosynthetic demands caused by deregulated proliferation of cancer
cells. This observation suggests that autophagy-disrupting small
molecules might be utilized as cancer chemotherapeutic
agents.[8,9] Over the past decade, considerable effort has been
devoted to discovering autophagy-inhibiting small molecules
for use as chemical probes in efforts aimed at obtaining a better
understanding of the complex autophagy process and as new
therapeutic agents to treat diseases, particularly tumors.[10,11]
These studies have shown that 3-methyladenine and wortmannin are upstream autophagy inhibitors that suppress the activity
of phosphoinositide 3-kinase (PI3 K).[12,13] In addition, bafilomycin A1 (BfA1) was demonstrated to be an autophagy inhibitor
that prevents vacuolar H+-ATPase (V-ATPase) and consequently
increases the lysosomal pH.[14] Other investigations have
demonstrated that hydroxychloroquine blocks autophagy by
increasing the lysosomal pH and thus generating nonfunctional
autolysosomes.[15] Although the mechanistic basis for its anticancer activity remains unknown, hydroxychloroquine is currently undergoing clinical trials for treatment of various types of
tumors.[16,17]
A cell-based screening approach[18–21] has assisted the
discovery of novel substances that target specific cellular
pathways associated with autophagy.[22] In the investigation
described below, we employed this method to identify
autophazole (Atz) as a new small molecule autophagy inhibitor
with anticancer activity. In this effort, we showed that Atz was
mainly internalized into lysosomes and disrupted their function,
thereby leading to generation of nonfunctional autolysosomes
and promotion of autophagy inhibition. In addition, Atz
induced LMP-mediated caspase-dependent apoptosis of cancer
cells.
Results and Discussion
Identification of an autophagy inhibitor
Cells, stably expressing a tandem mRFP EGFP LC3 fusion
protein (mRFP, monomeric red fluorescence protein; EGFP,
enhanced green fluorescence protein), have been frequently
utilized to assess the effect of substances on the autophagic
process.[23–25] Both RFP and GFP fluorescence signals are
observed in the autophagosome of transfected cells. However,
RFP but not GFP signals are detected in the acidic autolysosome
because fluorescence from the latter is very weak at low pH
[a] S.-H. Park, I. Shin, Prof. Dr. I. Shin
Department of Chemistry, Yonsei University
Seoul 03722 (South Korea)
E-mail: [email protected]
[b] Dr. G.-H. Kim, Dr. S.-K. Ko
Anticancer Agent Research Center
Korea Research Institute of Bioscience and Biotechnology (KRIBB)
Cheongju 28116 (South Korea)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbic.202100296
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values. As a result, cells expressing mRFP EGFP LC3 display
distinguishable fluorescence patterns depending on the state of
induction or inhibition of autophagy. Specifically, during
autophagy induction, merged images of cells exhibit red puncta
exclusively because GFP but not RFP fluorescence is very weak
at the low pH in autolysosomes. However, when autophagy is
blocked, merged images of cells display yellow puncta because
of co-localization of red and green fluorescence signals in
autophagosomes. On the contrary, under normal conditions
where a basal level of autophagy exists, negligible fluorescence
is observed in cells expressing the fusion protein.
These phenomena were utilized to identify novel small
molecules that disrupted the autophagic process. For screening,
HeLa cells stably transfected with a plasmid containing a
mRFP EGFP LC3 gene were exposed individually to ca. 1,000
imidazole derivatives (10 μM) for 12 h. BfA1 and torin-1 were
employed as controls for substances that inhibit and induce
autophagy, respectively.[14,26] The treated cells were then
analyzed using a high-content screening platform (CellomicsTM). Most of the imidazole derivatives were found to have
no effect on autophagy, as inferred from the observation of
very weak fluorescence in treated cells. Gratifyingly, one
substance in the library, autophazole (Atz, Figure 1a), greatly
increased the number of yellow puncta in the merged images
of cells, suggesting that it blocks autophagy (Figure S1). This
event was also observed in cells treated with BfA1.
To confirm this finding, a large quantity of Atz was
synthesized using the route shown in Scheme 1, and its
autophagy inhibitory activities at several concentrations were
assessed by incubation of HeLa cells stably expressing
mRFP EGFP LC3. The results of cell image analysis showed that
cells treated with Atz displayed strong yellow puncta at a
concentration as low as 1 μM (Figures S2 and 1b).
It is known that the level of LC3-II increases and that of p62
decreases during autophagy induction, and that levels of both
LC3-II and p62 increase when autophagy is disrupted.[27] Thus,
to verify that Atz disrupts autophagy, levels of LC3-II and p62
were determined by Western blotting of HeLa cells treated with
Atz, along with BfA1 and torin-1. The results of immunoblotting
analysis showed that the levels of both LC3-II and p62 increased
in cells exposed to Atz as well as those treated with BfA1
(Figure 1c).
The results of previous studies have also shown that
autophagy inhibition leads to the accumulation of p62
complexed with LC3-II on autophagosomes.[23,28,29] Thus, colocalization of p62 with LC3-II in cells provides additional
evidence for autophagy inhibition. On this basis, HeLa cells
stably expressing the EGFP LC3 protein were exposed to Atz
and then subjected to immunostaining with p62 antibody.
Analysis of cell images revealed that Atz treatment led to a
large increase in co-localization of p62 with LC3 (Figure 1d).
Taken together, the findings described above clearly indicate
that Atz inhibits autophagy.
Autophazole blocks autophagy by impairing lysosomal
function
Next, we investigated the mechanism underlying autophagy
inhibition promoted by Atz. One possible mode of action of Atz
in its inhibition of autophagy is impairment of lysosomal
function. To assess this possibility, we determined first if Atz
was internalized into lysosomes. For this purpose, lysosomes
were isolated and then treated with Atz along with the control
Atz OH, which contained OH instead of the terminal NH2 group
in Atz and had no effect on autophagy (Figure S3).[29] HPLC
analysis of contents inside lysosomes indicated that Atz entered
lysosomes but Atz OH did not (Figure S4). The findings suggest
that the terminal amine moiety in Atz is necessary for its
internalization and accumulation into lysosomes.
Figure 1. Autophazole disrupts autophagy. (a) Structure of Atz. (b) HeLa cells
stably expressing the mRFP EGFP LC3 fusion protein were treated with Atz
for 12 h. Cell images were obtained using confocal fluorescence microscopy
(scale bar: 10 μm). (c) HeLa cells were treated with Atz for 12 h, along with
BfA1 (5 nM) and torin-1 (0.5 μM). Expression levels of LC3 and p62 were
determined by Western blot analysis. β-Actin was used as a loading control.
(d) HeLa cells stably expressing the EGFP LC3 fusion protein were treated
with Atz for 12 h. Cell images were obtained using confocal fluorescence
microscopy. Scheme 1. Synthesis of Atz.
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We next determined the ability of Atz to induce lysosomal
membrane permeabilization (LMP) as part of the impairment of
lysosomal function. When the lysosomal membrane of cells is
disrupted to induce LMP, proton leakage takes place from acidic
lysosomes into the cytosol.[30] An increase in the lysosomal pH
can be readily detected using acridine orange, which stains
acidic lysosomes with red fluorescence but neutral lysosomes
with greatly diminished red fluorescence.[24,25,31] On this basis,
HeLa cells were exposed to several concentrations of Atz for
various times, followed by treatment with acridine orange.
Analysis of images of treated cells showed that Atz induced
LMP, as judged from the observation that red fluorescence
derived from acridine orange disappeared almost completely in
cells treated with Atz in a time- and concentration-dependent
manner (Figures 2a and S5).
It has been also shown that during induction of LMP,
lysosomal enzymes such as cathepsins are released into the
cytosol.[32] In order to determine if cathepsin B is released from
lysosomes into the cytosol when Atz induces LMP, the cytosolic
fraction of cells treated with Atz were subjected to Western
blotting with cathepsin B antibody. The results showed that as
a concentration of Atz increased, the amount of cathepsin B in
the cytosol increased (Figure 2b). Atz-promoted release of
lysosomal cathepsin B into the cytosol was also evaluated. For
this purpose, the cathepsin B activity in the cytosolic fraction of
HeLa cells treated with Atz was measured using the fluorogenic
substrate MR (RR)2 in the absence and presence of the
cathepsin B inhibitor leupeptin.[33] Atz promoted an increase in
cathepsin B activity in the cytosol, and the increase was almost
completely abrogated when leupeptin was present (Figure 2c).
These findings support the conclusion that Atz induces LMP in
its impairment of lysosomal function.
The effect of Atz on fusion of lysosomes with autophagosomes was explored next. HeLa cells were incubated with Atz
along with BfA1 as a control for 6 h, and then subjected to
immunostaining with LAMP2 and p62 antibodies that are
known markers of lysosomes and autophagosomes,
respectively.[15,34] The results of cell image analysis showed that
cells treated with Atz displayed only yellow puncta in the
merged images of cells (Figure 2d). This finding indicates that
fusion between autophagosomes and lysosomes takes place in
Atz treated cells.[15] In contrast, discrete LAMP2A and p62
puncta were seen in merged images of cells treated with BfA1,
indicating that BfA1 blocks the fusion of autophagosomes with
lysosomes.[34] Recent findings showed that fusion of autophagosomes with lysosomes was prevented by a high concentration
of intracellular Ca2+ regardless of whether lysosomes are
functional or nonfunctional.[34] Thus, the level of Ca2+ in the
cytosol of cells treated independently with Atz and BfA1 was
measured using the Ca2+-sensitive fluorescent probe Fluoro-
4NW.[24] As anticipated, the cytosolic Ca2+ concentration was
significantly higher in cells treated with BfA1 (Figure S6).
However, Atz did not affect the level of cytosolic Ca2+, and thus
it had no effect on fusion of autophagosomes with lysosomes.
Collectively, the findings suggest that Atz blocks the autophagic
process by disrupting lysosomal function and generating nonfunctional autolysosomes.
Autophazole induces LMP-mediated apoptotic cancer cell
death
It is known that LMP triggers caspase-dependent apoptosis of
cancer cells.[31,35] In this process, cathepsin B, liberated from
lysosomes into the cytosol, cleaves the pro-apoptotic protein
Bid to generate truncated Bid (tBid).[36] Resultant tBid in turn
activates the pro-apoptotic protein Bax for its translocation to
mitochondria for induction of mitochondrial outer membrane
permeabilization (MOMP). MOMP induction promotes cytochrome c release from mitochondria into the cytosol where it
binds to Apaf-1 and procaspase-9 to produce the
apoptosome.[37,38] The apoptosome complex activates caspase-9,
which subsequently promotes caspase-3 activation, leading to
apoptosis.[38,39]
With this sequence of events in mind, we designed an
investigation to assess the effect of Atz on LMP-dependent
caspase activation. The generation of tBid in cancer cells treated
with Atz was determined initially by Western blot analysis with
Bid and tBid antibodies. Atz promoted Bid cleavage to generate
tBid (Figure 3a). We then assessed if MOMP was induced in
cancer cells treated with Atz. The results of immunostaining of
cancer cells with Bax (active monomer) antibody showed that
Atz caused an increase in the level of active Bax which colocalized with MitoTracker red fluorescence (Figure 3b). Because
Atz induced MOMP, we next determined if it promoted release
Figure 2. Autophazole inhibits autophagy by impairing lysosomal function.
(a) HeLa cells were treated with 5 μM Atz for indicated times and then
100 nM acridine orange for 30 min. Cell images were obtained using
confocal fluorescence microscopy (scale bar: 10 μm). (b) Cathepsin B in a
cytosolic fraction of HeLa cells treated with Atz for 4 h was analyzed by
Western blotting. (c) Cathepsin B activity of a cytosolic fraction of HeLa cells
treated with Atz for 4 h was measured using MR (RR)2 in the absence and
presence of leupeptin (means.d., n=3). (d) HeLa cells were incubated with
10 μM Atz or 5 nM BfA1 for 10 h followed by incubation with LAMP2 and
p62 antibodies. Cell images were obtained using confocal fluorescence
microscopy (scale bar: 10 μm).
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of cytochrome c from mitochondria into the cytosol. As shown
in Figure 3c, Atz treatment of cancer cells indeed caused
liberation of mitochondrial cytochrome c into the cytosol.
Furthermore, additional studies showed that Atz enhanced
generation of an active form of caspase-3 and a cleaved
product of its cellular substrate, poly(ADP-ribose) polymerase
(PARP). However, when cancer cells were co-treated with Atz
and leupeptin, cytochrome c release from mitochondria,
activation of caspase-3 and cleavage of PARP were greatly
reduced. To obtain more evidence for the conclusion that Atz
enhanced caspase activation, caspase activities of lysates of
cells treated with Atz were determined by using the colorimetric peptide substrate for caspases, Ac DEVD pNA (pNA=pnitroaniline). Caspase activities in cells treated with Atz
increased and this increase was suppressed when either
Ac DEVD CHO, a known inhibitor of caspases, or leupeptin was
present in cells treated with Atz (Figure 3d).
Since Atz promoted LMP-mediated caspase activation, we
determined its cell death activity. In this study, several cancer
(HeLa, human cervical cancer cells; A549, human lung adenocarcinoma epithelial cells; HCT116, human colorectal carcinoma
cells; MCF-7, human breast adenocarcinoma cells) and normal
cell lines (HaCaT, human keratinocyte cells; MRC-5, human fetal
lung fibroblast cells) were incubated with increasing concentrations of Atz for 24 h and cell viabilities were then measured
using an MTT assay. The number of viable cells was found to
decrease in a dose-dependent manner with half maximum
inhibitory concentrations (IC50) of ca. 6 μM, regardless of the cell
types (Figure S7).
Finally, attempts were made to identify protein targets of
Atz using several techniques including affinity chromatography,
drug affinity responsive target stability (DARTS) and cellular
thermal shift assay (CETSA).[40–43] However, none of these
methods provided information on possible targets for Atz.
Because Atz is capable of disrupting lysosomes to induce LMP,
we also examined its possible inhibitory activities toward two
enzymes, acid spingomyelinase (ASM) and Hsp70, that are
involved in the lysosomal stabilization in cancer cells.[44,45] ASM
is the lysosomal enzyme responsible for degradation of
sphingomyelin to form phosphorylcholine and ceramide crucial
for the lysosomal stabilization. The results of studies using an
ASM assay kit showed that while the known inhibitor of ASM,
chlorpromazine,[46] blocked the activity of ASM, Atz did not
(Figure 4a). Furthermore, the results of an investigation using a
Pi-Colorlock assay for determining ATPase activity of Hsp70
revealed that although the known Hsp70 inhibitor, apoptozole
(Az), blocked the ATPase activity of Hsp70,[47] Atz did not
(Figure 4b). The combined findings indicate that Atz induces
LMP without affecting ASM activity and the ATPase activity of
Hsp70.
Conclusion
Because deregulation of autophagy is associated with the onset
of various diseases, several small molecules that modulate this
process have been developed. However, additional substances
are still needed for use in efforts aimed at elucidating the
complex autophagic process and discovering therapeutic
candidates. In the present study, we identified the novel small
molecule-based autophagy inhibitor Atz using cell-based highcontent screening of an imidazole library. Although failing to
Figure 3. Autophazole induces LMP-mediated apoptotic cancer cell death.
(a) HeLa cells were treated with Atz for 8 h. Immunoblotting was conducted
by using the corresponding antibodies. (b) HeLa cells were treated with Atz
for 12 h. Immunocytochemistry was performed using Bax (active monomer)
antibody (scale bar, 10 μm). (c) HeLa cells were treated with Atz for 12 h in
the absence and presence of 20 μM leupeptin. Samples were immunoblotted with the corresponding antibodies. (d) HeLa cells were treated for 18 h
with 10 μM Atz in the absence and presence of 20 μM leupeptin. Caspase
activities of cell lysates were determined using Ac DEVD pNA (200 μM) in
the absence and presence of 20 μM Ac DEVD CHO (means.d., n=3).
Figure 4. Autophazole does not affect ASM activity and ATPase activity of
Hsp70. (a) The activity of ASM in HeLa cell lysates was measured in the
present of various concentrations of Atz or chloropromazine (CPZ) by using
an ASM assay kit (means.d., n=3). (b) ATPase activity of Hsp70 was
measured in the present of various concentrations of Atz or apoptozole (Az)
by using a Pi-Colorlock assay (means.d., n =3).
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identify its target protein(s), we demonstrated how Atz
interfered with the autophagic process and promoted cancer
cell death. In this effort, we showed that Atz mainly located in
lysosomes, where it disrupted lysosomal function and induced
LMP (Figure 5). This event led to inhibition of autophagy by
preventing formation of functional autolysosomes. Also, we
found that Atz-induced LMP caused release of cathepsins from
lysosomes into the cytosol, which activated Bid to produce tBid,
thereby inducing MOMP. These events eventually promoted
cancer cell death via caspase activation. As a consequence, Atz
blocked autophagy by disrupting lysosomal function to induce
LMP, the key event which promoted MOMP-mediated caspase
activation. It is anticipated that Atz will be a useful chemical
probe for elucidating details about the autophagic process and
understanding the correlation between autophagy and apoptosis.
Experimental Section
Construction of the imidazole library: Members of the imidazole
library were prepared using the procedures we developed
previously.[48–50]
Compound 1: A mixture of 4-morpholinoaniline (200 mg,
1.36 mmol), 4-formylbenzoic acid (324 μL, 2.04 mmol), 4,4’-dimethybenzil (526 mg, 2.04 mmol) and ammonium acetate (619 mg,
8.16 mmol) in acetic acid (8.6 mL) was stirred for 12 h at 100 °C.
After cooling to room temperature, the mixture was diluted with
ethyl acetate, and washed with water, saturated aqueous NaHCO3
and brine. The organic layer was dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue was
subjected to flash column chromatography (CH2Cl2:MeOH=10:1)
to give 1 as a gray solid in 57% yield: 1
H NMR (400 MHz, CDCl3) δ
8.05 (s, 2 H), 7.81 (s, 1 H), 7.53 (d, 2 H), 7.39 (d, 2 H), 7.24 (d, 2 H),
7.03 (d, 2 H), 6.92 (d, 2 H), 6.82–6.78 (m, 4 H), 6.67 (d, 2 H), 3.83 (s, 3
H), 3.78 (s, 3 H), 3.48–3.46 (m, 4 H), 3.09 (s, 2 H), 2.92 (s, 2 H); 13C
NMR (100 MHz, CDCl3) δ 170.1, 145.3, 140.0, 139.5, 138.1, 134.0,
133.4, 133.2, 131.2, 130.1, 130.0, 129.9, 129.5, 129.1, 128.6, 128.3,
127.4, 126.8, 119.7, 66.3, 53.3, 21.6; ESI-MS calcd for C30H23N5O2
[M+H]+ 530.3, found 530.3.
Autophazole: To a stirred solution of 1 (100.0 mg, 0.18 mmol) in
DMF (1.5 mL) was added N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl, 43.3 mg, 0.21 mmol) and 1-hydroxybenzotriazole (HOBt, 87.5 mg, 0.21 mmol). After being stirred for
1 h at room temperature, t-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)
carbamate (140.7 mg, 0.54 mmol) was added to the mixture. After
stirring for 2 h, the mixture was diluted with EtOAc, washed with
brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was subjected to flash column
chromatography (CH2Cl2:EtOAc=20:1 to 10:1) to give a white solid
in 80% yield.
To a stirred solution of the above product (1.4 mmol) in CH2Cl2
(5 mL) was added TFA (5 mL). After stirring for 1 h, the reaction was
quenched by addition of water. After extraction of the solution
with CH2Cl2, the combined organic layers were washed with
saturated aqueous NaHCO3 and water, dried over anhydrous
Na2SO4, filtered and concentrated under reduced pressure. The
residue was used as autophazole without further purification (86%
yield): 1
H NMR (400 MHz, CD3OD) δ 7.64 (d, 2 H, J=8.2 Hz), 7.42 (d,
2 H, J=8.2 Hz), 7.27 (d, 2 H, J=7.3 Hz), 6.97–6.87 (m, 7 H), 6.73 (d, 2
H, J=7.3 Hz), 3.68 (t, 2 H, J=4.6 Hz), 3.57–3.45 (m, 10 H), 3.02 (t, 2
H, J=4.8 Hz), 2.95 (t, 2 H, J=4.8 Hz), 2.19 (d, 6 H, J=7.8 Hz); 13C
NMR (100 MHz, CD3OD) δ 171.2, 146.5, 143.4, 139.5, 138.3, 134.6,
134.2, 133.2, 131.2, 131.0, 130.7, 129.4, 129.3, 129.0, 128.9, 128.7,
127.4, 126.8, 119.7, 70.5, 70.2, 67.9, 66.7, 66.4, 58.1, 53.8, 48.4, 39.7,
21.3; HR ESI-MS calcd for C40H45N5O4 [M+H]+ 660.3564, found
660.3567.
Cell culture and transfection: All cell lines were purchased from
Korean Cell Line Bank. Cancer (HeLa, human cervical cancer cells;
A549, human lung carcinoma epithelial cells; HCT-116, human
colon cancer cells; MCF-7, human breast adenocarcinoma cells) and
normal cell lines (HaCaT, human keratinocyte cells; MRC-5, human
fetal lung fibroblast cells) were cultured in RPMI 1640 (Invitrogen)
or DMEM (Invitrogen) supplemented with 10% fetal bovine serum
(FBS), 50 units/mL penicillin and 50 units/mL streptomycin. Cells
were maintained at 37°C under a humidified atmosphere of 5%
CO2. HeLa cells stably expressing mRFP EGFP LC3 fusion protein
were constructed by transfecting with the tandem
mRFP EGFP LC3 plasmid using Lipofectamine 2000 and then
selecting with 600 μg/mL G418 over 3 weeks. Cells were maintained
at 37°C under a humidified atmosphere containing 5% CO2.
High-content cell-based screening: HeLa cells stably expressing
mRFP EGFP LC3 fusion protein was seeded in 96-well plates for
24 h and subjected to incubation with 10 μM of each member of an
imidazole library for 12 h. After washing cells with DPBS three
times, cell images were obtained and analyzed by using a
CellomicsTM high-content screening platform.
Western blot analysis: HeLa cells were incubated with Atz at the
indicated concentrations and for the indicated times. After washing
cells with PBS, RIPA buffer containing one table of the protease
inhibitor cocktail was added to the cells for lysis. After incubation
on ice for 10 min, cells were homogenized using a 1 mL syringe
with a 26-gauge needle. Cells were subjected to centrifugation at
15,000×g for 20 min at 4°C and the supernatant was collected.
Proteins were separated by using 6–12% SDS-PAGE. Rabbit Bid
polyclonal (Cell Signaling Technology, 1:1000), mouse cathepsin B
monoclonal (Santa Cruz Biotechnology, 1:1000), rabbit caspase-3
polyclonal (Santa Cruz Biotechnology, 1:1000), rabbit cleaved
caspase-3 polyclonal (Santa Cruz Biotechnology, 1:1000), rabbit
PARP polyclonal (Cell Signaling Technology, 1:1000), mouse
cytochrome c monoclonal (Biovision, 1:1000), rabbit LC3 (SigmaAldrich, 1:2000), mouse p62 (Santa Cruz Biotechnology, 1:1000)
and mouse β-actin (Santa Cruz Biotechnology, 1:1000) antibodies
were used as primary antibodies. Horseradish peroxidase (HRP)- Figure 5. Proposed mechanism of Atz to inhibit autophagy and to induce
caspase activation.
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conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, 1:2000)
and goat anti-mouse IgG (Santa Cruz Biotechnology, 1:2000) were
used as secondary antibodies. The immunoblots were developed
by using a West-ZOL® plus Western Blot Detection System (Intron
Biotechnology Inc., South Korea). Protein bands were analyzed by
using a G:BOX Chemi Fluorescent & Chemiluminescent Imaging
System.
Immunocytochemistry: HeLa cells were treated with Atz at the
indicated concentrations and for the indicated times. After washing
cells with PBS, the cells were fixed with PBS buffer containing 4%
formaldehyde and 0.1% triton X-100 for 15 min. Cells were
incubated with mouse active monomeric Bax monoclonal (Enzo Life
Science, 1:200), mouse LAMP2 monoclonal (Santa Cruz Biotechnology,1:200), guinea pig p62 polyclonal (Progen, 1:200) for 1 h at
room temperature followed by incubation with Alexa-Fluor 488
conjugated mouse IgG or Alexa-Fluor 588 conjugated guinea pig
IgG (Invitrogen, 1:200) for Autophagy Compound Library 1 h at room temperature. The cells were
imaged by using confocal fluorescence microscopy (Zeiss LSM 800).
Cell images were analyzed by using the ZEN 2011 software.
Acknowledgements
This study was supported financially by the National Research
Foundation of Korea (grant no. 2020R1A2C3003462 to I.S.) and
KRIBB Research Initiative Program funded by the Ministry of
Science ICT (MSIT) of Korea (S.-K.K.).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: autophagy · apoptosis · cancer cell death · highthroughput screening · small molecules
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Manuscript received: June 15, 2021
Revised manuscript received: July 14, 2021
Accepted manuscript online: July 15, 2021
Version of record online:
ChemBioChem
Full Papers
doi.org/10.1002/cbic.202100296
ChemBioChem 2021, 22, 1–7 www.chembiochem.org 6 © 2021 Wiley-VCH GmbH
These are not the final page numbers!
Wiley VCH Freitag, 23.07.2021
2199 / 213336 [S. 6/7] 1
FULL PAPERS A novel autophagy inhibitor (autophazole, Atz) is reported which was
identified by cell-based high-content
screening of an imidazole library. Autophazole was found to disrupt
lysosomal function, thereby leading
to inhibition of autophagy. In
addition, Atz induces lysosomal
membrane permeabilization-associated apoptotic cancer cell death.
S.-H. Park, I. Shin, Dr. G.-H. Kim, Dr. S.-
K. Ko, Prof. Dr. I. Shin* 1 – 7
An Autophagy-Disrupting Small
Molecule Promotes Cancer Cell
Death via Caspase Activation
Wiley VCH Freitag, 23.07.2021