Antiviral activity of an isatin derivative via induction of PERK-Nrf2-mediated suppression of cap-independent translation

Article abstract of DOI:10.1021/cb400775z

We report here an isatin derivative 45 (ID45) against coxsackievirus B3 (CVB3) replication, which was synthesized based on a high-throughput screen of a unique natural product library. ID45 showed the most potent anti-CVB3 activity among the four synthesized compounds. Treatment of cells with ID45 before or after infection significantly reduced viral particle formation, resulting in protection of cells from virus-induced apoptosis. In addition, ID45 treatment caused remarkable up-regulation of glucose-regulated protein 78 (GRP78), a hallmark of endoplasmic reticulum (ER) stress and an indicator of enhanced cell viability. In identifying the ER stress response pathway induced by ID45, we found that ID45 activated PKR-like ER protein kinase (PERK) but failed to up-regulate eIF2α phosphorylation. Instead ID45 activated transcription factor Nrf2 (NF-E2-related factor-2), which is evidenced by its nuclear translocation and upregulation of its downstream target genes NQO1 (NAD(P)H quinone-oxidoreductase 1) and GCLM (glutamate-cysteine ligase, modifier subunit). This observation was further verified by using siRNAs of GRP78 or Nrf2, which blocked both the translocation of Nrf2 and up-regulation of its target genes, leading to aggressive viral replication and enhanced cell apoptosis. Finally, we found that ID45-induced up-regulation of NQO1 protected eIF4GI, a eukaryotic cap-dependent translation initiation factor, from cleavage by CVB3 protease and degradation by proteasomes. Taken together, our findings established that a novel antiviral mechanism of isatin derivative ID45 inhibits CVB3 replication by promoting cell survival through a PERK/Nrf2-dependent ER stress pathway, which benefits host cap-dependent translation but suppresses CVB3 cap-independent translation.

Full text of DOI:10.1021/cb400775z

Articles
pubs.acs.org/acschemicalbiology
Antiviral Activity of an Isatin Derivative via Induction of PERK-Nrf2-
Mediated Suppression of Cap-Independent Translation
Huifang M. Zhang,†,¶ Huanqin Dai,‡,¶ Paul J. Hanson,† Huidong Li,§ Hui Guo,‡,∥ Xin Ye,†
Maged G. Hemida,† Luoqiang Wang,‡,⊥ Yaojun Tong,‡,∥ Ye Qiu,† Selina Liu,† Fengping Wang,†,#
Fuhang Song,‡ Buchang Zhang,⊥ Jian-Guo Wang,*,§ Li-Xin Zhang,*,‡,⊥ and Decheng Yang*,†
†Department of Pathology and Laboratory Medicine, University of British Columbia, Institute for Heart and Lung Health, St. Paul’s
Hospital, Vancouver, BC V6Z 1Y6, Canada
‡Key Laboratory of Pathogenic Microbiology & Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
§State-Key Laboratory and Institute of Elemento-Organic Chemistry, Nankai University, Tianjin, China
∥Graduate University, Chinese Academy of Sciences, Beijing, China
⊥School of Life Sciences, Anhui University, Hefei, China
#Department of Emergency, Harbin Medical University, Heilongjiang, China
S
* Supporting Information
ABSTRACT: We report here an isatin derivative 45 (ID45) against coxsackievirus B3
(CVB3) replication, which was synthesized based on a high-throughput screen of a unique
natural product library. ID45 showed the most potent anti-CVB3 activity among the four
synthesized compounds. Treatment of cells with ID45 before or after infection significantly
reduced viral particle formation, resulting in protection of cells from virus-induced
apoptosis. In addition, ID45 treatment caused remarkable up-regulation of glucose-
regulated protein 78 (GRP78), a hallmark of endoplasmic reticulum (ER) stress and an
indicator of enhanced cell viability. In identifying the ER stress response pathway induced
by ID45, we found that ID45 activated PKR-like ER protein kinase (PERK) but failed to
up-regulate eIF2α phosphorylation. Instead ID45 activated transcription factor Nrf2 (NF-
E2-related factor-2), which is evidenced by its nuclear translocation and upregulation of its
downstream target genes NQO1 (NAD(P)H quinone-oxidoreductase 1) and GCLM
(glutamate-cysteine ligase, modifier subunit). This observation was further verified by using
siRNAs of GRP78 or Nrf2, which blocked both the translocation of Nrf2 and up-regulation of its target genes, leading to
aggressive viral replication and enhanced cell apoptosis. Finally, we found that ID45-induced up-regulation of NQO1 protected
eIF4GI, a eukaryotic cap-dependent translation initiation factor, from cleavage by CVB3 protease and degradation by
proteasomes. Taken together, our findings established that a novel antiviral mechanism of isatin derivative ID45 inhibits CVB3
replication by promoting cell survival through a PERK/Nrf2-dependent ER stress pathway, which benefits host cap-dependent
translation but suppresses CVB3 cap-independent translation.
antifungal potentiator and abyssomicins as potent anti-TB agents
are good examples of the use of the library for drug discovery.5,6
CVB3 is a RNA virus, and its genome encodes 11 proteins
including two viral proteases 2A and 3C.7 The 5′ end of the
genome does not have a cap structure (a 7-methylguanosine
triphosphate group); instead, it is covalently linked to a small
viral protein VPg. This viral RNA also has an unusually long 5′
untranslated region (5′UTR) harboring an internal ribosome
entry site (IRES).8 Thus, CVB3 RNA, unlike cellular mRNAs or
many other viral genomes that start translation by a cap-
dependent manner relying on the participation of cap-binding
proteins and eukaryotic translation initiation factor 4GI
oxsackievirus B3 (CVB3), an enterovirus in the Picornavir-
Cidie family, is the primary causal agent of viral myocarditis,
particularly in children and young adolescents. This disease is
often associated with sudden unexpected death.1 Many synthetic
compounds have been reported to inhibit enteroviruses.2,3
Unfortunately, there is no approved antiviral drug for the
treatment of acute enteroviral infections, including those against
CVB3, in part because of the inadequacy of current chemical
libraries in providing small molecules with sufficient activity
against CVB3. Natural products have nevertheless historically
played a valuable role in drug discovery and development
because they occupy tremendous chemical structural space that is
unmatched by any other small molecule families. A marine
microbial extract library containing over 20,000 microbial
extracts has been established in our laboratory and been screened
for various biological activities.4,5,22 Identifying beauvericin as an
Received: October 9, 2013
Accepted: January 31, 2014
Published: January 31, 2014
© 2014 American Chemical Society
1015
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
(eIF4GI), initiates its translation by a cap-independent (IRES-
driven) mechanism.9
Mammalian cells possess a signaling network that senses the
accumulation of misfolded or unfolded proteins within the
endoplasmic reticulum (ER)10 and determines cell fate following
exposure to stress signals including virus infections.11,12 In
addition, the ER membrane system is closely associated with the
sites of picornaviral multiplication.13 In response to ER stress, a
coordinated, adaptive program called the unfolded protein
response (UPR) is activated to adapt to stress signals targeting
the ER. A master ER chaperon is the 78 kDa glucose regulated
protein (GRP78).14 Induction of GRP78 increases protein
folding capacity, which represents a major survival arm of UPR
and a host self-defense response. There are three major arms of
the UPR network.15 Among these, PKR-like ER protein kinase
(PERK)-dependent signaling is critical for cell survival following
the initiation of UPR. UPR-mediated PERK activation impedes
protein translation via phosphorylation-dependent inhibition of
eukaryotic translation initiation factor-2α (eIF2α).16,17 However,
independent of its translational regulatory capability, PERK-
dependent signals can also elicit the activation of pro-survival
transcription factor Nrf2 (NF-E2-related factor-2), leading to cell
survival.18 A number of viruses including CVB3 have been shown
to induce UPR during infection.11,12,19 This response is a host
self-defense mechanism against viral invasion. It is for this reason
that the UPR pathway is an ideal target for antiviral drug
screening and evaluation. In this study, based on our high
throughput screening of the library, four isatin derivatives (ID)
were synthesized. Antiviral evaluation using two well-established
cell lines identified ID45 as a potent anti-CVB3 agent. We further
revealed that ID45-induced up-regulation/activation of target
genes enhances the stability of eIF4GI protein, a critical factor
required for cap-dependent translation initiation of host mRNAs
but a competitor against CVB3 RNA translation. These data
suggest that ID45 inhibits CVB3 replication by activation of the
pro-survival UPR pathway, leading to suppression of cap-
independent translation of CVB3.
Figure 1. Chemical structure of isatin and its four derivatives.
we chose 0.1 MOI for this study. For the dose study, Figure 2B
shows that ID45 treatment decreased the expression of viral VP1
protein in a dose-dependent manner, and the dose of 10 μM
ID45 reduced VP1 expression to an undetectable level. These
data were further supported by a viral plaque assay (Figure 2C),
showing dramatic reduction of CVB3 particle formation after
treatment with ID45. The dose-dependent anti-CVB3 property
of ID45 was also tested in HL-1 cardiomyocytes using similar
conditions and produced similar results (Figure 2D,E).
On the basis of antiviral evaluation, we further confirmed ID45
1
1
structures by H NMR, ESI-MS, and elemental analysis. H
NMR analysis data of ID45 is provided in Supplementary Figure
S2. Compound ID45 was recrystallized from ethanol/dichloro-
methane to give yellow crystals suitable for X-ray single-crystal
diffraction, the structure of which was determined using graphite
monochromated Mo KR radiation (Supplementary Figure S3).
Bond lengths, angles, and torsion angles of ID45 are listed in the
Supplementary Table S1.
Assays for Cytotoxicity and Time of Treatment of ID45.
To understand the mode of anti-CVB3 action and test for
possible cytotoxicity caused by prolonged ID45 treatment, HeLa
cells were treated with ID45 for 30 min, and then the compound
was either removed by replacing fresh media without drug or
retained for continuous treatment. The cells were then infected
with CVB3 or sham-infected with phosphate-buffered saline
(PBS). Antiviral activity and cytotoxicity were detected by
Western blot for VP1 and MTS cell viability assay, respectively, at
16 h post infection (pi). Figure 3A shows that CVB3 VP1 levels
in ID45-removed cells were slightly higher than that in ID45-
retained cells, indicating that treatment with ID45 for 30 min is
long enough for drug internalization and full function. This also
indicates that longer treatment does not increase cytotoxicity. As
CVB3 infection causes cell apoptosis,24 we next investigated
whether the antiviral activity of ID45 is associated with the
induction of cell death. We first found that treatment with ID45
in the sham-infected controls (Figure 3A, lanes 1 and 2) did not
cause cleavage of pro-caspase-3, indicating that the compound is
not toxic and ID45 itself cannot induce cell apoptosis. We further
found that cleaved caspase-3 in ID45-treated cells (lanes 4 and 6)
is less than that in DMSO-treated cells (lanes 3 and 5), indicating
again that ID45 inhibits cell apoptosis and benefits cell survival
during CVB3 infection. This conclusion is supported further by
the cell viability assay, which demonstrated that in ID45-treated/
CVB3 infected cells, either removal or retention of the
compound could result in a high rate (81−86%) of cell viability.
Specifically, there was a 17−23% increase as compared to the
corresponding control cells treated with DMSO (Figure 3B).
To evaluate the potential for future clinical trial, we mimicked
the natural conditions for the treatment of the existing infection,
RESULTS AND DISCUSSION
■
Synthesis of Anti-CVB3 Isatin Derivatives Based on
High Throughput Screening. Isatin, a metabolite of marine
bacteria in the embryos of some shrimps, enables the embryos to
become remarkably resistant to infection by pathogenic fungus.20
Isatin derivatives such as methisazone (marboran, MIBT,1-
methylisatin 3-thiosemicarbazone) were reported to inhibit
vaccinia, cowpox, and herpes simplex virus infections.21,22Isatin
and its derivatives were also identified as inhibitors of human
mitochondrial monoamine oxidase B.23 Furthermore, no toxicity
is observed when using isatin. On the basis of this finding, we
synthesized four new isatin derivatives, ID45, 48, 52, and 146
(Figure 1). Synthetic routes are shown in the Supplementary
Figurer S1. In testing antiviral effects using CVB3-infected cells,
ID45 showed the strongest anti-CVB3 activity followed by
methisazone21 (a known ID control), ID52, and ID48. In
contrast, ID146 seems to enhance CVB3 replication (Figure 2A).
Meanwhile, we also used caspase-3 activation, an executive phase
of cell apoptosis, as a criterion to demonstrate the strongest
inhibition of pro-caspase-3 cleavage in the ID45-treated sample.
The anti-CVB3 activity of ID45 was further tested using different
MOIs (multiplicity of infection) and doses. Infections with 1 and
0.1 MOI all inhibited CVB3 replication. Considering the lower
MOI infection will benefit the collection of enough cell lyses for
analysis of signal transduction pathways in antiviral mechanism,
1016
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
Figure 2. In vitro validation of anti-CVB3 activity of isatin derivatives. (A) In vitro screen. HeLa cells were treated with four new isatin derivatives (ID146,
ID52, ID48, and ID45, respectively) at 10 μM for 30 min and then infected with CVB3 at 0.1 MOI for 16 h. This is the infection condition used
throughout this study unless otherwise specified. Methisazone- and DMSO-treated cells were used as positive and negative controls, respectively.
Antiviral activity was evaluated by Western analysis of CVB3 VP1 protein production and cleavage of pro-caspase 3. β-Actin was used as an equal loading
control. (B) Dose testing. HeLa cells were treated with ID45 at indicated doses and then infected with CVB3. Cell lysates were collected for Western
analysis using a VP1 antibody. (C) Plaque assay. CVB3 particles were measured by viral plaque assay using supernatants from 10 μM ID45-treated and
CVB3-infected cells. (D) Validation in HL-1 cardiomyocytes. HL-1 cells were cultured in Claycomb medium and treated with ID45 at different doses
and then infected with CVB3. Anti-CVB3 activity was validated by Western analysis of VP1. (E) Plaque assay. CVB3 particles were measured by plaque
assay using supernatants from HL-1 cells treated with 10 μM ID45 (n = 3, p < 0.05).
i.e., treatment of the cells after CVB3 infection. We treated the
cells with ID45 at 2 or 6 h after viral infection and found that the
levels of VP1 reduction as well as the activation of caspase-3 were
similar to that of cells treated for 30 min prior to infection (Figure
3C). This anti-CVB3 activity independent of time of treatment
was further verified by evaluation using HL-1 cardiomyocytes
(Figure 3D). We further showed that the reduction of VP1
expression (Figure 3D) was due to corresponding promotion of
HL-1 cell viability (Figure 3E). In the cell viability assay,
comparing to sham-infected control, cells pretreated with ID45
for 30 min and then infected with CVB3 showed 78% cell
survival, which is almost the same as that of the cells treated at 6 h
pi. These data suggest that the antiviral effect of ID45 is
independent of the time for addition of the compound. Most of
the previous antiviral drugs inhibit viral replication via their pro-
apoptotic activity, which kills the host cells supporting viral
replication. In a cytotoxicity assay, we noticed that the sham-
infected cells treated with ID45 did not show activation of
caspase-3; ID45 is not only nontoxic to the cells but also
enhances cell viability.
ID45 Treatment Induces GRP78 Up-regulation and
UPR in Both Dose-Dependent and Time-Dependent
1017
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
Figure 3. Assays for cytotoxicity and time of treatment. (A) HeLa cells were treated with ID45 for 30 min, and the drug was either removed by replacing
the medium or retained for continuing treatment. The cells were then infected with CVB3 or sham-infected. Antiviral activity was determined by
Western analysis of VP1 and cleavage of pro-caspase-3 as described in Figure 2. (B) Cytotoxicity of ID45 was evaluated with a MTS cell viability assay
using HeLa cells treated as described in panel A. Cell viability of nontreated/sham-infected cells was defined as 100% (control). Other data are presented
as percentage of the control (n = 3, p < 0.05). Time of treatment assay. HeLa cells (C) or HL-1 cells (D) were treated for 30 min prior to or 2 or 6 h pi
CVB3 infection. Anti-CVB3 evaluation was conducted by Western analysis as described above. Cytotoxicity of ID45 was evaluated with the MTS cell
viability assay using HL-1 cells (E) treated with ID45 for 30 min prior to or 6 h pi. The additional controls are cells nontreated/sham-infected as
indicated. Cell viability of the samples were determined as described in panel B (n = 3, p < 0.05).
Manners. While studying the underlying mechanism by which
ID45 promotes cell survival and inhibits CVB3 replication, we
first speculated that the ERK and/or PI3K/Akt pathways may be
involved in the pro-survival activity of ID45. However, we failed
to detect the activation of these two pathways (data not shown).
As our previous study demonstrated that CVB3 infection induces
GRP78 up-regulation and subsequent activation of UPR, a host
survival strategy,19 we asked whether ID45-induced cell survival
is due to its ability to up-regulate GRP78 and enhance UPR.30,31
To address this question, we treated cells with ID45 at doses
from 2 to 10 μM for 30 min. We also treated cells with ID45 at 10
μM for different durations ranging from 0.5 h to overnight. We
found that ID45 did induce up-regulation of GRP78 and cell
morphology changes in both dose-dependent and duration-
dependent manners, as detected by Western analysis (Figure
4A,B) and phase-contrast microscopy (Figure 4C). All these data
indicate that under the treatment of ID45 overnight most of the
cells showed the round-up shape and reduced size compared to
DMSO-treated cells (Figure 4C panel d). However, the
detection of caspase-3 activation did not show pro-caspase-3
1018
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
Figure 4. ID45 up-regulates GRP78 and induces UPR in both dose- and time-dependent manners. (A) HeLa cells were treated with ID45 at indicated
doses for 30 min. Cell lysates were collected for Western analysis of GRP78 expression. (B) HeLa cells were treated with ID45 for the indicated time
courses. GRP78 expression was determined by Western blot. β-Actin was an equal loading control. (C) The cell morphological changes were observed
after 30 min or overnight (ON) treatment by phase contrast microscopy. (D) Pro-caspase-3 cleavage was detected by Western blot. CVB3-infected cell
was used as a positive control.
cleavage in these cells (Figure 4D), suggesting that this
morphology change is not due to cell apoptosis but due to cell
responses to ER stress.
survival transcription factor Nrf218 by up-regulation and nuclear
translocation of this protein.27 To reveal the underlying signal
pathway activated by ID45, HeLa cells were treated with ID45 for
16 h, and cellular proteins were extracted for Western analysis.
Figure 5A shows that ID45 induced up-regulation of
phosphorylated PERK (p-PERK) following GRP78 induction
of UPR but did not significantly change the levels of
phorsphorylated eIF2α (p-eIF2α) and total eIF2α. This implies
that ID45-induced UPR cannot induce the p-eIF2α-mediated
apoptosis pathway. Thus, we turned our attention to detect the
up-regulation of Nrf2, a transcription factor that can be activated
directly by p-PERK.18 We found that Nrf2 was significantly up-
regulated after ID45 treatment in both HeLa cells and
cardiomyocytes. This Nrf2 activation was further verified by
the facts that (i) there was significant up-regulation of the target
genes, NQO1 (NAD(P)H quinone-oxidoreductase 1) and
GCLM (glutamate-cysteine ligase, modifier subunit) (Figure
5A) and (ii) similar results were obtained by experiments using
murine HL-1 cells (Figure 5B). Since the antibody against
human GCLM did not work for murine HL-1 cells, we excluded
this protein from the detections in HL-1 cells.
Nrf2 activity is usually suppressed by Keap1 protein in unstress
conditions through interactions to form an Nrf2-Keap1
complex.28 To determine if Keap1 protein is involved in ID45-
mediated regulation of Nrf2 expression, we knocked down
Keap1 with specific siRNAs and then treated the cells with ID45.
Supplementary Figure S5 shows that silencing Keap1 with
siRNAs increased Nrf2 levels compared to the cells treated with
scrambled control siRNAs. However, when comparing the ID45-
treated cells with the DMSO-treated samples, although the Nrf2
levels were dramatically increased by ID45 treatment in
Silencing of GRP78 with siRNAs Relieves ID45-Induced
UPR and Attenuates CVB3 VP1 Reduction. To further verify
that ID45-induced antiviral effect occurs through induction of
GRP78 and activation of UPR, we transfected cells with siRNAs
targeting GRP78 and then treated with ID45 but without CVB3
infection. To confirm that silencing of GRP78 would suppress
ID45-induced inhibition of VP1 expression, HeLa cells were
transfected with GRP78 siRNAs, treated with ID45, and then
infected with CVB3. Western analysis (Supplementary Figure
S4C) showed that silencing of GRP78 with siRNAs reduced
GRP78 expression. However, only when cells were transfected
with scrambled siRNA and treated with ID45, the reduction of
the VP1 protein production reached the lowest level (lane 4)
compared to other treatments. These data suggest that ID45-
induced VP1 reduction is through up-regulation of GRP78 and
activation of the pro-survival pathway of ER stress response.
ID45 Induces Cell Survival via PERK-Nrf2 but Not PERK-
eIF2α Pathway of UPR. Having demonstrated that ID45 could
promote cell survival via activating the GRP78-mediated UPR,
our next step was to identify the specific branch of the UPR
pathway that leads to cell survival. During ER stress, PERK
activation has been recognized as one of the central mediators
determining cell fate during ER stress. Although PERK activation
can suppress translation of certain proteins, it can also promote
translation of certain proteins involved in cell growth or death. In
addition to inducing cell apoptosis via phosphorylation of
eIF2α25 and subsequent up-regulation of proapoptotic tran-
scription factor CHOP,26 p-PERK can also directly activate pro-
1019
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
Figure 5. ID45-induced GRP78 up-regulation activates PERK-Nrf2
pathway of UPR. HeLa cells (A) and HL-1 cells (B) were treated with
ID45 as described above. Whole cell lysates were collected for Western
analysis to detect up-regulation of GRP78 and Nrf2, phosphorylation of
PERK and eIF2a, total level of eIF2α, as well as up-regulation of Nrf2
and its target genes NQO1 and GCLM. β-Actin was used as an equal
loading control.
scrambled siRNA-treated cells, this increase was not obvious in
Keap1 siRNA-treated samples. In addition, NQO1 expression
level also showed a similar pattern as that of Nrf2 in the
corresponding cells. These data suggest that ID45-induced Nrf2
activation is largely through the induction of dissociation of Nrf2-
Keap1 complex.
ID45 Treatment Induces Nrf2 Nuclear Translocation.
Nrf2 has been well studied in antioxidative stress signaling.
During oxidative stress, Nrf2 dissociates from the Keap1/Nrf2
complex and is transported into the nucleus, where it promotes
expression of its downstream target genes involved in cell
growth.27 Thus, the function of Nrf2 in cytoprotection is
documented in promoting longevity,29 disease prevention,30,31
and shaping immune response to hepatitis B virus infection.32
The ID45-induced nuclear translocation of Nrf2 protein was
demonstrated in both CVB3- and sham-infected HeLa cells by
Western analysis using cytosolic and nuclear proteins. The purity
of these proteins was monitored by detecting tubulin and histone
protein, respectively. Figure 6A shows that ID45-treated cells
showed an increased amount of Nrf2 in nuclear extract compared
to that in the cytosolic fraction. A similar result was observed in
PBS sham-infected cells (Figure 6B). It seems that CVB3
infection enhanced ID45-induced Nrf2 nuclear import. These
data were further solidified in HL-1 cells (Figure 6C,D). To
directly observe the up-regulation and translocation of Nrf2 in
the cells, immunocytochemical staining of the proteins was
conducted, and the result of this showed that Nrf2 protein
translocated to the nucleus after ID45 treatment overnight
(Figure 6E).
Figure 6. ID45 treatment induces Nrf2 nuclear translocation. HeLa cells
were treated with ID45 and then infected with CVB3 (A) or sham-
infected with PBS (B). The cytosolic and nuclear proteins were isolated,
and the Nrf2 protein distribution in these fractions was detected by
Western analysis. α-Tubulin and histone were used for purity control of
cytosolic and nuclear extractions, respectively. β-Actin was the loading
control. The experiment was repeated to verify the findings using HL-1
cells and the corresponding data are shown in panels C and D,
respectively. (E) Immunostaining. HeLa cells culturing on glass
coverslips were treated with ID45 overnight. The cells were stained
with an anti-Nrf2 primary antibody and then a green-fluorescent Alexa
Fluor 488 dye-conjugated second antibody. The nuclei were counter-
stained with DAPI. Nrf2 protein distribution in cytosol and nucleus was
visualized by confocal microscopy. HeLa cells treated with tunicamycin
(1 μg/mL) overnight were visualized as a control.
1020
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
Silencing GRP78 or Nrf2 with siRNAs Attenuates ID45-
Induced Nrf2 Up-regulation and Nuclear Translocation.
To further confirm that ID45-induced up-regulation of Nrf2 and
its target gene NQO1 as well as the subsequent nuclear
translocation of Nrf2 is due to GRP78-mediated UPR, total
cellular proteins were isolated from HeLa cells transfected with
siRNAs targeting GRP78 or Nrf2 and then treated with ID45.
Western analysis showed that GRP78 siRNAs suppressed both
GRP78 and Nrf2 expression, while Nrf2 siRNAs suppressed only
Nrf2 expression compared to GRP78 siRNA. These data imply
that GRP78 is located upstream of Nrf2 in this pathway. The
GRP78-mediated activation of Nrf2 was further solidified by the
suppression of its target gene NQO1 expression via siRNA
silencing of either Nrf2 or GRP78 (Supplementary Figure S6A).
To further demonstrate the activation of Nrf2 by observation of
its nuclear translocation, cytosolic and nuclear proteins were
extracted. Western analysis showed that both siRNAs attenuated
the gene expression and nuclear translocation of Nrf2
(Supplementary Figure S6B). These data suggest that ID45
induces UPR-mediated cell survival signaling via activation of
GRP78 and induction of Nrf2 nuclear translocation.
Silencing Nrf2 Suppresses Antiviral Effect of ID45 and
Increases CVB3-Induced Cell Death. We have shown that
activation of Nrf2 is an important step toward cell survival and
inhibition of CVB3 replication. Thus, we reasoned that
inactivation of Nrf2 signaling would sensitize cells to CVB3-
induced cell apoptosis. To verify this speculation, HeLa cells
were transfected with Nrf2 siRNAs, treated with ID45, and then
infected with CVB3. Western analysis using cell lysates showed
that Nrf2 siRNAs suppressed expression of Nrf2 and its target
gene NQO1 compared to the controls (Supplementary Figure
S7A). This suppression in CVB3-infected cells in turn inhibited
VP1 production. This phenomenon is largely due to the host cell
death induced by Nrf2 siRNAs, which could not support CVB3
replication. This notion is further supported by the activation of
caspase-3, up-regulation of pro-apoptotic transcription factor
CHOP (C/EBP homologous protein), decrease of cell viability
(Supplementary Figure S7B), and increase of CVB3 particle
release from the dead cells (Supplementary Figure S7C). These
data confirmed that the antiviral effect of ID45 is achieved
through enhancing cell viability, which requires the activation of
the Nrf2-NQO1 signal cascade.
ID45-Induced NQO1 Up-regulation Protects eIF4GI
from Cleavage and Degradation. NQO1 has been reported
to play a role in regulating mRNA translation via protection of
eIF4GI from degradation by the proteasome during oxidative
stress.33 It is also known that intact eIF4GI is required for cap-
dependent translation initiation of cellular mRNAs but not for
cap-independent translation initiation of picornaviruses34 and
that CVB3 infection shuts down host protein translation by
cleavage of eIF4GI by its protease 2A.24 To further explore the
underlying mechanism by which ID45 inhibits CVB3 replication,
the role of ID45-induced NQO1expression in regulating eIF4GI
stability during ER stress was analyzed. After treatment of the
cells with ID45 followed by infection with CVB3, cellular
proteins were used to detect eIF4GI protein. As shown in Figure
7A, when comparing the CVB3-infected samples, the ID45-
treated cells (lanes 7 and 8) produced less cleavage/degradation
products (100−150 kDa) than the DMSO-treated cells (lanes 3
and 4) at 6−8 h pi, suggesting that ID45 protects eIF4GI from
cleavage by CVB3 protease and degradation by proteasomes.
The role of NQO1 in enhancing the stability of eIF4GI during
CVB3 infection was further supported by an experiment using
Figure 7. ID45-induced NQO1 up-regulation protects eIF4GI from
cleavage and degradation. (A) HeLa cells were treated with ID45 and
then infected with CVB3 or sham-infected with PBS. Cell lysates
collected at indicated time points pi were analyzed for eIF4GI cleavage
and degradation by Western blot using 7% SDS-PAGE. The molecular
mass markers are indicated. β-Actin was used as a loading control. (B)
HeLa cells were transfected with NQO1 siRNA or control siRNA,
treated with ID45 or DMSO, and then infected with CVB3. NQO1
expression and eIF4GI cleavage/degradation were detected by Western
blot using10% SDS-PAGE. Levels of NQO1 and cleavage/degradation
products were determined by densitometry analysis of data from three
independent experiments using the NIH ImageJ program, normalized to
β-actin, and presented as mean SD. p < 0.05 (lower panel).
NQO1 siRNAs. Figure 7B demonstrates that transfection with
NQO1 siRNA led to more cleavage and further degradation of
the cleaved fragments (i.e., fewer 100−150 kDa fragments) of
1021
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
eIF4GI than the scrambled siRNA-transfected group. Note that
in order to clearly show the cleavage/degradation products, a
more concentrated gel (10%) was used in Figure 7B. Our data, to
the best of our knowledge, provide the link for the first time
between the up-regulation of Nrf2 and anti-CVB3 activity. In
general, promotion of host cell survival will create favorable
conditions for viral replication. However, in our experimental
system, surprisingly Nrf2-mediated cell survival led to strong
inhibition of CVB3 replication. In revealing this unique
mechanism, our data show that the antiviral effect is attributed
to the up-regulation of Nrf2 target gene NQO1 and to the unique
structure of CVB3 RNA. It has been reported that NQO1,
through the reduction of intracellular quinones, plays a role in the
cellular response to oxidative stress.35 It is also known that
NQO1 protein expression protects eIF4GI from degradation by
the proteasome. This protection mechanism relies on the
interaction of NQO1 and eIF4GI, which blocks the access of
proteasome to eIF4GI.33 eIF4GI is a critical component of the
eukaryotic translation initiation complex eIF4F, which serves as a
scaffolding protein for the assembly of eIF4F, which is composed
of eIF4E (the mRNA cap-binding protein) and eIF4A (an
ATPase-dependent RNA helicase). Thus, via its association with
the mRNA cap-binding protein eIF4E and with other translation
initiation factor (e.g., eIF3) that are bound to the 40S ribosomal
subunit, eIF4GI creates a physical link between the cap structure
and the ribosome, thus facilitating cap-dependent translation
initiation of host cellular mRNAs.36 eIF4GI also functions in cap-
independent (IRES-driven) translation initiation, but this
function is carried out by its cleavage forms. Upon infection,
eIF4GI is attacked by viral proteases. The resulting eIF4GI
cleavage products serve to reprogram the cell’s translation
machinery, as the N-terminal product inhibits cap-dependent
translation of host cell mRNAs by sequestering eIF4E while the
C-terminal product stimulates IRES-mediated translation
initiation of viral RNAs.37 CVB3 RNA, like that of other
picornaviruses, does not have a cap structure at its 5′ end but
contains an IRES within the long 5′ UTR. Thus CVB3
translation initiation is driven by an IRES mechanism. For this
reason, virus RNAs and host mRNAs will compete with each
other for translational machineries. However, during treatment
with ID45, the up-regulated NQO1 can interact with eIF4GI and
protect it from degradation by proteasomes. Interestingly, we
found that this interaction protects eIF4GI from cleavage by
CVB3 proteases. The stabilized intact eIF4GI then maintains
high protein translation rates for host mRNAs and therefore
limits cap-independent translation of viral RNAs, which
contributes to the antiviral effect of ID45.
Figure 8. Proposed mechanism of ID45 action. Compound ID45 causes
GRP78-mediated ER stress response, leading to phosphorylation of
PERK, up-regulation and nuclear translocation of transcription factor
Nrf2, and subsequent up-regulation of target gene NQO1. The
produced NQO1 protein protects eIF4GI from cleavage by viral
protease and degradation by proteosomes. The enhanced stability of
eIF4GI promotes cap-dependent translation of host mRNAs but
suppresses cap-independent translation of coxsackievirus.
thaw followed by centrifugation to remove cell debris and was stored at
−80 °C. HeLa cells (ATCC), a cell line that has been well established to
study CVB3 pathogenesis, were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 100 μg/mL penicillin-
streptomycin, 2 mM glutamine, and 10% Clontech fetal bovine serum
(FBS). The HL-1 cell line, a cardiac muscle cell line established from an
AT-mouse atrial cardiomyocyte tumor lineage, was a gift from Dr.
William C. Claycomb (Louisiana State University Medical Centre, New
Orleans, USA).38 The cells were maintained in Claycomb medium (JRH
Biosciences) supplemented with 10% FBS, 100 μg/mL penicillin-
streptomycin, 0.1 mM norepinephrine (Sigma), and 2 mM L-glutamine
(Invitrogen).
Isatin Derivative Synthesis. The synthesis of the compounds and
confirmation of the ID45 structure were conducted by following
methods described previously.39 5,7-Dibromoindoline-2,3-dione (A, 3.0
g, 9.8 mmol) and anhydrous potassium carbonate (1.25 g, 9 mmol) were
added to 40 mL of DMF in a 100 mL flask, and then the mixture was
stirred for 1 h at 65 °C. Next, m-fluorobenzyl chloride (B, 1.72 g, 12
mmol) was added, and the reactant solution was stirred for 4 h at the
same temperature. Water (50 mL) was added, and the mixture was
filtered and washed with cold water. Red crystals were collected and
dried to give 5,7-dibromo-1-(3-fluoro-benzyl)-indole-2,3-dione (C, 3.25
g, yield 80%). 1H NMR (CDCl3, 400 MHz): δ 7.831 (s, 1H, isatin-ArH),
7.739 (s, 1H, isatin-ArH), 7.314 (m, 1H, isatin-ArH and ArH), 7.066−
6.932 (m, 3H, isatin-ArH and ArH), 5.400 (s, 2H, Ar-CH2N).
5,7-Dibromo-1-(3-fluoro-benzyl)-indole-2,3-dione (C, 2.1 g, 5
mmol) was mixed with 2-fluorobenzohydrazide (D, 0.77 g, 5 mmol)
and acetic acid (2.5 mL). The mixture was refluxed for 6 h before being
cooled to RT. The target compound was filtered, washed with water, and
dried to give a crude orange product. Recrystallization from ethanol
yielded an orange solid, 2-fluoro-benzoic acid [5,7-dibromo-1-(3-fluoro-
benzyl)-2-oxo-1,2-dihydro-indol-3-ylidene]-hydrazide (E, yield 77%,
mp 242−244 ◦C). The same method was used to prepare all of the other
analogues of ID45.
In summary, this study has identified a new anti-CVB3
compound, which inhibits CVB3 replication through a
previously nondocumented novel mechanism (Figure 8). Since
the inhibition of CVB3 replication is achieved via suppression of
cap-independent translation initiation of viral RNA, ID45 has the
potential capability to inhibit other members of picornaviruses as
they all utilize the IRES-mediated mechanisms for initiating
protein synthesis. In addition, as the induced activation of Nrf2
and up-regulation of its target gene NQO1 are pro-survival
markers for the host but not for CVB3, the detection of these
markers may serve as a suitable model system for screen of
noncytotoxic broad-spectrum antiviral agents for picornaviruses.
METHODS
■
Virus and Cells. CVB3 (Kandolf strain) was propagated in HeLa
cells. The virus stock was isolated from cells by three cycles of freeze−
Western Analysis. Western blotting was performed by standard
protocols as described previously40 with minor modification. Briefly, the
1022
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
cells were washed with cold PBS before the addition of the lysis buffer.
After incubation for 20 min on ice, the supernatant containing the
proteins was collected by centrifugation at 15,000 × g for 15 min at 4 °C,
and the protein concentration was determined by the Bradford assay
(Bio-Rad). Equal amounts of protein were separated by 7−10% SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto
nitrocellulose membranes. The membranes were blocked with PBS
containing 0.1% Tween-20 and 5% nonfat dry milk and then probed
with one of the following primary antibodies: monoclonal anti-mouse
caspas-3, polyclonal anti-mouse histone H1, monoclonal anti-mouse
GRP78, polyclonal anti-human Nrf2, polyclonal anti-human Keap1, and
monoclonal anti-mouse CHOP from Santa Cruz; polyclonal anti-mouse
α/β-tubulin, anti-human p-PERK, anti-human p-eIF2α, and anti-human
total eIF2α from Cell Signaling; monoclonal anti-mouse NQO1 and
polyclonal anti-mouse GCLM from Abcam; monoclonal anti-mouse β-
actin from Sigma-Aldrich; or monoclonal anti-mouse VP1 from Leica
Microsystem NCL-ENTERO. After washing, each membrane was
incubated with a secondary antibody (goat anti-mouse or goat anti-
rabbit) conjugated to horseradish peroxidase. Signals were detected by
the ECL method according to the manufacturer’s instructions
(Amersham).
Virus Plaque Assay. Virus titers were determined by plaque assay as
described previously.41 Briefly, HeLa cells were seeded onto 6-well
plates (8 × 105 cells/well) and incubated at 37 °C for 20 h. When the cell
confluence reached approximately 90%, cells were washed with PBS and
then overlaid with 500 μL of diluted viral supernatant. The cells were
incubated at 37 °C for 60 min, and the supernatant was removed.
Finally, cells were overlaid with 2 mL of sterilized soft Bacto-agar-
minimal essential medium. The cells were cultured at 37 °C for 72 h,
fixed with Carnoy’s fixative for 30 min, and stained with 1% crystal violet.
The plaques were counted, and the plaque forming unit per milliliter
(pfu/mL) was calculated.
Cell Viability Assay. Cell viability assays were conducted by using a
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium salt (MTS) assay kit (Promega) following the
manufacturer’s instructions. Briefly, cells were incubated with MTS
solution for 4 h, and the absorbance was measured at 492 nm using an
enzyme-linked immunosorbent assay (ELISA) plate reader (TECAN).
The absorbance of nontreated and sham-infected cells was defined as the
value of 100% survival (control), and the remaining data were converted
to a percentage of the control.
Nuclear and Cytosolic Protein Extraction. These proteins were
extracted by using a NE-PER nuclear and cytoplasmic extraction kit
(Pierce) according to the manufacturer’s instructions. Briefly, the cells
were treated with ID45 at a dose of 10 μM overnight, washed with cold
PBS, and harvested by centrifugation of the collected cell suspensions.
The cell pellet was dissolved in Buffer 1 provided in the kit by strong
vortex, followed by incubation on ice. After incubation, cells were
resuspended in Buffer 2 by vortex and incubating. The cell suspension
was centrifuged at 5,000 × g for 5 min to collect the supernatant
containing cytosolic proteins, and the resulting pellet was used for
nuclear protein extraction. The pellet was resuspended in buffer NER by
vortex. After centrifugation at 16,000 × g for 10 min, the supernatant was
collected for nuclear proteins.
post transfection the medium was replaced with serum-containing
DMEM and the cells were continually cultured for 24 h.
Statistic Analysis. Student’s t test was employed to analyze the data.
The results are expressed as means
standard deviations of three
independent experiments. A p value of less than 0.05 was considered
statistically significant.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed methods, supplementary figures, and NMR spectra.
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: nkwjg@nankai.edu.cn.
*E-mail: lzhang03@gmail.com.
*E-mail: decheng.yang@hli.ubc.ca.
Author Contributions
¶These two authors contribute equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the China-Canada Joint Health
Research Initiative grants (30911120483) and in part by China
National Funds for Distinguished Young Scientists (31125002),
the National Program on Key Basic Research Project (973
program, 2013CB734000), Canada Institute of Health Research
(DC0190GP), National Great New Drug Research and
Development Project (No. 2011ZX09102-011-11), and Key
Natural Science Foundation from Tianjin S&T (No.
12JCZDJC25700). M.G.H. is a recipient of the CIHR-IMPACT
and Heart & Stroke Foundation of Canada postdoctoral
fellowship. X.Y. is a recipient of the UGF Award of University
of British Columbia. We would like to thank G. Fung and D.
Kayra for their assistance on confocal microscopy and C.
Vavricka for his comments on the manuscript.
REFERENCES
■
(1) Sagar, S., Liu, P. P., and Cooper, L. T., Jr. (2012) Myocarditis.
Lancet 379, 738−747.
(2) Aguado, L., Thibaut, H. J., Priego, E. M., Jimeno, M. L., Camarasa,
M. J., Neyts, J., and Perez-Perez, M. J. (2010) 9-Arylpurines as a novel
class of enterovirus inhibitors. J. Med. Chem. 53, 316−324.
(3) Ji, X. Y., Zhong, Z. J., Xue, S. T., Meng, S., He, W. Y., Gao, R. M., Li,
Y. H., and Li, Z. R. (2010) Synthesis and antiviral activities of synthetic
glutarimide derivatives. Chem. Pharm. Bull. 58, 1436−1441.
(4) Zhang, L. X., An, R., Wang, J. P., Sun, N., Zhang, S., Hu, J. C., and
Kuai, J. (2005) Exploring novel bioactive compounds from marine
microbes. Curr. Opin. Microbiol. 8, 276−281.
(5) Zhang, L., Yan, K., Zhang, Y., Huang, R., Bian, J., Zheng, C., Sun, H.,
Chen, Z., Sun, N., An, R., Min, F., Zhao, W., Zhuo, Y., You, J., Song, Y.,
Yu, Z., Liu, Z., Yang, K., Gao, H., Dai, H., Zhang, X., Wang, J., Fu, C., Pei,
G., Liu, J., Zhang, S., Goodfellow, M., Jiang, Y., Kuai, J., Zhou, G., and
Chen, X. (2007) High-throughput synergy screening identifies
microbial metabolites as combination agents for the treatment of fungal
infections. Proc. Natl. Acad. Sci. U.S.A. 104, 4606−4611.
Immunocytochemistry and Confocal Microscopy. HeLa cells
proliferating on glass coverslips in a 6-well plate at 70% confluence were
treated with ID45 at 10 μM for 16 h and then washed with PBS. The cells
were stained according to methods described previously.42 The cells
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-
X-100 for 10 min, and stained with an anti-Nrf2 primary antibody and
then with a goat anti-mouse IgG labeled with green-fluorescent Alexa
Fluor 488 dye (Invitrogen). Nuclei were stained with 4′,6′-diamidine-2′-
phenylindole dihydrochloride (DAPI). Cells were observed under a
Leica SP2 AOBS confocal fluorescence microscope.
(6) Wang, Q., Song, F., Xiao, X., Hung, P., Li, L., Monte, A., Abdel-
Mageed, W. M., Wang, J., Guo, H., He, W., Xie, F., Dai, H., Liu, M.,
Chen, C., Xu, H., Liu, M., Piggott, A. M., Liu, X., Capon, R. J., and Zhang,
L. (2013) Abyssomicins from a South China Sea deep-sea sediment
Verrucosispora sp.: Natural thioether Michael addition adducts as
potential antitubercular prodrugs. Angew. Chem., Int. Ed., 1231−1234.
siRNAs and Transfection. All siRNAs (Santa Cruz) used in this
study were transfected with oligofectamine using a kit from Invitrogen.
Briefly, 2 × 105 cells were grown at 37 °C overnight in 6-well plates
containing serum-free DMEM. When the cells reached 60−70%
confluence, they were washed with 1 × PBS and overlaid with
transfection complexes containing siRNAs and oligofectamine. At 6 h
1023
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024

ACS Chemical Biology
Articles
(7) Klump, W. M., Bergmann, I., Muller, B. C., Ameis, D., and Kandolf,
R. (1990) Complete nucleotide sequence of infectious Coxsackievirus
B3 cDNA: two initial 5′ uridine residues are regained during plus-strand
RNA synthesis. J. Virol. 64, 1573−1583.
(8) Yang, D., Wilson, J. E., Anderson, D. R., Bohunek, L., Cordeiro, C.,
Kandolf, R., and McManus, B. M. (1997) In vitro mutational and
inhibitory analysis of the cis-acting translational elements within the 5′
untranslated region of coxsackievirus B3: potential targets for antiviral
action of antisense oligomers. Virology 228, 63−73.
(9) Yang, D., Cheung, P., Sun, Y., Yuan, J., Zhang, H., Carthy, C. M.,
Anderson, D. R., Bohunek, L., Wilson, J. E., and McManus, B. M. (2003)
A shine-dalgarno-like sequence mediates in vitro ribosomal internal
entry and subsequent scanning for translation initiation of coxsack-
ievirus B3 RNA. Virology 305, 31−43.
(10) Kaufman, R. J. (1999) Stress signaling from the lumen of the
endoplasmic reticulum: coordination of gene transcriptional and
translational controls. Genes Dev. 13, 1211−1233.
(11) Qian, Z., Xuan, B., Chapa, T. J., Gualberto, N., and Yu, D. (2012)
Murine cytomegalovirus targets transcription factor ATF4 to exploit the
unfolded protein response. J. Virol. 88, 6712−6723.
(12) Ambrose, R. L., and Mackenzie, J. M. (2013) ATF6 signaling is
required for efficient West Nile virus replication by promoting cell
survival and inhibition of innate immune responses. J. Virol. 87, 2206−
2214.
(13) Jezequel, A. M., and Steiner, J. W. (1966) Some ultrastructural and
histochemical aspects of Coxsackie virus-cell interactions. Lab. Invest. 15,
1055−1083.
(14) Li, J., and Lee, A. S. (2006) Stress induction of GRP78/BiP and its
role in cancer. Curr. Mol. Med. 6, 45−54.
(15) Wu, J., Rutkowski, D. T., Dubois, M., Swathirajan, J., Saunders, T.,
Wang, J., Song, B., Yau, G. D., and Kaufman, R. J. (2007) ATF6alpha
optimizes long-term endoplasmic reticulum function to protect cells
from chronic stress. Dev. Cell 13, 351−364.
(16) Harding, H. P., Zhang, Y., and Ron, D. (1999) Protein translation
and folding are coupled by an endoplasmic-reticulum-resident kinase.
Nature 397, 271−274.
(17) Shi, Y., Vattem, K. M., Sood, R., An, J., Liang, J., Stramm, L., and
Wek, R. C. (1998) Identification and characterization of pancreatic
eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in
translational control. Mol. Cell. Biol. 18, 7499−7509.
(18) Cullinan, S. B., Zhang, D., Hannink, M., Arvisais, E., Kaufman, R.
J., and Diehl, J. A. (2003) Nrf2 is a direct PERK substrate and effector of
PERK-dependent cell survival. Mol. Cell. Biol. 23, 7198−7209.
(19) Zhang, H. M., Ye, X., Su, Y., Yuan, J., Liu, Z., Stein, D. A., and
Yang, D. (2010) Coxsackievirus B3 infection activates the unfolded
protein response and induces apoptosis through downregulation of
p58IPK and activation of CHOP and SREBP1. J. Virol. 84, 8446−8459.
(20) Gilturnes, M. S., Hay, M. E., and Fenical, W. (1989) Symbiotic
marine-bacteria chemically defend crustacean embryos from a
pathogenic fungus. Science 246, 116−118.
(21) Quenelle, D. C., Keith, K. A., and Kern, E. R. (2006) In vitro and
in vivo evaluation of isatin-beta-thiosemicarbazone and marboran
against vaccinia and cowpox virus infections. Antiviral Res. 71, 24−30.
(22) Kang, I. J., Wang, L. W., Hsu, T. A., Yueh, A., Lee, C. C., Lee, Y. C.,
Lee, C. Y., Chao, Y. S., Shih, S. R., and Chern, J. H. (2011) Isatin-beta-
thiosemicarbazones as potent herpes simplex virus inhibitors. Bioorg.
Med. Chem. Lett. 21, 1948−1952.
(23) Medvedev, A., Buneeva, O., and Glover, V. (2007) Biological
targets for isatin and its analogues: Implications for therapy. Biol.:
Targets Ther. 1, 151−162.
(24) Chau, D. H., Yuan, J., Zhang, H., Cheung, P., Lim, T., Liu, Z., Sall,
A., and Yang, D. (2007) Coxsackievirus B3 proteases 2A and 3C induce
apoptotic cell death through mitochondrial injury and cleavage of
eIF4GI but not DAP5/p97/NAT1. Apoptosis 12, 513−524.
(25) Harding, H. P., Zhang, Y., Zeng, H., Novoa, I., Lu, P. D., Calfon,
M., Sadri, N., Yun, C., Popko, B., Paules, R., Stojdl, D. F., Bell, J. C.,
Hettmann, T., Leiden, J. M., and Ron, D. (2003) An integrated stress
response regulates amino acid metabolism and resistance to oxidative
stress. Mol. Cell 11, 619−633.
(26) Harding, H. P., Novoa, I., Zhang, Y., Zeng, H., Wek, R., Schapira,
M., and Ron, D. (2000) Regulated translation initiation controls stress-
induced gene expression in mammalian cells. Mol. Cell 6, 1099−1108.
(27) Cullinan, S. B., and Diehl, J. A. (2004) PERK-dependent
activation of Nrf2 contributes to redox homeostasis and cell survival
following endoplasmic reticulum stress. J. Biol. Chem. 279, 20108−
20117.
(28) Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarashi, K., Engel, J.
D., and Yamamoto, M. (1999) Keap1 represses nuclear activation of
antioxidant responsive elements by Nrf2 through binding to the amino-
terminal Neh2 domain. Genes Dev. 13, 76−86.
(29) Kwon, J., Han, E., Bui, C. B., Shin, W., Lee, J., Lee, S., Choi, Y. B.,
Lee, A. H., Lee, K. H., Park, C., Obin, M. S., Park, S. K., Seo, Y. J., Oh, G.
T., Lee, H. W., and Shin, J. (2012) Assurance of mitochondrial integrity
and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade.
EMBO Rep. 13, 150−156.
(30) Chan, K., and Kan, Y. W. (1999) Nrf2 is essential for protection
against acute pulmonary injury in mice. Proc. Natl. Acad. Sci. U.S.A. 96,
12731−12736.
(31) Lee, J. M., Shih, A. Y., Murphy, T. H., and Johnson, J. A. (2003)
NF-E2-related factor-2 mediates neuroprotection against mitochondrial
complex I inhibitors and increased concentrations of intracellular
calcium in primary cortical neurons. J. Biol. Chem. 278, 37948−37956.
(32) Schaedler, S., Krause, J., Himmelsbach, K., Carvajal-Yepes, M.,
Lieder, F., Klingel, K., Nassal, M., Weiss, T. S., Werner, S., and Hildt, E.
(2010) Hepatitis B virus induces expression of antioxidant response
element-regulated genes by activation of Nrf2. J. Biol. Chem. 285,
41074−41086.
(33) Alard, A., Fabre, B., Anesia, R., Marboeuf, C., Pierre, P., Susini, C.,
Bousquet, C., and Pyronnet, S. (2010) NAD(P)H quinone-oxy-
doreductase 1 protects eukaryotic translation initiation factor 4GI
from degradation by the proteasome. Mol. Cell. Biol. 30, 1097−1105.
(34) Jackson, R. J., Hellen, C. U., and Pestova, T. V. (2010) The
mechanism of eukaryotic translation initiation and principles of its
regulation. Nat. Rev. Mol. Cell. Biol. 11, 113−127.
(35) Moscovitz, O., Tsvetkov, P., Hazan, N., Michaelevski, I., Keisar,
H., Ben-Nissan, G., Shaul, Y., and Sharon, M. (2012) A mutually
inhibitory feedback loop between the 20S proteasome and its regulator,
NQO1. Mol. Cell 47, 76−86.
(36) Prevot, D., Darlix, J. L., and Ohlmann, T. (2003) Conducting the
initiation of protein synthesis: the role of eIF4G. Biol. Cell 95, 141−156.
(37) Ohlmann, T., Rau, M., Pain, V. M., and Morley, S. J. (1996) The
C-terminal domain of eukaryotic protein synthesis initiation factor (eIF)
4G is sufficient to support cap-independent translation in the absence of
eIF4E. EMBO J. 15, 1371−1382.
(38) Claycomb, W. C., Lanson, N. A., Jr., Stallworth, B. S., Egeland, D.
B., Delcarpio, J. B., Bahinski, A., and Izzo, N. J., Jr. (1998) HL-1 cells: a
cardiac muscle cell line that contracts and retains phenotypic
characteristics of the adult cardiomyocyte. Proc. Natl. Acad. Sci. U.S.A.
95, 2979−2984.
(39) Shang, J. L., Guo, H., Li, Z. S., Ren, B., Li, Z. M., Dai, H. Q., Zhang,
L. X., and Wang, J. G. (2013) Synthesis and evaluation of novel
sulfenamides as novel anti Methicillin-resistant Staphylococcus aureus
agents. Bioorg. Med. Chem. Lett. 23, 724−727.
(40) Liu, Z., Zhang, H. M., Yuan, J., Lim, T., Sall, A., Taylor, G. A., and
Yang, D. (2008) Focal adhesion kinase mediates the interferon-gamma-
inducible GTPase-induced phosphatidylinositol 3-kinase/Akt survival
pathway and further initiates a positive feedback loop of NF-kappaB
activation. Cell Microbiol. 10, 1787−1800.
(41) Yuan, J., Liu, Z., Lim, T., Zhang, H., He, J., Walker, E., Shier, C.,
Wang, Y., Su, Y., Sall, A., McManus, B., and Yang, D. (2009) CXCL10
inhibits viral replication through recruitment of natural killer cells in
coxsackievirus B3-induced myocarditis. Circ. Res. 104, 628−638.
(42) Sall, A., Zhang, H. M., Qiu, D., Liu, Z., Yuan, J., Lim, T., Ye, X.,
Marchant, D., McManus, B., and Yang, D. (2010) Pro-apoptotic activity
of mBNIP-21 depends on its BNIP-2 and Cdc42GAP homology (BCH)
domain and is enhanced by coxsackievirus B3 infection. Cell Microbiol.
12, 599−614.
1024
dx.doi.org/10.1021/cb400775z | ACS Chem. Biol. 2014, 9, 1015−1024