Discovery and Mechanistic Elucidation of a Class of Protein Disulfide Isomerase Inhibitors for the Treatment of Glioblastoma

Article abstract of DOI:10.1002/cmdc.201700629

Protein disulfide isomerase (PDI) is overexpressed in glioblastoma, the most aggressive form of brain cancer, and folds nascent proteins responsible for the progression and spread of the disease. Herein we describe a novel nanomolar PDI inhibitor, pyrimidotriazinedione 35G8, that is toxic in a panel of human glioblastoma cell lines. We performed a medium-throughput 20 000-compound screen of a diverse subset of 1 000 000 compounds to identify cytotoxic small molecules. Cytotoxic compounds were screened for PDI inhibition, and, from the screen, 35G8 emerged as the most cytotoxic inhibitor of PDI. Bromouridine labeling and sequencing (Bru-seq) of nascent RNA revealed that 35G8 induces nuclear factor-like 2 (Nrf2) antioxidant response, endoplasmic reticulum (ER) stress response, and autophagy. Specifically, 35G8 upregulated heme oxygenase 1 and solute carrier family 7 member 11 (SLC7A11) transcription and protein expression and repressed PDI target genes such as thioredoxin-interacting protein 1 (TXNIP) and early growth response 1 (EGR1). Interestingly, 35G8-induced cell death did not proceed via apoptosis or necrosis, but by a mixture of autophagy and ferroptosis. Cumulatively, our data demonstrate a mechanism for a novel PDI inhibitor as a chemical probe to validate PDI as a target for brain cancer.

Full text of DOI:10.1002/cmdc.201700629

Accepted Article
Title: Discovery and Mechanistic Elucidation of a Class of PDI
Inhibitors for the Treatment of Glioblastoma
Authors: Anahita Kyani, Shuzo Tamura, Suhui Yang, Andrea Shergalis,
Soma Somanta, Yuting Kuang, Mats Ljungman, and Nouri
Neamati
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10.1002/cmdc.201700629
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Discovery and Mechanistic Elucidation of a Class of PDI
Inhibitors for the Treatment of Glioblastoma
Anahita Kyani, Ph.D.[a], ‡, Shuzo Tamura, M.D.[a], ‡, Suhui Yang, Ph.D.[a], ‡, Andrea Shergalis, M.S.[a], ‡
Soma Samanta, Ph.D.[a], Yuting Kuang, Ph.D.[a], Mats Ljungman, Ph.D.[b], Nouri Neamati, Ph.D.[a]*
,
[a]
[b]
Dr. A. Kyani, Dr. S. Tamura, Dr. S. Yang, A. Shergalis, M.S., Dr. S. Somanta, Dr. Y Kuang, Prof. M. Ljungman,
Department of Medicinal Chemistry, College of Pharmacy
University of Michigan
North Campus Research Complex, 1600 Huron Parkway, Ann Arbor, Michigan 48109, United States
E-mail: neamati@umich.edu
Prof. M. Ljungman
Departments of Radiation Oncology and Environmental Health Sciences
University of Michigan
North Campus Research Complex, 1600 Huron Parkway, Ann Arbor, Michigan 48109, United States
Supporting information for this article is given via a link at the end of the document.
Abstract: Protein disulfide isomerase (PDI) is overexpressed in
glioblastoma, the most aggressive form of brain cancer, and folds
nascent proteins responsible for the progression and spread of the
disease. Herein, we describe a novel, nanomolar PDI inhibitor,
pyrimidotriazinedione 35G8, that is toxic in a panel of human
glioblastoma cell lines. We performed a medium throughput 20,000-
compound screen of a diverse subset of 1,000,000 compounds to
identify cytotoxic small molecules. Cytotoxic compounds were
screened for PDI inhibition, and, from the screen, 35G8 emerged as
the most cytotoxic inhibitor of PDI. Bromouridine-labeling and
sequencing (Bru-seq) of nascent RNA revealed that 35G8 induced
Nrf2 (nuclear factor-like 2) antioxidant response, endoplasmic
reticulum stress response, and autophagy. Specifically, 35G8
upregulated heme oxygenase 1 and SLC7A11 (solute carrier family 7
member 11) transcription and protein expression and repressed PDI
target genes such as TXNIP (thioredoxin-interacting protein 1) and
EGR1 (early growth response 1). Interestingly, 35G8-induced cell
death did not proceed via apoptosis or necrosis, but by a mixture of
autophagy and ferroptosis. Cumulatively, our data demonstrate a
mechanism for a novel PDI inhibitor as a chemical probe to validate
PDI as a target for brain cancer.
Protein disulfide isomerase (PDI; EC 5.3.4.1) is a 57-kDa
endoplasmic reticulum (ER) oxidoreductase of the thioredoxin
superfamily that assists protein folding in the ER by catalyzing
disulfide rearrangements (isomerase activity), disulfide formation
(oxidase activity), and disulfide reduction (reductase activity).[3, 4]
PDI is overexpressed in several cancers but most significantly in
glioblastoma.[3] Previously, we demonstrated that PDI knockdown
by siRNA leads to substantial cytotoxicity in ovarian cancer
cells.[5] PDI inhibitors and modulators are being developed to
combat cancer and neurological diseases (for a comprehensive
review of PDI inhibitors, see ref. 3 and 4a). The PDI inhibitor
bacitracin inhibits migration and invasion of glioblastoma cells[6]
and enhances apoptosis caused by ER stress-inducing agents in
melanoma cells.[7] Another class of compounds, T8, are weak
inhibitors of PDI and, at moderately high concentrations, sensitize
several cancer cell lines to etoposide treatment.[8] A reversible,
selective, non-toxic PDI inhibitor, ML359, was developed as a
probe to study thrombosis-related diseases.[9] Modulators of PDI
have also been shown to be neuroprotective. A reversible PDI
modulator, LOC14 (EC50 = 500 nM), has neuroprotective effects
in cellular and rat models of Huntington’s disease.[10] Furthermore,
PDI inhibitor CCF642 was demonstrated to be effective in a
mouse xenograft model of multiple myeloma.[11] Mounting
evidence highlights PDI as an important target against several
diseases including cancer, emphasizing the need for potent,
clinically relevant PDI inhibitors for cancer treatment.
Introduction
Glioblastoma is the most common type of malignant central
nervous system (CNS) tumor. Prevalence increases with age with
peak incidence in individuals aged 60-79 years.[1] Despite the
treatment options available – surgical resection followed by
chemoradiotherapy and adjuvant chemotherapy (temozolomide)
Herein, we report on the development of 35G8 as a novel and
potent PDI inhibitor that demonstrates activity in brain cancer cells
and has drug-like properties. The activity of 35G8 in a diverse set
of robust assays confirmed that the initial observation of activity
was not a consequence of its redox-cycling status. Results from
nascent RNA Bru-seq[12] analysis showed that the transcription of
498 genes increased and 238 genes decreased at least 2-fold
following a 4-hour incubation with 35G8 in U87MG glioblastoma
cells. Gene set enrichment analysis demonstrated the
upregulated genes to be involved in the Nrf2 antioxidant response
and the unfolded protein response (UPR). Genes with decreased
transcription involved histone and DNA repair pathways. In
–
the five-year survival rate of patients diagnosed with
glioblastoma is only 5.0 %.[1, 2] Current treatments are marginally
effective and the number of cases is expected to grow with the
aging population, emphasizing the urgent need for the
development of novel and effective therapies for glioblastoma.
Disease recurrence and drug resistance remain the major
challenges for a successful cure.
1
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10.1002/cmdc.201700629
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known stabilizing ligands, such as estradiol.[17] To further validate
35G8 binding to PDI, we performed the cellular thermal shift
assay (CETSA) and drug affinity responsive target stability
(DARTS) assay. 35G8 also destabilized PDI via CETSA (Figure
2C). 35G8 had little effect on a related molecular chaperone,
GRP78, but did seem to stabilize the cysteine-containing
glutathione-transferase Omega 1 (GSTO1). In the DARTS assay,
U87MG cell lysates were subjected to pronase degradation in the
presence or absence of PACMA31 or 35G8 (Figure 2D). Both
compounds protected PDI from proteolysis, but had no effect on
the degradation of GRP78 or GSTO1. These results established
35G8 as a potent, selective inhibitor of PDI.
addition, 35G8 upregulates two key genes, SLC7A11 and
HMOX1, and may kill cells through an iron-dependent form of cell
death independent of apoptosis and necrosis, called
ferroptosis.[13] The alterations in the transcriptional landscape
induced by 35G8 provide a more comprehensive understanding
of the mechanisms of PDI inhibition in brain cancer therapy.
Results and Discussion
35G8 is a nanomolar inhibitor of PDI
We further validated 35G8 as a bona fide PDI inhibitor by
examining several of its close derivatives. Of the 16 analogues
reported in the National Cancer Institute (NCI) database (Table
S1), we pursued a refined group of eight compounds from the
Developmental Therapeutics Program and tested their purity
using UPLC-MS. Three of the eight compounds (NSC 67078,
99733 and 280172; hereafter referred to as NC72, NC75, and
NC79, respectively) were over 95% pure and were tested in the
insulin turbidity assay (Table 1).
To identify cytotoxic small molecules, we screened a highly
diverse library of 20,000 compounds, representing over one
million compounds, in the colon cancer cell line HCT116 (Figure
1). From the initial screen, we identified 443 cytotoxic compounds
with IC50 values under 10 μM. These 443 compounds were tested
for PDI inhibition in an insulin turbidity assay.[14] Eight compounds
demonstrated potent inhibition (IC50 < 1.0 μM), and after
confirming the activity with re-purchased compound stocks and
verifying a dose-dependent response, the most potent compound,
1,3,6-trimethylpyrimido[5,4-e] [1,2,4] triazine-5,7(1H,6H)-dione
(35G8), was selected for further analysis and optimization.
We also synthesized several analogues of 35G8 to validate the
above findings. The lead compound, 35G8, contains methyl
substituents at the three N1, C3, and N6 positions (Figure 1). We
incorporated various substituents at the C3 position while
maintaining the methyl groups at N1 and N6 due to the efficient
introduction of the N1 and N6 methyl groups early in the synthesis
(Scheme 1). Nucleophilic attack of methylhydrazine on 6-chloro-
3-methyl uracil (1) led to hydrazinylpyrimidine-2,4(1H,3H)-dione
(2a).[18] Further condensation with aldehydes furnished the
corresponding hydrazones (3a-f). Each hydrazone was cyclized
by treatment with sodium nitrite in acetic acid/water to afford a
mixture of pyrimidotriazinediones (4a-f) and the corresponding N-
oxide derivative (5d).
All 35G8 analogues had strong PDI inhibitory activity with
submicromolar IC50 values, except NC75 (> 120 μM) and NC79
(6.55 ± 1.19 μM) in the insulin turbidity assay (Table 1). The
pyrimidotriazinedione compound (35G8, IC50: 0.17 ± 0.01 μM)
was more potent than the corresponding N-oxide compound
(NC79). A similar trend was observed between 4d (IC50: 0.36 ±
0.05 μM) and 5d (IC50: 0.42
± 0.07 μM). Among the
pyrimidotriazinediones, the compounds containing a methyl group
(4a) or no substituent (NC72) at R1 had enhanced activity
compared to those with an aromatic moiety (4b-f), likely due to
steric effects (Figure S1A). Interestingly, the PDI inhibitory
activity was abolished upon removal of the methyl substituent at
R2 (NC75: IC50 > 120 µM) compared to NC72 (IC50: 0.11 µM),
indicating that the methyl group at R2 may be necessary to retain
PDI inhibitory activity (Figure S1B). Furthermore, the removal of
PDI inhibitory activity abolished the cytotoxicity of the compound.
Figure 1. Discovery of 35G8. Work flow summarizing the screening process
that identified 35G8 as a potent PDI inhibitor. 20,000 compounds were screened
in an MTT assay with HCT116 cells and 443 compounds were cytotoxic in these
cells. The 443 compounds were tested further in an insulin turbidity assay; 35G8
had the most potent IC50 value and was taken for further biochemical analysis
and optimization.
We next used the thermal shift assay[15] to validate whether 35G8
stabilizes its presumed target, PDI. Intriguingly, 35G8 destabilized
PDI, indicated by the decrease in melting temperature of the
protein (Figure 2A). The dose-dependence of the negative
thermal shifts at all concentrations tested (ΔTm: −3.64 °C at 100
µM; −2.94 °C at 10 µM; −1.43 °C at 1 µM) (Figure 2B) provides
further evidence that 35G8 associates with and destabilizes PDI.
The melting temperature of a protein shifts positively or negatively
in the presence of a ligand, and this change in melting
temperature parallels the stability of the protein.[16] These results
suggest 35G8 interacts with PDI at a unique site compared to
35G8 analogues inhibit glioblastoma cell proliferation
All synthesized compounds demonstrated potent cytotoxicity in
four glioblastoma cell lines, U87MG, U118MG, A172 and NU04,
with IC50 values under 10 μM, except 4c (Table 2). The IC50 value
of 35G8 in U87MG cells is 1.1 ± 0.2 μM. NC72 demonstrated the
most potent cytotoxicity (IC50 = 0.5 ± 0.1 μM), complementing its
potency in the PDI assay. NC75 and NC79 had little effect on cell
growth. Interestingly, this suggests that the methyl substituent is
2
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Figure 2. 35G8 destabilizes PDI. (A) Thermal shifts observed for recombinant PDI (0.3 mg/ml) with various concentrations of 35G8. DMSO was used as a control.
(B) Apparent melting temperatures (Tm) and change in melting temperature derived from ThermoFluor assay (C) Protein expression of PDI, GRP78, GSTO1, and
actin (loading control) in the absence or presence of 35G8 at varying temperatures in the cellular thermal shift assay (D) Western blot analysis of DARTS assay
with PDI, GRP78, and GSTO1 subjected to 100 μM PACMA31 (P), 100 μM 35G8 (G), or DMSO (-). Samples were subjected to varying concentrations of pronase.
Data are means from three independent experiments.
important for both PDI activity (as seen in the dramatic IC50 value
increase from NC72 to NC75) and cytotoxicity.
an iron chelator (Figure S2B). 35G8-induced cell death was
rescued in the presence of DFO, suggesting ferroptosis may play
a role in 35G8-induced cell death.
35G8 may bind in the catalytic site of PDI
The pyrimidotriazinedione class may bind PDI catalytic sites
preferentially and interact with the cysteine residues (Figure 3A-
C). Interestingly, the compounds are predicted to bind the C-
terminal C397 site over the sequence-identical N-terminal C53
site, likely due to the composition of a binding pocket around
C397. Docking pose conformation of 35G8 in the C397 catalytic
site and the corresponding PoseView26 representation illustrate
hydrogen bonding and pi-pi interactions with important residues
of the active site: K424, E391 and Y393 (Figure 3D). The
synthesized analogues, an additional 409 analogues of 35G8,
and the NC compounds also bind in the N-terminal active site
pocket, forming similar interactions (Figure S1B). The docking
poses of the synthesized analogues (4b-5d) indicate that the
larger substitution at R1 is subject to steric hindrance that
decreases potency (Figure S1A). NC75 has the lowest docking
score for binding to the catalytic site (Figure S1B), in agreement
with its inactivity in vitro. This suggests that the presence of a
methyl group at the R2 position is important for the hydrophobic
interaction in the binding site.
Scheme 1. Synthesis of 3-substituted 35G8 analogues. Reagents and
conditions: (a) methylhydrazine, EtOH, reflux; (b) aldehyde (R-CHO),
anhydrous EtOH, room temperature; (c) NaNO2, AcOH/H2O, room temperature.
Pretreatment with Z-VAD-FMK, an irreversible caspase
inhibitor,[19] and necrostatin-1, a necroptosis inhibitor,[20] did not
protect the cells from 35G8-induced cell death (Figure S2A).
These results indicate that neither necrosis nor apoptosis are the
main pathways responsible and another pathway may be
implicated in cell death. To assess the role of ferroptosis upon
35G8 treatment, we treated the cells with deferoxamine (DFO),
3
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Table 1. PDI inhibitory activity of 35G8 analogues. IC50 values obtained in insulin turbidity assay. Data are means ± standard deviation from three independent
experiments.
Compound
35G8 (4a)[a]
4b[b]
Basic Module
R1
R2
IC50 (μM)
0.17 ± 0.01
0.39 ± 0.03
A
A
CH3
CH3
CH3
4c[c]
4d[d]
A
A
CH3
CH3
0.33 ± 0.04
0.36 ± 0.05
4e[e]
4f[f]
A
CH3
0.32 ± 0.01
A
B
CH3
CH3
0.24 ± 0.04
0.42 ± 0.07
5d[g]
NC72 (NSC67078)
A
A
B
H
H
CH3
H
0.105 ± 0.004
> 120
NC75
(NSC99733)
NC79
(NSC280172)
CH3
CH3
6.55 ± 1.19
PACMA31
-
-
-
5.81 ± 1.23
[a] 1,3,6-Trimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione. [b] 1,6-Dimethyl-3-phenylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione. [c] 3-Benzyl-1,6-
dimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione. [d] 3-(4-Methoxyphenyl)-1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione. [e] 3-(3-
Methoxyphenyl)-1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione. [f]1,6-Dimethyl-3-(4-nitrophenyl)pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione. [g] 3-
(4-Methoxyphenyl)-1,6-dimethyl-5,7-dioxo-1,5,6,7-tetrahydropyrimido[5,4-e][1,2,4]triazine 4-oxide.
35G8 induces the Nrf2 antioxidant pathway and ER stress
response
Enrichment Analysis) (Figure 4B, Table S5 and S6). GSEA
snapshots of enriched gene sets are reported in Figure S3 and
S4. GSEA revealed enrichment of the Nrf2-mediated oxidative
stress response upon 35G8 treatment (Figure 4B). Treatment
To better elucidate the cellular response to the
pyrimidotriazinediones, we performed nascent RNA sequencing
using the Bru-seq[22] method and analyzed changes in gene
transcription rates in response to 35G8 in U87MG cells. Four
hours after 35G8 treatment, 498 genes were upregulated at least
two-fold and 238 genes were downregulated at least two-fold.
Many of the top upregulated genes are implicated in the Nrf2
antioxidant response, ER stress response, and autophagy. We
identified the top 20 upregulated and downregulated gene sets
(Table S2 and S3) and analyzed the genes that were upregulated
or downregulated at least two-fold with IPA (Ingenuity Pathway
Analysis) (Figure 4A and Table S4) and GSEA (Gene Set
also
correlates
with
set,
KOBAYASHI_EGFR_SIGNALING_24HR_DN
gene
suggesting 35G8 may inhibit EGFR signaling. DAVID (the
Database for Annotation, Visualization and Integrated Discovery)
analysis and GSEA identified functional terms related to ER and
redox-active disulfide, providing further evidence for PDI inhibition
by 35G8 (Figure 4C and 4D).
The upregulation of Nrf2 response genes, including HMOX1 (19-
fold increase), SLC7A11 (63-fold increase), AKR1C1 (59-fold
increase), and LOC344887 (23-fold increase), is likely
a
protective response to the insults caused by 35G8 (Figure 4E).
4
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Figure 3. Docking of 35G8 analogues on PDI reveals their interaction with catalytic cysteine 397. (A) Location of the three binding pockets on the domain architecture
of PDI. (B) Heat map plot for docking of 409 analogues of 35G8 in three binding pockets of PDI. (C) Structural overview of ten 35G8 analogues docked in PDI
binding sites. The catalytic cysteines are colored by atom in a space-filling representation. The rest of the protein is depicted in grey and orange. The docked
structures are shown in purple, green and blue for the C53, H256, and C397 site, respectively. (D) Docking pose of 35G8 in the C397 catalytic site of the PDI along
with a PoseView representation showing its interactions with the binding site residues.
mTORC1 pathway.[26] IRS2 (12-fold increase) activation induces
We also confirmed parallel increases in HMOX1 and SLC7A11
protective autophagy to clear unwanted protein aggregates[27] and
protein expression (Figure 4F). The Nrf2 antioxidant pathway
may also help remove damaged cells. TMEM74 (28-fold
mitigates oxidative stress by inducing antioxidant response
increase), a transmembrane protein localized to the lysosome
elements.[23] PDI is vital in the UPR, and inhibiting this key protein
and autophagosome, regulates autophagy.[28] The increased
disrupts proteostasis, ultimately leading to ER stress and cell
transcription of these autophagy-related genes prompted us to
death when the cell cannot cope with the accumulation of
misfolded proteins. ER stress target genes downstream the
PERK-ATF4 ER stress response pathway, CHAC1 (46-fold
increase), DDIT3 (4-fold increase), and HSPA5 (8-fold increase)
increased as a result of 35G8 treatment (Figure 4E). Protein
expression of GRP78 (HSPA5) and DDIT3 increased upon 24-
hour treatment of 2 μM 35G8 (Figure 4G); however, CHAC1
results indicate that 35G8 induces the ER stress and Nrf2
protein was undetectable, likely because the CHAC1 protein is
rapidly degraded by the proteasome (data not shown).[24] mRNA
measure protein expression of several autophagy markers
(Figure 4H). Cleaved LC3B expression increased significantly
after 24-hour treatment with 2 μM 35G8, however expression
levels of other autophagy markers, including ATG3, ATG5, ATG7,
and beclin 1, did not change (data not shown), suggesting that
autophagy may play a more protective role in this case. These
response in brain cancer cells to contribute to cell death. GSEA
also showed that 35G8 treatment repressed many genes involved
expression of other downstream targets of the PERK-ATF4 ER
in DNA repair pathways such as mismatch repair, homologous
stress response pathway, including TRIB3 and ASNS,[25] also
recombination, base excision repair and nucleotide excision
increased in response to 35G8 (Figure 4E). These results
suggest that brain cancer cells rely on PDI to maintain redox
homeostasis, and when PDI is inhibited, cells undergo
irremediable ER stress that leads to cell death.
We also identified several autophagic signaling genes that
respond to ER stress triggered by 35G8, including TRIB3, IRS2,
and TMEM74 (Figure 4E). TRIB3 (23-fold increase), as a
downstream target of ATF4, mediates autophagy by inhibiting the
repair (Figure S5). Even though not all pathways showed
significance individually from GSEA, the fact that all of them were
suppressed suggests that the expression of these DNA repair
genes is regulated by a common transcription factor that requires
PDI-mediated folding for proper activity. These findings open up
the interesting possibility that 35G8 could act synergistically with
DNA-damaging agents and have therapeutic implications.
5
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Table 2. In vitro cytotoxicity of 35G8 analogues in a panel of human glioblastoma cell lines. Cytotoxicity measured in the MTT assay. Data are means from at least
three independent experiments.
IC50 (μM)
Compound
U87MG
U118MG
NU04
A172
35G8
4b
1.1 ± 0.2
3.0 ± 0.3
12.7 ± 3.7
1.2 ± 0.2
1.1 ± 0.2
1.8 ± 0.7
1.9 ± 0.7
0.5 ± 0.1
> 100
3.9 ± 0.1
4.6 ± 0.5
24.0 ± 7.4
3.9 ± 0.6
2.4 ± 0.6
6.2 ± 1.6
4.3 ± 0.1
-
0.8 ± 0.2
3.7 ± 1.2
> 30
2.0 ± 0.6
1.8 ± 0.4
8.2 ± 2.5
1.5 ± 0.4
1.5 ± 0.1
1.1 ± 0.2
1.7 ± 0.1
-
4c
4d
0.86 ± 0.04
0.76 ± 0.22
4.9 ± 1.2
1.5 ± 0.7
-
4e
4f
5d
NC72
NC75
NC79
PACMA31
-
-
-
> 100
-
-
-
0.13 ± 0.07
0.28 ± 0.04
0.4 ± 0.1
0.12 ± 0.10
Bru-seq analysis identifies novel glioblastoma markers
35G8 induces ferroptosis
AKR1C1, IL-6, CHAC1 and TNFSF9 are among the top 20
upregulated genes with significantly decreased expression in
brain cancer compared to normal brain tissues (Figure 5A-C).
Conversely, genes that were downregulated upon 35G8
treatment, including TXNIP (−7.40-fold change), EGR1 (−5.65-
fold change), and ITGA3 (−3.89-fold change) are often
overexpressed in brain cancer (Figure 5D-F). Additional genes
affected include HMOX1, IRS2, SLC7A11, and mir181A2HG
(Figure S6). These data suggest 35G8 inhibits transcription of
these mRNA, or inhibits an upstream regulator of ITGA3 and
EGR1. The results also indicate a gene such as IL6 may be used
as a biomarker of 35G8 inhibition in future studies and EGR1 may
be a novel glioblastoma marker.
Both transcription and protein expression of HMOX1 and
SLC7A11 are highly upregulated by 35G8 (Figure 4E and 4F).
These proteins have been implicated in the non-apoptotic cell
death mechanism, ferroptosis. HMOX1 is necessary for
ferroptosis and is a major source of iron in the body.[29] Inhibition
of cysteine-glutamate exchange through system xc-, of which
SLC7A11 is a component, induces iron-dependent cell death.[30]
To determine whether 35G8 induces ferroptosis in U87MG cells,
we treated the cells in the presence or absence of deferoxamine
(DFO), an iron chelator (Figure S2B).[31] In the presence of DFO,
35G8 is almost three times less potent (IC50 = 5.8 ± 1.0 μM) than
when used alone (IC50 = 2.2 ± 0.7 μM). These data suggest that
PDI may play an important role in preventing ferroptosis in brain
cancer.
35G8 induces ROS formation
Because the cells responded to 35G8 by upregulating the Nrf2-
mediated oxidative stress response, we investigated the
production of reactive oxygen species (ROS) by 35G8 and its
analogues to determine whether the cytotoxicity of these
compounds is dependent on ROS induction. We observed
significant ROS induction by all 35G8 analogues tested at 5 μM
as early as four hours after treatment, except for 4c (Figure 6).
ROS accumulation with these compounds was time-dependent.
At 24 hours, 5 μM 35G8 treatment achieved maximal ROS
induction, comparable to 100 μM hydrogen peroxide treatment
(Figure 6C). No change in the fluorescent signal in the samples
containing 35G8 without H2DCFDA dye was observed,
eliminating the possibility of endogenous fluorescence affecting
the assay. N-acetyl-cysteine (NAC) did not affect the cytotoxicity
of 35G8 significantly (Table S7). This suggests 35G8-induced cell
death is not solely dependent on ROS induction.
35G8 is expected to cross the blood-brain barrier
The likelihood of blood-brain barrier (BBB) permeation, AlogP,
water solubility, polar surface area, and number of rotatable
bonds of 35G8 and its synthesized analogues were determined
with a qualitative model in the ADMET predictor (Version 7.0)
(Table S8; Figure S7). The AlogP of the compounds is between
-1.1 and 1.1 and the likelihood of BBB permeation is high. The
polar surface area of 35G8 is less than 90 Å2, the cutoff for
predicted CNS penetration.[32] The average molecular weight of
marketed CNS compounds is 310, and the 35G8 analogues range
in molecular weight from 207 – 315. Similarly, TMZ has a
molecular weight of 194 Da, ClogP of -0.82, and a polar surface
area of 108 Å2. These data demonstrate that 35G8 will be able to
cross the blood-brain barrier.
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Figure 4. Effects of 35G8 treatment on cellular pathways. (A) Pathways from the Bru-seq analysis of 35G8-treated cells. (B) GSEA for “NFE2L2.V2,” the top gene
set matched with upregulated genes from Bru-seq results. Functional terms represented by genes upregulated (C) and downregulated (D) at least 2-fold by 35G8
treatment. Pathway analysis was performed using DAVID (left) and GSEA (right). (E) Histograms of differentially expressed proteins between 35G8-treated and
DMSO-treated U87MG cells. Fold change bars are in black for UPR genes, dark grey for autophagy-related genes, and light grey for Nrf2-related genes. (F) Western
blot showing Nrf2-regulated proteins SLC7A11 and HMOX1 expression upon 24-hour treatment of U87MG cells with 1 or 2 μM 35G8. (G) Western blot of ER stress-
induced proteins DDIT3 and GRP78 expression upon 24-hour treatment of U87MG cells with 1 and 2 μM 35G8. (H) Western blot of autophagy-related proteins
LC3B, beclin 1, ATG3, ATG5, and ATG7 expression upon 24-hour treatment of U87MG cells with 1 (+) and 2 (++) μM 35G8. -: vehicle-treated control. GAPDH used
as a loading control. Experiments repeated in triplicate.
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Discussion
the UPR and silenced by JNK, is expressed to remove damaged
cells.[42] 35G8 treatment initiates a protective response by
upregulating the UPR and inducing autophagy to combat ER
stress. Ultimately, unbalanced homeostatic mechanisms
overwhelm the cellular machinery, and this leads to cell death.
ROS induction is likely responsible for the increased expression
levels of TXNRD1 (9-fold increase) and TXN (2-fold increase).
TXNIP inhibits TXN activity, and TXNIP expression is significantly
inhibited by 35G8 treatment (7.4-fold decrease). ER stress
activates the ERK1/2 MAP kinase signaling pathway, repressing
TXNIP expression leading to thioredoxin nuclear translocation.[43]
Interestingly, TXNIP is overexpressed in brain cancer patients.
Furthermore, TXNIP can bind PDI and increase its activity.[43]
Lower TXNIP levels allow TRX to bind ASK1 and prevent
apoptosis.[44] Therefore, decreased expression of TXNIP may
contribute to the absence of apoptosis signaling observed upon
35G8 treatment.
Another class of genes that were repressed by 35G8 are involved
in DNA repair (Figure S5). While this repression was not
dramatic, GSEA analysis showed that several genes involved in
mismatch repair, homologous recombination, base excision
repair and nucleotide excision repair had reduced transcription
following 35G8 treatment. It is possible that these genes share a
common transcription factor that requires PDI-assisted protein
folding for optimal function. Importantly, these findings suggest
that 35G8 may be used in combination with DNA damaging
agents or PARP1 inhibitors to augment their therapeutic
effectiveness.
The screen of 20,000 diverse compounds in a growth inhibition
assay produced 35G8 as the most potent inhibitor of proliferation
of the colon cancer cell line HCT116. 35G8 destabilizes PDI and
blocks its reductase activity. As a consequence, 35G8 likely
causes cell death via continuous activation of ER stress and
disruption of homeostatic balance, among other factors. 35G8
was validated in orthogonal assays to rule out that activity was not
a consequence of its redox-cycling status. 35G8 generates H2O2
in the presence of DTT at the concentrations used in the PDI
assay (Figure S8A), however, H2O2 does not interfere with insulin
reduction catalyzed by PDI (Figure S8B). The reactive nature of
the pyrimidotriazinedione class underlines the importance of
testing activity in a wide variety of assays, including non-
fluorescent methods, in order to eliminate false positive results.
Therefore, we performed several assays with various output
methods to test our novel compounds.
The Bru-seq results revealed that 35G8 promoted the activation
of the Nrf2 pathway. Of the top 20 upregulated genes following a
4-hour 35G8 treatment, four are implicated in the Nrf2 pathway
(SLC7A11, HMOX1, AKR1C1, and LOC344887). Nrf2 is a
transcription factor that normally is kept at low levels due to
degradation mediated via Keap1.[33] Following exposure to ROS,
Keap1 is inactivated and Nrf2 induces transcription of genes to
counteract the oxidative insult.[34] SLC7A11 is part of a cysteine-
glutamate transporter (system xc-) that is regulated by Nrf2 as well
as ATF4.[35] HMOX1, another Nrf2-regulated gene, increased
over 19-fold upon 35G8 treatment. We also found that
transcription of the AKR1C1 gene, which is induced by ROS but
expressed at low levels in gliomas, increased significantly
following 35G8 treatment. Furthermore, the lncRNA LOC344887
has been shown to be activated by Nrf2.[36] Nrf2-regulated genes
may be responsible for treatment resistance in glioblastoma,
providing further evidence that inhibiting PDI could be a sound
strategy to treat glioblastoma. [37]
Several ER stress markers were induced in response to 35G8
treatment, including CHAC1, DDIT3, ASNS, and ATF3. Due to the
strong upregulation of CHAC1, a pro-apoptotic marker regulated
by ATF4, we hypothesize that the PERK-ATF4-DDIT3 branch of
the UPR is likely activated upon PDI inhibition by 35G8 treatment.
The ER stress response and autophagy are closely linked, and
ER stress may induce autophagy in 35G8-treated cells.
Autophagy is the process of protein and organelle degradation by
lysosomes, used as a survival mechanism to provide energy for
the cell.[38] The ER stress response protein ATF4 promotes
autophagy[39] by upregulating genes like TRIB3.[40] While
autophagy can be protective as a survival mechanism, increased
autophagic signaling causes cell death. It is still unclear whether
TMEM74 is regulated by ATF4, but upregulation of TMEM74
mRNA may lead to autophagic PI3K signaling. The increase of
ARG2 expression upon 35G8 treatment may be a result of the
activation of the UPR and lower cellular levels of arginine, leading
to autophagy.[41] IRS2, a key insulin signaling protein regulated by
The key Nrf2-regulated genes SLC7A11 and HMOX1 are
essential
markers
for
iron-dependent,
erastin-induced
ferroptosis.[29, 30] SLC7A11 is a negative regulator of ferroptosis
and upregulation of SLC7A11 occurs as a response to system xc-
inhibition.[13] Efforts to treat glioma patients by inhibiting system
xc- have failed;[45] however, combining SLC7A11 inhibition with a
PDI inhibitor may be a promising new strategy.
System xc- imports cystine for glutathione synthesis[13] to maintain
intracellular redox balance, and the expression of this system is
often elevated in several cancers, including gliomas.[46] System xc-
inhibitors, in particular sulfasalazine, as single agents for the
treatment of gliomas have been unsuccessful,[47] but have been
shown to sensitize glioma cells to radiation therapy.[48] Similarly,
the ferroptosis inducer erastin sensitizes glioblastoma cells to
temozolomide by inhibiting system xc-.[49] These studies provide
evidence that system xc- is an important target for combating
resistance in brain cancer. Importantly, 35G8-induced cell death
can be rescued by deferoxamine, suggesting that ferroptosis is
occurring. Interestingly, Bru-seq analysis of 35G8-treated cells
revealed a pattern of gene expression similar to that of erastin-
treated cells (Figure S9), including induction of the ER stress
response, unfolded protein response, and expression of the
erastin-exposure pharmacodynamic marker, CHAC1.[50]
This indicates that as a consequence of PDI inhibition, 35G8 is
causing blockade of system xc-. However, a link between PDI and
SLC7A11 expression has not yet been established and further
investigation is warranted.
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Figure 5. Effect of 35G8 treatment on RNA synthesis in U87MG cells. 35G8 induces transcription of (A) AKR1C1, (B) CHAC1 and (C) TNFSF9 while corresponding
box plots show downregulation of these genes in brain cancer. 35G8 inhibits the transcription of (D) TXNIP, (E) EGR1 and (F) ITGA3 while corresponding box plots
show upregulation of these genes in brain cancer. FC: fold change; GBM: glioblastoma
Conclusions
We identified 35G8 as a markedly potent PDI inhibitor that may
have therapeutic potential as a single agent and in combination
with SLC7A11 inhibitors or DNA-damaging agents. 35G8 and its
analogues demonstrate activity in human brain cancer cells likely
through upregulation of ER stress and UPR that leads to
autophagy-mediated ferroptosis. Taken together, our data
suggest 35G8 is a useful investigational PDI inhibitor, expected to
easily cross the blood brain barrier, that can be optimized to
develop novel therapeutic agents to treat malignant glioma.
Experimental Section
Chemistry. All commercial chemicals and solvents were reagent grade
and were used without further purification unless otherwise specified.
Analytical thin layer chromatography was performed on Merck pre-coated
plates (silica gel 60 F254) to follow the course of reactions. Proton nuclear
magnetic resonance (1H NMR) spectroscopy was performed on Bruker
Ascend 400 MHz spectrometer or Varian 500 MHz spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm) units relative to
residual undeuterated solvent. The following abbreviations are used to
describe peak splitting patterns when appropriate: s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), bs (broad singlet), dd (doublet of
doublets), dt (doublet of triplets). Coupling constants (J) are expressed in
Hertz (Hz). Low-resolution mass spectra were recorded on a Thermo-
Scientific LCQ Fleet mass spectrometer or a Micromass LCT time-of-flight
instrument utilizing the electro spray ionization (ESI) mode. HPLC was
used to determine the purity of biologically tested compounds using the
Shimadzu HPLC Test Kit C18 column (3 μm, 4.6 × 50 mm) under the
A solution of 6-chloro-3-methyluracil (2.01 g, 12.5 mmol), methylhydrazine
following gradient elution conditions: mobile phase A of acetonitrile/water
(2.87 g, 62.3 mmol) and absolute ethanol (30 mL) was heated at reflux for
(10-95%) or mobile phase B of methanol/water (10-95%). The purity of
3 hours. The reaction mixture was cooled to room temperature, and the
three NCI compounds (NC72, NC75 and NC79) was determined by ultra-
precipitate was collected, washed with ethanol, and dried to give 2a as a
white solid (819 mg, 39 %). 1H NMR (400 MHz, DMSO-d6) δ: 7.64 (bs,
performance liquid chromatography (UPLC). UPLC was carried out using
Acquity UPLC BEH (C18-1.7 μm, 2.1 mm × 50 mm) with a gradient elution
1H), 4.75 (s, 1H), 3.33 (bs, 2H), 3.03 (s, 3H), 2.43 (s, 3H). MS (ESI) m/z =
of acetonitrile/water (10-100%). The purity was established by integration
171 (M+H)+.
of the areas of major peaks detected at 254 nm, and all tested compounds
including three NC compounds have ≥ 95% purity.
3-Methyl-6-(1-methylhydrazinyl)pyrimidine-2,4(1H,3H)-dione (3a)
Figure 6. ROS induction activity of synthesized 35G8 analogues at (A) 4 hours,
(B) 6 hours, and (C) 24 hours. In (C), hydrogen peroxide concentration is 500,
To a suspension of 2a (3.18 g, 18.7 mmol) in absolute ethanol (30 mL)
was added acetaldehyde (1.65 g, 37.4 mmol) at room temperature with
stirring. The reaction mixture was stirred for 2 hours, and the precipitate
was filtered off by suction, washed with ethanol, and dried to give 3a as an
off-white solid (823 mg, 22 %). 1H NMR (400 MHz, CDCl3) δ: 9.21 (bs, 1H),
7.08 (q, J = 5.2 Hz, 1H), 5.00 (d, J = 2.4 Hz, 1H), 3.31 (s, 3H), 3.16 (s, 3H),
2.09 (s, 3H). MS (ESI) m/z = 197 (M+H)+.
100, and 20 μM, from left to right. Data are means from three independent
experiments; error bars show standard deviation.
3-Methyl-6-(1-methylhydrazinyl)pyrimidine-2,4(1H,3H)-dione (2a)
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6-(2-Benzylidene-1-methylhydrazinyl)-3-methylpyrimidine-
1.65 mmol) as reactants. The crude compound was immediately used in
the next step.
2,4(1H,3H)-dione (3b)
The same procedure for the synthesis of compound 3a was followed using
compound 2a (500 mg, 2.94 mmol) and benzaldehyde (636 mg, 5.88
mmol) as reactants to yield 3b as a beige solid (590 mg, 78 %). 1H NMR
R (500 MHz, DMSO-d6) δ: 10.64 (s, 1H), 7.97 (s, 1H), 7.96 (d, J = 6.5 Hz,
2H), 7.44 – 7.38 (m, 3H), 5.24 (s, 1H), 3.34 (s, 3H), 3.10 (s, 3H). MS (ESI)
m/z = 259 (M+H)+.
1,3,6-Trimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione (4a)
A stirring solution of the hydrazone 3a (823 mg, 4.19 mmol) in glacial acetic
acid (10 mL)/water (0.6 mL) cooled to 0 °C was treated with sodium nitrite
(895 mg, 12.6 mmol). The reaction mixture was allowed to warm to room
temperature while stirring. Stirring continued until TLC indicated
consumption of the starting material, thereby furnishing a mixture of the
pyrimidotriazinedione (4a) and the corresponding N-oxide derivative. The
reaction mixture was diluted with water and extracted with
dichloromethane. The combined organic layers were dried over sodium
sulfate, and the solvent was evaporated in vacuo. The resulting residue
was chromatographed on silica to afford the product 4a as a brilliant yellow
solid (272 mg, 31 %). 1H NMR (500 MHz, CDCl3) δ: 4.11 (s, 3H), 3.49 (s,
3H), 2.75 (s, 3H). MS (ESI) m/z = 208 (M+H)+. HPLC (mobile phase A):
purity 99.9%.
3-Methyl-6-(1-methyl-2-(2-phenylethylidene)hydrazinyl)pyrimidine-
2,4(1H,3H)-dione (3c)
The same procedure for the synthesis of compound 3a was followed using
compound 2a (500 mg, 2.94 mmol) and phenyl acetaldehyde (744 mg,
5.88 mmol) as reactants. The crude compound was further purified by
recrystallization from ethanol to yield 3c as a white brilliant solid (427 mg,
53 %). 1H NMR (500 MHz, CDCl3) δ: 9.18 (s, 1H), 7.36 (t, J = 7.3 Hz, 2H),
7.29 (t, J = 7.4 Hz, 1H), 7.21 (d, J = 7.3 Hz, 2H), 7.09 (t, J = 5.7 Hz, 1H),
5.02 (d, J = 2.5 Hz, 1H), 3.70 (d, J = 5.7 Hz, 2H), 3.31 (s, 3H), 3.14 (s, 3H).
MS (ESI) m/z = 273 (M+H)+.
1,6-Dimethyl-3-phenylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione
(4b)
6-(2-(4-Methoxybenzylidene)-1-methylhydrazinyl)-3-
The same procedure for the synthesis of compound 4a was followed using
the hydrazone 3b (300 mg, 1.16 mmol) and sodium nitrite (247 mg, 3.48
mmol) as reactants to afford the product 4b as an orange solid (112 mg,
36 %). 1H NMR (500 MHz, CDCl3) δ: 8.32 (dd, J = 8.1, 1.6 Hz, 2H), 7.56 –
7.52 (m, 3H), 4.24 (s, 3H), 3.53 (s, 3H). MS (ESI) m/z = 270 (M+H)+. HPLC
(mobile phase A): purity 95.0%.
methylpyrimidine-2,4(1H,3H)-dione (3d)
The same procedure for the synthesis of compound 3a was followed using
compound 2a (300 mg, 1.76 mmol) and 4-methoxybenzaldehyde (490 mg,
3.52 mmol) as reactants. The crude compound was further purified by
recrystallization from ethanol to yield 3d as a light beige solid (241 mg,
47 %). 1H NMR (500 MHz, CDCl3) δ: 9.18 (s, 1H), 7.66 (s, 1H), 7.63 – 7.60
(m, 2H), 6.98 – 6.94 (m, 2H), 5.10 (d, J = 2.5 Hz, 1H), 3.87 (s, 3H), 3.32
(s, 6H). MS (ESI) m/z = 289 (M+H)+.
3-Benzyl-1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione
(4c)
The same procedure for the synthesis of compound 4a was followed using
the hydrazone 3c (300 mg, 1.10 mmol) and sodium nitrite (235 mg, 3.30
mmol) as reactants. The crude product was purified by column
chromatography and further recrystallized from ethanol to afford the
product 4c as an oil (100 mg, 32 %). 1H NMR (500 MHz, CDCl3) δ: 7.38 –
7.25 (m, 5H), 4.29 (s, 2H), 4.11 (s, 3H), 3.47 (s, 3H). MS (ESI) m/z = 284
(M+H)+. HPLC (mobile phase A): purity 98.6%.
6-(2-(3-Methoxybenzylidene)-1-methylhydrazinyl)-3-
methylpyrimidine-2,4(1H,3H)-dione (3e)
The same procedure for the synthesis of compound 3a was followed using
compound 2a (200 mg, 1.17 mmol) and 3-methoxybenzaldehyde (320 mg,
2.35 mmol) as reactants. The crude compound was further purified by
recrystallization from ethanol to yield 3e as an off-white solid (90 mg,
27 %). 1H NMR (500 MHz, CDCl3) δ: 9.16 (s, 1H), 7.66 (s, 1H), 7.35 (t, J =
7.9 Hz, 1H), 7.26 – 7.23 (m, 1H), 7.18 – 7.17 (m, 1H), 6.97 (dt, J = 8.2, 1.7
Hz, 1H), 5.13 (d, J = 2.5 Hz, 1H), 3.86 (s, 3H), 3.34 (s, 3H), 3.32 (s, 3H).
MS (ESI) m/z = 289 (M+H)+.
3-(4-Methoxyphenyl)-1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-
5,7(1H,6H)-dione (4d)
The same procedure for the synthesis of compound 4a was followed using
the hydrazone 3d (220 mg, 0.763 mmol) and sodium nitrite (163 mg, 2.29
mmol) as reactants. The crude product was purified by column
chromatography and further recrystallized from ethanol to afford the
product 4d as a red solid (81 mg, 35 %). 1H NMR (500 MHz, CDCl3) δ:
8.25 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 4.21 (s, 3H), 3.89 (s, 3H),
3.52 (s, 3H). MS (ESI) m/z = 300 (M+H)+. HPLC (mobile phase A): purity
96.1%.
3-Methyl-6-(1-methyl-2-(4-nitrobenzylidene)hydrazinyl)pyrimidine-
2,4(1H,3H)-dione (3f)
The same procedure for the synthesis of compound 3a was followed using
compound 2a (140 mg, 0.82 mmol) and 4-nitrobenzaldehyde (254 mg,
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3-(3-Methoxyphenyl)-1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-
Growth Inhibition Assay. Cell growth inhibition was assessed by MTT
assay as previously described.[51] Cells were seeded in duplicate in 96-well
plates at 7000 - 10000 cells/well. After overnight incubation at 37 °C and
5 % CO2, cells were treated with indicated compounds for 72 hours. For
the combination therapies, NAC was added to the well at the same time
as 35G8 (24 hours after plates were seeded), and Z-VAD-FMK and
Necrostatin-1 were added to the well 1 hour prior to 35G8 addition. The
plates were incubated with drug or vehicle control for 72 hours at 37 °C
and 5 % CO2. MTT solution (20 μL 3 mg/mL) was added to the wells, and
the cells were incubated for 4 hours at 37 °C. Supernatant was removed
and DMSO (100 μl) was added to each well. The plates were shaken for
15 minutes at room temperature and absorbance of the formazan crystals
was measured at 570 nm. Cell growth inhibition was assessed by the cell
viability rate as [1-(At-Ab)/(Ac- Ab)]×100 (At , Ac and Ab were the
absorbance values from cells which were treated with compound, cells
which were not treated with compound, and blank, respectively). Cell
viability was determined with the MTT assay. U87MG cells were seeded
at 5000 cells per well in 96-well plates. Deferoxamine (Sigma Aldrich, St.
Louis, MO) was added to cells in a five-point, three-fold dilution series from
400 μM. 35G8 was added immediately after in a five-point, three-fold
dilution series from 100 μM. Cells were incubated with compounds for 12
hours at 37 °C, and MTT assay was performed as stated above.
5,7(1H,6H)-dione (4e)
The same procedure for the synthesis of compound 4a was followed using
the hydrazone 3e (77 mg, 0.267 mmol) and sodium nitrite (70 mg, 0.801
mmol) as reactants to afford the product 4e as a yellow solid (15 mg, 19 %).
1H NMR (500 MHz, CDCl3) δ: 7.91 (d, J = 7.7 Hz, 1H), 7.79 (s, 1H), 7.42
(t, J = 7.9 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H), 4.23 (s, 3H), 3.90 (s, 3H), 3.52
(s, 3H). MS (ESI) m/z = 300 (M+H)+. HPLC (mobile phase B): purity
97.2%.
1,6-Dimethyl-3-(4-nitrophenyl)pyrimido[5,4-e][1,2,4]triazine-
5,7(1H,6H)-dione (4f)
The same procedure for the synthesis of compound 4a was followed using
the crude hydrazone 3f (69 mg, 0.227 mmol) and sodium nitrite (60 mg,
0.683 mmol) as reactants to afford the product 4f as an orange solid (18
mg, 25 %). 1H NMR (500 MHz, CDCl3) δ: 8.51 (d, J = 8.9 Hz, 2H), 8.37 (d,
J = 8.9 Hz, 2H), 4.27 (s, 3H), 3.53 (s, 3H). MS (ESI) m/z = 313 (M-H)-.
HPLC (mobile phase A): purity 96.8%.
PDI Protein Purification. The expression vector of recombinant human
PDI protein with N-terminal His tag was a gift from Dr. Lloyd W. Ruddock
(University of Oulu, Oulu, Finland). PDI expression and purification were
performed as previously described[5] with slight modifications. In brief,
protein production was carried out in Escherichia Coli strain BL21 (DE3)
pLysS grown in LB medium with 200 μg/ml ampicillin (EMD Biosciences,
La Jolla, CA) at 37°C and incubated at an A600 of 0.5 for 4 hours with 1
mM isopropyl β-D-1-thiogalactopyranoside (GoldBio, St. Louis, MO). Cells
were harvested by centrifugation (4000 × g for 15 min) and were re-
suspended in one-tenth volume Buffer A (20 mM sodium phosphate, pH
7.3). Cells were lysed by sonication and the cell debris was removed by
centrifugation (16000 × g for 30 min). The supernatant was applied to a
bed of Ni-nitrilotriacetic acid in a histidine-binding column (Qiagen, Hilden,
Germany), equilibrated with 10 ml of Buffer A and incubated at 4 °C,
overnight. After incubation, the column was washed in Buffer A and then
in Buffer B (20 mM sodium phosphate, 0.5 M sodium chloride and 50 mM
imidazole, pH 7.3). His-tagged proteins were eluted using Buffer C (20 mM
sodium phosphate and 50 mM EDTA, pH 7.3) and eluent was dialyzed in
100 mM sodium phosphate buffer (pH 7.0) with 2 mM EDTA.
3-(4-Methoxyphenyl)-1,6-dimethyl-5,7-dioxo-1,5,6,7-
tetrahydropyrimido[5,4-e][1,2,4]triazine 4-oxide (5d)
Following the same procedure for the synthesis of the
pyrimidotriazinedione (4d), the corresponding N-oxide derivative was
isolated by column chromatography to afford the product 5d as an orange
solid (90 mg, 37 %). 1H NMR (500 MHz, CDCl3) δ: 7.86 (d, J = 9.0 Hz, 2H),
6.99 (d, J = 8.9 Hz, 2H), 4.07 (s, 3H), 3.89 (s, 3H), 3.42 (s, 3H). MS (ESI)
m/z = 316 (M+H)+. HPLC (mobile phase A): purity 98.1%.
Reagents. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) was purchased from Amresco (Solon, OH). N-acetylcysteine (NAC)
was purchased from Sigma-Aldrich (St. Louis, MO). Methyl (3S)-5-fluoro-
3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)
butanoyl]
amino] propanoyl] amino]-4-oxopentanoate (Z-VAD-FMK) was purchased
from Tocris Bioscience (Bristol, UK). 5-(1H-indol-3-ylmethyl)-3-methyl-2-
sulfanylideneimidazolidin-4-one (Necrostatin-1) was purchased from
Cayman Chemical Company (Ann Arbor, MI). Phenol red, H2O2, and
horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St.
Louis, MO). Hank’s Balanced Salt Solution (HBSS) was purchased from
Hyclone, Logan, UT, and sodium hydroxide was purchased from EMD,
Gibbstown, NJ.
Measurement of PDI Activity. PDI activity was assessed by measuring
the PDI-catalyzed reduction of insulin as described previously.[14] In brief,
recombinant PDI protein (0.4 μM) was incubated with indicated
compounds at 37 °C for 1 hour in sodium phosphate buffer (100 mM
sodium phosphate, 2 mM EDTA, 8 μM DTT, pH 7.0). A mixture of sodium
phosphate buffer, DTT (500 μM), and bovine insulin (130 μM; Gemini
BioProducts, West Sacramento, CA) was added to the incubated PDI
protein. The reduction reaction was catalyzed by PDI at room temperature,
and the resulting aggregation of reduced insulin B chains was measured
at 620 nm. PDI activity was calculated with the formula, PDI activity (%) =
Cell Culture. The human glioblastoma cells, U87MG, U118MG, NU04 and
A172 (ATCC, Manassas, VA), were obtained in 2013, and were
maintained in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA) with
10 % fetal bovine serum (Thermo Fisher Scientific, Waltham, MA). Cells
were grown as monolayer cultures at 37 °C in a humidified atmosphere of
5 % CO2 and tested for mycoplasma contamination with the Mycoplasma
Detection Kit, PlasmoTest (InvivoGen, San Diego, California).
[(ODT80[PDI+DTT+compound]
-
ODT0[PDI+DTT+compound])
-
(ODT80[DTT] - ODT0[DTT])] / [(ODT80[PDI+DTT] - ODT0[PDI+DTT]) -
(ODT80[DTT] - ODT0[DTT])] × 100 (ODT0 and ODT80 were the
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absorbance values at 0 min and 80 min after the reduction reaction,
respectively).
knowledge-based conformer generator.[21] The docking site was defined
for all residues within 10 Å around the center, defined as the sulfur atoms
of the catalytic residues C53 and C397 and nitrogen NE2 of H256 for the
hydrophobic site. Docking studies were performed using the standard
default settings with 100 GA (genetic algorithm) runs on each molecule.
Gold Score was used to quantify the interactions between molecules and
PDI and the annealing parameters with cutoff values of 3.0 Å for hydrogen
bonds and 4.0 Å for van der Waals interactions were used as default.
When the top three solutions attained rmsd values within 1.5 Å, docking
was terminated. During the docking process, a maximum of 10 conformers
was considered for each compound.
Thermal Shift Assay. Thermal shift of purified PDI (0.3 mg/ml in 100 mM
NaPO4, pH 7.0) in the presence or absence of 35G8 was determined as
described.[15] Briefly, 5 µl protein-dye (1,8-ANS, 0.3 mM; Sigma Aldrich, St.
Louis, MO) solutions were dispensed in each well of a 384-well microplate
(Thermo Scientific, AB1384K) and equal volumes of the test compound
solutions were dispensed to each well. Then, 3 µl of silicone oil (Sigma
Aldrich, St. Louis, MO) was added to each well to prevent evaporation.
DMSO (2 % in buffer) was used as control. Fluorescence emission was
detected by measuring light intensity using a CCD camera. The plate was
heated at a temperature range from 25 to 90 °C at 1°C/minute in the
ThermoFluor instrument (Johnson & Johnson, New Brunswick, NJ).
Compounds were replicated three times in a 384-well plate.
Bru-seq Analysis. Bru-seq experiments[12] and analysis were performed
as previously reported. Briefly, U87MG cells were placed in dishes on Day
1. Cells were changed to fresh media on Day 5 and treated with DMSO or
35G8 at 1 μM for 4 hours. Bromouridine was added into the media to a
final concentration of 2 mM to label newly synthesized nascent RNA in the
last 30 minutes of treatment. Cells were then collected in TRIzol (Thermo
Fisher Scientific, Waltham, MA) and total RNA was isolated. The
bromouridine-containing RNA population was further isolated and
sequenced. Sequencing reads were mapped to a reference genome.
Cellular Thermal Shift Assay. The cellular thermal shift assay was
performed following previously established procedure.[52] U87MG cells
were seeded at 2 x 106 cells/100 mm dish and allowed to attach overnight.
Cells were treated with 0.5, 1.0, or 2.0 μM 35G8, or DMSO as the negative
control, for 2 hours at 37 °C, 5 % CO2. After treatment, cells were
trypsinized, washed with DPBS twice, and suspended in 600 μL DPBS.
The cells were split into 100 μL aliquots, heated at indicated temperatures
for 3 minutes in the Veriti Thermal Cycler (Applied Biosystems), and
incubated for 3 minutes at room temperature. The cells were flash-frozen
twice and spun at 14 x g for 20 minutes at 4 °C. Supernatants were
collected and loaded onto a 10 % polyacrylamide gel at a volume of 16 μL,
with 4 μL 4X SDS loading dye. Subsequently, Western blotting was run
following the procedure reported herein.
Bioinformatic Analysis. Bru-seq data of 35G8 treatment was filtered
using the cut off value of gene size > 300 bp and mean (RPKM) > 0.5 and
a total of 7,770 genes were ranked based on the fold change values versus
control (DMSO). DAVID functional annotation analysis[56] was performed
on 460 upregulated and 220 downregulated genes with fold change ≥ 2
and ≤ -2. IPA of Bru-seq data was performed using the IPA web-based
application (Ingenuity Systems, Inc.) on the list of 680 up- and
downregulated genes (fold change ≥ 2 and ≤ -2) (Table S4). Top canonical
pathways were ranked based on the P-value of significance and maximum
number of genes in the pathway. Gene Set Enrichment Analysis (GSEA)
of Bru-seq data was done on a pre-ranked gene list of 7,770 genes of
35G8 treatment based on the Kolmogorov–Smirnov statistic.[57] Table S4
and S5 show the top 20 gene sets for up- and downregulated genes of the
Bru-seq dataset of 35G8 treatment, respectively. The snapshots of the
enrichment profiles of these 20 gene sets are provided in Figure S3 and
S4.
Drug Affinity Responsive Target Stability. The DARTS assay was
performed following previously established procedure.[53] U87MG cells
were grown to approximately 80-85% confluence, washed with ice-cold
DPBS, and lysed with lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5%
sodium deoxycholate, 50 mM Tris, pH 8.0). Cells were collected and lysis
was allowed to occur for 10 minutes on ice. Cells were spun at 18,000 x g
for 20 minutes at 4 °C to collect the supernatant. Protein concentration was
determined via BCA assay. 100 μM PACMA31 or 35G8 or 1% DMSO were
incubated with aliquots of cell lysate at 5 mg/ml for 30 minutes with shaking
at room temperature. Pronase (Sigma Aldrich, St. Louis, MO) was added
to 20 μL aliquots of cell lysates at 0, 1:1000 (0.005 μg/μL), 1:500 (0.01
μg/μL), or 1:250 (0.02 μg/μL) for 30 minutes at room temperature.
Digestion was stopped by adding 1X protease inhibitor cocktail (Sigma
Aldrich, St. Louis, MO) and incubating the reactions on ice for 10 minutes.
SDS-PAGE loading buffer (6 μL of 5X) was added to the samples, and
samples were heated for 10 minutes at 70 °C. Samples were spun down
briefly and 20 μg of protein was loaded into acrylamide gels (10%) for
Western blot analysis.
ROS Detection Assay. U87MG cells were detached with 0.05% trypsin-
EDTA (Thermo Fisher Scientific, Waltham, MA), neutralized, centrifuged
and resuspended in cell culture media. Suspension was treated with 20
μM cell-permeable H2DCFDA (Thermo Fisher Scientific, Waltham, MA) for
30 minutes at 37 °C. Cells were centrifuged again and washed with cell
culture media to remove excess probe. After washing, cells were placed in
a black-wall 384-well plate at 20,000 cells/well, incubated for 30 minutes
and treated with compounds at designated conditions. Fluorescent signals
were read at 493 nm/523 nm for ROS detection at designated time points
(4, 6, and 24 hours).
Docking Study. Docking studies were performed using GOLD, version
4.0 (Cambridge Crystallographic Data Centre).[54] The crystal structure of
human PDI in its reduced state (PDB ID: 4EKZ)[55] was used for all
calculations. 3D structures of the ligands were created and energy
minimized using MMFF94 forcefield implemented in OMEGA 2.5.1.4
(OpenEye Scientific Software, http://www.openeye.com), a systematic,
Western Blot. Primary antibodies for GRP78, HMOX1, CHAC1, CHOP,
LC3B, GSTO1, and SLC7A11 and secondary antibodies were purchased
from Cell Signaling (Danvers, MA). Primary antibody for P4HB was
purchased from Protein Tech (Rosemont, IL). U87MG cells were treated
with DMSO or 2 μM 35G8 for 1, 3, 6, 12, or 24 hours. Cells were harvested
13
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10.1002/cmdc.201700629
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FULL PAPER
[4]
a) L. Ellgaard, L. W. Ruddock, EMBO Rep. 2005, 6(1), 28-32; b) A.
with a lysis buffer (25 mM tris(hydroxymethyl)aminomethane, 150 mM
NaCl, 17 mM Triton X-100, 3.5 mM SDS, pH 7.4), lysed via sonication, and
spun in a centrifuge at 13,500 × g at 4 °C for 10 minutes. Supernatant was
collected and protein concentration determined with the BCA assay
(Thermo Fisher Scientific, Waltham, MO). Samples were prepared with 50
μg protein and loaded onto 10 % (or 12 % for LC3B and DDIT3) acrylamide
(Bio-Rad, Hercules, CA) gels. Protein from gels was electro-transferred to
methanol-activated immobilon-FL PVDF membranes (EMD Millipore, La
Jolla, CA). Membranes were blocked for 1 hour with Odyssey Blocking
Buffer (LI-COR Biosciences, Lincoln, NE). Membranes were probed for
proteins using primary antibodies (P4HB, 1:1000; GRP78, 1:1000;
GSTO1, 1:1000; HMOX1, 1:1000; CHAC1, 1:1000; CHOP, 1:500; LC3B,
1:2000; SLC7A11, 1:2000) overnight at 4 °C. Membranes were incubated
with secondary antibodies (anti-rabbit, 1:7500, or anti-mouse, 1:7500) and
fluorescence was imaged by Odyssey Imaging Systems (LI-COR
Biosciences).
Shergalis, N. Neamati, in Encyclopedia of Signaling Molecules (Ed.: S. Choi),
Springer New York, 2016, pp. 1-12.
[5]
Duncan, E. Zandi, N. A. Petasis, N. Neamati, Proc. Natl. Acad. Sci. U S A
2012, 109(40), 16348-16353.
[6]
Laerum, R. Bjerkvig, Cancer Res. 2006, 66(20), 9895-9902.
[7] P. E. Lovat, M. Corazzari, J. L. Armstrong, S. Martin, V. Pagliarini,
D. Hill, A. M. Brown, M. Piacentini, M. A. Birch-Machin, C. P. Redfern, Cancer
Res. 2008, 68(13), 5363-5369.
[8]
Hecht, S. Fulda, S. Zahler, I. Antes, U. Kazmaier, S. A. Sieber, A. M. Vollmar,
Angew. Chem. Int. Ed. Engl. 2014, 53(47), 12960-12965.
S. Xu, A. N. Butkevich, R. Yamada, Y. Zhou, B. Debnath, R.
D. Goplen, J. Wang, P. Enger, B. B. Tysnes, A. J. Terzis, O. D.
J. Eirich, S. Braig, L. Schyschka, P. Servatius, J. Hoffmann, S.
[9]
C. Khodier, L. VerPlank, P. P. Nag, J. Pu, J. Wurst, T. Pilyugina, C.
Dockendorff, C. N. Galinski, A. A. Scalise, F. Passam, L. van Hessem, J.
Dilks, D. R. Kennedy, R. Flaumenhaft, M. A. J. Palmer, S. Dandapani, B.
Munoz, S. L. Schrieber, in Probe Reports from the NIH Molecular Libraries
Program, Bethesda (MD), 2010.
[10]
A. Kaplan, M. M. Gaschler, D. E. Dunn, R. Colligan, L. M. Brown,
A. G. Palmer, D. C. Lo, B. R. Stockwell, Proc. Natl. Acad. Sci. U S A 2015,
112(17), E2245-E2252.
[11]
S. Vatolin, J. G. Phillips, B. K. Jha, S. Govindgari, J. Hu, D.
Grabowski, Y. Parker, D. J. Lindner, F. Zhong, C. W. Distelhorst, M. R. Smith,
C. Cotta, Y. Xu, S. Chilakala, R. R. Kuang, S. Tall, F. J. Reu, Cancer Res.
2016, 76(11), 3340-3350.
[12]
Magnuson, T. E. Wilson, M. Ljungman, Methods 2014, 67(1), 45-54.
[13] S. J. Dixon, K. M. Lemberg, M. R. Lamprecht, R. Skouta, E. M.
Zaitsev, C. E. Gleason, D. N. Patel, A. J. Bauer, A. M. Cantley, W. S. Yang, B.
Morrison, III, B. R. Stockwell, Cell 2012, 149(5), 1060-1072.
M. T. Paulsen, A. Veloso, J. Prasad, K. Bedi, E. A. Ljungman, B.
Redox Cycling Assay. The redox cycling assay was adapted from a
previously published experiment.[58] In duplicate in a 384-well plate, 20 μL
of HBSS buffer, 100 U of catalase, 100 μM H2O2, 100 μM H2O2 + 100 U
catalase, 0.5% DMSO, 500 μM DTT, 10 μM 35G8, 10 μM 35G8 + 500 μM
DTT, or 10 μM 35G8 + 500 μM DTT + 100 U of catalase was added to a
reaction mixture with HBSS to a final volume of 60 μL. The reaction was
incubated at room temperature for 30 minutes, and phenol red-HRP
detection reagent was added to a final concentration of 100 μg/ml phenol
red and 60 μg/ml HRP in each well. The reaction was incubated for an
hour at room temperature. Sodium hydroxide (10 μL, 1 N) was added to
wells and absorbance was measured at 610 nm.
[14]
M. M. Khan, S. Simizu, N. S. Lai, M. Kawatani, T. Shimizu, H.
Osada, ACS Chem. Biol. 2011, 6(3), 245-251.
[15]
M. W. Pantoliano, E. C. Petrella, J. D. Kwasnoski, V. S. Lobanov,
J. Myslik, E. Graf, T. Carver, E. Asel, B. A. Springer, P. Lane, F. R. Salemme,
J. Biomol. Screen. 2001, 6(6), 429-440.
[16]
P. Cimmperman, L. Baranauskienė, S. Jachimovičiūtė, J. Jachno,
J. Torresan, V. Michailovienė, J. Matulienė, J. Sereikaitė, V. Bumelis, D.
Matulis, Biophys. J. 2008, 95(7), 3222-3231.
[17]
[18]
T. P. Primm, H. F. Gilbert, J. Biol. Chem. 2001, 276(1), 281-286.
G. D. Daves, C. C. Cheng, R. K. Robins, J. Am. Chem. Soc. 1961,
83(18), 3904-3905; T. Nagamatsu, H. Yamasaki, T. Hirota, M. Yamato, Y.
Kido, M. Shibata, F. Yoneda, Chem. Pharm. Bull. 1993, 41(2), 362-368.
[19]
A. Degterev, Z. H. Huang, M. Boyce, Y. Q. Li, P. Jagtap, N.
Mizushima, G. D. Cuny, T. J. Mitchison, M. A. Moskowitz, J. Y. Yuan, Nat.
Chem. Biol. 2005, 1(2), 112-119.
Statistical Analysis. IC50 values were calculated using GraphPad Prism
7 software (GraphPad Software, Inc.). The error bars indicate mean ±
standard deviation.
[20]
A. Degterev, J. Hitomi, M. Germscheid, I. L. Ch'en, O. Korkina, X.
Teng, D. Abbott, G. D. Cuny, C. Yuan, G. Wagner, S. M. Hedrick, S. A.
Gerber, A. Lugovskoy, J. Yuan, Nat. Chem. Biol. 2008, 4(5), 313-321.
[21]
T. Stahl, J. Chem. Inf. Model. 2010, 50(4), 572-584.
[22] M. T. Paulsen, A. Veloso, J. Prasad, K. Bedi, E. A. Ljungman, Y.
P. C. D. Hawkins, A. G. Skillman, G. L. Warren, B. A. Ellingson, M.
C. Tsan, C. W. Chang, B. Tarrier, J. G. Washburn, R. Lyons, D. R. Robinson,
C. Kumar-Sinha, T. E. Wilson, M. Ljungman, Proc. Natl. Acad. Sci. U S A
2013, 110(6), 2240-2245.
Acknowledgements
[23]
K. Itoh, T. Chiba, S. Takahashi, T. Ishii, K. Igarashi, Y. Katoh, T.
The authors would like to thank Michelle Paulsen for running the
Bru-seq experiments. The expression vector of recombinant
human PDI was a generous gift from Dr. Lloyd W. Ruddock
(University of Oulu, Oulu, Finland). This work was supported in
part by a grant from NIH (CA193690).
Oyake, N. Hayashi, K. Satoh, I. Hatayama, M. Yamamoto, Y. Nabeshima,
Biochem. Biophys. Res. Commun. 1997, 236(2), 313-322.
[24]
Kiuchi, Mol. Cell. Biochem. 2013, 380(1-2), 97-106.
[25] Y. Y. Shang, M. Zhong, L. P. Zhang, Z. X. Guo, Z. H. Wang, Y.
Zhang, J. T. Deng, W. Zhang, Clin. Exp. Pharmacol. Physiol. 2010, 37(1), 51-
55; F. Siu, P. J. Bain, R. LeBlanc-Chaffin, H. Chen, M. S. Kilberg, J. Biol.
Chem. 2002, 277(27), 24120-24127.
K. Oh-hashi, Y. Nomura, K. Shimada, H. Koga, Y. Hirata, K.
[26]
M. Salazar, A. Carracedo, I. J. Salanueva, S. Hernandez-Tiedra,
Keywords: drug discovery • cancer • oxidoreductases • protein
disulfide isomerase • unfolded protein response
M. Lorente, A. Egia, P. Vazquez, C. Blazquez, S. Torres, S. Garcia, J. Nowak,
G. M. Fimia, M. Piacentini, F. Cecconi, P. P. Pandolfi, L. Gonzalez-Feria, J. L.
Iovanna, M. Guzman, P. Boya, G. Velasco, J. Clin. Invest. 2009, 119(5), 1359-
1372.
[27]
A. Yamamoto, M. L. Cremona, J. E. Rothman, J. Cell Biol. 2006,
References:
172(5), 719-731.
[28]
C. F. Yu, L. Wang, B. F. Lv, Y. Lu, L. Zeng, Y. Y. Chen, D. L. Ma,
[1]
Q. T. Ostrom, H. Gittleman, P. Liao, C. Rouse, Y. W. Chen, J.
T. P. Shi, Biochem. Biophys. Res. Commun. 2008, 369(2), 622-629.
[29]
6(27), 24393-24403.
[30]
Dowling, Y. L. Wolinsky, C. Kruchko, J. Barnholtz-Sloan, Neuro-Oncology
2014, 16, 1-63.
[2]
M.-Y. Kwon, E. Park, S.-J. Lee, S. W. Chung, Oncotarget 2015,
R. Stupp, W. P. Mason, M. J. van den Bent, M. Weller, B. Fisher,
S. J. Dixon, D. N. Patel, M. Welsch, R. Skouta, E. D. Lee, M.
M. J. Taphoorn, K. Belanger, A. A. Brandes, C. Marosi, U. Bogdahn, J.
Curschmann, R. C. Janzer, S. K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe, J.
G. Cairncross, E. Eisenhauer, R. O. Mirimanoff, R. European Organisation for,
T. Treatment of Cancer Brain, G. Radiotherapy, G. National Cancer Institute of
Canada Clinical Trials, N. Engl. J. Med. 2005, 352(10), 987-996.
Hayano, A. G. Thomas, C. E. Gleason, N. P. Tatonetti, B. S. Slusher, B. R.
Stockwell, eLife 2014, 3, e02523.
[31]
J. M. C. Gutteridge, R. Richmond, B. Halliwell, Biochem. J. 1979,
184(2), 469-472.
[32]
H. van de Waterbeemd, G. Camenisch, G. Folkers, J. R. Chretien,
[3]
S. Xu, S. Sankar, N. Neamati, Drug Discov. Today 2014, 19(3),
O. A. Raevsky, J. Drug Target. 1998, 6(2), 151-165.
222-240.
[33]
Engel, M. Yamamoto, Genes Dev. 1999, 13(1), 76-86.
[34] G. T. Wondrak, Antioxid. Redox Signal. 2009, 11(12), 3013-3069.
K. Itoh, N. Wakabayashi, Y. Katoh, T. Ishii, K. Igarashi, J. D.
14
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700629
ChemMedChem
FULL PAPER
[35]
a) R. J. Bridges, N. R. Natale, S. A. Patel, Br. J. Pharmacol. 2012,
165(1), 20-34; b) M. Koritzinsky, B. G. Wouters, Semin. Radiat. Oncol. 2013,
23(4), 252-261.
[36]
G. Johnson, L. Beaver, D. E. Williams, E. Ho, R. H. Dashwood, in
Thirteenth Annual AACR International Conference on Frontiers in Cancer
Prevention Research, Vol. 8, Can. Prev. Res., New Orleans, LA, 2015.
[37]
a) J. H. Zhu, H. D. Wang, Y. W. Fan, Y. X. Lin, L. Zhang, X. J. Ji,
M. L. Zhou, Oncol. Rep. 2014, 32(2), 443-450; b) J. H. Zhu, H. D. Wang, Q.
Sun, X. J. Ji, L. Zhu, Z. X. Cong, Y. Zhou, H. D. Liu, M. L. Zhou, BMC Cancer
2013, 13, 380.
[38]
[39]
B. Levine, Nature 2007, 446(7137), 745-747.
W. B'chir, A. C. Maurin, V. Carraro, J. Averous, C. Jousse, Y.
Muranishi, L. Parry, G. Stepien, P. Fafournoux, A. Bruhat, Nucleic Acids Res.
2013, 41(16), 7683-7699.
[40]
M. Salazar, M. Lorente, A. Orea-Soufi, D. Dávila, T. Erazo, J.
Lizcano, A. Carracedo, E. Kiss-Toth, G. Velasco, Biochem. Soc. Trans. 2015,
43(5), 1122-1126.
[41]
R. García-Navas, M. Munder, F. Mollinedo, Autophagy 2012,
8(11), 1557-1576.
[42]
643-651.
[43]
L. Xu, G. A. Spinas, M. Niessen, Horm. Metab. Res. 2010, 42(9),
F. T. Ogata, W. L. Batista, A. Sartori, T. F. Gesteira, H. Masutani,
R. J. Arai, J. Yodoi, A. Stern, H. P. Monteiro, PLoS One 2013, 8(12), e84588.
[44]
S. Lee, S. M. Kim, R. T. Lee, Antioxid. Redox Signal. 2013, 18(10),
1165-1207.
[45]
P. A. Robe, D. H. Martin, M. T. Nguyen-Khac, M. Artesi, M.
Deprez, A. Albert, S. Vanbelle, S. Califice, M. Bredel, V. Bours, BMC Cancer
2009, 9(19), 372.
[46]
G. Y. Gillespie, H. Sontheimer, J. Neurosci. 2005, 25(31), 7101-7110.
[47] P. W. Gout, A. R. Buckley, C. R. Simms, N. Bruchovsky, Leukemia
2001, 15(10), 1633-1640.
W. J. Chung, S. A. Lyons, G. M. Nelson, H. Hamza, C. L. Gladson,
[48]
L. Sleire, B. S. Skeie, I. A. Netland, H. E. Forde, E. Dodoo, F.
Selheim, L. Leiss, J. I. Heggdal, P. H. Pedersen, J. Wang, P. O. Enger,
Oncogene 2015, 34(49), 5951-5959.
[49]
L. Chen, X. Li, L. Liu, B. Yu, Y. Xue, Y. Liu, Oncol. Rep. 2015,
33(3), 1465-1474.
[50]
S. J. Dixon, D. Patel, M. Welsch, R. Skouta, E. Lee, M. Hayano, A.
G. Thomas, C. Gleason, N. Tatonetti, B. S. Slusher, B. R. Stockwell, eLife
2014, 3, e02523.
[51]
J. Carmichael, W. G. DeGraff, A. F. Gazdar, J. D. Minna, J. B.
Mitchell, Cancer Res. 1987, 47(4), 936-942.
[52]
R. Jafari, H. Almqvist, H. Axelsson, M. Ignatushchenko, T.
Lundback, P. Nordlund, D. M. Molina, Nat. Protoc. 2014, 9(9), 2100-2122.
[53]
34-46.
[54]
B. Lomenick, R. W. Olsen, J. Huang, ACS Chem. Biol. 2011, 6(1),
G. Jones, P. Willett, R. C. Glen, A. R. Leach, R. Taylor, J. Mol.
Biol. 1997, 267(3), 727-748.
[55]
C. Wang, W. Li, J. Ren, J. Fang, H. Ke, W. Gong, W. Feng, C. C.
Wang, Antioxid. Redox Signal. 2013, 19(1), 36-45.
[56]
a) G. Dennis, B. T. Sherman, D. A. Hosack, J. Yang, W. Gao, H.
C. Lane, R. A. Lempicki, Genome Biol. 2003, 4(5), 3; b) S. B. a. L. R. Huang
da W, Nat. Protoc. 2009, 4(1), 44-57.
[57]
A. Subramanian, P. Tamayo, V. K. Mootha, S. Mukherjee, B. L.
Ebert, M. A. Gillette, A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, J.
P. Mesirov, Proc. Natl. Acad. Sci. U S A 2005, 102(43),15545-15550.
[58]
P. A. Johnston, K. M. Soares, S. N. Shinde, C. A. Foster, T. Y.
Shun, H. K. Takyi, P. Wipf, J. S. Lazo, Assay Drug Dev. Technol. 2008, 6(4),
505-518.
15
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Entry for the Table of Contents
We describe a nanomolar, cytotoxic protein disulfide isomerase
inhibitor, 35G8, that is potent in a panel of human glioblastoma
cell lines. Bromouridine-labeling and sequencing of nascent
RNA revealed that 35G8 induced Nrf2 antioxidant response, ER
stress response, and autophagy, and may induce cell death via
ferroptosis.
16
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