Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch

Article abstract of DOI:10.7554/eLife.11878

The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6β or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination.

Full text of DOI:10.7554/eLife.11878

RESEARCH ARTICLE
Ceapins are a new class of unfolded
protein response inhibitors, selectively
targeting the ATF6a branch
1*,
Ciara M Gallagher Carolina Garri1,2, Erica L Cain1, Kenny Kean-Hooi Ang3,
Christopher G Wilson3, Steven Chen3, Brian R Hearn3, Priyadarshini Jaishankar3,
1
3
3
1
*
Andres Aranda-Diaz , Michelle R Arkin , Adam R Renslo , Peter Walter
1Department of Biochemistry and Biophysics, Howard Hughes MedicaI Institute,
2
University of California, San Francisco, United States; Fundacio´ n Ciencia Para la
3
Vida, Santiago, Chile; Small Molecule Discovery Center, Department of
Pharmaceutical Chemistry, University of California, San Francisco, San Francisco,
United States
Abstract The membrane-bound transcription factor ATF6a plays a cytoprotective role in the
unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6a
promotes cell survival in cancer models. We used cell-based screens to discover and develop
Ceapins, a class of pyrazole amides, that block ATF6a signaling in response to ER stress. Ceapins
sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly
specific inhibitors of ATF6a signaling, not affecting signaling through the other branches of the
UPR, or proteolytic processing of its close homolog ATF6b or SREBP (a cholesterol-regulated
transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that
can be used to explore both the mechanism of activation of ATF6a and its role in pathological
settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches
either singly or in combination.
*For correspondence: ciara@
walterlab.ucsf.edu (CMG); peter@
walterlab.ucsf.edu (PW)
DOI: 10.7554/eLife.11878.001
Competing interests: The
authors declare that no
competing interests exist.
Funding: See page 30
Introduction
Most secreted and transmembrane proteins utilize the endoplasmic reticulum (ER) as a dedicated
folding compartment. It is estimated that about one third of all newly synthesized proteins pass
through the ER, where they fold and assemble into multi-subunit complexes, and where post-transla-
tional modifications such as disulfide bridge formation and glycosylation occur (Braakman and
Hebert, 2013). Dedicated quality control mechanisms ensure that only properly folded proteins exit
the ER. These mechanisms are part of the cell’s ’proteostasis network’ and include chaperone sys-
tems to aid in protein folding and ER associated degradation (ERAD) to remove terminally misfolded
proteins (Ruggiano et al., 2014). In addition, the ER has the ability to adjust its folding capacity
upon demand through activation of a homeostatic signaling network, called the unfolded protein
response (UPR). The UPR directs cell fate - cells that cannot restore homeostasis then initiate apo-
ptosis to prevent secretion or cell surface presentation of misfolded, non-functional proteins
(Lin et al., 2007; Lu et al., 2014).
Received: 25 September 2015
Accepted: 26 May 2016
Published: 20 July 2016
Reviewing editor: Davis Ng,
Temasek Life Sciences
Laboratory and National
University of Singapore,
Singapore
Copyright Gallagher et al. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Three principal transmembrane sensors of the UPR independently monitor folding stress in the
ER – IRE1, PERK and ATF6 (Gardner et al., 2013). These UPR branches function cooperatively to
decrease the load of incoming polypeptides and to increase both the protein folding and degrada-
tive capacity of the ER through regulation of transcription and translation.
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eLife digest Newly made proteins must be folded into specific three-dimensional shapes before
they can perform their roles in cells. Many proteins are folded in a cell compartment called the
endoplasmic reticulum. The cell closely monitors the quality of the work done by this compartment.
If the endoplasmic reticulum has more proteins to fold than it can handle, unfolded or misfolded
proteins accumulate and trigger a stress response called the unfolded protein response. This
increases the capacity of the endoplasmic reticulum to fold proteins to match the demand.
However, if the stress persists, then the unfolded protein response instructs the cell to die to protect
the rest of the body.
A protein called ATF6a is one of three branches of the unfolded protein response. This protein is
found in the endoplasmic reticulum where it is inactive. Endoplasmic stress causes ATF6a to move
from the endoplasmic reticulum to another compartment called the Golgi apparatus. There, two
enzymes cut ATF6a to release a fragment of the protein that then moves to the nucleus to increase
the production of the machinery needed to fold proteins in the endoplasmic reticulum.
Errors in protein folding can cause serious diseases in humans and other animals. Drugs that
target ATF6a might be able to regulate part of the unfolded protein response to treat these
diseases. However, no drugs that act on ATF6a had been identified. Now, two groups of
researchers have independently identified small molecules that specifically target ATF6a.
Gallagher et al. screened over 100,000 compounds for their ability to reduce the activity of
ATF6a-regulated genes. The experiments reveal that a class of small molecules termed Ceapins can
selectively block the activity of ATF6a during endoplasmic reticulum stress, but had no effect on
other proteins involved in the unfolded protein response. Furthermore, when human cells
experiencing stress were treated with Ceapins, a greater number of cells died in comparison to cells
that had not received Ceapins. An accompanying study by Gallagher and Walter reports on the
mechanism by which Ceapins act on ATF6a.
Independently, Plate et al. identified a type of small molecule that can activate ATF6. Together,
the findings of Gallagher et al. and Plate et al. may lead to the development of new drugs for
treating diseases associated with incorrect protein folding in the endoplasmic reticulum.
DOI: 10.7554/eLife.11878.002
ATF6 and IRE1 increase the folding capacity of the ER by upregulating transcription of UPR target
genes. ATF6 is a membrane-tethered transcription factor activated by regulated trafficking and pro-
teolysis producing ATF6-N, the ATF6 fragment that constitutes the functional transcription factor
(Haze et al., 1999; Ye et al., 2000; Chen et al., 2002). In contrast, the highly conserved kinase-
endoribonuclease IRE1 removes an intron from the mRNA encoding the UPR effector XBP1 allowing
translation of XBP1s (’s’ for spliced), the functional transcription factor variant of this protein
(Yoshida et al., 2001). ATF6-N and XBP1s bind to ER stress response (ERSE) (Yoshida et al., 1998;
2000; Roy and Lee, 1999) and unfolded protein response (UPRE) elements (Yamamoto et al.,
2004), respectively in the promoters of UPR target genes. ATF6 upregulates transcription of chaper-
ones, foldases and lipid synthesis genes (Wu et al., 2007; Yamamoto et al., 2007; Adachi et al.,
2008), while XBP1 upregulates ER chaperones and the ERAD machinery (Lee et al., 2003; Acosta-
Alvear et al., 2007). Decreasing the load of proteins entering the ER is coordinated by IRE1 and
PERK. Regulated IRE1-dependent mRNA decay (RIDD) cleaves ER-targeted mRNAs leading to their
degradation (Hollien et al., 2009). PERK, a second transmembrane kinase, phosphorylates itself and
the a-subunit of the initiation factor (eIF2-a) leading to transient inhibition of cap-dependent transla-
tion and an increase in translation of UPR effectors with upstream open reading frames
(Harding et al., 1999; Sidrauski et al., 2015), including the transcription factor ATF4 and the apo-
ptotic effector CHOP. The balance between survival and death is controlled temporally. Both IRE1
and ATF6 signaling attenuate after prolonged ER stress, removing most of the cytoprotective func-
tions of the UPR (Lin et al., 2007; Lu et al., 2014; Haze et al., 2001). PERK signaling is maintained
and, through CHOP, commits the cell to apoptosis (Zinszner et al., 1998; Palam et al., 2011).
This dual capacity of the UPR to boost the protein folding capacity or drive cell death has been
implicated in many disease models (Ryno et al., 2013). Many small molecule inhibitors and
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activators of the PERK and IRE1 enzymes have been isolated (Maly and Papa, 2014; Maurel et al.,
2015; Mendez et al., 2015). In contrast, no pharmacological agents promoting the selective modu-
lation of ATF6 have been developed, in part due to the fact that, unlike IRE1 and PERK, ATF6 is not
an enzyme.
Two closely related homologs define the ATF6 family of ER stress sensors; ATF6a and ATF6b,
which respond to the same stress inducers and are activated with similar kinetics. ATF6a and ATF6b
act redundantly during development as single knockout mice are viable and fertile while double
knockout animals are pre-implantation lethal (Yamamoto et al., 2007). Conversely, ATF6a and
ATF6b do not appear to act redundantly during ER stress as ATF6a knockout cells or animals die
when challenged with ER stressors (Wu et al., 2007; Yamamoto et al., 2007). The transcriptional
targets of ATF6b and its role during ER stress remain poorly defined.
Regulation of ATF6 signaling is by spatial separation of the substrate, ATF6, in the ER and the
proteases, site-1 and site-2 proteases (S1P and S2P, respectively), in the Golgi apparatus: upon ER
stress ATF6 moves from the ER to the Golgi apparatus. The mechanism by which ATF6 trafficking is
regulated is poorly understood but the transport requires the COPII coat (Nadanaka et al., 2004;
Schindler and Schekman, 2009), which is not unique to ATF6. Inhibition of ATF6 was achieved by
inhibiting the proteases that release it from the membrane – S1P and S2P, respectively (Ye et al.,
2000; Okada et al., 2003). These proteases do not uniquely process ATF6 but also play essential
roles in regulating cholesterol homeostasis via processing of SREBP (Brown and Goldstein, 1997)
and lysosome biogenesis via processing of the a/b-subunit precursor of the N-acetylglucosamine-1-
phosphotransferase complex (Marschner et al., 2011). Their pleiotropic engagement limits the use-
fulness of S1P and S2P inhibitors for studies of the UPR.
To date, the ATF6 signaling pathway was considered ’undruggable’. Here we developed cell-
based screens to identify a series of pyrazole amides as the first selective inhibitors of the ATF6a
branch of the UPR. We show in the accompanying manuscript that these compounds trap ATF6a in
the ER in discrete foci, which inspired us to name the compounds ’Ceapins’ from the Irish verb
’ceap’ meaning ’to trap’ (Gallagher and Walter, 2016). Ceapins do not inhibit activation of either
IRE1 or PERK in response to ER stress, nor do they inhibit trafficking and cleavage of SREBP in
response to low sterols or ATF6b in response to ER stress. Through structure-activity studies we
increased the potency of the series ten-fold to an IC50 of 600 nM. Inhibition of ATF6a with Ceapin
analogs has no toxicity in unstressed cells but increases the sensitivity of cells to ER stress inducers,
closely mimicking the genetic ablation of ATF6a in mice. This makes Ceapins the most selective class
of ATF6a inhibitors identified to date and the first to act through a mechanism distinct from prote-
ase inhibition or general trafficking between the ER and the Golgi.
Results
Isolation of small molecule inhibitors of ATF6 mediated transcription of
UPR targets
To isolate small molecule modulators of ATF6 signaling, we used an assay based on the activation of
transcription of ATF6 target genes. To this end, we cloned two copies of the ER stress response ele-
ment (ERSE) upstream of a minimal promoter driving expression of luciferase (Figure 1A) into a ret-
roviral vector, which we used to generate a HEK293T-based ERSE-luciferase reporter stable cell line.
To induce ER stress, we treated the reporter cells with thapsigargin (Tg), an inhibitor of the ER cal-
cium pump. ER stress causes a 3.8 Æ 0.2-fold induction of luciferase activity (Figure 1B). The ER
stress-induced luciferase was not affected by inhibition of IRE1 (Figure 1—figure supplement 1)
(Patterson et al., 2011), indicating that the cell line reported selectively on the ATF6 branch. We
screened 106,281 compounds at a single concentration for their ability to inhibit Tg-induced lucifer-
ase activity in the reporter cells (Figure 1C, see also Figure 3—figure supplement 2 for overview of
screen workflow). About 1% of the compounds (1142) showed >69% inhibition (amounting to three
standard deviations from the mean of stressed controls). To focus on ATF6 pathway specific modula-
tors, we next removed from consideration compounds that showed inhibitory activity in analogous
assays based on luciferase reporters induced by either IRE1-dependent mRNA splicing (Figure 1—
figure supplement 1) (Mendez et al., 2015) or PERK-dependent translational control
(Sidrauski et al., 2013). When fresh stocks were retested in dose response assays, all 598 remaining
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Figure 1. Isolation of small molecule inhibitors of ATF6 mediated transcriptional response induced by ER stress. (A) Schematic representation of ERSE-
luciferase construct used to make screening cell line. Two copies of the ER Stress Response Element (ERSE) were cloned in front of a minimal CMV
promoter (MCP) driving expression of luciferase. (B) Luciferase activity is induced 3.84 0.16 fold upon ER stress (100 nM Tg) in ERSE-Luciferase 293T
reporter cell line. Mean of three independent experiments with at least duplicate wells is plotted; error bars are standard error of the mean. (C) Primary
screen data from ERSE-luciferase transcriptional reporter cell line. Each plate was internally normalized from 0–100% inhibition using stressed and
unstressed controls respectively. 106,281 compounds were added in combination with ER stressor and assayed for their ability to inhibit stress-induced
production of ERSE-luciferase. Plot shows % inhibition for each control and compound tested - blue lines denote mean and standard deviation of each
population, black dots indicate those wells more than two standard deviations away from the mean of the population.1142 compounds scoring more
than three standard deviations from the mean (>69% inhibition, orange line) were classified as hits. (D–J). 293 cells expressing doxycycline inducible
Figure 1 continued on next page
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Figure 1 continued
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MPZ-GFP were uninduced (D) or induced with 50 nM doxycycline without (E) or with inhibitors (F–I) for seven hours and then fixed and stained for GFP
(green), actin (red) and DNA (blue). Inhibitors tested were the protein synthesis inhibitor cycloheximide at either 0.01 mg/mL (F) or 0.1 mg/mL (G), the ER
stressor thapsigargin (100 nM, H) or the S1P inhibitor (50 mM Pf-429242, I). (J) Mean induction of GFP per cell per image was quantified and plotted as
fold induction relative to uninduced controls.
DOI: 10.7554/eLife.11878.003
The following figure supplement is available for figure 1:
Figure supplement 1. IRE1 inhibitor blocks induction of luciferase activity through XBP-luciferase but not ERSE-luciferase.
DOI: 10.7554/eLife.11878.004
compounds showed inhibitory activity with an IC50 < 13.5 mM against Tg-induced luciferase activity
in the reporter cells.
We further triaged the reconfirmed compounds to remove non-specific inhibitors of transcription
or translation. To this end, we developed a high-throughput assay to determine if the compounds
blocked expression of green fluorescent protein-tagged myelin protein zero (MPZ-GFP) under the
control of a doxycycline-inducible promoter (Figure 1D–I). Addition of doxycycline induced the
MPZ-GFP reporter 2.6
0.06-fold (Figure 1E) compared to uninduced controls (Figure 1D). As
expected, the translation inhibitor cycloheximide (’CHX’, Figure 1F,G) prevented MPZ-GFP expres-
sion, and treatment of the cells with an ER stressor (Figure 1H) or with an S1P inhibitor (Figure 1I)
(Hay et al., 2007; Hawkins et al., 2008) did not block doxycycline-induced MPZ-GFP expression.
We quantified the fold-induction of MPZ-GFP by doxycycline (Figure 1J). Sixty of the 598 com-
pounds isolated in the primary screen inhibited doxycycline-induced MPZ-GFP expression and were
removed from further consideration, yielding a collection of 538 compounds that inhibited ER stress-
induced ATF6 signaling without inhibiting either transcription or translation or inhibiting signaling
through other UPR branches.
Isolation of small molecule inhibitors of ER stress-induced nuclear
translocation of GFP-ATF6a
Each step of ATF6a activation occurs in a different organelle – stress-sensing in the ER, proteolytic
processing in the Golgi and activation of transcription in the nucleus. To begin mapping the action
of the inhibitors to the steps of ATF6 activation, we next determined the subcellular localization of
ATF6a using a cell line that stably expresses GFP-ATF6a (Figure 2A–D). Using an antibody against
GRP94 to mark the ER and a DNA stain to mark the nucleus, we examined the ratio of GFP intensity
between the nucleus and the ER. In unstressed cells, GFP-ATF6a colocalized with GRP94, indicating
its predominant localization in the ER (Figure 2A). Upon ER stress, GFP-ATF6a translocated to the
nucleus and colocalized with DNA (Figure 2B, yellow in merged image). As a positive control, when
we inhibited ATF6 cleavage by S1P, GFP-ATF6a no longer translocated to the nucleus but accumu-
lated in perinuclear punctae, corresponding to the Golgi apparatus (Figure 2C, see also Figure 3 in
Gallagher and Walter (2016)). We classified compounds that decreased or inhibited nuclear translo-
cation as ’Class 1 inhibitors’. Figure 2D shows Ceapin-A1 as an example in this class showing
decreased nuclear GFP signal and perinuclear GFP-ATF6a punctae. We classified compounds that
allowed nuclear translocation but inhibited reporter transcription as ’Class 2 inhibitors’. By our defini-
tion, Class 2 inhibitors act downstream of ER-Golgi trafficking, proteolysis, and nuclear import of
ATF6. These inhibitors may act by preventing DNA binding or interaction with transcriptional co-acti-
vators, such as NF-Y (Yoshida et al., 2000; Li et al., 2000).
To quantify GFP-ATF6a localization, we defined a threshold for activated cells – i.e. cells that
responded to ER stress and show nuclear translocation of GFP-ATF6. Using CellProfiler
(Carpenter et al., 2006), we used the GRP94 and DNA images to generate masks for the ER and
nuclei, respectively (Figure 2—figure supplement 1). We next calculated the ratio of nuclear to ER
GFP signal (nuc:ER ratio) for each cell and plotted the nuc:ER ratios for the unstressed and stressed
cells as histograms (Figure 2E). The distributions showed a wide range of responses within the pop-
ulation of cells in each well. To compare wells treated with different inhibitors, we extracted from
these distributions a single metric representing the percentage of stressed cells for each well, as
described in the Methods. This allowed us to convert single cell measurements of GFP-ATF6a from
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Figure 2. Isolation of small molecule inhibitors of ER stress induced nuclear translocation of ATF6. (A–D) Nuclear translocation assay in U2-OS GFP-
ATF6a cells. U2-OS cells expressing GFP-ATF6a were treated with either vehicle (unstressed, DMSO, A) or ER stressor in the absence (ER stress, 100
Figure 2 continued on next page
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Figure 2 continued
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nM Tg, B) or presence of S1P inhibitor (20 mM Pf-429242, C) or screen hit (6.6 mM Ceapin-A1, D). After five hours, cells were fixed and stained for GFP
(green), GRP94 (ER marker, blue) and DNA (nuclear marker, DAPI, red). (E–F) Quantification of nuclear translocation assay. (E) Single cell ratios of
nuclear: ER GFP intensity were calculated for four images per well for each treatment (unstressed and stressed control wells are present seven times per
plate) and plotted as histograms. For each plate, the minimum nuclear: ER ratio where the percentage of stressed cells is greater than the percentage
unstressed cells is calculated and annotated as the threshold for activation by ER stress (light grey vertical dashed line). (F) For each plate, the percent
activation by ER stress is calculated for the control wells (unstressed n = 1904, ER stress n = 2095, unstressed + S1P inhibitor n = 366, stressed + S1P
inhibitor n = 330 cells) and used to generate a Z’ score for the plate.
DOI: 10.7554/eLife.11878.005
The following figure supplements are available for figure 2:
Figure supplement 1. Annotation of nuclear and ER regions used for calculation of the ratio of nuclear to ER GFP-ATF6a signal for each cell.
DOI: 10.7554/eLife.11878.006
Figure supplement 2. Example of a heat map for a plate from the nuclear translocation assay secondary screen showing percent inhibition of test
compounds compared to controls.
DOI: 10.7554/eLife.11878.007
Figure supplement 3. Ceapin-A1 inhibits nuclear translocation of GFP-ATF6.
DOI: 10.7554/eLife.11878.008
Figure supplement 4. Combining data from high content assays identified non-specific inhibitors of trafficking and toxic compounds.
DOI: 10.7554/eLife.11878.009
the images to compare across plates. We performed these analyses in biological triplicates. Statisti-
cal evaluation of unstressed and stressed controls yielded a mean Z’ of 0.7 +/- 0.18, which is an
exceptionally robust read-out for a cell- and image-based high-throughput assay. We further vali-
dated the analyses using S1P inhibitor as a positive control (Figure 2E and F). An example of a heat
map for one plate assessing the inhibitory activity of different compounds is shown in Figure 2—fig-
ure supplement 2. An example of a hit from this assay, Ceapin-A1 is shown in Figure 2—figure sup-
plement 3. Analysis of the data using t-tests instead of the threshold method gave the same results,
indicating that thresholding did not introduce a bias into the data analysis. Of 598 compounds
tested in this way, 85 showed robust inhibition of ER stress-induced nuclear translocation of GFP-
ATF6a above three standard deviations from the mean of the negative (Tg-alone) control.
We further triaged top-scoring compounds to remove false positives. Toxic compounds scored as
hits in the ERSE-luciferase assay but identified in image-based assays as compounds that reduced
the cell number per well below 50% of stressed controls (e.g., wells E5, G5 and G10 in Figure 2—
figure supplement 2; sample images in Figure 2—figure supplement 4E and J). They were
removed from further consideration. Likewise, we removed 18 compounds that inhibited nuclear
translocation of GFP- ATF6a by globally inhibiting protein trafficking from the ER by examining the
image data from our MPZ-GFP assay (Figure 2—figure supplement 4C,D, H and I). MPZ-GFP is tar-
geted to the ER where it is folded prior to export to the plasma membrane (Figure 2—figure sup-
plement 4G) (Pennuto et al., 2008). Furthermore, we applied a potency cut-off of IC50 < 5 mM to
Class 2 (transcriptional) inhibitors. We next analyzed the chemical structures of the compounds to
remove pan-assay interference compounds (PAINS) (Baell and Holloway, 2010; Dahlin et al.,
2015). After completion of these assays, we were left with 38 Class 1 and 128 Class 2 inhibitors, of
which 29 and 70 were repurchased.
Ceapins, a class of pyrazole amides that inhibit ATF6 but not SREBP
processing
We performed dose-response ERSE-luciferase assays on the repurchased compounds using two dif-
ferent ER stressors – Tg or tunicamycin (Tm). Tm inhibits N-linked glycosylation and induces the
UPR. Of 99 repurchased compounds, 98 showed inhibitory activity with Tg, of which 82 also inhib-
ited tunicamycin induced ERSE-luciferase. We next assessed compounds displaying well-shaped
dose-response curves and IC50’s < 19 mM in both Tg and Tm ERSE-luciferase assays for their ability
to inhibit induction of endogenous ATF6a target genes, GRP78 (encoding the ER HSP70 BiP) and
HERPUD1 (encoding a ubiquitin-domain-containing protein involved in ERAD) (Figure 3A, and Fig-
ure 3—figure supplement 1) (Wu et al., 2007; Yamamoto et al., 2007; Adachi et al., 2008). This
qPCR assay did not rely on the presence of reporters and proved the most stringent (a workflow for
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Figure 3. Isolation of Ceapin-A1, a small molecule inhibitor of ATF6 but not SREBP processing. (A) ERSE-luciferase assay in HEK293T cells. Cells were
treated without (DMSO) or with ER stressor (100 nM Tg) in the presence or absence of inhibitors for nine hours. Increasing concentrations of either S1P
Figure 3 continued on next page
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Figure 3 continued
inhibitor (Pf-429242, red) or Ceapin-A1 (green) but not IRE1 inhibitor (4 m8C, grey) block ER stress-induced luciferase activity. Plotted is one
representative experiment showing mean and standard deviation for each inhibitor concentration (triplicate wells per point). Dashed grey lines indicate
the relative luciferase activity of unstressed and stressed controls. (B) ER stress induced upregulation of the endogenous ATF6a target gene GRP78 in
U2-OS cells. Cells were treated without (DMSO, open circles) or with ER stress (100 nM Tg, black squares) in the absence or presence of inhibitors for
four hours prior to isolation of mRNA. Upregulation of GRP78 mRNA was measured using qPCR. mRNA levels for GRP78 were normalized to GAPDH
for each well and then compared to unstressed and stressed controls. ER stress induced GRP78 mRNA induction is inhibited by co-incubation with
either S1P inhibitor (2.3 mM Pf-429424, red) or Ceapin-A1 (10 mM, green) but not the inactive Ceapin analog A5 (10 mM, blue). Inhibition of the ISR (200
nM or 400 nM ISRIB, orange) partially inhibits GRP78 induction while inhibition of IRE1 (10 mM 4 m8C, grey) has only minor effects. Data plotted is the
mean percent activation of GRP78 transcription relative to unstressed (0%) and stressed (100%) controls from two or three independent experiments,
each with duplicate reactions carried out on duplicate wells. (C) Induction of SREPB processing by lipoprotein depletion in HeLa cells. HeLa cells were
grown in lipoprotein deficient media for 16.5 hr prior to addition of either sterols or inhibitors for five hours. One hour prior to lysis proteasome
inhibitor (25 mg/mL ALLN) was added to prevent the degradation of the cleaved SREBP-N fragment. Whole cell lysates were analyzed by Western
blotting for SREPB1 and GAPDH. Arrowheads denote positions of full-length (SREBP) and cleaved (SREBP-N) variants of SREBP1. Lipoprotein depletion
induces cleavage of SREBP (lanes 1, 11) that is inhibited by addition of sterols (10 mg/mL cholesterol, 1 mg/mL 25-hydroxycholesterol, lanes 2, 12) or
increasing concentrations of a S1P inhibitor (Pf-429242, lanes 7–10) but not increasing concentrations of Ceapin-A1 (lanes 3–6). Data shown is
representative of three independent experiments. (D) Induction of ATF6a processing by ER stress in T-Rex cells expressing FLAG-tagged ATF6a.
Arrowheads denote positions of full-length (ATF6a), cleaved membrane-bound (ATF6a-M) and cleaved nuclear (ATF6a-N) variants of ATF6. Cells were
treated without (lanes 1,6,11) or with (lanes 2,7,12) ER stressor (100 nM Tg) alone or in combination with either S1P inhibitor (0.75 mM Pf-429242, lanes
3,4,8,9,13,14) or Ceapin-A1 (14.95 mM, lanes 5,10,15) for two hours prior to harvesting. One hour prior to lysis proteasome inhibitor (MG132, 10 mM) was
added. Cells were harvested and separated by centrifugation into total, membrane and nuclear fractions and analyzed by Western blot for ATF6a (anti-
FLAG), PERK (membrane control), ATF4 (nuclear control) and GAPDH (loading control). Note that totals were run on 10% gels while membrane and
nuclear fractions were run on gradient gels to visualize the migration differences between ATF6a-N and ATF6a-M and between PERK and
phosphorylated PERK respectively. Data shown is representative of two independent experiments.
DOI: 10.7554/eLife.11878.010
The following figure supplements are available for figure 3:
Figure supplement 1. Identification of Ceapin-A1, a small molecule that inhibits ATF6 processing in response to ER stress.
DOI: 10.7554/eLife.11878.011
Figure supplement 2. Screening workflow Summary of screening workflow that lead to the identification of Ceapin-A1 consisting of primary (yellow),
secondary (orange) and tertiary (green) screens.
DOI: 10.7554/eLife.11878.012
Figure supplement 3. Ceapin-A1 inhibits ER stress induced ERSE-luciferase activity ERSE-luciferase assay in HEK293T cells.
DOI: 10.7554/eLife.11878.013
Figure supplement 4. Mutation of S1P cleavage site in ATF6a leads to production of ATF6a-M upon ER stress.
DOI: 10.7554/eLife.11878.014
the screen is shown in Figure 3—figure supplement 2). Of the Class 1 inhibitors, only one consis-
tently blocked ATF6a target gene upregulation: Ceapin-A1 (’A1’ standing for Analog 1 the founding
compound in the series described below).
Ceapin-A1 inhibited both ERSE-luciferase induction by both Tg (Figure 3A) and Tm (Figure 3—
figure supplement 3) and induction of GRP78 mRNA (Figure 3B) with an IC50 = 4.7 1.1 mM and
to the same extent as the S1P inhibitor. Inhibition of IRE1 (Cross et al., 2012) had little effect in the
same assays (Figure 3A and B). In contrast, inhibition of the integrated stress response (ISR)
(Sidrauski et al., 2013) showed a decrease in ATF6a target gene induction, as previously reported
for PERK knockout cells (Wu et al., 2007) (Figure 3B). By these criteria, Ceapin-A1 behaved like a
selective inhibitor of ATF6a.
Since Ceapin-A1 treatment of stressed cells resulted in the same punctate localization of GFP-
ATF6a as the S1P inhibitor in the nuclear translocation assay (Figure 2C and D), we next investi-
gated if Ceapin-A1 was in fact an S1P inhibitor. To this end, we monitored the processing of endog-
enous SREBP1 in HeLa cells (Figure 3C). Cells grown in lipoprotein-deficient media activate SREBP
processing, indicated by the presence of faster migrating, cleaved SREBP-N (Figure 3C, lanes 1 and
11). This cleavage was blocked by the addition of either sterols (Figure 3C, lanes 2 and 12) or
increasing concentrations of the S1P inhibitor (Figure 3C, lanes 7–10) to the cell culture media. Addi-
tion of Ceapin-A1 to 25 mM (> five times its IC50) had no effect on lipoprotein depletion-mediated
SREBP processing (Figure 3C, lanes 3–6). Therefore, Ceapin-A1 neither inhibits S1P nor S2P and
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emerges as the first small molecule inhibitor of ATF6 that does not inhibit SREBP or other pathways
known to use these proteases.
We then analyzed the cleavage of ATF6a directly, using a stable HEK293 based cell line that
expressed FLAG-tagged ATF6a from an inducible promoter. Induction of ER stress led to cleavage
of full-length ATF6a to produce the faster migrating, active transcription factor ATF6a-N
(Figure 3D, lanes 1 and 2). Surprisingly, in the presence of the S1P inhibitor, ER stress-induced cleav-
age of ATF6a was not blocked (Figure 3D, lanes 3 and 4). This was unexpected, given that inhibition
of S1P prevented both nuclear translocation of GFP- ATF6a (Figure 2C and F) and upregulation of
ATF6a target genes (Figure 3A and B, and Figure 3—figure supplement 1 and 3). As a first clue
on how to resolve the paradox, we observed that the cleavage product, henceforth referred to as
ATF6a-M, produced in the presence of the S1P inhibitor migrated more slowly on SDS polyacryl-
amide gels than the product ATF6a-N produced by ER stress alone.
We next analyzed the subcellular localization of ATF6a-M using differential centrifugation. In con-
trast to ER stressed cells, where ATF6a was recovered in the membrane fraction (Figure 3D, lane
12) and ATF6a-N is in the nuclear fraction (Figure 3D, lane 7), both ATF6a and ATF6a-M were
recovered in in the membrane fraction in cells treated with ER stress inducer and the S1P inhibitor
(Figure 3D, lane 14). We therefore denoted this fragment as ATF6a-M, since it remains membrane-
bound and as such is incapable of entering the nucleus and activating ATF6a target genes. The
transmembrane protein PERK and the transcription factor ATF4 served as controls for membrane
and nuclear fractions respectively (Figure 3D). We observed the same fragment in cells expressing a
variant of ATF6a, in which the S1P cleavage site was mutated (Figure 3—figure supplement 4),
indicating that an alternate protease generated ATF6a-M and that ATF6a-M was not a substrate for
subsequent membrane-release by S2P cleavage. Importantly, cells treated with both ER stress
inducer and Ceapin-A1 did not show any ATF6a cleavage product (Figure 3D, lanes 5, 10, 15). This
difference strongly suggested that the mechanism of inhibition of ATF6a processing by Ceapin-A1 is
not via protease inhibition, but that ATF6a is trapped in a place or state where it cannot be cleaved.
For this reason, we chose to name the chemical scaffold of the inhibitor ’Ceapin’, after the Irish verb
‘ceap’ meaning to trap.
Synthesis of a more potent Ceapin scaffold analog: Ceapin-A7
The original hit Ceapin-A1 is an N-benzyl pyrazol-4-yl amide (Figure 4A). We used the ERSE-lucifer-
ase assay to guide structure-activity relationship (SAR) studies aimed at defining the essential phar-
macophore within the Ceapin scaffold. We found that all four rings present in Ceapin-A1 are
necessary for activity (Figure 4B). Thus, deletion of the furan ring (as in Ceapin-A2) led to a loss of
detectable activity in the ERSE-luciferase assay. Among several aryl and heteroaryl rings examined in
place of the furan, only a simple phenyl ring as in Ceapin-A3 (IC50 6.9 0.7) afforded activity compa-
rable to Ceapin-A1 (IC50 = 4.9 1.2 mM). The benzyl substituent on the pyrazole ring was similarly
sensitive to modification. Thus, analogs lacking the ortho and para chloro substituents (Ceapin-A4)
or bearing methyl in place of the benzyl group (Ceapin-A5) were inactive (Figure 4B and D). Further
SAR of ring substitution in the benzyl side chain revealed that substitution at both the ortho and
para position with spheroid hydrophobes was optimal for activity (Figure 4C). Thus, the 2,4-dibro-
mobenzyl analog Ceapin-A6 was about two-fold more potent than Ceapin-A1, while the correspond-
ing bis-trifluoromethyl congener Ceapin-A7 (IC50 = 0.59
0.17 mM) was approximately ten-fold
more potent than Ceapin-A1 (Figure 4C and D). In contrast, analogs bearing a single trifluoromethyl
group at either the para (Ceapin-A8) or ortho position (Ceapin-A9) were notably less potent than
Ceapin-A7. Additional studies were performed with Ceapin-A7, representing an improved lead from
the Ceapin series and with the inactive Ceapin analog A5 as a negative control.
We next confirmed the potency of Ceapin analogs on endogenous ATF6a target induction using
qPCR analysis of GRP78, HERPUD1, and ERO1B, an ER oxidoreductase (Wu et al., 2007;
Yamamoto et al., 2007; Adachi et al., 2008) (Figure 4E). The mean IC50 values for the S1P inhibi-
tor, Ceapin-A1 and Ceapin-A7 calculated from dose-response qPCR assays correlated well with the
values obtained using the ERSE-luciferase reporter (Figure 4F), validating both the analogs and the
use of the ERSE-luciferase assay for SAR studies. As expected, the inactive Ceapin analog A5 does
not inhibit induction of these targets (Figure 3B). The increased IC50 values for HERPUD1 reflect the
contribution from the IRE1/XBP1 and PERK/ATF4 branches to transcriptional upregulation of this
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Figure 4. SAR studies of Ceapin-A1 improve potency by an order of magnitude. (A–C) Summary of structure activity relationship for Ceapin analogs. (A)
Chemical structure of the initial screen hit, Ceapin-A1. SAR of rings (B) or substituents on bis-substituted phenyl ring (C) of Ceapin scaffold. IC50 values
were obtained using ERSE-luciferase assay in 293T cells where compounds were tested in dose-response in combination with ER stressor (100 nM Tg).
IC50 values are from at least four independent experiments for each compound. (D) ERSE-luciferase assay showing improved potency of Ceapin-A7
Figure 4 continued on next page
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Figure 4 continued
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(purple) and lack of activity of Ceapin-A5 (blue) compared to Ceapin-A1 (green). 293T cells with stably integrated ERSE-luciferase reporter were treated
with ER stressor (100 nM Tg) and increasing concentrations of Ceapin analogs for nine hours prior to reading luciferase activity. Data plotted are mean
values from a representative experiment with each point done in triplicate, error bars are standard deviation. (E) qPCR analysis of endogenous ATF6a
target genes. U2-OS cells were treated without or with ER stressor in the presence of increasing concentrations of Ceapin analogs for four hours prior
to harvesting of mRNA for qPCR analysis. mRNA levels for GRP78, HERPUD1 and ERO1B were normalized to GAPDH for each well and then compared
to unstressed controls. Data plotted are mean IC50 values calculated from duplicate experiments, each with duplicate qPCR reactions from duplicate
wells for S1P inhibitor (Pf-429242, red), Ceapin-A1 (green) and Ceapin-A7 (purple). Error bars are standard deviation. (F) Calculated mean IC50 values
and standard deviations from qPCR analysis described above.
DOI: 10.7554/eLife.11878.015
target (Lee et al., 2003; Ma and Hendershot, 2004), further underscoring the selectivity of Ceapin
for the ATF6 branch of the UPR.
Ceapin specifically inhibits ATF6a but not IRE1 or PERK branches of the
UPR
Inhibition of IRE1 did not significantly alter ATF6 signaling as seen using ERSE-luciferase or GRP78
mRNA induction upon ER stress (Figure 3A and B). We next validated that Ceapins selectively
inhibit the ATF6 over the IRE1 and PERK branches of the UPR monitoring the activation of each UPR
branch directly. To this end, we used a polyclonal antibody against ATF6a developed by the Mori
lab (Haze et al., 1999), which allowed us to look at endogenous ATF6a in U2-OS cells (Figure 5A).
As expected, treatment of cells with Tm produced both a faster migrating unglycosylated form of
ATF6a (ATF6a*) and cleaved ATF6a-N (Figure 5A, compare lanes 1 and 2). Cells treated with both
Tm and Ceapin-A7 contained the unglycosylated form of ATF6a but not ATF6a-N (Figure 5A, lane
3), indicating that despite the accumulation of unglycosylated proteins, ATF6a was not cleaved.
ATF6a–derived bands from cells treated with Tm and the inactive Ceapin analog A5 were identical
to Tm alone (Figure 5A, lane 4). ATF6a–derived bands from cells treated with Tg alone or in combi-
nation with the inactive Ceapin analog A5 (inactive analog) showed both ATF6a and ATF6a-N
(Figure 5A, lanes 6 and 8) while cells treated with both Tg and active Ceapin-A7 were indistinguish-
able from those derived from unstressed cells (Figure 5A, lanes 5, 7). Induction of ER stress using
either Tg or Tm led to upregulation of BiP protein levels (Figure 5B, compare lanes 2 and 6 to lanes
1 and 5). Consistent with our qPCR analysis of its mRNA levels (GRP78 mRNA), Ceapin-A7 but not
the inactive Ceapin analog A5 inhibited ER stress-induced upregulation of BiP (Figure 5B, compare
lanes 3 and 7 to lanes 4 and 8). Thus consistent with our analyses above, Ceapins inhibit cleavage
and functional activation of endogenous ATF6a in response to ER stress.
The same lysates were also analyzed for activation of the other branches of the UPR (Figure 5A,
bottom panels). Ceapin-A7 did not inhibit activation of either the PERK (shown by a slower migrating
band representing the phosphorylated form of PERK [Harding et al., 1999]) or the IRE1 (shown by
production of XBP1s protein) branches of the UPR. These results validate and extend our analyses of
the UPR in the 293T-based FLAG-ATF6a reporter cell line. Ceapin-A1 inhibited cleavage of FLAG-
ATF6a without inhibiting induction of ATF4 (Figure 3D, compare lanes 6, 7 and 10) or autophos-
phorylation of PERK (indicated by the shift in mobility; Figure 3D, compare lanes 11, 12 and 15).
There is cross-talk between UPR branches. Specifically, effectors of both the IRE1 and PERK
branches, XBP1 and CHOP respectively, are non-exclusive transcriptional targets of ATF6 (Wu et al.,
2007; Adachi et al., 2008). Treatment of U2-OS cells with ER stressors showed upregulation of both
spliced XBP1 mRNA (XBP1s, Figure 5C) and DDIT3 mRNA (encodes CHOP, Figure 5C). Consistent
with Ceapin inhibiting the ATF6-branch selectively, co-incubation of cells with ER stressor and either
S1P inhibitor or Ceapin-A1 showed only a partial decrease in upregulation of XBP1s and DDIT3
mRNA (Figure 5C, Figure 5—figure supplement 1), in agreement with data from ATF6a knockout
mouse embryonic fibroblasts (MEF) (Wu et al., 2007; Adachi et al., 2008). Taken together, these
data show that Ceapins are selective inhibitors of the ATF6 branch of the UPR and do not inhibit
either IRE1 or PERK signaling.
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Figure 5. Ceapins are selective inhibitors of the ATF6a branch of the UPR. (A) U2-OS cells were treated without (DMSO) or with ER stressor (100 nM Tg
or 2.5 mg/mL Tm) in the absence or presence of Ceapin analogs (6 mM each) for two hours. One hour prior to lysis, proteasomal inhibitor (10 mM
Figure 5 continued on next page
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Figure 5 continued
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MG132) was added to prevent the degradation of the cleaved ATF6a-N fragment. Cells were harvested and analyzed by Western Blot for ATF6a,
PERK, XBP1 and GAPDH (loading control). Arrowheads denote the positions of full-length (ATF6a), unglycosylated full-length (ATF6a*) and cleaved
(ATF6a-N) variants of ATF6a and also PERK and phospho-PERK. Data shown is representative of three independent experiments. (B) U2-OS cells were
treated without (DMSO) or with ER stressor (100 nM Tg or 2.5 mg/mL Tm) in the absence or presence of Ceapin analogs (6 mM each). After eight hours,
cells were harvested and analyzed by Western Blot for BiP and GAPDH (loading control). (C) U2-OS cells were treated without (DMSO) or with ER
stressor (100 nM Tg, black) in the absence or presence of ten-fold the IC50 of either S1P inhibitor (Pf-429242, 3.2 mM, red) or Ceapin-A1 (35.7 mM,
green). Four hours later cells were harvested and mRNA extracted. mRNA levels for XBP1s or DDIT3 were normalized to GAPDH for each well and then
compared to unstressed controls. Data plotted are from duplicate qPCR reactions from duplicate wells, error bars are standard deviation. (D) U2-OS
cells were treated without (DMSO) or with ER stressor (100 nM Tg or 2.5 mg/mL Tm) in the absence or presence of increasing concentration of Ceapin-
A7 (0.6, 1.89, 6, 18.9 mM) or S1P inhibitor (5 mM Pf-429242) for four and a half hours. One hour prior to lysis, proteasomal inhibitor (10 mM MG132) was
added to prevent the degradation of the cleaved ATF6a-N and ATF6b-N fragments. Cells were harvested and analyzed by Western Blot for ATF6a,
ATF6b and GAPDH (loading control). Arrowheads denote the positions of full-length (ATF6a, ATF6b), unglycosylated full-length (ATF6a*, ATF6b*),
cleaved membrane bound (ATF6a-M) and cleaved (ATF6a-N, ATF6b-N) variants of ATF6a and ATF6b. Data shown is representative of three
independent experiments.
DOI: 10.7554/eLife.11878.016
The following figure supplements are available for figure 5:
Figure supplement 1. Induction of XBP1s and DDIT3 mRNA is only partially inhibited by either the S1P inhibitor or Ceapin-A1 U2-OS cells were treated
without (DMSO) or with ER stressor (2.0 mg/mL Tm, black) in the absence or presence of ten-fold the IC50 of either S1P inhibitor (Pf-429242, 3.2 mM, red)
or Ceapin-A1 (35.7 mM, green).
DOI: 10.7554/eLife.11878.017
Figure supplement 2. ATF6b-N is generated in ER stressed cells treated with Ceapin-A7 but not ER stressed cells treated with the S1P inhibitor.
DOI: 10.7554/eLife.11878.018
Ceapin selectively inhibits ATF6a but not ATF6b
The ER contains two related bZip proteins – ATF6a and ATF6b (Haze et al., 2001). ATF6a and
ATF6b show 41% identity in their amino acid sequences, with both S1P and S2P cleavage sites con-
served. ATF6b is activated with similar kinetics and in response to the same stressors as ATF6a and,
as ATF6a, moves from the ER to the Golgi apparatus where it is processed by S1P and S2P to
release ATF6b-N from the membrane allowing its nuclear translocation. Given the similarity between
these related proteins, we next tested if Ceapins inhibit processing of ATF6b in response to ER
stress. To this end, we treated U2-OS cells either with Tg or Tm in the absence or presence of
increasing concentrations of Ceapin-A7 or with S1P inhibitor and analyzed the migration pattern of
both endogenous ATF6a and ATF6b-derived bands (Figure 5D). Consistent with our previous
results, we observed no ATF6a-N in lysates from Ceapin-A7 treated cells (Figure 5D, lanes 3–6 (Tg)
and lanes 10–13 (Tm)) but observed ATF6a-M in lysates from S1P inhibitor-treated cells (Figure 5D,
lane 7 (Tg) and lane 14 (Tm)). In contrast, Ceapin-A7 had no effect on the production of ATF6b-N,
even at the highest concentration (18.9 mM; corresponding to 33x its IC50 for ATF6a) (Figure 5D,
lanes 17–20 and 24–27). We further confirmed that while ATF6b-N is not present in nuclear extracts
of ER stressed cells treated with S1P inhibitor, cells subjected to ER stress in the absence or pres-
ence of Ceapin-A7 both have nuclear localized ATF6b-N (Figure 5—figure supplement 2).
Ceapin sensitizes cells to ER stress
ATF6a knockout mice and MEF cells show impaired survival in the face of acute (Yamamoto et al.,
2007) or chronic (Wu et al., 2007) ER stress. We tested if Ceapins would similarly impair survival of
human cells treated with an ER stressor. To this end, we treated U2-OS cells with increasing concen-
trations of Tg and monitored cell viability over a seventy-two hour time course (Figure 6A). From
the survival curve, we measured an IC50 of 7.1 nM for Tg alone in U2-OS cells (Figure 6B). Cells co-
incubated with both Tg and Ceapin-A7 showed an almost two-fold increase in sensitivity to ER stress
(IC50 = 4.5 nM), whereas the inactive Ceapin analog A5 showed no difference (Figure 6B). This two-
fold difference is consistent with the data from genetic ablation of ATF6a in mice (Yamamoto et al.,
2007).
Since changes in cell viability may be due to cytostatic and/or cytotoxic effects, we next deter-
mined whether Ceapin-A7 displayed increased apoptosis in response to ER stress. To this end, we
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Figure 6. Ceapin-A7 sensitizes cells to ER stress. (A–B) U2-OS cells were treated with increasing concentrations of ER stressor (Tg) in the absence
(black) or presence of six micromolar Ceapin analogs - Ceapin-A5 (inactive, blue) or Ceapin-A7 (purple). (A) After seventy-two hours reducing potential
of living cells was assayed to determine cell viability. Data plotted are the means of four independent experiments performed in triplicate, error bars
represent the standard error of the mean. (B) EC50 values calculated for ER stressor in the absence or presence of Ceapin analogs showing mean and
95% confidence limits. (C). U2-OS cells were treated with increasing concentrations of ER stressor (Tg) in the absence (black) or presence of 6 mM
Ceapin-A7 (purple). To analyze cell death, cells were stained with Annexin V and 7AAD and analyzed by flow cytometry. Data plotted are the means
from three independent experiments performed in duplicate; error bars represent standard deviation.
DOI: 10.7554/eLife.11878.019
stained cells with annexin V, which measures phosphoserine flipping as a marker for apoptotic cells,
and 7-aminoactinomycin D (7AAD), a membrane impermeable dye taken up only by cells with com-
promised plasma membranes as a marker for late apoptotic / necrotic cell death. We treated cells
with or without ER stressor at different concentrations in the absence or presence of Ceapin-A7 and
analyzed the cells by flow cytometry (Figure 6C). Cells treated with Ceapin-A7 alone showed no dif-
ference in cell death compared to vehicle alone, consistent with previous work demonstrating that
homozygous ATF6a knockout mice are viable and fertile (Wu et al., 2007; Yamamoto et al., 2007).
At low concentrations of ER stress (10 nM Tg), inhibition of ATF6a did not enhance cytotoxicity,
however as the concentration of ER stressor was increased (30 nM and 90 nM), ATF6a inhibition
resulted in a two-fold increase in apoptotic cells compared to cells treated with ER stressor alone.
Thus human cells treated with ER stress and Ceapin-A7 phenocopy the results obtained using
genetic ablation of ATF6a in mouse models. Ceapins therefore define a first-in-class series of ATF6a
inhibitors that selectively blocks ATF6a and not ATF6b, SREBP or other UPR branches without rely-
ing on inhibition of the proteases that are also used by other critical signaling pathways.
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Discussion
In this work we describe the isolation, specificity and chemical refinement of the Ceapin scaffold, the
first selective and highly potent pharmacological inhibitors of the ATF6a branch of the UPR. The
ATF6a pathway is the least well-understood UPR branch – its mechanism of activation in response to
ER stress remains unknown, and the lack of unique enzymes in the pathway precluded development
of biochemical assays for screening purposes. Prior to this work, the few compounds shown to
inhibit ATF6a signaling (PDI and S1P inhibitors) target important housekeeping enzymes that are
shared among multiple pathways and hence elicit pleiotropic effects (Maurel et al., 2015). Indeed,
ATF6 was considered ’undruggable’ (Maly and Papa, 2014). Our study highlights the value of com-
bining chemical biology with cell-based screening to identify precise tools for ‘intractable’ pathways,
as amply demonstrated in other pioneering work (Cassidy-Stone et al., 2008; Schreiber et al.,
2015).
To identify Ceapins, we combined cell-based screens to isolate a highly selective inhibitor of
ATF6a. Using a broad range of secondary assays for different steps of pathway activation, we built a
toolbox of small molecules with which to interrogate ATF6a signaling at different points. We suc-
cessfully isolated compounds that act both upstream of proteolytic cleavage, corresponding to the
early stages of activation of ATF6a, and downstream of nuclear import, corresponding to transcrip-
tional activation. From over 100,000 compounds screened, we found one, Ceapin-A1, that is not
only selective for ATF6a but acts precisely at its initial activation stage that currently is the least
understood step. As we detail in the accompanying manuscript, Ceapins induce rapid, reversible
clustering of ATF6a, preventing exit of ATF6a from the ER (Gallagher and Walter, 2016).
Ceapins inhibit activation of ATF6a in response to ER stress without inhibiting ATF6b or SREBP
activation, two other ER-bound transcription factors that are similarly trafficked and processed by
S1P and S2P in the Golgi apparatus. Ceapins also do not inhibit the IRE1 or PERK branches of the
UPR, indicating that the compounds do not generally interfere with sensing ER stress. Further under-
scoring their high selectivity, Ceapins show no toxicity to unstressed cells, consistent with the fact
that ATF6a knockout mice are viable and fertile. Loss of ATF6a becomes detrimental when cells or
animals are treated with ER stressors, and Ceapin action mimics this effect. Thus Ceapins promise to
be invaluable, first-in-class tools to investigate the role of ATF6a independently or in combination
with inhibition of other UPR branches in models of human disease.
Efforts to study the role of ATF6a in disease models have been limited to knockdown or overex-
pression experiments. ATF6a was shown to be required for the survival of dormant tumor cells
(Schewe and Aguirre-Ghiso, 2008). In a cystic fibrosis model, knockdown of ATF6a was shown to
increase delivery of the poorly folded 4F508 variant of the cystic fibrosis transmembrane conduc-
tance regulator to the plasma membrane where it could function (Kerbiriou et al., 2007). Thus
ATF6a inhibitors could be beneficial in diseases where decreased ER quality control would amelio-
rate cell function. In contrast, enhancing ATF6a activity using inducible activation of ATF6a-N
increased quality control of protein folding in the ER and decreased secretion and extracellular
aggregation of amyloidogenic proteins involved in light chain amyloidosis (Cooley et al., 2014).
Ceapins offer a new strategy to investigate the role of ATF6a in existing disease models without
requiring the introduction of knockdown or overexpression constructs. While the pharmacokinetic
properties of current Ceapin analogs limit their usefulness in animal studies, continued SAR studies
to improve metabolic stability offer promise for the future.
Much is made in reviews about the therapeutic potential of modulating the UPR and proteostasis
networks (Ryno et al., 2013; Lindquist and Kelly, 2011; Brandvold and Morimoto, 2015). While
there has been an explosion in small molecule modulators of the UPR, it is still unclear which kinds
of modulations open therapeutic windows for individual disease states. Some cell types exclusively
activate one branch, others all three, and often it is not even clear whether down- or up-regulation
of one branch or the other would lead to the desired outcome. In the UPR network, extensive cross-
talk between the three branches further complicates the issue, with both compensation and interde-
pendence between the UPR branches having implications for how to manage UPR modulation for
therapeutic benefit (Wu et al., 2007). For example, both PERK and ATF6 act downstream of vascular
endothelial growth factor (VEGF) mediated cell survival and angiogenesis (Karali et al., 2014). Addi-
tionally, inhibition of IRE1 alone in models of diabetes led to hyper-activation of ATF6 that led to
severe nephropathy (Madhusudhan et al., 2015). As these examples illustrate, appreciating the
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level of cross talk and compensation between UPR branches is essential to the design of successful
UPR-based therapeutic strategies. To date, this was only possible for IRE1 and PERK. With the iden-
tification of Ceapins, we finally have tools to modulate all three branches and to unleash the full
potential of UPR modulation that has remained theoretical up to this point.
Materials and methods
Plasmid construction
ERSE-luciferase reporter
A DNA sequence containing 5 copies of the ERSE element: CCAATCGGCGGCCTCCACG (red =
NF-Y binding, blue = ATF6 binding) spaced by nine nucleotides was synthesized, PCR amplified with
5’ BglII and 3’ Acc65I overhangs and cloned using BglII / Acc65I into pGL4.28 (Promega, E846A)
which contains a minimal CMV promoter upstream of the luc2CP gene, a synthetically derived lucif-
erase sequence with humanized codon optimization and hCL1 and PEST destabilization sequences.
After sequence verification, clones containing two (D9 (=pCGG008), D10), three (D5) or four (D1,
D7) copies of the ERSE element were recovered.
These ERSE promoter variants driving luciferase were excised from pGL4.28 by digesting with
FseI (to exclude the SV40 polyA terminator), blunting with T4 DNA polymerase, purifying and subse-
quent digestion with BglII. They were ligated into the retroviral vector pQCXIP (Clontech, 631516)
that had been digested with XbaI, blunted with T4 DNA polymerase, purified, digested with BglII
and then dephosphorylated. Plasmids were verified by sequencing and two were selected for gener-
ation of stable cell lines – 2xERSE-Luciferase (D9 clone 3,) and 3xERSE-luciferase (D5 clone 5).
MPZ-GFP
The coding region for myelin protein zero (MPZ) was amplified from a pINCY plasmid containing
MPZ (Open Biosystems # IHS1380-97434176, LIFESEQ 3361858 NM_000530 - incyte full length
human cDNA clone) using oligonucleotides containing 5’ HindIII and 3’ BamHI sites. Purified PCR
product was digested and ligated into HindIII / BamHI linearized pEGFP-N3 (Clontech). The resulting
MPZ-monomeric-EGFP fusion was subcloned using HindIII / NotI into HindIII / PspOMI digested
dephosphorylated pDEST-FRT-TO (kind gift from Andrew N. Krutchinsky).
6xHis-3xFLAG-HsATF6a - wild-type and R416A alleles
The coding region for 3xFLAG-HsATF6a was obtained from pCMV7-3xFLAG-HsATF6a (kind gift
from Ron Prywes) (Shen and Prywes, 2004). The R416A mutation was introduced by site-directed
mutagenesis using a single oligonucleotide 5’ - gtgagccctgcaaatcaaaggGCgcaccttctaggattttctgc –
3’. Wild-type or R416A alleles were amplified by PCR using a 5’ oligonucleotide containing 6xHIS
and attB1 site and 3’ oligo with attB1 site and recombined using Gateway technology firstly into the
entry vector pDONR-221 using BP clonase (Life Technologies # 11789020) and from there into the
destination vector pDEST-FRT-TO using LR clonase (Life Technologies # 11791020).
Cell line construction and culture conditions
Growth media was DMEM with high glucose (Sigma D5796) supplemented with 10% FBS (Life tech-
nologies # 10082147), 2 mM L-glutamine (Sigma G2150), 100 U penicillin 100 mg/mL streptomycin
(Sigma P0781). Additional cell line specific supplements are detailed below. Cells were incubated at
37 C, 5% CO2 unless stated otherwise.
˚
Human bone osteosarcoma (U2-OS) cells (ATCC HTB-96) and human embryonic kidney (HEK)
293T cells (ATCC CRL-3216) were obtained from the American Type Culture Collection. U2-OS cells
stably expressing GFP-ATF6a were purchased from Thermo Scientific (084_01). Growth media was
supplemented with 500 mg/mL G418 (Roche 04 727 878 001) to maintain expression of GFP-ATF6.
HeLa-NF cells were a generous gift from Paul Wade (NIH) (Fujita et al., 2003). The XBP1 reporter
cell line (HEK293T XBP1-Luciferase) was derived from the HEK 293T cell line (ATCC CRL-3216) and
was described previously (Mendez et al., 2015). The ERSE-luciferase reporter cell line was also
derived from the HEK 293T cell line (ATCC CRL-3216) and is described below. 293 T-REx cells
expressing doxycycline-inducible 6xHis-3xFLAG-HsATF6a (wild type (Sidrauski et al., 2013) or
mutant) or MPZ-GFP are derived from (Tet)-ON 293 human embryonic kidney (HEK) cells (Clontech)
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containing a ferritin-like protein (Flp) recombination target (FRT) site (Cohen and Panning, 2007)
and are described below. Commercially available cell lines were authenticated by DNA fingerprint
STR analysis by the suppliers. All cell lines were visually inspected using DAPI DNA staining and
tested negative for mycoplasma contamination.
ERSE-luciferase reporter cell line (293T-D9)
Retroviral ERSE-luciferase vectors were used to produce recombinant retroviruses using standard
methods. Briefly, pQCXIP-ERSE-Luciferase vectors were co-transfected with a VSV-G envelope on a
separate plasmid (Clontech Retro-X Universal Packaging System, 631512) using lipofectamine and
optiMem into the GP2-293 packaging cell line grown in antibiotic free, high glucose (4.5 g/L) DMEM
supplemented with 1 mM sodium pyruvate, 10% fetal bovine serum and 4 mM L-glutamine. The
resulting viral supernatant was harvested at 24 hr and 48 hr and used to transduce HEK293T (ATCC
CRL-3216) cells that were then selected with puromycin. The stable cell line generated from the
2xERSE-luciferase construct (D9, PWM112) showed the best fold induction in response to ER stress
and was used for the screen and all ERSE-luciferase assays in this manuscript. An early passage of
293T-D9 was expanded and frozen in aliquots such that the same passage of cells was used for each
screening day.
MPZ-GFP and 6xHis-3xFLAG-HsATF6-alpha-R416A T-Rex cell lines
pcDNA5-FRT-TO plasmids were co-transfected with pOG44 into (Tet)-ON 293 human embryonic
kidney (HEK) cells containing a ferritin like protein (Flp) recombination target (FRT) site (Flp-In T-Rex
cells) (Cohen and Panning, 2007) according to manufacturers instructions (Invitrogen). After selec-
tion with 100 mg/mL Hygromycin B (Gold Biotechnology) single colonies were isolated, expanded
and tested for expression of either tagged MPZ or tagged ATF6-alpha. Expression of MPZ-GFP or
6xHis-3xFLAG-HsATF6-alpha variants was induced with 50 nM doxycycline.
High throughput small molecule screening
384 well ERSE-luciferase assays
106,281 compounds from the UCSF Small Molecule Discovery Center diversity collection (Chem-
Bridge Diverse, ChemDiv Diversity and SPECS) were stored as 10 mM stocks in 384w plates at -
80 C. 293T-D9 cells were thawed 3–4 days and split once prior to assay. For the primary screen,
˚
10,000 cells in 50 mL were plated per well in poly-D-lysine coated white 384 well plates (Greiner bio-
one CELLCOAT 781945) using either Biomek FXp liquid handler (Beckman Coulter) with V&P Scien-
tific Tumble Stir reservoir or WellMate bulk dispenser. Plates were sealed with breathable seals (E&K
Scientific AeraSeal T896100-S) and incubated in humidified chambers (Corning 431301 Square Dish
with wet tissue lining) for 18–24 hr. 50 nL compound per well was added from DMSO stocks in com-
pound plates (Greiner 784201 E1312150 384w plate PP conical V bottom) using Biomek FXp with
384 pin tool for 10 mM final compound concentration in 0.1% DMSO final. 2 mL ER stress inducer
was added and wells were mixed using 384w tips (Fluotics #P30-384FX.NS) for 100 nM thapsigargin
final. Plates were sealed with breathable seals and incubated for 9 hr in humidified chambers. Plates
were cooled to room temperature for 10 min prior to addition of 13.4 mL One-Glo reagent (Promega
#E6130) per well using BioTek EL406 bulk dispenser. Plates were shaken to improve cell lysis and
after 5 min incubation were read by Analyst HT plate reader (Molecular Devices). For comparison
between days each plate was normalized using unstressed (DMSO, 100% inhibition) and ER stressed
(Tg, 0% inhibition) controls (32 wells of each per plate) and the percent inhibition of ER stress
induced luciferase for each compound was calculated. Controls were used to calculate fold induction
(S/B) and z’ score for each plate as follows:
S=B ¼ ðmean ER stressed controlsÞ=ðmean unstressed controlsÞ:
Z0 ¼ 1 À ð3Ãðstandard deviation stressed ctrls þ standard deviation unstressed ctrlsÞ=
ðmean stressed controls À mean unstressed controlsÞÞ
For dose-response assays, compounds were plated in 16 point two-fold serial dilution in DMSO
starting at 50 mM in 384w plates and the assay was run as described for the primary screen. Average
S/B and z’ for secondary screen DR assays were 2.87 0.19 and 0.56 0.09 respectively. Average S/
B and z’ for tertiary screen DR assays were 2.75 0.13 and 0.64 0.05 (100 nM Tg) and 1.59 0.04
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and 0.25 0.16(2 mg / mL Tm) respectively. For structure-activity relationship (SAR) studies cell num-
ber was reduced to 6250 cells / well which improved both S/B and z’ for the assay.
96 well ERSE-luciferase assays
For SAR studies a subset of assays were run in 96w format. The assay was run essentially as
described for 384w plates with the following adjustments. Cell plating and compound or one-glo
addition was done manually. 20,000 cells in 100 mL final were plated per well (poly-D-lysine coated
96w, Greiner bio-one CELLCOAT 655945). To keep final DMSO concentration at 0.21% compounds
were first plated as 500x stocks in DMSO in 16 point quarter log dilution series in microplates (com-
pounds, Applied Biosystems MicroAmp Optical 96w N8010560), diluted to 6x in media containing
either ER stress inducer or vehicle in plates (Fisherbrand 96 w 14-230-232) immediately prior to addi-
tion to cells for 1x final (20 mL per well). After 9-hr incubation, 31.2 mL of one-glo was added per
well and plates were read on a SpectraMax M5 plate reader (Molecular Devices) using Softmax Pro
v.5.4.1. The IRE1 inhibitor (4 m8C) was purchased from Matrix Chemicals.
96 well high content assays (nuclear translocation assay and inducible GFP
assay)
Image based secondary screens were run in 96 well format. Cells were plated 18–24 hr before induc-
tion of ER stress, covered with breathable seals (E&K Scientific AeraSeal T896100-S) and incubated
in a humidified chamber at 37 C, 5% CO2. Compounds were added using a quad-mapping protocol
˚
from 384w compound plates into 96 well assay plates using Biomek FXp with 96w pin tool. Inducers
were added and wells were mixed using 96w Biomek tips (250 mL tip for Biomek FX, USA Scientific #
1062–2410). Plates were imaged in an IN Cell Analyzer 2000 (GE Healthcare Life Sciences) using 20x
air objective and Cy3, DAPI and FITC excitation and emission filtersets. Images were analyzed using
CellProfiler and MATLAB (see below).
Inducible GFP assay (MPZ-GFP in 293 T-Rex cells) – compound treatment
and imaging
For MPZ-GFP T-Rex cell-line, 96 well plates (Greiner Costar 3595) were coated overnight with
100 mL of 10 mg/mL fibronectin in PBS (Sigma F1141) at 37 C. 150 mL of 0.1 Â 106 cells/mL were
˚
plated per well. For inducible GFP assay compounds were tested at three doses – 6.6, 2.2 and
0.7 mM final – in the presence of 50 nM doxycycline (Sigma D9891) and incubated for eight hours
prior to fixation. Average z’ for duplicate experiments was 0.74 0.12. Inhibitors used were cyclo-
heximide (Sigma C7698) and S1P inhibitor (Pfizer Pf-429242).
Media was aspirated and 100 mL of fixative (4% paraformaldehyde in PBS, P6148, Sigma) was
added per well and incubated 10 min at room temperature (RT). After fixation, cells were washed
three times with PBS, permeabilized with PBS containing 0.1% Triton-X100, washed with PBS and
then blocked with PBS containing 2% normal goat serum (Jackson ImmunoResearch Laboratories #
005-000-121) for one hour at RT. Primary antibody (anti-GFP, mAb 3E6 Invitrogen # A11120) was
used at 1:1000 dilution in blocking solution and incubated at 4 C overnight. Cells were washed three
˚
times with PBS then incubated with secondary antibody (anti-mouse-Alexa-488, Life Technologies #
A11029) and rhodamine phalloidin in blocking solution for two hours RT. Cells were washed three
times with PBS, the first wash containing Hoechst 33,342 (Invitrogen # H1399) at 1:1000 dilution.
Washing and permeabilization steps were five minutes each.
Inducible GFP assay (MPZ-GFP in 293 T-Rex cells) – image analysis
Image analysis used CellProfiler. After importing of images, illumination correction for each channel
was performed using background algorithm with block size of 120 without smoothing. Nuclei were
identified from the Hoechst image as primary objects with a diameter of 15–40 pixel units. Cells
were identified from the actin image as secondary objects using propagation from the primary
objects using global Otsu three-class thresholding with weighted variance – middle intensity pixels
were assigned to the foreground. GFP intensity for each cell was calculated from the original MPZ-
GFP image and exported as a MATLAB file for analysis.
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Nuclear translocation assay (GFP-ATF6a in U2-OS cells) – compound
treatment and imaging
GFP-ATF6a U2-OS cells were plated in media containing G418 selection agent in 96 well plates –
100 mL of 0.032 Â 106 cells per mL in ibidi 96-well ibiTreat m-plate (ibidi 89626). Compounds were
tested at 6.6 mM final in the presence of ER stress inducer (100 nM Tg) and incubated for five hours
prior to fixation. Average z’ for triplicate experiments was 0.70 0.18.
After 5 hr, media was removed and cells were fixed in 4% PFA (Electron Microscopy Sciences
15714) in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9) for 15
min RT. Cells were permeabilized with PHEM-Tx (PHEM containing 0.1% Triton X-100, two washes,
5 min RT), washed twice in PHEM, blocked in PHEM containing 2% normal goat serum (Jackson
Immunoresearch Laboratories 005-000-121) for 1 hr at RT. Primary antibodies were incubated in
blocking solution overnight at 4 C. Cells were washed three times in PHEM-Tx then incubated with
˚
secondary antibodies and nuclear stain (DAPI, Molecular Probes D-1306, 5 mg/mL) in blocking solu-
tion for 2 hr RT protected from light. Cells were washed three times PHEM-Tx, twice PHEM. Anti-
bodies used were rat anti-GRP94 9G10 (abcam ab2791), mouse anti-GFP 3E6 (Invitrogen A11120),
anti-rat-Alexa-555 (Invitrogen A21434), anti-mouse-Alexa-488 (Invitrogen A11029), each at 1:1000
dilution.
Nuclear translocation assay (GFP-ATF6a in U2-OS cells) – image analysis
Image analysis used CellProfiler. After importing of images, illumination correction for each channel
was performed using background algorithm with block size of 120 with smoothing using median fil-
ter of 50 pixel unit size. Nuclei were identified from the DAPI image as primary objects with a diame-
ter of 23–43 pixel units. Clumped objects were distinguished based on fluorescence intensity.
Resulting objects were shrunk by 1 pixel to prevent the nuclear envelope being counted as nuclear
signal. The ER for each cell was identified from the GRP94 image as secondary objects using propa-
gation from the primary objects using global Otsu two-class thresholding with weighted variance.
The nuclear area of each cell was subtracted from the ER area to give the final ER mask (see Fig-
ure 2—figure supplement 1). GFP intensity for the nucleus and ER of each cell was calculated from
the original GFP-ATF6a image and the nuclear to ER ratio of GFP intensity was calculated per cell
and exported as a MATLAB file for analysis.
Automated image quantification and hit determination for high-content
assays
Data from CellProfiler analysis were exported to MATLAB (R2009a, MathWorks) for further
computation.
Nuclear translocation assay – GFP-ATF6
The values of the ratio of nuclear intensity to endoplasmic reticulum intensity (nuclear-to-ER ratio)
for each well and plate were re-ordered to compile all data from one experiment in one single
matrix. Both negative (untreated cells (vehicle only)) and positive (thapsigargin treated cells) controls
were included in each experiment. Since these conditions are the reference for the rest of the wells
in each plate, outliers were removed in order to make the analysis more reliable. Control wells that
had a p-value of 0.1 or greater in a two-tailed two-sample t-test (each particular sample tested
against the group of controls) were considered as outliers and were removed from further analysis.
While most of the experiments showed very homogeneous controls, there was a single plate in
which 1 well was disregarded.
To quantify the degree of activation (nuclear localization) of each compound, we calculated a
nuclear-to-ER ratio threshold above which a cell is classified as activated. The minimum and maxi-
mum values in a whole plate were used to define the number of intervals (each one of a range of
0.025 units) used to generate histograms of the distribution of values in the controls. The data from
the individual positive (thapsigargin treated) and negative (untreated) control wells were merged
into two groups of measurements and the relative frequency of values in each interval for each
group was calculated. The intersection of the histograms (relative frequency vs. interval mean value)
was defined as the mean value of the interval in which the counting in the positive control was
greater than the negative control. This threshold was used to classify each cell in a well as activated
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(with nuclear to cytoplasm ratio greater than the threshold) or non-activated (nuclear to cytoplasm
ratio less or equal to the threshold). The percentage of activation under each treatment was calcu-
lated as the percentage of cells in the activated state in each well.
The percentages of activation in the controls allowed us to define a percentage of inhibition. The
percentage of activation in the positive control was set to 0% inhibition and the percentage of acti-
vation in the negative control was set to 100%. The percentage of inhibition (%inh) was calculated
for each plate (i) as
ꢀ
ꢁ
%acti À %actu
%actt À %actu
%inhi ¼ 1 À
 100
(1)
where %acti is the percentage of activation in well i; %actu and %actt are the mean percentage of
activation in the untreated controls or the treated controls, respectively.
While the percentage of inhibition can be used for scoring, a binary classification was necessary
to identify hits in the screening. Conditions in which the inhibition percentage was at least 3 stan-
dard deviations greater than the negative control (cutoff value) were flagged as inhibition hits.
Inducible GFP assay – MPZ-GFP
The mean fluorescence intensity for each well was imported to Matlab and similar computations
were conducted. The mean fluorescence was log-transformed in order to get Gaussian distributions.
A two-tailed two-sample t-test was performed in each group of controls (positive and negative) to
detect outliers (p-value of 0.05 or grater) and remove them from the pool of control wells.
A percentage of inhibition was calculated by assuming the mean of the logarithm of the mean of
the controls to be 0% (untreated cells) or 100% (treated cells) as
ꢀ
ꢁ
logðFÞiÀlogðFÞu
logðFÞtÀlogðFÞu
%inhi ¼ 1 À
 100
(2)
where log(F)i is the logarithm of mean fluorescence in well i; log(F)u and log(F)t are the mean loga-
rithm of mean fluorescence in the untreated controls or the treated controls, respectively.
In order to identify the compounds that inhibited the expression of MPZ-GFP, we performed a
one-tailed two-sample t-test on the logarithm of the fluorescence of each of the wells against the
positive (doxycycline induced) control wells (left-tailed and 0.5% confidence). The p-values for each
test were used to score compounds by general inhibition. An approximate value for the percentage
of inhibition that was detected as significantly different in each well (cutoff value) was calculated as
the mean of the percentage of inhibition of the minimum hit and the maximum non-hit.
Determination of toxicity – both nuclear translocation and inducible GFP
assays
To detect toxic compounds in both assays, the number of cells in each well was used as a proxy for
cell survival. The relative number of cells in each well to the mean of the number of cells in the nega-
tive control wells was calculated. A condition was flagged as toxic if the relative number of cells was
less than 0.5.
Assessment of the quality of each assay
In order to assess the robustness of each assay plate, we calculated the z’ factor as
ꢀ
ꢁ
ðst þ suÞ
ðꢀt þ ꢀuÞ
z0 ¼ 1 À 3 Â
(3)
where st and su are the standard deviations of the treated and untreated controls; mt and mu are the
means of the controls (percentage of activation in ATF6-inhibition screening and logarithm of mean
fluorescence in general inhibition screening).
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Quantitative PCR analysis of mRNA expression to measure ATF6a
target gene induction
At 18–24 hr prior to drug treatment, 12,000 U2-OS cells were plated per well of a 96 well plate
(Costar 3595), covered with breathable seals (Aeraseal) and incubated in a humidified chamber
37 C, 5% CO2. Each compound was tested at in duplicate wells using either 100 nM Tg or 2 mg/mL
˚
Tm as ER stress inducer. Eight unstressed and stressed wells (four per inducer) per plate were
included as controls. Quarter-log serial dilutions of inhibitors in DMSO at 500x assay concentration
were prepared in 96w plates = ’compound plate’ (Applied Biosystems MicroAmp Optical 96-well
reaction plate N8010560). Media without or with inducer was prepared to 6.073x and added to 96w
plates to which 500x inhibitor stocks were added to 6x final = ’inducer plate’. Media without inducer
contained DMSO as vehicle for unstressed control. Media containing either vehicle, ER stress inducer
or ER stress inducer and inhibitor was added to cells to 1x final, covered with breathable seals and
incubated for four hours at 37 C, 5% CO2. The final volume of DMSO was equal between all wells
˚
(0.2%). Cells were lysed, RNA was prepared and PCR reactions were assembled using the Power
SYBR Green Cells-to-CT kit (Life Technologies #4402955) according to manufacturers instructions.
Oligos used for qRT-PCR were as follows:
GRP78 (BiP): 5’-CATGGTTCTCACTAAAATGAAAG-3’ and 5’-GCTGGTACAGTAACAACTG-3’.
Herpud1: 5’-CAGAAATCAACGCCAAGGTG-3’ and 5’-GAACTTCCCTTTGCCTTAAACC-3’
Ero1LB: 5’-AATCTGAAGCGACCTTGTCC-3’ and 5’-GCCCAGCTTTTATTCCAACC-3’
XBP1s: 5’-GGAGTTAAGACAGCGCTTGG-3’ and 5’-CCTGCACCTGCTGCG-3’
DDIT3 (CHOP): 5’-AGCCAAAATCAGAGCTGGAA-3’ and 5’-TGGATCAGTCTGGAAAAGCA-3’
GAPDH: 5’-TGGAAGATGGTGATGGGATT-3’ and 5’-AGCCACATCGCTCAGACAC-3’
For each experiment, duplicate reactions were performed on duplicate wells giving four values
for each dose. qRT-PCR reactions were run using a CFX96 Real Time System (Bio-Rad). Expression
was normalized first to GAPDH internal control and then compared to stressed controls using CFX
Manager 3.0 software (Bio-Rad). For each inhibitor data was log transformed, dose-response curves
(log(inhibitor) versus response, variable slope, four parameter) were plotted and IC50 were calcu-
lated using Prism 5.Of (GraphPad Software, Inc).
Analysis of SREBP cleavage in response to lipoprotein depletion
Cleavage of SREBP in HeLa cells was analyzed essentially as described (Hua et al., 1995;
Espenshade et al., 1999; Sakai et al., 1996). Briefly, two days prior to drug treatment HeLa cells
were plated in growth media at a density of 4 Â 104 cells per well of a six-well plate. The following
day, 16.5 hr prior to drug treatment, growth media was replaced with lipoprotein deficient media –
DMEM with high glucose (Sigma D5796), 10% lipoprotein deficient serum from fetal calf (LPDS,
Sigma S5394), 50 mM compactin (aka mevastatin, a HMG-CoA reductase inhibitor, Santa Cruz Bio-
technology sc-200853), 50 mM mevalonolactone to facilitate non-sterol isoprenoids (Sigma 68519)
(Goldstein and Brown, 1990), 2 mM L-glutamine (Sigma G2150), 100 U penicillin 100 mg /mL strep-
tomycin (Sigma P0781).
After 16.5 hr in lipoprotein deficient media, sterols or inhibitors were added to cells. Sterols
added were cholesterol (10 mg/mL, Sigma C3045) and 25-hydroxycholesterol (1 mg/mL, Sigma
H1015). Serial dilutions of inhibitors in DMSO at 1000x were prepared for both Ceapin and the S1P
inhibitor, Pf-429242. Final concentrations of inhibitors on cells were 0.5, 5, 15 and 25 mM respec-
tively. For each well not receiving either sterols or inhibitors the corresponding vehicle was added
such that the final concentration of ethanol and DMSO was equal for all wells. Four hours after addi-
tion of inhibitors, a proteasome inhibitor (25 mg/mL ALLN, Sigma A6185) was added to prevent deg-
radation of cleaved SREBP-N. One hour later cells were harvested and protein lysates were
prepared.
For lysis, 0.5 mL of scraping buffer was added per well. Cells from each well were scrapped into
eppendorf tubes and centrifuged at 3000 g for five minutes at four degrees. Each cell pellet was
resuspended in 10 mL of lysis buffer and incubated on ice for twenty minutes. Tubes were then vor-
texed for five minutes at four degrees to shear genomic DNA, incubated on ice for five minutes, cen-
trifuged at 1000 g for two minutes at four degrees. Total protein concentration per sample was
determined from 2 mL of each sample using the BCA assay according to manufacturers instructions
(Thermo Scientific 23225). For each sample, 8.92 mg total protein was loaded per lane of a fifteen
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well SDS-PAGE gel. After western blotting, membranes were probed with anti-SREBP1 (abcam
ab3259) or anti-GAPDH (abcam ab9485).
Scraping buffer is 10 mM MG132 (Sigma C2211), 1x complete protease inhibitor (Roche Diagnos-
tics 05056489001) in phosphate buffered saline (Sigma D8537). Lysis buffer is 200 mM Tris pH 8.0,
1% SDS, 100 mM NaCl, 10 mM MG132, 1x complete protease inhibitor. Loading buffer was added
to each sample from a 5x stock to 1x final. 1x loading buffer is 40 mM Tris pH 8.0, 0.2% SDS, 8 mM
DTT, 6% glycerol, 10 mM MG132, 1x complete protease inhibitor, bromophenol blue.
Differential centrifugation of 6xHis-3xFLAG-HsATF6-alpha expressing
T-Rex cells
Cell plating and drug treatment
Two days prior to drug treatment 2.1 Â 106 6xHis-3xFLAG-HsATF6a T-Rex cells per plate were
plated in 100 mm dishes. The following day, expression of tagged ATF6a was induced using 50 nM
doxycycline. 22.5 hr later ER stressor (100 nM Tg in DMSO, Sigma T9033) with or without inhibitors
was added to cells and incubated for one hour. Vehicle was added to ensure the final concentration
of DMSO was the same for all samples. Inhibitors used were S1P inhibitor (0.75 mM Pf-429242,
Pfizer) or Ceapin-A1 (5 mM Ceapin-A1).
Differential centrifugation
To harvest cells, 2.9 mL of scraping buffer was added per plate and cells were scraped into
15 mL Falcon tubes. Samples were centrifuged at 3000 rpm in Beckman GH3.8 rotor at four
degrees for fifteen minutes. Cell pellets were resuspended in 1.6 mL Buffer A and incubated for
ten minutes on ice. Cells were lysed by passing through a 22.5 gauge needle attached to a 1 mL
syringe thirty times. 0.1 mL sample was taken (= total). Samples were spun 1000 g for seven
minutes at four degrees. The pellet from this spin is the nuclear fraction and the supernatant
contains membranes and cytosol. The nuclear pellet was washed once with Buffer A, resus-
pended in 0.29 mL buffer B and incubated rotating for one hour at four degrees. Nuclear sam-
ples were centrifuged at 100,000 g in Beckman TLA 100.2 rotor for 30 min at four degrees. The
supernatant from this spin is the nuclear extract. The membrane and cytosol samples were centri-
fuged at 100,000 g in Beckman TLA 100.2 rotor for 30 min. The pellet from this step contains
membranes, the supernatant is the cytosol. The membrane pellet was washed once in Buffer B
and resuspended in 0.29 mL Buffer B. The cytosol containing supernatant was precipitated with
five volumes of ice-cold acetone for ten minutes on ice then centrifuged at 3500 rpm in Beck-
mann GS-6KR for fifteen minutes at four degrees. The pellet from this step was resuspended in
0.29 mL Buffer B. Protein concentration for membrane, nuclear and cytosolic samples was deter-
mined using the BCA assay according to manufacturers instructions (Thermo Scientific 23225).
For western blot analysis, 15 mL of total, 5 mg membranes or 15 mg nuclear extract was loaded
per lane of a fifteen well SDS-PAGE gels (Tg – Any kD, BFA – 10% Bio-rad TGX minigels). After
blotting, membranes were probed with the following antibodies: FLAG (1:1000, Sigma M2
F1804), PERK (1:500, Cell Signaling Technology C33E10), ATF4 (1:1000, Santa Cruz sc-200),
GAPDH (1:1000, abcam ab-9485).
Buffers
Scraping buffer: 10 mM MG132 (Sigma C2211), 1x complete protease inhibitor (Roche Diagnostics
05056489001) in phosphate buffered saline (Sigma D8537).
Buffer A: 10 mM HEPES-KOH pH 7.4, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-
EDTA, 1 mM Na-EGTA, 1x complete protease inhibitor.
Buffer B: 10 mM HEPES-KOH pH 7.6, 2.5% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 1 mM Na-
EDTA, 1 mM Na-EGTA, 1x complete protease inhibitor.
SDS loading buffer (5x): 150 mM Tris-HCL pH 7.4, 3% SDS, 5% glycerol, 2.5% b-mercaptoethanol,
pinch bromophenol blue.
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Differential centrifugation of U2-OS cells for endogenous ATF6a and
ATF6b
Cell plating and drug treatment
Two days prior to drug treatment 4.13 Â 105 U2-OS cells per plate were plated in 100mm dishes,
four dishes per drug treatment. Two days later, ER stressor (100 nM Tg in DMSO, Sigma T9033) with
or without inhibitors was added to cells and incubated for four hours. Vehicle was added to ensure
the final concentration of DMSO was the same for all samples. Inhibitors used were S1P inhibitor
(5 mM Pf-429242, Pfizer) or Ceapin-A7 (6 mM Ceapin-A7). One hour prior to lysis, proteasomal inhibi-
tor (10 mM MG132 (Sigma C2211)) was added to prevent the degradation of the cleaved ATF6a-N
and ATF6b-N fragments.
Differential centrifugation
Differential centrifugation was performed as described above for 6xHis-3xFLAG-ATF6a expressing
T-Rex cells except the buffer volumes were quartered. For western blot analysis, 10 mL of total
or 15 mg nuclear extract was loaded per lane of a fifteen well SDS-PAGE gels, one gel each for
ATF6a and ATF6b. After blotting, membranes were probed with antibodies against ATF6a
(Haze et al., 1999) (1:1000 in 5% BSA) or ATF6b (Wu et al., 2007) (1:1000 in 5% milk) at four
degrees overnight. After developing for ATF6a and ATF6b membranes were stripped for sixty
seconds shaking at room temperature in stripping buffer (7 M Guanidine hydrochloride (Sigma
G4505), 50 mM Glycerol (MP Biomedicals 800689), 50 mM EDTA (Fisher BP120-1), 100 mM potas-
sium chloride (Fisher P217-3), 20 mM b-mercaptoethanol (Sigma M6250), washed twice in dis-
tilled water, stripped again for sixty seconds, washed extensively with distilled water, PBS-Tween,
blocked in 5% milk before cutting the membranes to probe for PERK (1:500, Cell Signaling Tech-
nology C33E10) or ATF4 (1:1000, Santa Cruz sc-200) each in 5% milk in PBS-Tween.
Synthesis of ceapin analogs
N-{1-[(2,4-Dichlorophenyl)methyl]-1H-pyrazol-4-yl}-5-(furan-2-yl)-1,2-oxazole-3-carboxamide (Ceapin-
A1) was purchased from Chemdiv. N-{1-[(2,4-Dichlorophenyl)methyl]-1H-pyrazol-4-yl}-5-methyl-1,2-
oxazole-3-carboxamide (Ceapin-A2) andN-{1-[(2,4-dichlorophenyl)methyl]-1H-pyrazol-4-yl}-5-phenyl-
1,2-oxazole-3-carboxamide(Ceapin-A3) were purchased from ChemBridge. N-(1-Benzyl-1H-pyrazol-
4-yl)-5-(furan-2-yl)-1,2-oxazole-3-carboxamide (Ceapin-A4) and 5-(furan-2-yl)-N-(1-methyl-1H-pyra-
zol-4-yl)-1,2-oxazole-3-carboxamide(Ceapin-A5) were purchased from Enamine. Reagents and sol-
vents were purchased from Sigma- Aldrich, Acros, Combi-Blocks, AK Scientific, ChemBridge,
Enamine or TCI America and used as received unless otherwise indicated. Flash column chroma-
tography was carried out using a Biotage Isolera Four system and SiliaSep silica gel cartridges
from Silicycle. Hydrogenation reactions were carried out in ThalesNano H-Cube reactor using
30 mm 10% Pt/C catalyst cartridges. 1H NMR spectra were recorded on a Varian INOVA-400
400MHz spectrometer. Chemical shifts are reported in d units (ppm) relative to residual solvent
peak. Coupling constants (J) are reported in hertz (Hz). Characterization data are reported as fol-
lows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, br=broad, m=multi-
plet), coupling constants, number of protons, mass to charge ratio. LC/MS analyses were
performed on a Waters Micromass ZQ/Waters 2795 Separation Module/Waters 2996 Photodiode
Array Detector/Waters 2424 Evaporative Light Scattering Detector system. Separations were car-
ried out on an XTerra MS C18 5 mm 4.6 Â 50 mm column at ambient temperature using a mobile
phase of water-methanol containing 0.1% formic acid.
Method A for alkylation
To a solution of 4-nitro-1H-pyrazole (1 equiv) in N,N-dimethylformamide, were added potassium
carbonate (2 equiv) and the benzyl bromide (1 equiv). The mixture was stirred at ambient tem-
perature until judged complete by LC/MS. The reaction mixture was then diluted with ethyl ace-
tate (10 mL), washed with saturated ammonium chloride solution (10 mL), water (10 mL) and
brine (10 mL). The organic layer was dried over magnesium sulfate, concentrated under reduced
pressure and purified by flash column chromatography (ethyl acetate/hexanes) to obtain the
product.
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Method B for hydrogenation
A methanolic solution of the nitro compound was passed through 10% Pt/C catalyst at a rate of
1 mL/min in the H-Cube reactor under atmospheric pressure and ambient temperature until the
reaction was judged complete by LC/MS. The reaction mixture was concentrated under reduced
pressure to obtain the crude amine that was used without further purification.
Method C for amide coupling
To a solution of the carboxylic acid (1 equiv) in N,N-dimethylformamide, were added HATU (1.1
equiv), the amine (1 equiv), and N,N-diisopropylethylamine (2 equiv). The mixture was stirred at
ambient temperature until the reaction was judged complete by LC/MS. The reaction mixture was
then diluted with ethyl acetate (10 mL), washed with saturated ammonium chloride solution (10 mL),
water (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, concentrated
under reduced pressure and purified by flash column chromatography (ethyl acetate/hexanes) to
obtain the product.
1-{[2,4-Dibromophenyl]methyl}-4-nitro-1H-pyrazole
Chemical structure 1. 1-{[2,4-Dibromophenyl]methyl}-4-nitro-1H-pyrazole.
DOI: 10.7554/eLife.11878.020
To a cooled (0 C) solution of 2,4-dibromobenzyl alcohol (0.1 g, 0.37 mmol) in dichloromethane
˚
(1.0 mL) and N,N-diisopropylethylamine (0.13 mL, 0.75 mmol) was added dropwise methanesulfonyl
chloride (0.032 mL, 0.41 mmol). The mixture was stirred at 0 C for 15 min followed by addition of
˚
potassium carbonate (0.1 g, 0.75 mmol) and a solution of 4-nitro-1H-pyrazole (0.042 g, 0.37 mmol)
in N,N-dimethylformamide (0.5 mL). The mixture was stirred at 50 C for 18 hr. The reaction mixture
˚
was then diluted with ethyl acetate (10 mL), washed with saturated ammonium chloride solution
(10 mL), water (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, con-
centrated under reduced pressure and purified by flash column chromatography (20% ethyl acetate/
1
hexanes) to obtain 0.43 g (82%) of the title compound as a cream colored solid. H NMR (400 MHz,
CDCl3) d 8.15 (s, 1H), 8.08 (s, 1H), 7.79 (S, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 5.37
(s, 2H). LCMS m/z 361 (MH+).
N-(1-{[2,4-Dibromophenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-1,2-
oxazole-3-carboxamide (Ceapin-A6)
Chemical structure 2. N-(1-{[2,4-Dibromophenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-1,2-oxazole-3-carboxamide
(Ceapin-A6).
DOI: 10.7554/eLife.11878.021
To a refluxing mixture of 1-{[2,4-dibromophenyl]methyl}-4-nitro-1H-pyrazole (0.06 g, 0.17 mmol)
and ammonium chloride (0.09 g, 1.7 mmol) in a 2:1 mixture of ethanol/water (6.0 mL), was added
in portions, iron (0.028 g, 0.5 mmol) over a period of 30 min. After refluxing for an additional
5 hr and cooling to ambient temperature, dichloromethane (20 mL) was added to the reaction
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mixture. The organic layer was washed with brine, dried over magnesium sulfate and concen-
trated to obtain 1-{[2,4-dibromophenyl]methyl}-1H-pyrazol-4-amine which was used without further
purification.
To a solution of 5-(furan-2-yl)-1,2-oxazole-3-carboxylic acid (0.016 g, 0.09 mmol) in N,N-dimethyl-
formamide (0.5 mL), were added HATU (0.038 g, 0.099 mmol), 1-{[2,4-dibromophenyl]methyl}-1H-
pyrazol-4-amine (0.03 g, 0.09 mmol), and N,N-diisopropylethylamine (0.031 mL, 0.18 mmol). The
mixture was subjected to conditions described in method C and purified by flash column chromatog-
raphy (20% ethyl acetate/hexanes) to obtain 0.016 g (36%) of the title compound as a pink colored
solid. 1H NMR (400 MHz, CDCl3) d 8.52 (s, 1H), 8.09 (s, 1H), 7.74 (S, 1H), 7.62 (s, 1H), 7.58 (s, 1H),
7.39 (dd, J = 8.3, 1.9 Hz, 1H), 6.98 (d, J = 3.4 Hz, 1H), 6.90 (s, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.56–
6.68 (m, 1H), 5.34 (s, 2H); LCMS m/z 492 (MH+).
1-{[2,4-Bis(trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole
Chemical structure 3. 1-{[2,4-Bis(trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole.
DOI: 10.7554/eLife.11878.022
To a solution of 4-nitro-1H-pyrazole (0.2 g, 1.8 mmol) in N,N-dimethylformamide (2.0 mL), were
added potassium carbonate (0.489 g, 3.5 mmol) and 2,4-bis(trifluoromethyl)benzyl bromide
(0.332 mL, 1.8 mmol). The mixture was subjected to conditions described in method A and purified
by flash column chromatography (15% ethyl acetate/hexanes) to obtain 0.43 g (72%) of the title
1
compound as a white solid. H NMR (400 MHz, CDCl3) d 8.15 (d, J = 8.1 Hz, 2H), 7.98 (s, 1H), 7.82
(d, J = 8.1 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 5.57 (s, 2H).
N-(1-{[2,4-bis(trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-
yl)-1,2-oxazole-3-carboxamide (Ceapin-A7)
Chemical structure 4. N-(1-{[2,4-bis(trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-1,2-oxazole-3-
carboxamide (Ceapin-A7).
DOI: 10.7554/eLife.11878.023
A solution of 1-{[2,4-bis(trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole (0.04 g, 0.1 mmol) in
methanol (20 mL) was subjected to hydrogenation conditions described in general method B to
obtain about 37 mg of crude 1-{[2,4-bis(trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-amine as a col-
orless oil which was used without further purification. LCMS m/z 310 (MH+).
To a solution of 5-(furan-2-yl)-1,2-oxazole-3-carboxylic acid (0.018 g, 0.1 mmol) in N,N-dimethyl-
formamide (0.5 mL), were added HATU (0.042 g, 0.11 mmol), 1-{[2,4-bis(trifluoromethyl)phenyl]
methyl}-1H-pyrazol-4-amine (0.031 g, 0.1 mmol), and N,N-diisopropylethylamine (0.035 mL, 0.2
mmol). The mixture was subjected to conditions described in general method C to obtain 0.045 g
(95%) of the title compound as a white powder. 1H NMR (400 MHz, CDCl3) d 8.52 (s, 1H), 8.11 (s,
1H), 7.94 (s, 1H), 7.72 (d, J = 8 Hz, 1H), 7.67 (s, 1H), 7.58 (s, 1H), 7.09 (d, J = 8.1 Hz, 1H), 6.98 (d, J =
3.5 Hz, 1H), 6.90 (s, 1H), 6.56–6.57 (m, 1H), 5.58 (s, 2H); LCMS m/z 471 (MH+).
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1-{[4-(Trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole
Chemical structure 5. 1-{[4-(Trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole.
DOI: 10.7554/eLife.11878.024
To a solution of 4-nitro-1H-pyrazole (0.25 g, 2.21 mmol) in N,N-dimethylformamide (2.5 mL), were
added potassium carbonate (0.61 g, 4.42 mmol) and 4-(trifluoromethyl)benzyl bromide (0.34 mL,
2.21 mmol). The mixture was stirred at ambient temperature for 18 hr and then subjected to condi-
tions described in method A to obtain 0.61 g of the crude product as a white solid which was used
without further purification. 1H NMR (400 MHz, CDCl3) d 8.11 (d, J = 2.7 Hz, 2H), 7.66 (d, J = 8.0 Hz,
2H), 7.39 (d, J = 8.0 Hz, 2H), 5.37 (s, 2H).
N-(1-{[4-(Trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-
1,2-oxazole-3-carboxamide (Ceapin-A8)
Chemical structure 6. N-(1-{[4-(Trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-1,2-oxazole-3-
carboxamide (Ceapin-A8).
DOI: 10.7554/eLife.11878.025
A solution of 1-{[4-(trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole (0.029 g, 0.1 mmol) in metha-
nol (15 mL) was subjected to hydrogenation conditions described in method B to obtain about
0.026 g of crude 1-{[4-(trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-amine as a colorless oil which
was used without further purification
To a solution of 5-(furan-2-yl)-1,2-oxazole-3-carboxylic acid (0.019 g, 0.1 mmol) in N,N-dimethyl-
formamide (0.5 mL), were added HATU (0.042 g, 0.11 mmol), 1-{[4-(trifluoromethyl)phenyl]methyl}-
1H-pyrazol-4-amine (0.026 g, 0.1 mmol), and N,N-diisopropylethylamine (0.035 mL, 0.2 mmol). The
mixture was subjected to conditions described in method C and purified by flash column chromatog-
raphy (30% ethyl acetate/hexanes) to obtain 0.017 g (43%) of the title compound as a white powder.
1H NMR (400 MHz, CDCl3) d 8.50 (s, 1H), 8.07 (s, 1H), 7.58–7.60 (m, 3H), 7.33 (d, J = 7.9 Hz, 2H),
6.97 (d, J = 3.5 Hz, 1H), 6.90 (s, 1H), 6.56 (dd, J = 3.5, 1.7 Hz, 1H), 5.34 (s, 2H); LCMS m/z 403
(MH+).
1-{[2-(Trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole
Chemical structure 7. 1-{[2-(Trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole.
DOI: 10.7554/eLife.11878.026
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To a solution of 4-nitro-1H-pyrazole (0.25 g, 2.21 mmol) in N,N-dimethylformamide (2.5 mL), were
added potassium carbonate (0.61 g, 4.42 mmol) and 2-(trifluoromethyl)benzyl bromide (0.53 g, 2.21
mmol). The mixture was stirred at ambient temperature for 18 hr and then subjected to conditions
described in method A to obtain 0.55 g of the crude 1-{[2-(trifluoromethyl)phenyl]methyl}-4-nitro-
1
1H-pyrazole as a white solid which was used without further purification. H NMR (400 MHz, CDCl3)
d 8.07–8.10 (m, 2H), 7.74 (d, J = 7.7 Hz, 1H), 7.48–7.59 (m, 2H), 7.28 (d, J = 8.0 Hz, 1H), 5.52 (s, 2H).
LCMS m/z 272 (MH+).
N-(1-{[2-(Trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-
1,2-oxazole-3-carboxamide (Ceapin-A9)
Chemical structure 8. N-(1-{[2-(Trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-yl)-5-(furan-2-yl)-1,2-oxazole-3-
carboxamide (Ceapin-A9).
DOI: 10.7554/eLife.11878.027
A solution of 1-{[2-(trifluoromethyl)phenyl]methyl}-4-nitro-1H-pyrazole (0.032 g, 0.1 mmol) in metha-
nol (15 mL) was subjected to hydrogenation conditions described in method B to obtain about
0.029 g of crude 1-{[2-(trifluoromethyl)phenyl]methyl}-1H-pyrazol-4-amine as a colorless oil which
was used without further purification. LCMS m/z 242 (MH+).
To a solution of 5-(furan-2-yl)-1,2-oxazole-3-carboxylic acid (0.019 g, 0.1 mmol) in N,N-dimethyl-
formamide (0.5 mL), were added HATU (0.042 g, 0.11 mmol), 1-{[2-(trifluoromethyl)phenyl]methyl}-
1H-pyrazol-4-amine (0.026 g, 0.1 mmol), and N,N-diisopropylethylamine (0.035 mL, 0.2 mmol). The
mixture was subjected to conditions described in method C and purified by flash column chromatog-
raphy (25% ethyl acetate/hexanes) to obtain 0.028 g (70%) of the title compound as a white powder.
1H NMR (400 MHz, CDCl3) d 8.50 (s, 1H), 8.04 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.66 (s, 1H), 7.58 (s,
1H), 7.45–7.49 (m, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.97–6.99 (m, 1H), 6.90 (s,
1H), 6.55–6.57 (m, 1H), 5.52 (s, 2H); LCMS m/z 403 (MH+).
Cell viability assay
U2-OS cells were detached using Accutase (Innovative Cell Technologies #AT-104-500) and the cell
number was determined with the Scepter Cell Counter as described by the manufacturer (Millipore).
2000 cells were seeded into each well of a 96 well black walled optical bottom plate (Corning 3882)
in normal growth medium. After 16–18 hr, the media was removed from the cells and 120 mL of
fresh media with increasing concentrations of thapsigargin plus or minus 6 uM Ceapin 2 or 3. The
highest concentration of thapsigargin was 90 nM and six 1:3 serial dilutions were performed. The
final DMSO concentration for all samples including the DMSO only control was 0.034%. Breathable
seals were used to seal the plate and placed in the incubator in a humidified chamber. At 72 hr,
PrestoBlue Cell Viability Reagent (Life Technologies #A13262) was added to each well and incubated
at 37C for 10 min and read on a plate reader as described by the manufacturer. PrestoBlue Cell Via-
bility Reagent is a Resazurin based cell viability indicator that is reduced to a highly fluorescent mole-
cule by viable cells. For data analysis the background (wells with media without cells) was subtracted
from the experimental wells and viability relative to the DMSO treated wells was calculated in Micro-
soft Excel. The means of four independent experiments performed in triplicate were graphed using
GraphPad Prism using the non-linear regression Sigmoidal, 4PL, X is log (Concentration) equation.
The absolute EC50 was calculated in GraphPad Prism to interpolate X at 50% with 95% confidence
intervals.
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Cell death assay
US-OS cells were detached with Accutase (Innovative Cell Technologies #AT-104-500) and cells
counted using the Sceptor Cell Counter as described by the manufacture (Millipore). 20,000 cells in
0.5 mL of growth medium were added to each well of a 24 well plate (Corning 3526). After 16–18 hr
the media was carefully removed from the wells and fresh medium with DMSO, 10, 30 or 90 nM
Thapsigargin plus or minus 6 mM Ceapin 3 was added. Final DMSO concentration for all wells was
0.034%. After 72 hr, media was removed from each wells and placed in 1.5 mL Eppendorf tubes.
250 mL of Accutase was used to detach cells in the well and the entire volume was added to Eppen-
dorf tube containing the kept media. 100 mL of the media and cell mixture in the Eppendorf tubes
were transferred to 2 mL microtubes (VWR #16466–030) containing 100 mL of room temperature
Muse Annexin V and Dead Cell Reagent (EMD Millipore #MCH100105). The kit contains Annexin V
to detect phosphatidylserine for use as an early apoptotic marker and the membrane impermeable
DNA dye, 7-Aminoactinomycin D (7AAD), to detect late apoptotic and necrotic cells. Tubes were
gently vortexed for 5 s and incubated at room temperature for 20 min in the dark. Flow cytometry
was performed using the Muse Cell Analyzer (EMD Millipore) and gated as directed by the manufac-
turer. To minimize reading times between samples, 1000 events were read for each sample and the
percentage of live and apoptotic/dead cells was calculated as described by the manufacturer. The
means of three independent experiments performed in duplicate were graphed in GraphPad Prism.
Abbreviations used
7AAD: 7-aminoactinomycin D
ATF: Activating Transcription Factor
CFTR: Cystic Fibrosis transmembrane conductance regulator
CHOP: CCAAT-enhancer binding proteins (C/EBP) Homologous Protein
CMV: Cytomegalovirus
ERSE: ER Stress response Element
GFP: Green Fluorescent Protein
GRP: Glucose Regulated Protein
HEK293T: Human embryonic Kidney 293T cells.
IRE1: Inositol Requiring Enzyme One
MCP: Minimal cytomegaloviral promoter
PERK: Protein Kinase R (PKR)-like Endoplasmic Reticulum Kinase
RIDD: Regulated IRE1-Dependent Decay
Tg: Thapsigargin
Tm: Tunicamycin
U2-OS: U2 Osteosarcoma cells
UPRE: Unfolded Protein Response Element
VEGF: Vascular Endothelial Growth Factor
XBP1: X-box Binding Protein One
Acknowledgements
The authors thank Carmela Sidrauski and Diego Acosta-Alvear for valuable input on high-throughput
screening of UPR pathways and Samantha Zeitlin for input on high-content screening. We also thank
Margaret Elvekrog, Mable Lam, Sandra Torres and Britt Adamson for critical reading of the manu-
script and the members of the Walter lab for discussion and support. We also thank Pablo Valen-
zuela for his strong support. The S1P inhibitor Pf-429242 was a generous gift from Pfizer. pDEST-
FRT-TO was a kind gift from Andrew N. Krutchinsky. Antibodies against ATF6a and ATF6b were
kind gifts from Kazutoshi Mori and Thomas Rutkowski.
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Cell Biology
Additional information
Funding
Funder
Grant reference number
Author
Howard Hughes Medical
Institute
Collaborative Innovation
Award
Ciara M Gallagher
Carolina Garri
Erica L Cain
Kenny Kean-Hooi Ang
Christopher G Wilson
Steven Chen
Brian R Hearn
Priyadarshini Jaishankar
Michelle R Arkin
Adam R Renslo
Peter Walter
European Molecular Biology
Organization
Long Term Fellowship
F32CA1777128
Ciara M Gallagher
National Cancer Institute
Erica L Cain
Fundacion Ciencia Para La
Vida
Project Basal PFB-16 and
CONICYT
Carolina Garri
QB3 Malaysia Program
Kenny Kean-Hooi Ang
Peter Walter
Howard Hughes Medical
Institute
The funders had no role in study design, data collection and interpretation, or the decision to
submit the work for publication.
Author contributions
CMG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or
revising the article; CG, High-throughput screening and qPCR (data acquisition), Analysis and inter-
pretation of data, Editing the manuscript, Acquisition of data; ELC, Viability assays (design), Data
acquisition, Analysis and interpretation of data, Writing the methods for viability assays, Editing the
manuscript, Drafting or revising the article; KK-HA, CGW, High-throughput screening data acquisi-
tion, Analysis and interpretation of data, Acquisition of data; SC, High-content screening data acqui-
sition, Acquisition of data; BRH, Chemistry design (scaffold analysis for secondary screen hits), Initial
SAR of Ceapins, Acquisition of data, Analysis and interpretation of data; PJ, Chemistry design (SAR
of Ceapins), Writing chemistry methods, Acquisition of data, Analysis and interpretation of data,
Drafting or revising the article; AA-D, High-content screening (data analysis), Writing the methods
for HCS data analysis, Analysis and interpretation of data, Drafting or revising the article; MRA,
High-throughput screening data analysis and interpretation, Editing the manuscript, Analysis and
interpretation of data, Drafting or revising the article; ARR, Chemistry design (SAR of Ceapins), Writ-
ing the manuscript, Analysis and interpretation of data, Drafting or revising the article; PW, Concep-
tion and design, Drafting or revising the article
Author ORCIDs
Peter Walter, http://orcid.org/0000-0002-6849-708X
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