Development of a Chemical Toolset for Studying the Paralog-Specific Function of IRE1

Article abstract of DOI:10.1021/acschembio.9b00482

The dual kinase endoribonuclease IRE1 is a master regulator of cell fate decisions in cells experiencing endoplasmic reticulum (ER) stress. In mammalian cells, there are two paralogs of IRE1: IRE1α and IRE1β. While IRE1α has been extensively studied, much less is understood about IRE1β and its role in signaling. In addition, whether the regulation of IRE1β's enzymatic activities varies compared to IRE1α is not known. Here, we show that the RNase domain of IRE1β is enzymatically active and capable of cleaving an XBP1 RNA mini-substrate in vitro. Using ATP-competitive inhibitors, we find that, like IRE1α, there is an allosteric relationship between the kinase and RNase domains of IRE1β. This allowed us to develop a novel toolset of both paralog specific and dual-IRE1α/β kinase inhibitors that attenuate RNase activity (KIRAs). Using sequence alignments of IRE1α and IRE1β, we propose a model for paralog-selective inhibition through interactions with nonconserved residues that differentiate the ATP-binding pockets of IRE1α and IRE1β.

Full text of DOI:10.1021/acschembio.9b00482

Articles
pubs.acs.org/acschemicalbiology
Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
Development of a Chemical Toolset for Studying the Paralog-
Specific Function of IRE1
Hannah C. Feldman,† Venkata Narayana Vidadala,† Zachary E. Potter,† Feroz R. Papa,§,∥,⊥,#,∇
Bradley J. Backes,§,∥ and Dustin J. Maly*,†,‡
†Department of Chemistry, University of Washington, Seattle, Washington, United States
‡Department of Biochemistry, University of Washington, Seattle, Washington, United States
∥
⊥
#
§Department of Medicine, Lung Biology Center, Department of Pathology, Diabetes Center, ∇California Institute for
Quantitative Biosciences, University of CaliforniaSan Francisco, San Francisco, California,United States
S
* Supporting Information
ABSTRACT: The dual kinase endoribonuclease IRE1 is a
master regulator of cell fate decisions in cells experiencing
endoplasmic reticulum (ER) stress. In mammalian cells, there
are two paralogs of IRE1: IRE1α and IRE1β. While IRE1α has
been extensively studied, much less is understood about IRE1β
and its role in signaling. In addition, whether the regulation of
IRE1β’s enzymatic activities varies compared to IRE1α is not
known. Here, we show that the RNase domain of IRE1β is
enzymatically active and capable of cleaving an XBP1 RNA
mini-substrate in vitro. Using ATP-competitive inhibitors, we
find that, like IRE1α, there is an allosteric relationship between
the kinase and RNase domains of IRE1β. This allowed us to
develop a novel toolset of both paralog specific and dual-
IRE1α/β kinase inhibitors that attenuate RNase activity (KIRAs). Using sequence alignments of IRE1α and IRE1β, we propose
a model for paralog-selective inhibition through interactions with nonconserved residues that differentiate the ATP-binding
pockets of IRE1α and IRE1β.
arious perturbations within the cell can cause endoplas-
mic reticulum (ER) stress, resulting in unfaithful folding
for an active transcription factor that upregulates targets that
aid in ER adaptation.14−16 Conversely, prolonged ER stress
leads to the cleavage of ER-localized mRNA by IRE1α’s RNase
in an event called regulated IRE1 dependent decay, or
RIDD.17−19 RIDD increases ER stress and promotes apoptosis
through the cleavage of mRNA encoding for adaptive ER
proteins and antiapoptotic proteins, as well as cleaving miRNA
functioning to suppress pro-apoptotic and pro-inflammatory
proteins.20
In mammalian cells, IRE1α has a highly homologous paralog
called IRE1β. While IRE1α is ubiquitously expressed, IRE1β’s
expression is mainly isolated to epithelial cells in the
gastrointestinal tract and bronchia.21 IRE1β’s domain
architecture is the same as IRE1α’s, but much less is known
about its RNase activity or its functional role in the cell. While
it has been shown that IRE1β’s kinase retains phosphotransfer-
ase function, there have been conflicting reports about the
ability of its RNase domain to cleave XBP1 and the efficiency
of this cleavage event compared to IRE1α.16,22 It has been
suggested that under ER stress, rather than prioritizing XBP1
V
and the accumulation of proteins within the lumen of the
ER.1,2 In mammalian cells, these perturbations result in the
unfolded protein response (UPR), which consists of three
transmembrane sensor proteins: IRE1, PERK, and ATF6.3−7
The initial aim of the UPR is to restore ER homeostasis, where
all three UPR sensors initiate signals to upregulate genes that
aid in ER stress adaptation.8 Additionally, PERK mediates a
global translation block to help decrease the ER’s protein
folding burden.5 If these initial adaptive responses fail, the
UPR switches to terminal outputs that result in apoptosis.9
IRE1α is the most ancient and conserved component of the
UPR and is thought to be one of the dominant factors in cell
fate decision making in cells experiencing ER stress.4,10 IRE1α
contains a stress sensing N-terminal lumenal domain that is
connected to cytosolic protein kinase and RNase domains
through a transmembrane linker.11 Upon ER stress, IRE1α
becomes active through lumenal domain dimerization and
kinase trans-autophosphorylation. Phosphorylation of the
kinase promotes IRE1α dimerization on the cytosolic face of
the ER, an event required for RNase domain activity.12,13
Under an adaptive UPR, the RNase domain of IRE1α
facilitates the cleavage of XBP1 mRNA, which results in the
subsequent generation of a mature form of XBP1 that encodes
Received: June 17, 2019
Accepted: September 26, 2019
© XXXX American Chemical Society
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Figure 1. In vitro activity of IRE1β’s RNase domain. (A) Schematic of the XBP1 cleavage assay with IRE1α* and IRE1β*. An increase in
fluorescence is observed upon cleavage of a FRET-quenched XBP1 mini-substrate. (B) Real time fluorescence curves for 75 nM IRE1α* and 75
nM IRE1β* with 250 nM of the XBP1 mini-substrate. (C) Michaelis−Menten curves of IRE1α* and IRE1β* for the XBP1 mini-substrate. Data
shown are mean SEM, n = 3.
cleavage, IRE1β’s RNase primarily cleaves ER-localized mRNA
encoding secretory proteins.23 Furthermore, there is evidence
that IRE1β might have a distinct set of mRNA substrates from
IRE1α.23−25 These differences in enzymatic activity have been
highlighted in vivo, where IRE1β but not IRE1α has been
shown to be functionally required for mucin production.25
This relationship suggests that selective IRE1β inhibition may
be an attractive target in diseases characterized by the
overproduction of mucin, like cystic fibrosis, chronic
obstructive pulmonary disease, and asthma.26 Additionally,
mice that lack IRE1β develop colitis at an advanced rate and
have more pronounced hyperlipidemia, revealing that IRE1β
may play an essential role in lipid metabolism and
inflammation responses in gut and bronchial tissues.21,27
Here, we demonstrate that IRE1β’s RNase domain is
similarly efficient as IRE1α in cleaving an XBP1 mini-substrate
in vitro. Using different ATP-competitive inhibitors that have
been shown to either activate or inhibit IRE1α’s RNase
activity, we investigated the allostery between IRE1β’s kinase
and RNase domains. We find that an ATP-competitive
inhibitor that is a potent activator of IRE1α’s RNase activity
has a similar effect on IRE1β. Furthermore, we demonstrate
that, like IRE1α, IRE1β’s RNase activity can be allosterically
inhibited with ATP-competitive ligands called kinase inhibitor
RNase attenuators (KIRAs). This result motivated us to
determine whether we could develop KIRAs that demonstrate
paralog selectivity. By performing medicinal chemistry around
the scaffold of a KIRA developed to target IRE1α, we were able
to identify both dual IRE1α/β and paralog-selective KIRAs.
Using a kinobead-based profiling method, we demonstrate that
these inhibitors are selective for IRE1 on the kinome level.
Together, we expect our IRE1-targeting toolset will provide
valuable reagents for defining paralog-specific function in cells
and disease models.
dephosphorylated versions of IRE1 are monomeric and RNase
inactive.28,29 To assess the RNase activities of IRE1α* and
IRE1β*, we used a fluorescence-quenched XBP1 mini-
substrate labeled with a 5′-AlexaFluor647 and a 3′-IowaBlack
fluorescence quencher (Figure 1A). We tested the activity of
IRE1β*’s RNase by monitoring the real-time cleavage of a
XBP1 mini-substrate in vitro. Despite previous reports that
IRE1β’s RNase is less capable or incapable of cleaving XBP1,22
a comparison of equal concentrations of IRE1α* and IRE1β*
reveal that both are able to efficiently cleave the XBP1 mini-
substrate (Figure 1B). Further inspection of the XBP1 mini-
substrate product revealed that the RNase domains of IRE1α*
and IRE1β* generate fragments of similar size, supporting the
notion that these paralogs cleave XBP1 at the same site
(Supporting Information Figure 1). Next, we measured the
rate of IRE1α* and IRE1β* cleavage for varying concen-
trations of the XBP1 mini-substrate to determine their
Michaelis constant (KM) values for this RNA substrate. The
KM values for the XBP1 mini-substrate between the two
paralogs were nearly identical (Figure 1C). This result reveals
that IRE1β* can form back-to-back dimers, which are
necessary for RNase activity. Additionally, when dimerized,
the RNase domains of IRE1α* and IRE1β* are similarly able
to bind and cleave XBP1.
Previously, it has been established that the kinase and RNase
domains of IRE1α are allosterically coupled.28−30 The basis of
this allostery relies on the conformation of a structural element
in the ATP-binding site called helix-αC. When IRE1α is
unphosphorylated and inactive, helix-αC adopts a conforma-
tion that is incompatible with the back-to-back dimer required
for RNase activity. Phosphorylation of IRE1α’s activation loop
stabilizes the active conformation of helix-αC, which promotes
back-to-back dimerization and RNase activity.29 It has been
shown that ATP-competitive inhibitors can have divergent
effects on IRE1α’s RNase activity by preventing or promoting
formation of the back-to-back dimer through modulation of
helix-αC’s conformation.13,28 For example, AT9283 potently
activates IRE1α’s RNase activity by promoting IRE1α
dimerization, presumably though stabilization of the active
conformation of helix-αC.29 In contrast, inhibitor 1 (KIRA8)
inactivates IRE1α’s RNase domain by stabilizing an inactive
helix-αC conformation and preventing IRE1α dimerization.31
We refer to ATP-competitive inhibitors that dually inhibit
kinase and RNase activity, like 1, as kinase inhibitor RNase
attenuators (KIRAs). To examine the allostery between the
kinase and RNase domain of IRE1β, we tested the effects of
AT9283 and 1 on IRE1β’s RNase activity. To allow us to
RESULTS AND DISCUSSION
■
Activity and Allosteric Regulation of IRE1β’s RNase
Domain. We first performed biochemical characterization of
IRE1β with a construct that contains just the cytosolic kinase
and RNase domains, which we refer to as IRE1β*. Previous
studies have demonstrated that a similar construct of IRE1α,
IRE1α*, possesses both kinase and RNase activity.28,29 For in
vitro studies of IRE1, IRE1α* and IRE1β* were expressed in
baculovirus-infected insect cells and purified in the activation
loop phosphorylated form. Activation loop phosphorylation of
IRE1 promotes the formation of an RNase active dimer,
commonly referred to as the back-to-back dimer.13 In contrast,
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Figure 2. Divergent modulation of IRE1β’s RNase domain with ATP-competitive inhibitors. (A) Concentration regimes for testing the allosteric
modulation of IRE1β*’s RNase domain. (Top) Schematic of the oligomeric states of Low [IRE1β*] and High [IRE1β*]. (Bottom) Real time
fluorescence curves and initial rates for Low [IRE1β*] and High [IRE1β*]. Data shown are mean SEM, n = 3. (B) Activation of IRE1β*’s RNase
activity. (Top) Structure of the ATP-competitive RNase activator AT9283. (Bottom) Real time fluorescence curves and rates of XBP1 mini-
substrate cleavage for Low [IRE1β*] treated with DMSO (light gray) or AT9283 (purple) in the in vitro RNase assay. Data shown are mean
SEM, n = 3. (C) Inhibition of IRE1β*‘s RNase activity. (Top) Structure of KIRA 1. (Bottom) Real time fluorescence curves and rates of XBP1
mini-substrate cleavage for High [IRE1β*] treated with DMSO (dark gray) or 1 (coral) in the in vitro RNase assay. Data shown are mean SEM,
n = 3.
Figure 3. SAR of ATP-competitive KIRAs. (A) Chemical structure of KIRA 1 (top). Structural elements of 1 that are varied in our study are
colored. RNase IC50 values for 1 against both IRE1α* and IRE1β* and the fold selectivity for IRE1α (bottom). Inhibitor data are shown as the
mean SEM, n = 3. (B) LigPlot map detailing the binding interactions (yellow sticks) between inhibitor 2, a close analog of 1, and the ATP-
binding site of IRE1α (PDB: 4U6R). Residues involved in hydrogen-bond interactions are shown as sticks. Residues involved in hydrophobic
interactions are shown as gray eyelashes. (C) LigPlot map detailing the hypothesized binding pocket of IRE1β and its interactions with pyridine−
pyrimidine-based ligands. Residues that are conserved between IRE1β and IRE1α are shown in gray. Conservative mutations are shown in purple,
and nonconservative mutations are shown in red.
measure both activation and inhibition of RNase activity, we
tested these inhibitors under two different IRE1β* concen-
tration regimes. We used a concentration of IRE1β* (Low
[IRE1β*]) that is mainly monomeric and has low RNase
activity to measure activation of IRE1β’s RNase activity by
AT9283 (Figure 2A). A higher concentration of IRE1β* (High
[IRE1β*]), which contains a substantial amount of the RNase
active dimer, was used to test the inhibitory properties of KIRA
1. We found that Low [IRE1β*] treated with AT9283
demonstrated a 12-fold higher rate of XBP1 mini-substrate
cleavage than the apo form (Figure 2B). In contrast, treatment
of High [IRE1β*] with 1 led to an almost complete loss of its
ability to cleave the XBP1 mini-substrate (Figure 2C). The
ability of these two classes of ATP-competitive inhibitors to
divergently modulate IRE1β*’s RNase activity suggests that
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a
Scheme 1. Introduction of R1−R3 Groups into the Conserved Pyrimidine−Pyridine Scaffold of 1
a
Reagents and conditions: (i) R1-amine, TEA, DMSO, 80 °C, 18 h; (ii) 4-aminophenols, 5-aminonaphthols, or 4-aminonaphthols, K2CO3, DMF,
155 °C, μW, 2 h; (iii) sulfonyl chloride, pyridine, DCM, RT, 18 h; (iv) TFA/DCM (1:1), RT, 2 h.
the allosteric communication between its kinase and RNase
domains is similar to that of IRE1α.
tion (SNAr) of 2-chloro-4-(2-fluoro-3-pyridinyl)pyrimidine
with mono boc-protected diaminocycloalkanes. A subsequent
SNAr with commercially available 4-amino-phenols or amino-
naphthols was used to generate R2 group diversity. Diverse R3
groups were introduced by sulfonylation with various sulfonyl
chlorides. Following sulfonylation, final inhibitors were
generated by boc-deprotection with TFA (Scheme 1).
We screened KIRAs for the ability to inhibit the RNase
activities of IRE1α* and IRE1β* with our fluorescence-
quenched XBP1 mini-substrate assay (Figure 1A). We first
generated and tested an analog of 2 (inhibitor 3) that contains
identical trans-1,4-cyclohexanediamine R1 and 2-chlorophenyl
R3 groups but a slightly different 1,4-substituted 4-amino-1-
naphthol R2 group (Table 1). Inhibitor 3 is almost equipotent
against IRE1α* and IRE1β*. Replacement of the trans-1,4-
Paralog-Selective KIRAs. Having demonstrated that the
RNase activity of IRE1β can be modulated through its ATP-
binding site, we next determined whether it would be possible
to develop KIRAs with enhanced paralog selectivity. To do
this, we performed a structure−activity-relationship (SAR)
study around the pyrimidine−pyridine scaffold of KIRA 1 and
a close analog (KIRA 2; Figure 3A,B). Although KIRAs 1 and
2 were optimized for IRE1α, they were selected as starting
points for derivatization because of the demonstrated marked
selectivity of KIRA 1 on the kinome level.31,35 To visualize
potential differences in KIRA-binding residues between IRE1α
and IRE1β, we used a cocrystal structure of KIRA 2 bound to
IRE1α to generate a model of potential inhibitor contact
residues with IRE1β (Figure 3B,C). The basic trans-1,4-
cyclohexanediamine moiety of 2 makes several hydrophobic
contacts with residues lining the ATP-binding site of IRE1α
and forms a salt bridge with Glu651 located in IRE1α’s helix
αH. While IRE1β contains a Glu at the same position
(Glu600), an alanine residue (Ala646) in IRE1α that makes
hydrophobic contacts with the piperidine ring is substituted
with an Arg (Arg595) in IRE1β (Figure 3B,C). Therefore, we
introduced various R1 alicyclic groups that contain a basic
amine in our SAR to potentially exploit these differences
(Figure 3A). The naphthyl group of 2 bridges the core
pyrimidine−pyridine scaffold and the sulfonamide group.
While most of the residues in the ATP-binding site of
IRE1α that form contacts with the bridging naphthyl group of
2 are conserved in IRE1β, the identity of the gatekeeper
residueIle in IRE1α (Ile642) and Leu in IRE1β (Leu591)
is a clear difference between the two paralogs in this region of
the binding pocket. We thus varied the naphthyl group of 2
with various naphthyl and substituted phenyl R2 groups to see
if inhibitors could differentiate between IRE1α and IRE1β in
this region of the kinase active site. The arylsulfonamide of 2
occupies a pocket created by movement of helix-αC to an
inactive conformation. A notable difference in this pocket
between IRE1α and IRE1β includes a change from an Ala
(Ala609) in the helix-αC of IRE1α to a Val residue in IRE1β
(Val558). Therefore, we also generated analogs that contain
various sulfonamides at the R3 position to see if the pocket
formed by the movement of helix-αC could be used to gain
selectivity between IRE1β and IRE1α.
a
Table 1. SAR of KIRAs Containing Naphthyl R2 Groups
To facilitate the rapid generation of inhibitors, we
introduced diverse R1−R3 groups into the conserved
pyrimidine−pyridine scaffold of 1 (Scheme 1). A four-step
synthetic route was used to diversify the commercially available
2-chloro-4-(2-fluoro-3-pyridinyl)pyrimidine scaffold. R1 groups
were introduced through the nucleophilic aromatic substitu-
a
RNase IC50 data are shown as mean SEM, n = 3. RNase selectivity
was determined by dividing the IRE1α* RNase IC50 by the IRE1β*
RNase IC50 value for each inhibitor (individual IC50 curves provided
in Supporting Information Figure 2).
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a
cyclohexanediamine R1 group with a 6-amino-2-aza-spiro[3.3]
heptane moiety (inhibitor 4), while maintaining the same R1
and R3 groups as inhibitor 3, led to decreased inhibition of
both IRE1β*’s and IRE1α*’s RNase domains, albeit
disproportionately, resulting in 4.2-fold selectivity for
IRE1β*. Next, we explored whether R3 sulfonamide groups
affect paralog selectivity. Varying the R3 group of 3 from a 2-
chlorophenyl to a 2-chlorobenzyl group (inhibitor 5) led to a
∼30-fold reduction in potency against IRE1β*’s RNase activity
with minimal influence on IRE1α* potency. We speculated
that the Ala to Val substitution present in the helix-αC of
IRE1β would create a more restricted binding pocket that
would favorably accommodate smaller R3 groups. However, we
found that inhibitor 6, which contains identical R1 and R2
substituents as inhibitor 3 but a smaller cyclobutyl R3 group, is
remarkably selective (>2000-fold) for IRE1α*. Finally, we
determined how changing the display of the R3 sulfonamide
from the R2 naphthyl group would affect paralog potency and
selectivity. We observed that inhibitor 7, which contains the
same R1 and R3 groups as 6 but a 1,5-naphthyl substitution
instead of a 1,4, was also highly selective (>300-fold) for
IRE1α*.
Table 2. KIRAs Containing 4-aminophenol R2 Groups
We found that a 4-amino-1-naphthol R2 group provided
inhibitors that are IRE1α selective or equipotent for IRE1α
and IRE1β while maintaining reasonably potent RNase
inhibition (RNase IC50 < 20 nM). To develop KIRAs that
are potent and selective for IRE1β, we next focused on
optimizing the R2 position for this paralog (Table 2). A notable
difference between IRE1α and IRE1β is the identity of their
gatekeeper residues. IRE1α has an isoleucine residue (Ile642),
while IRE1β has a leucine (Leu591) at the gatekeeper position.
This led us to reason that it may be possible to selectively
target IRE1β over IRE1α by using smaller R2 substituents,
specifically substituted 4-aminophenol groups. First, we
generated inhibitor 8, which contains a trans-1,4-cyclo-
hexanediamine R1 group, a 2-chlorophenyl R3 group, and a
4-amino-3-fluorophenol at the R2 position. Inhibitor 8, which
contains the same R1 and R3 groups as IRE1α/IRE1β
equipotent inhibitor 3, demonstrated single-digit nanomolar
potency against IRE1β* and is 5-fold selective for this paralog
over IRE1α*. Thus, replacing the 4-amino-1-naphthol R2
group of 3 with a 4-amino-3-fluorophenol increased IRE1β*
selectivity. Replacing the trans-1,4-cyclohexanediamine R1
group of 8 with an (S)-3-aminopiperidine (inhibitor 9)
resulted in less potent inhibition of IRE1α* and IRE1β*. An
analog of 9 that contains a 2-chlorobenzylsulfonamide (10)
was found to be almost 10-fold selective for IRE1α* over
IRE1β*.
For all inhibitors tested, we found that varying away from a
2-chlorophenylsulfonamide R3 group was detrimental to
IRE1β* potency. Thus, for the remainder of inhibitors
generated in this SAR study, a 2-chlorophenylsulfonamide
group was maintained at the R3 position. Because the
introduction of a 4-amino-3-fluorophenol R2 group modestly
improved selectivity for IRE1β* (compare inhibitor 3 to
inhibitor 8), we next determined whether increasing the size of
the halogen at this position could enhance the ability to
discriminate between the two paralogs. Unfortunately,
inhibitors containing a 4-amino-3-chlorophenol (11 and 12)
were markedly less potent against IRE1β*. We next
determined the effect of changing the position of the halogen
from the 3-position to the 2-position on the 4-aminophenol R2
group by generating inhibitors 13 and 14, which contain 4-
a
RNase IC50 data are shown as mean SEM, n = 3. RNase selectivity
was determined by dividing the IRE1α RNase IC50 by the IRE1β
RNase IC50 value for each inhibitor (individual IC50 curves provided
in Supporting Information Figure 2).
amino-2-fluorophenols. Both inhibitors demonstrated promis-
ing selectivity for IRE1β*, with inhibitor 13, which contains a
trans-1,4-cyclohexanediamine R1 group, exhibiting single-digit
nanomolar potency against IRE1β* and 60-fold selectivity over
IRE1α*. Finally, we looked at how adding an additional
halogen to the 4-amino-2-fluorophenol R2 group affected the
potency and paralog selectivity of inhibitors. Inhibitor 15,
which contains a 4-amino-5-chloro-2-fluorophenol R2 group
and a trans-1,4-cyclohexanediamine R1 group, demonstrated
potent inhibition of IRE1β* and >100-fold selectivity over
IRE1α*. Replacing the trans-1,4-cyclohexanediamine R1 group
of 15 with an (S)-3-aminopiperidine (16) led to reduced
potency against IRE1β* and diminished selectivity. The
addition of a fluorine to the 3- or 5-positions (inhibitor 17
and 18, respectively) of the 4-amino-2-fluorophenol R2 group
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Figure 4. Kinase inhibition by exemplary KIRAs. (A) Schematic of the in vitro IRE1 kinase assay. Phosphotransferase activity of IRE1 was
measured by monitoring the IRE1α*/IRE1β*-mediated phosphorylation of the exogenous substrate myelin basic protein (MBP) by ATP[γP32/33].
(B) Ki values for IRE1α* and IRE1β* kinase activity. All Ki values were calculated using the Cheng-Prusoff equation. For IRE1α*, the Ki values
shown are mean SEM, n = 3. For IRE1β, the Ki values shown are the mean SEM, n = 3 (denoted by an asterisk (∗)) or the average values from
two measurements (individual inhibitory values provided in Supporting Information Table 1). Inhibitors with Ki values lower than the
concentration of IRE1α* or IRE1β* used in the assay are denoted with a double dagger (‡). Individual IC50 curves provided in Supporting
Information Figure 2.
was found to be generally detrimental to IRE1β* inhibition.
From this SAR, it is clear that employing 4-amino-2-
halophenol R2 groups and a trans-1,4-cyclohexanediamine R1
substituent can impart high IRE1β RNase selectivity.
method to determine if representative inhibitors from these
three classes are selective on the kinome level. Specifically, we
measured the abilities of representative inhibitors to compete
for binding of lysate kinases to an affinity matrix containing
seven different nonselective ATP-competitive inhibitors
(kinobeads).32−34 Kinobeads enrich kinases through their
ATP-binding sites and allow inhibitors to be quantitatively
profiled against a large percentage of the human kinome.
Binding of an inhibitor prevents enrichment with the kinobead
matrix, and kinome selectivity can be determined by
comparing the relative enrichment of a kinase target between
DMSO and inhibitor-treated lysates using quantitative mass
spectrometry (Supporting Information Figure 3). In total, we
profiled five inhibitors (1 (IRE1α-selective), 4 and 9 (dual-
IRE1), and 13 and 15 (IRE1β-selective)) with an HCT116/
HEK293 cell lysate mixture spiked with exogenous recombi-
nant IRE1β* (or IRE1α* for profiling of 1). The addition of
exogenous IRE1 was required because we were not able to
reproducibly quantify the endogenous protein.
The kinome selectivity of 1 was previously assessed with in
vitro activity assays against a large panel of recombinant
kinases, where it was shown to be highly selective across the
kinome.31,35 These results were verified in our kinobead assay,
where IRE1α was the only kinase identified as a target of 1
(Supporting Information Figure 4). Out of ∼150 kinases
quantified in the assay, IRE1β was the primary target of 3, 9,
13, and 15, with only one off-target identified for each
respective inhibitor (Figure 5). Inhibitor 3, which contains a
naphthol R2 group, prevented the enrichment of PKD2 in
addition to IRE1β. Compounds 9, 13, and 15, which all
contain 4-amino-halophenol R2 groups, share the same off
target, CDK2. To determine whether the level of competition
observed in our kinobead assay would lead to potent inhibition
of CDK2 activity, we tested the ability of 9, 13, and 15 to
inhibit the CDK2/cyclin A complex in vitro. Despite the ability
of 9, 13, and 15 to moderately prevent CDK2 binding to the
kinobead matrix in our lysate profiling experiments (Support-
KIRAs Inhibit the Kinase Activity of IRE1α/β. KIRA 1
was previously confirmed to be an ATP-competitive inhibitor
of IRE1α, and we assumed that all of the analogs generated in
this study inhibit IRE1 RNase activity through the ATP-
binding site.31 To verify that our inhibitors also inhibit the
kinase activities of IRE1α* and IRE1β*, we measured the
propensity of a representative set of compounds to block IRE1-
mediated phosphorylation of an exogenous substratemyelin
basic protein (MBP; Figure 4A). As expected, we found that all
compounds tested also inhibited the kinase activity of IRE1α*
and IRE1β* (Figure 4B). While we were not able to accurately
determine kinase IC50 values for a number of our more potent
inhibitors (3, 11, 13, 14, 15, and 18) against IRE1β* due to
constraints of the assay, we found that kinase IC50 values were,
in general, several-fold lower than RNase IC50 values for this
paralog. This is in contrast to IRE1α*, where inhibitors
generally exhibited similar IC50 values against kinase and
RNase activity. The reason for this discrepancy between the
two paralogs is unknown but may reflect differences in the
energetic penalties required to disrupt the dimeric states of
IRE1α* and IRE1β*, and, in turn, RNase inhibition. Thus,
inhibitors typically demonstrate slightly greater selectivity for
IRE1β* in the kinase assay than in the RNase assay.
Collectively, these results show that trends in inhibitor
selectivity are consistent in both kinase and RNase assays.
Kinome Selectivity of Paralog-Selective KIRAs. From
our optimization efforts, we were able to generate inhibitors
with three different profiles: IRE1α-selective inhibitors, dual-
IRE1 inhibitors that are equipotent against IRE1α and IRE1β,
and IRE1β-selective inhibitors. Inhibitors with these profiles
would be excellent tools for examining the contributions of
IRE1α and/or IRE1β activity in cells if they possess sufficient
general kinase selectivity. Therefore, we used a lysate profiling
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Figure 5. Kinome profiling of KIRAs 3, 9, 13, and 15. The kinome selectivities of 3, 9, 13, and 15 were determined with a previously described
kinobead lysate profiling method. Kinases that were quantified in the experiment are shown as circular nodes, where node size and color have been
scaled to the log2 difference (difference in LFQ intensity between DMSO treated and inhibitor treated lysates) between DMSO and treatment with
10 μM of KIRA 3 (A), 9 (B), 13 (C), or 15 (D). Gray nodes represent kinases that were quantified, but no competition with an inhibitor was
observed. Values shown are the mean of four replicates.
ing Information Figure 5A), all three KIRAs showed weak
inhibition of CDK2/cyclin A kinase activity (IC50 values > 5
μM; Supporting Information Figure 5B). For 9, 13, and 15, the
discrepancy between the activity assay with CDK2/cyclin A
and our kinobead profiling experiment could possibly be due
to the high concentrations (10 μM) of these inhibitors tested
in the profiling experiment, which allows sufficient occupation
of CDK2’s ATP-binding site despite their weak affinities for
this kinase. Another possibility is that these inhibitors can only
interact with inactive CDK2, but not active CDK2-cyclin
complexes. Previously, it has been shown that cyclin binding
causes a conformational change in CDK2’s ATP-binding site
that makes it inaccessible to certain types of ATP-competitive
inhibitors.36
Structural Model of Inhibitor Paralog Selectivity.
While extensive structural characterization has been performed
with IRE1α, equivalent information is not available for
IRE1β.29,31,37−42 To build a structural model of IRE1β
inhibition and selectivity, we used a sequence alignment of
the kinase and RNase domains of IRE1α and IRE1β. The
kinase domains of IRE1α and IRE1β share 81% sequence
identity, while the RNase domains are 61% identical (Figure
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Figure 6. Sequence alignment and structural comparison of IRE1α and IRE1β ATP-binding sites. (A) Sequence alignment of IRE1α and IRE1β.
(Top) Sequence comparison between IRE1α and IRE1β mapped onto the crystal structure of IRE1α (PDB: 4U6R). Residues that are conserved
are shown in gray, residues that have a conservative replacement are shown in purple, and residues with nonconservative replacements are shown in
red. (Bottom) Sequence alignment of IRE1α and IRE1β shows 80% sequence identity of the kinase domain and 61% sequence identity of the
RNase domain. (B) Interactions between 2, a close structural analog of KIRA 1, and the ATP-binding site of IRE1α. Compound 2 is shown as
yellow sticks, key interacting residues are shown as gray sticks, and interactions are denoted with green dashed lines. (C) Hypothesized binding
mode of KIRA 2 by mapping of nonidentical residues of IRE1β onto the structure of 4U6R. Conservative replacement nonidentical residues are
shown as purple sticks; nonconservative nonidentical residues are shown as red sticks. Residue numbering is for IRE1β.
6A).24 Due to the high degree of sequence identity between
the kinase domains of the two paralogs, we predicted that
KIRAs would have similar binding modes in their ATP-binding
sites. From our sequence alignment, we mapped sequence
differences between IRE1α and IRE1β onto a cocrystal
structure of IRE1α bound to the pyrimidine−pyridine KIRA
2 (PDB: 4U6R).31 KIRA 2 is nearly identical to potent dual-
IRE1 inhibitor 3, differing only by the presence of a methyl
group at the 2-position of the R2 naphthyl ring. Using the
mutagenesis tool in PyMol, residue side chain differences
between IRE1α and IRE1β were visualized for the cocrystal
structure of IRE1α bound to 2. We next looked at residues
within 5 Å of inhibitor 2. Of the 23 residues identified, only
four residues are not identical in IRE1α and IRE1β: Ala609
(Val558 in IRE1β), Ile642 (Leu591 in IRE1β), Ala646
(Arg595 in IRE1β), and Thr648 (Ser597 in IRE1β; Figure
6B,C). Although the specific interactions that each paralog
makes with inhibitors cannot be known in the absence of high-
resolution structural information, these sequence differences
likely play a major role in paralog selective inhibition. The
IRE1α residues Ala646 (Arg595 in IRE1β) and Thr648
(Ser597 in IRE1β) are directed toward the alicyclic portion of
2’s trans-1,4-cyclohexanediamine R1 group, which forms a salt
bridge with Glu651 in IRE1α and most likely Glu600 in
IRE1β. While we were unsuccessful in identifying R1 groups
that were optimal for IRE1β’s Arg595 and Ser597 residues, our
SAR data show that IRE1α is more permissive of variability at
this position. The stabilization of an inactive helix-αC “out”
conformation by KIRAs leads to IRE1 monomerization and
RNase inhibition. The 2-chlorophenyl aryl sulfonamide R3
group of 2 occupies the pocket created by movement of helix-
αC. In this inactive conformation, Ala609 in IRE1α (Val559 in
IRE1β) from helix-αC is projected toward inhibitor R3 groups.
Our SAR data demonstrate that the smaller Ala residue of
IRE1α allows it to favorably accommodate a greater diversity
of R3 substituents than IRE1β. Finally, the side chain of
IRE1α’s gatekeeper residue, Ile642, points directly toward the
methyl naphthyl R2 group of 2 (Figure 6B). Interestingly,
selectivity for IRE1β, which possesses a Leu gatekeeper, over
IRE1α was achieved most effectively by introducing 4-amino-
2-fluorophenol R2 groups. Thus, we hypothesize that 4-amino-
2-fluorophenol R2 groups are able to form more favorable
interactions with the Leu residue of IRE1β than with the Ile
residue of IRE1α and that paralog selectivity can be achieved
by optimizing gatekeeper/R2 group interactions.
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Conclusion. Pharmacological modulation of IRE1α’s
RNase domain using ATP-competitive inhibitors has proven
to be a useful method for examining the allosteric relationship
between the kinase and RNase domains of IRE1α, and for
better understanding the functional outputs of IRE1α’s RNase
domain. While IRE1α’s close paralog IRE1β shares the same
domain architecture, much less is known about the enzymatic
activities of IRE1β and its function within the cell. Under-
standing IRE1β’s role in the cell has been particularly difficult
to determine because, while IRE1β expression is limited to
epithelial cells of the gut and bronchia, all cells that express
IRE1β also express IRE1α. Pharmacological tools that are able
to discriminate between IRE1α and IRE1β will be useful
reagents for defining paralog-specific function in cells.
There have been conflicting reports on how efficiently the
RNase domain of IRE1β is able to cleave XBP1 mRNA.16,22
Here, we show that a cytosolic construct of IRE1β (IRE1β*)
has RNase activity comparable to IRE1α* for an XBP1 mini-
substrate. Therefore, IRE1β, like IRE1α, appears to form
dimers that bind and cleave XBP1 mRNA in vitro. A reason for
the observed discrepancies in IRE1β’s ability to cleave XBP1
may stem from differences in the phosphorylation state of the
activation loop, which promotes formation of the RNase-active
dimer, in the recombinant constructs used. The recombinant
IRE1β* construct used in our study contains a glutathione S-
transferase (GST) tag at its N-terminus. GST has been
demonstrated to form dimers,43 which likely enhances IRE1β*
activation loop autophosphorylation when being expressed in
insect cells. The N-terminal GST tag may also directly
promote formation of RNase-active back-to-back dimers in
the absence of activation loop phosphorylation. Regardless of
the source of these discrepancies, our data clearly show that
IRE1β can efficiently cleave an XBP1 mini-substrate when it is
dimerized. The implications of our results for the ability of full-
length IRE1β to cleave XBP1 mRNA in cells is unclear. In
cells, IRE1β’s ability to undergo activation loop autophosphor-
ylation appears to be hampered compared to its paralog
IRE1α.44 Therefore, XBP1 mRNA may be a poor substrate for
IRE1β in cells because the kinase domain does not undergo
activation loop phosphorylation under ER stress. Differences in
the ability of IRE1α and IRE1β to undergo autophosphor-
ylation may also help explain previously reported disparities in
the mRNA substrate preference between these two paralogs in
cells. Under ER stress, XBP1 mRNA may be cleaved by
activation loop phosphorylated IRE1α while IRE1β functions
to aid in later stages of the UPR by cleaving ER-localized
mRNAs as a part of RIDD.23−25
Our SAR studies suggest that the key to discriminating
between the very similar ATP-binding sites of IRE1α and
IRE1β is optimizing the KIRA substituent that is in close
proximity to their gatekeeper residues, which is Ile in IRE1α
and Leu in IRE1β. Altogether, KIRAs with differing selectivity
profiles (highly IRE1α-selective, dual-IRE1, and highly IRE1β-
selective) will serve as useful tools for work characterizing the
specific outputs of IRE1 in cells and, potentially, in animal
models of gastrointestinal and pulmonary ER stress-related
diseases.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.9b00482.
■
S
Supplemental Figures S1−S12, methods, protein amino
acid sequence and DNA sequence, synthetic procedures,
and inhibitor characterization (PDF)
Compound characterization checklist (XLSX)
Kinobead profiling data (XLSX)
AUTHOR INFORMATION
Corresponding Author
*E-mail: djmaly@uw.edu.
■
ORCID
Dustin J. Maly: 0000-0003-0094-0177
Notes
The authors declare the following competing financial
interest(s): B.J.B., F.R.P., and D.J.M. are founders, equity
holders, and consultants for OptiKIRA, LLC (Cleveland, OH),
a biotech company focused on treating ER-stress-induced
retinal degeneration.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health
(R01DK116064 and R01DK100623 (F.R.P. and D.J.M.)).
■
REFERENCES
■
(1) Scheuner, D., and Kaufman, R. J. (2008) The Unfolded Protein
Response: A Pathway That Links Insulin Demand with β-Cell Failure
and Diabetes. Endocr. Rev. 29, 317−333.
(2) van Anken, E., Pena, F., Hafkemeijer, N., Christis, C., Romijn, E.
P., Grauschopf, U., Oorschot, V. M., Pertel, T., Engels, S., Ora, A.,
Lastun, V., Glockshuber, R., Klumperman, J., Heck, A. J., Luban, J.,
and Braakman, I. (2009) Efficient IgM assembly and secretion require
the plasma cell induced endoplasmic reticulum protein pERp1. Proc.
Natl. Acad. Sci. U. S. A. 106, 17019−17024.
(3) Walter, P., and Ron, D. (2011) The unfolded protein response:
from stress pathway to homeostatic regulation. Science 334, 1081−
1086.
(4) Tirasophon, W., Welihinda, A. A., and Kaufman, R. J. (1998) A
stress response pathway from the endoplasmic reticulum to the
nucleus requires a novel bifunctional protein kinase/endoribonuclease
(Ire1p) in mammalian cells. Genes Dev. 12, 1812−1824.
(5) Harding, H. P., Zhang, Y., and Ron, D. (1999) Protein
translation and folding are coupled by an endoplasmic-reticulum-
resident kinase. Nature 397, 271−274.
To examine the allosteric relationship between IRE1β’s
kinase and RNase domains, we report the first ATP-
competitive pharmacological modulators of IRE1β. We show
that ATP-competitive inhibitors that activate IRE1α’s RNase
domain also activate IRE1β’s RNase domain. Additionally, we
find that ATP-competitive KIRAs are capable of inhibiting
IRE1β’s RNase activity like IRE1α’s. The ability of ATP-
competitive inhibitors to divergently modulate the RNase
activity of IRE1β suggests that it shares all or most of the
allosteric features of IRE1α.
By generating analogs of a KIRA that are moderately
selective for IRE1α, we were able to identify inhibitors with
two new paralog selectivity profiles: KIRAs that are equipotent
against IRE1α and IRE1β and KIRAs that are highly selective
for IRE1β. Lysate profiling experiments confirmed that all
three classes of KIRAs are selective across the human kinome.
(6) Haze, K., Yoshida, H., Yanagi, H., Yura, T., and Mori, K. (1999)
Mammalian transcription factor ATF6 is synthesized as a trans-
membrane protein and activated by proteolysis in response to
endoplasmic reticulum stress. Mol. Biol. Cell 10, 3787−3799.
(7) Hetz, C., Chevet, E., and Oakes, S. A. (2015) Proteostasis
control by the unfolded protein response. Nat. Cell Biol. 17, 829−838.
I
DOI: 10.1021/acschembio.9b00482
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
(8) Travers, K. J., Patil, C. K., Wodicka, L., Lockhart, D. J.,
Weissman, J. S., and Walter, P. (2000) Functional and genomic
analyses reveal an essential coordination between the unfolded
protein response and ER-associated degradation. Cell 101, 249−258.
(9) Shore, G. C., Papa, F. R., and Oakes, S. A. (2011) Signaling cell
death from the endoplasmic reticulum stress response. Curr. Opin.
Cell Biol. 23, 143−149.
(10) Han, D., Lerner, A. G., Vande Walle, L., Upton, J. P., Xu, W.,
Hagen, A., Backes, B. J., Oakes, S. A., and Papa, F. R. (2009)
IRE1alpha kinase activation modes control alternate endoribonuclease
outputs to determine divergent cell fates. Cell 138, 562−575.
(11) Wang, X. Z., Harding, H. P., Zhang, Y., Jolicoeur, E. M.,
Kuroda, M., and Ron, D. (1998) Cloning of mammalian Ire1 reveals
diversity in the ER stress responses. EMBO J. 17, 5708−5717.
(12) Ali, M. M., Bagratuni, T., Davenport, E. L., Nowak, P. R., Silva-
Santisteban, M. C., Hardcastle, A., McAndrews, C., Rowlands, M. G.,
Morgan, G. J., Aherne, W., Collins, I., Davies, F. E., and Pearl, L. H.
(2011) Structure of the Ire1 autophosphorylation complex and
implications for the unfolded protein response. EMBO J. 30, 894−
905.
(13) Lee, K. P., Dey, M., Neculai, D., Cao, C., Dever, T. E., and
Sicheri, F. (2008) Structure of the dual enzyme Ire1 reveals the basis
for catalysis and regulation in nonconventional RNA splicing. Cell
132, 89−100.
(14) Lu, Y., Liang, F. X., and Wang, X. (2014) A synthetic biology
approach identifies the mammalian UPR RNA ligase RtcB. Mol. Cell
55, 758−770.
(15) Kosmaczewski, S. G., Edwards, T. J., Han, S. M., Eckwahl, M. J.,
Meyer, B. I., Peach, S., Hesselberth, J. R., Wolin, S. L., and
Hammarlund, M. (2014) The RtcB RNA ligase is an essential
component of the metazoan unfolded protein response. EMBO Rep.
15, 1278−1285.
(16) Calfon, M., Zeng, H., Urano, F., Till, J. H., Hubbard, S. R.,
Harding, H. P., Clark, S. G., and Ron, D. (2002) IRE1 couples
endoplasmic reticulum load to secretory capacity by processing the
XBP-1 mRNA. Nature 415, 92−96.
(17) Hollien, J., Lin, J. H., Li, H., Stevens, N., Walter, P., and
Weissman, J. S. (2009) Regulated Ire1-dependent decay of messenger
RNAs in mammalian cells. J. Cell Biol. 186, 323−331.
(18) Hollien, J., and Weissman, J. S. (2006) Decay of endoplasmic
reticulum-localized mRNAs during the unfolded protein response.
Science 313, 104−107.
(19) Lerner, A. G., Upton, J. P., Praveen, P. V., Ghosh, R.,
Nakagawa, Y., Igbaria, A., Shen, S., Nguyen, V., Backes, B. J., Heiman,
M., Heintz, N., Greengard, P., Hui, S., Tang, Q., Trusina, A., Oakes, S.
A., and Papa, F. R. (2012) IRE1alpha induces thioredoxin-interacting
protein to activate the NLRP3 inflammasome and promote
programmed cell death under irremediable ER stress. Cell Metab.
16, 250−264.
(20) Upton, J. P., Wang, L., Han, D., Wang, E. S., Huskey, N. E.,
Lim, L., Truitt, M., McManus, M. T., Ruggero, D., Goga, A., Papa, F.
R., and Oakes, S. A. (2012) IRE1alpha cleaves select microRNAs
during ER stress to derepress translation of proapoptotic Caspase-2.
Science 338, 818−822.
(21) Bertolotti, A., Wang, X., Novoa, I., Jungreis, R., Schlessinger, K.,
Cho, J. H., West, A. B., and Ron, D. (2001) Increased sensitivity to
dextran sodium sulfate colitis in IRE1beta-deficient mice. J. Clin.
Invest. 107, 585−593.
(22) Imagawa, Y., Hosoda, A., Sasaka, S.-I., Tsuru, A., and Kohno, K.
(2008) RNase domains determine the functional difference between
IRE1α and IRE1β. FEBS Lett. 582, 656−660.
(25) Martino, M. B., Jones, L., Brighton, B., Ehre, C., Abdulah, L.,
Davis, C. W., Ron, D., O'Neal, W. K., and Ribeiro, C. M. P. (2013)
The ER stress transducer IRE1β is required for airway epithelial
mucin production. Mucosal Immunol. 6, 639−654.
(26) Hauber, H.-P., Foley, S. C., and Hamid, Q. (2006) Mucin
Overproduction in Chronic Inflammatory Lung Disease. Can. Respir.
J. 13, 327−335.
(27) Iqbal, J., Dai, K., Seimon, T., Jungreis, R., Oyadomari, M.,
Kuriakose, G., Ron, D., Tabas, I., and Hussain, M. M. (2008) IRE1β
Inhibits Chylomicron Production by Selectively Degrading MTP
mRNA. Cell Metab. 7, 445−455.
(28) Wang, L., Perera, B. G., Hari, S. B., Bhhatarai, B., Backes, B. J.,
Seeliger, M. A., Schurer, S. C., Oakes, S. A., Papa, F. R., and Maly, D.
J. (2012) Divergent allosteric control of the IRE1alpha endoribonu-
clease using kinase inhibitors. Nat. Chem. Biol. 8, 982−989.
(29) Feldman, H. C., Tong, M., Wang, L., Meza-Acevedo, R.,
Gobillot, T. A., Lebedev, I., Gliedt, M. J., Hari, S. B., Mitra, A. K.,
Backes, B. J., Papa, F. R., Seeliger, M. A., and Maly, D. J. (2016)
Structural and Functional Analysis of the Allosteric Inhibition of
IRE1α with ATP-Competitive Ligands. ACS Chem. Biol. 11, 2195−
2205.
(30) Papa, F. R., Zhang, C., Shokat, K., and Walter, P. (2003)
Bypassing a kinase activity with an ATP-competitive drug. Science 302,
1533−1537.
(31) Harrington, P. E., Biswas, K., Malwitz, D., Tasker, A. S., Mohr,
C., Andrews, K. L., Dellamaggiore, K., Kendall, R., Beckmann, H.,
Jaeckel, P., Materna-Reichelt, S., Allen, J. R., and Lipford, J. R. (2015)
Unfolded Protein Response in Cancer: IRE1alpha Inhibition by
Selective Kinase Ligands Does Not Impair Tumor Cell Viability. ACS
Med. Chem. Lett. 6, 68−72.
(32) Golkowski, M., Vidadala, R. S. R., Lombard, C. K., Suh, H. W.,
Maly, D. J., and Ong, S.-E. (2017) Kinobead and Single-Shot LC-MS
Profiling Identifies Selective PKD Inhibitors. J. Proteome Res. 16,
1216−1227.
(33) Bantscheff, M., Eberhard, D., Abraham, Y., Bastuck, S., Boesche,
M., Hobson, S., Mathieson, T., Perrin, J., Raida, M., Rau, C., Reader,
V., Sweetman, G., Bauer, A., Bouwmeester, T., Hopf, C., Kruse, U.,
Neubauer, G., Ramsden, N., Rick, J., Kuster, B., and Drewes, G.
(2007) Quantitative chemical proteomics reveals mechanisms of
action of clinical ABL kinase inhibitors. Nat. Biotechnol. 25, 1035−
1044.
(34) Golkowski, M., Perera, G. K., Vidadala, V. N., Ojo, K. K., Van
Voorhis, W. C., Maly, D. J., and Ong, S. E. (2018) Kinome
chemoproteomics characterization of pyrrolo[3,4-c]pyrazoles as
potent and selective inhibitors of glycogen synthase kinase 3. Mol.
Omics 14, 26−36.
(35) Morita, S., Villalta, S. A., Feldman, H. C., Register, A. C.,
Rosenthal, W., Hoffmann-Petersen, I. T., Mehdizadeh, M., Ghosh, R.,
Wang, L., Colon-Negron, K., Meza-Acevedo, R., Backes, B. J., Maly,
D. J., Bluestone, J. A., and Papa, F. R. (2017) Targeting ABL-IRE1α
Signaling Spares ER-Stressed Pancreatic β Cells to Reverse Auto-
immune Diabetes. Cell Metab. 25, 1207.
̈
(36) Alexander, L. T., Mobitz, H., Drueckes, P., Savitsky, P.,
Fedorov, O., Elkins, J. M., Deane, C. M., Cowan-Jacob, S. W., and
Knapp, S. (2015) Type II Inhibitors Targeting CDK2. ACS Chem.
Biol. 10, 2116−2125.
(37) Wiseman, R. L., Zhang, Y., Lee, K. P., Harding, H. P., Haynes,
C. M., Price, J., Sicheri, F., and Ron, D. (2010) Flavonol Activation
Defines an Unanticipated Ligand-Binding Site in the Kinase-RNase
Domain of IRE1. Mol. Cell 38, 291−304.
(38) Sanches, M., Duffy, N. M., Talukdar, M., Thevakumaran, N.,
Chiovitti, D., Canny, M. D., Lee, K., Kurinov, I., Uehling, D., Al-awar,
R., Poda, G., Prakesch, M., Wilson, B., Tam, V., Schweitzer, C., Toro,
A., Lucas, J. L., Vuga, D., Lehmann, L., Durocher, D., Zeng, Q.,
Patterson, J. B., and Sicheri, F. (2014) Structure and mechanism of
action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease
inhibitors. Nat. Commun. 5, 4202.
(23) Nakamura, D., Tsuru, A., Ikegami, K., Imagawa, Y., Fujimoto,
N., and Kohno, K. (2011) Mammalian ER stress sensor IRE1β
specifically down-regulates the synthesis of secretory pathway
proteins. FEBS Lett. 585, 133−138.
(24) Iwawaki, T., Hosoda, A., Okuda, T., Kamigori, Y., Nomura-
Furuwatari, C., Kimata, Y., Tsuru, A., and Kohno, K. (2001)
Translational control by the ER transmembrane kinase/ribonuclease
IRE1 under ER stress. Nat. Cell Biol. 3, 158−164.
(39) Korennykh, A. V., Egea, P. F., Korostelev, A. A., Finer-Moore,
J., Zhang, C., Shokat, K. M., Stroud, R. M., and Walter, P. (2009) The
J
DOI: 10.1021/acschembio.9b00482
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
Articles
unfolded protein response signals through high-order assembly of
Ire1. Nature 457, 687−693.
(40) Joshi, A., Newbatt, Y., McAndrew, P. C., Stubbs, M., Burke, R.,
Richards, M. W., Bhatia, C., Caldwell, J. J., McHardy, T., Collins, I.,
and Bayliss, R. (2015) Molecular mechanisms of human IRE1
activation through dimerization and ligand binding. Oncotarget 6,
13019−13035.
(41) Colombano, G., Caldwell, J. J., Matthews, T. P., Bhatia, C.,
Joshi, A., McHardy, T., Mok, N. Y., Newbatt, Y., Pickard, L., Strover,
J., Hedayat, S., Walton, M. I., Myers, S. M., Jones, A. M., Saville, H.,
McAndrew, C., Burke, R., Eccles, S. A., Davies, F. E., Bayliss, R., and
Collins, I. (2019) Binding to an Unusual Inactive Kinase
Conformation by Highly Selective Inhibitors of Inositol-Requiring
Enzyme 1α Kinase-Endoribonuclease. J. Med. Chem. 62, 2447−2465.
(42) Concha, N. O., Smallwood, A., Bonnette, W., Totoritis, R.,
Zhang, G., Federowicz, K., Yang, J., Qi, H., Chen, S., Campobasso, N.,
Choudhry, A. E., Shuster, L. E., Evans, K. A., Ralph, J., Sweitzer, S.,
Heerding, D. A., Buser, C. A., Su, D.-S., and Deyoung, M. P. (2015)
Long-Range Inhibitor-Induced Conformational Regulation of Human
IRE1 Endoribonuclease Activity. Mol. Pharmacol. 88, 1011−1023.
(43) Fabrini, R., De Luca, A., Stella, L., Mei, G., Orioni, B., Ciccone,
S., Federici, G., Lo Bello, M., and Ricci, G. (2009) Monomer−Dimer
Equilibrium in Glutathione Transferases: A Critical Re-Examination.
Biochemistry 48, 10473−10482.
(44) Grey, M. J., Cloots, E., Simpson, M. S., LeDuc, N., Serebrenik,
Y. V., De Luca, H., De Sutter, D., Luong, P., Thiagarajah, J. R., Paton,
A. W., Paton, J. C., Seeliger, M. A., Eyckerman, S., Janssens, S., and
Lencer, W. I. (2019) IRE1β negatively regulates IRE1α signaling in
response to endoplasmic reticulum stress, BioRxiv, 586305, https://
www.biorxiv.org/content/10.1101/586305v1.
K
DOI: 10.1021/acschembio.9b00482
ACS Chem. Biol. XXXX, XXX, XXX−XXX