Long-range inhibitor-induced conformational regulation of human IRE1α endoribonuclease activity

Article abstract of DOI:10.1124/mol.115.100917

Activation of the inositol-requiring enzyme-1 alpha (IRE1α) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and

Full text of DOI:10.1124/mol.115.100917

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2015/10/05/mol.115.100917.DC1.html
1521-0111/88/6/1011–1023$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.115.100917
Mol Pharmacol 88:1011–1023, December 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Long-Range Inhibitor-Induced Conformational Regulation of
Human IRE1a Endoribonuclease Activity s
Nestor O. Concha, Angela Smallwood, William Bonnette, Rachel Totoritis, Guofeng Zhang,
Kelly Federowicz, Jingsong Yang, Hongwei Qi, Stephanie Chen, Nino Campobasso,
Anthony E. Choudhry, Leanna E. Shuster, Karen A. Evans, Jeff Ralph, Sharon Sweitzer,
Dirk A. Heerding, Carolyn A. Buser, Dai-Shi Su, and M. Phillip DeYoung
Oncology R&D (K.F., J.Y., L.E.S., K.A.E., J.R., D.A.H., C.A.B., D.S.S, M.P.D.), Biological Sciences (R.T., G.Z., H.Q., S.C., A.E.C.,
S.S.), and Chemical Sciences, GlaxoSmithKline Research and Development, Collegeville, Pennsylvania (N.O.C., A.S., W.B., N.C.)
Received July 20, 2015; accepted September 25, 2015
ABSTRACT
Activation of the inositol-requiring enzyme-1 alpha (IRE1a) pro- kinase activation loop to the DFG-out conformation. Inactiva-
tein caused by endoplasmic reticulum stress results in the tion of IRE1a RNase activity appears to be caused by a
homodimerization of the N-terminal endoplasmic reticulum conformational change, whereby the aC helix is displaced,
luminal domains, autophosphorylation of the cytoplasmic ki- resulting in the rearrangement of the kinase domain-dimer
nase domains, and conformational changes to the cytoplasmic interface and a rotation of the RNase domains away from each
endoribonuclease (RNase) domains, which render them func- other. In contrast, staurosporine binds at the ATP-binding site
tional and can lead to the splicing of X-box binding protein 1 of IRE1a, resulting in a dimer consistent with RNase active
(XBP 1) mRNA. Herein, we report the first crystal structures of the yeast Ire1 dimers. Activation of IRE1a RNase activity appears
cytoplasmic portion of a human phosphorylated IRE1a dimer to be promoted by a network of hydrogen bond interactions
in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)- between highly conserved residues across the RNase dimer
2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1a- interface that place key catalytic residues poised for reaction.
selective kinase inhibitor, and staurosporine, a broad spectrum These data implicate that the intermolecular interactions be-
kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)- tween conserved residues in the RNase domain are required for
2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the ki- activity, and that the disruption of these interactions can be
nase and RNase activities of IRE1a. The inhibitor interacts with achieved pharmacologically by small molecule kinase domain
the catalytic residues Lys599 and Glu612 and displaces the inhibitors.
(Harding, et al., 2002; Ron, 2002; Feldman et al., 2005). UPR
signaling also regulates cell survival by modulating apoptosis and
autophagy and can induce cell death under prolonged ER stress if
teins, hypoxia, glucose deprivation, depletion of endoplasmic
the misfolded protein burden is too high (Ma and Hendershot,
reticulum (ER) calcium levels, and changes in ER redox status
2004; Rouschop et al., 2010; Woehlbier and Hetz, 2011).
Introduction
Cellular stresses, such as accumulation of unfolded pro-
activate the unfolded protein response (UPR), an intracellular
signal transduction network involved in restoring protein
homeostasis [reviewed by Walter and Ron (2011)]. To allevi-
ate these types of stress responses, the UPR responds by
halting protein translation, activating transcription of UPR-
associated target genes, and degrading misfolded proteins
Three key ER membrane proteins have been identified as
primary effectors of the UPR: protein kinase R–like ER kinase
(PERK), inositol-requiring enzyme-1 (IRE1) a/b, and activating
transcription factor 6 (Schroder and Kaufman, 2005). IRE1a is a
transmembrane protein that functions both as an ER stress
sensing receptor via its N-terminal ER luminal domain and as a
signal transducer via its cytoplasmic C-terminal kinase and
endoribonuclease (RNase) domains (Tirasophon et al., 1998).
Upon sensing ER stress, the extracellular portion of the IRE1a
protein will homodimerize, allowing for transautophosphoryla-
tion, which, in turn, induces a conformational change, resulting
All authors are past or present employees of GlaxoSmithKline. No potential
conflicts of interest were disclosed by the authors.
dx.doi.org/10.1124/mol.115.100917.
This article has supplemental material available at molpharm.
aspetjournals.org.
s
ABBREVIATIONS: APY29, 2-N-(3H-benzimidazol-5-yl)-4-N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine; BHQ-1, Black Hole quencher-1;
ER, endoplasmic reticulum; FAM, 6-carboxyfluorescein fluorescent reporter; FBS, fetal bovine serum; GSK2850163, (R)-2-(3,4-dichlorobenzyl)-N-(4-
methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide; ID, identity; IRE1, inositol-requiring enzyme-1; KIRA6, 1-(4-[8-amino-3-tert-butylimidazo(1,5-a)
pyrazin-1-yl]naphthalen-1-yl)-3-[3-(trifluoromethyl)phenyl]urea; PCR, polymerase chain reaction; PDB, Protein Data Bank; PERK, protein kinase R–like
endoplasmic reticulum kinase; pIRE1a, phosphorylated inositol-requiring enzyme-1 alpha; RNase, endoribonuclease; RT, real time; SAR, structure
activity relationship; STS, staurosporine; TEV, tobacco etch virus; UPR, unfolded protein response; UPRE, unfolded protein response element; XBP 1,
X-box binding protein 1.
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Concha et al.
in activation of the RNase domains (Ali et al., 2011). Phosphor- that this process is conserved evolutionarily. Here, we present
ylation within the kinase activation loop is an essential step for a proposed structure of the final state of a human phosphor-
RNase activation (Prischi et al., 2014). Mammalian IRE1a ylated (active) IRE1a dimer that is cocrystallized with two
excises a 26–base pair intron from the mRNA of X-box binding kinase inhibitors that have opposing effects to the RNase
protein 1 (XBP 1), which causes a translational frame shift activity of the protein.
downstream of the splice site to produce XBP 1s, the active form
of the transcription factor (Yoshida et al., 2001; Calfon et al.,
Materials and Methods
IRE1a Protein Expression and Purification. The cytosolic
domain of human IRE1a (NM_001433), encompassing amino acids
547–977, was cloned into pENTR/tobacco etch virus (TEV)/D-TOPO
2002; Lee et al., 2002). XBP 1 is responsible for the activation of
key UPR target genes, including molecular chaperones and
components of the ER-associated protein degradation machin-
ery (Lee at al., 2003). Activation of IRE1a is also reported to
result in the induction of regulated IRE1a-dependent degrada- (Life Technologies, Carlsbad, CA) and subsequently transferred to a
pDest8 (Life Technologies) vector backbone containing the N-terminal
Flag epitope tag followed by the 6xHis tag and TEV cleavage site
(ENLYFQG/S). Baculovirus generation was accomplished using
the Bac-to-Bac baculovirus generation system (Life Technologies).
Flag-His6-TEV-IRE1a (547–977) protein expression in baculovirus-
infected insect cells was accomplished following established procedures
(Wasilko and Lee, 2006). Briefly, a proprietary Sf9 insect cell line
was grown to the early log phase, infected with 1 Â 107 baculovirus-
infected insect cells/10 l culture, and incubated at 27°C. Cell paste was
harvested at 66–72 hours postinfection. A human phosphorylated or
tion of a subset of mRNAs encoding secretory proteins or the
induction of apoptosis via IRE1a signaling through its kinase
domain and downstream effectors ASK1, JNK1, and Caspase-12
(Urano et al., 2000; Hollien and Weissman, 2006).
Loss of ER homeostasis (i.e., loss or hyperactivation of
UPR signaling) has been attributed to a number of dis-
eases, including cancer, diabetes, cardiovascular diseases,
liver diseases, and neurodegenerative disorders, and the UPR
is increasingly becoming an attractive pathway in drug
discovery (Hetz et al., 2013; Maly and Papa, 2014). To this dephosphorylated IRE1a protein containing N-terminal Flag-His6
tags and a TEV protease cleavage site between the tags and an IRE1a
protein was purified from ∼150 g of cells from a 10-l culture [lysed in
1.5 l of lysis buffer (50 mM Hepes, pH 7.5, 10% glycerol, and 300 mM
NaCl) by the EmulsiFlex-C50 homogenizer (Avestin, Ottowa, ON,
Canada)]. The protein in the clear supernatant from centrifugation at
30,000g for 30 minutes at 4°C was first captured in 20 ml of NiNTA-SF
beads (Qiagen, Venlo, Netherlands) in batch mode for 4 hours at 4°C.
The beads were poured into a column and washed with 20 mM
imidazole in lysis buffer, and the IRE1a protein was eluted from the
column by 300 mM imidazole in 50 mM Hepes, pH 7.5, and 150 mM
end, an increasing body of work has been performed to identify
potent and selective molecules of IRE1a and better under-
stand how these molecules bind and affect IRE1a activa-
tion mechanisms. Crystal structures of the C-terminal region
of phosphorylated (active) yeast Ire1 were the first to be char-
acterized and revealed that Ire1 forms dimers arranged in
a “back-to-back” configuration, with the kinase active sites
facing outward [Protein Data Bank (PDB) identity (ID) 2RIO;
Lee at al., 2008; PDB ID 3FBV; Korennykh et al., 2009; PDB
ID 3LJ0; Wiseman et al., 2010). This dimer arrangement also NaCl buffer. The Ni elution pool was concentrated using a 10-kDa
molecular weight cutoff filter concentrator to about 25 ml, to which
3 mg of TEV protease was added to remove the His6 tag. This mixture
was then transferred into dialysis tubing (8-kDa molecular weight
cutoff) and dialyzed overnight against 3 l of MonoQ buffer A (50 mM
Hepes, pH 7.5, 50 mM NaCl, 5 mM DTT, and 1 mM EDTA),
passed through a second 20-ml MonoQ column (GE Healthcare,
Piscataway, NJ), and eluted with a 50–500 mM NaCl gradient over
10 column volumes. The eluted samples were analyzed by liquid
chromatography–mass spectrometry, and the major peak with a mass
consistent with the expected molecular weight for the protein plus
formed the basis of a rod-shaped helical structure, represent-
ing high-order oligomeric Ire1 structure in complex
a
with the kinase inhibitor 2-N-(3H-benzimidazol-5-yl)-4-N-(5-
cyclopropyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine (APY29)
(3FBV) (Korennykh et al., 2009). These yeast back-to-back
structures contrast with the first reported human IRE1a
dimer: a dephosphorylated C-terminal IRE1a- Mg21-ADP
complex, possibly representing an early state prior to phos-
phoryl transfer (PDB ID 3P23; Ali et al., 2011). In this
structure, the kinase active sites are facing each other and three phosphates (80 mass units per phosphate) was purified in a
Hiload Superdex 200 sizing column (GE Healthcare), with a buffer of
50 mM Hepes, pH 7.5, 200 mM NaCl, 5 mM DTT, and 1 mM EDTA.
The eluted protein (2–3 mg protein in 1-ml aliquots) was stored at
280°C and later used in assays and crystallography. The remaining
fractions from the Mono Q column were pooled and treated with
l-phosphatase to produce the fully dephosphorylated enzyme before
it was further purified and stored in the same way as the triply
phosphorylated protein.
are in a suitable orientation and proximity for transautophos-
phorylation, but the RNase domains are far from each other
and inactive. A similar “face-to-face” structure was recently
reported for mouse IRE1a, but this structure was phosphor-
ylated (PDB ID 4PL3; Sanches et al., 2014). More recently, a
few other dephosphorylated human crystal structures were
reported. One is of a cocrystal structure containing a kinase
domain inhibitor bound to an IRE1a monomer (PDB ID 4U6R;
Harrington et al., 2014). Here, no dimers were found with the
Phosphorylated IRE1a RNase Activity Assay. The nuclease
enzymatic activity of phosphorylated IRE1a (pIRE1a) was measured
inhibitor-bound structure, suggesting that the compound may using a dual labeled 36-mer RNA substrate that contained the IRE1a
recognition sequence, with a 6-carboxyfluorescein fluorescent reporter
(FAM) at the 39 end, and the Black Hole quencher-1 (BHQ-1) at the
59 end (59/6-FAM/rCrArG rUrCrC rGrCrA rGrCrA rCrUrG/BHQ-1/39;
Integrated DNA Technologies, Coralville, IA). Upon cleavage, the
release of FAM results in an increase in fluorescent signal measured
at lex/lem 5 485/535 nm. A typical enzymatic reaction was carried out
with 10 nM pIRE1a and a 500 nM substrate in a buffer containing
20 mM Hepes, pH 7.5, 5 mM MgCl2, 10 mM NaCl, 1 mM DTT, 0.05%
Tween 20, and 0.02% heat-treated casein (heated for 20 minutes at
60°C prior to use each time), and the fluorescence change was followed
either prevent dimerization or stabilize a monomeric IRE1a.
The second report presented two back-to-back dimers of
IRE1a, one in the apo form and one in an inhibitor-bound
form (PDB ID 4Z7G and 4Z7H; Joshi et al., 2015). These
structures are consistent with yeast Ire1 dimers, but are
distinct in that the apo structure contains a twisted interface
between the dimers across the RNase domains, which may
represent an additional intermediate of IRE1a prior to full
activation. The culmination of all these structures may depict
the IRE1 protein at various levels of activation and suggest using an Envision plate reader (PerkinElmer, Waltham, MA).

Structure of Human Phosphorylated (Active) IRE1a Dimer
1013
To identify inhibitors of IRE1a nuclease activity, pIRE1a was 90 minutes. Following the incubation, the reaction was terminated
screened against the GlaxoSmithKline compound collection. The RNA with 0.02% SDS solution in nuclease-free water. The plates were
oligomer substrate was added to the assay plates containing 10 mM of centrifuged for 3 minutes at 1000 rpm prior to measuring product
compound. The reaction was initiated immediately by the addition of
the enzyme, and the plates were centrifuged for 1 minute at 500 rpm.
formation using the Viewlux imager (PerkinElmer).
Competitive Inhibition Studies. In single compound inhibition
The final reaction mixture contained 10 nM pIRE1a, 25 nM RNA studies, the concentrations of the substrate or noncleavable substrate
oligomer, 20 mM Hepes, pH 7.5, 5 mM MgCl2, 1 mM DTT, 0.05%
Tween 20, 10 mM NaCl, and 0.02% casein. The reaction plates were
incubated at room temperature for 90 minutes before the reaction was
terminated with 0.015% SDS in 20 mM Hepes, pH 7.5. The plates were
(analog in which the ribose linked to the guanine at the cleavage site
was replaced by deoxyribose) and GSK2850163 were varied and the
enzyme concentration was fixed at 10 nM. The modes of inhibition
and the inhibition constants were determined by fitting the initial
centrifuged for 3 minutes at 1000 rpm prior to measuring product velocities to different models (competitive, uncompetitive, and non-
formation using the Viewlux imager (PerkinElmer).
competitive) using Grafit software (Erithacus Software). In a double
pIRE1a Kinase Assays. In the ADP-Glo assay, a compound’s inhibition experiment, the concentration of the first inhibitor was
potency toward pIRE1a kinase activity was measured as its inhibition varied at several different concentrations of the second inhibitor, and
of an intrinsic, slow ATP hydrolysis activity. One hundred nanoliters the concentrations of the enzyme and substrate were kept constant at
of dimethylsulfoxide solution of (R)-2-(3,4-dichlorobenzyl)-N-(4-
10 and 100 nM, respectively. The reactions were monitored kineti-
methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide (GSK2850163) cally, and the initial reaction velocities were analyzed using the
at various concentrations was added into a white Greiner low volume Yonetani-Theorell equation.
384-well plate. The reaction was carried out with 5 nM pIRE1a and
Protein Crystallization and Structure Determination. Crys-
60 mM ATP in 10 ml of 50 mM Hepes buffer, pH 7.5, containing 30 mM tals of pIRE1a (547–977) with GSK2850163 (PDB ID 4YZ9) were
NaCl, 10 mM MgCl2, 1 mM DTT, 0.02% Chaps, and 0.01 mg/ml bovine prepared by mixing pIRE1a with 0.5 mM GSK2850163 and incubating
serum albumin. The reaction was stopped after 2 hours by adding 5 ml
overnight on ice. The crystals were grown at 20°C by vapor diffusion in
of ADP-Glo reagent I, which also depletes the remaining ATP. Follow- sitting drops containing 2 ml of protein (13 mg/ml in 50 mM Hepes, pH
ing a 1-hour incubation, 5 ml of ADP-Glo reagent II was added 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.5 mM GSK2850163,
into the reaction, which converts the ADP product into ATP to serve
and 0.25% dimethylsulfoxide) and 2 ml of reservoir solution containing
as the substrate for the coupled luciferin/luciferase reaction. After polyethylene glycol (PEG) 3350 (16–22%), 100 mM Hepes, pH 7.0, and
30 minutes, the plate was read on a Wallac ViewLux microplate imager 200 mM Ca21 acetate. The crystals were thick rods that appeared over
(PerkinElmer).
2–5 days and reached full size (0.05 Â 0.025 Â 0.3 mm) in 2 weeks.
Seeding was used to improve crystal quality. The pIRE1a-GSK2850163
crystals were frozen in a solution of 20% ethylene glycol, 22% PEG 3350,
and 0.2 M calcium acetate added in a stepwise manner to the protein
drop before mounting the crystal on the loop.
In the fluorescence polarization assay, a compound’s affinity was
measured by its ability to compete with a fluorescent-labeled ATP site
ligand for binding to the kinase domain. The fluorescence polarization
assay was carried out in the same reaction buffer described above. A
10-ml reaction contained 5 nM pIRE1a, 5 nM fluorescent ligand
The crystals of pIRE1a with Mg21-ADP (PDB ID 4YZD) were grown
(GSK369716A), and GSK2851063 at various concentrations. After by mixing 2 ml of protein solution [10 mg/ml pIRE1a (547–977) in
the addition of reagents, the plate was incubated at room temperature
for 15 minutes and then read on an Analyst GT multimode reader
50 mM Hepes, pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA, 1 mM
ADP (100 mM ADP stock was pH ∼7.0), and 1 mM MgCl2] with 2 ml of
(Molecular Devices, Sunnyvale, CA) at an excitation/emission of 485/ reservoir solution (16% PEG 3350 and 200 mM Na1 malonate, pH 6.0)
530 nm, with a dichroic of 505 nm. The IC50 values were calculated in sitting drops at room temperature. Seeding was used to initiate
using the data fitting software GraFit (Erithacus Software, Horley, crystal growth. Crystals appeared the next day and grew to full size in
UK).
3 weeks. For data collection, the crystals were frozen in a solution of
PERK Kinase Assay. Recombinant PERK protein (Life Technol- 20% ethylene glycol, 22% PEG 3350, and 200 mM Na1 malonate, pH
ogies) was purchased for use in a selectivity assay to test the pIRE1a 6.0, and added to the protein drop before mounting the crystals on the
inhibitors. A solution of PERK (0.40 nM) in assay buffer containing loop.
10 mM HEPES, pH 7.5, 2 mM CHAPS, 5 mM MgCl2, and 1 mM DTT was
preincubated with the compound for 30 minutes at room temperature.
The reaction was initiated by the addition of the substrate mixture,
which contained 0.040 mM biotinylated eukaryotic translation initiation
factor 2a peptide and 5 mM ATP. The reaction plates were incubated at
room temperature for 60 minutes, followed by termination with a
The complex of pIRE1a (547–977) with staurosporine (STS) (PDB
ID 4YZC) was prepared by mixing the protein with 0.5 mM staur-
osporine and incubating overnight on ice. The crystals were grown at
20°C by vapor diffusion in a sitting drop containing 2 ml of protein
(13 mg/ml in 50 mM Hepes, pH 7.5, 200 mM NaCl, 5 mM DTT, and
1 mM EDTA) and 2 ml of reservoir solution containing PEG 300 (30–
detection/quench mixture. The detection/quench mixture contained 15 mM 40%), 100 mM Hepes, pH 7.5, and 200 mM KCl. Small hexagonal
EDTA, 4 nM eukaryotic translation initiation factor 2a phosphoanti- plates appeared over 5–10 days and reached full size (0.05 Â 0.75 Â
body (Millipore, Billerica, MA), 4 nM europium-labeled anti-rabbit 0.15 mm) in ∼20 days. The crystals were flash frozen in liquid N2
antibody (PerkinElmer), and 40 nM streptavidin APC (PerkinElmer).
The plates were incubated for 10 minutes at room temperature prior to
directly from the crystallization drop. All diffraction data were
collected at the Advanced Photon Source, Argonne National Labora-
measuring the homogeneous time resolved fluorescence signal (exci- tories, Life Sciences Collaborative Access Team, Sector 21.
tation at 610–640 nm and emission at 660 nm) using the Viewlux
imager (PerkinElmer).
All structures were determined by molecular replacement with
Phaser (Afonine et al., 2010), as implemented in CCP4 (Winn et al.,
RNase L Activity Assay. A solution of 0.1 nM RNase L, 1 mM 2011), using the human dephosphorylated IRE1a- Mg21-ADP complex
ATP, and 8 nM 2-5A [29,59-linked oligoadenylate, p1–3(A29p59A)n $ 2] in (3P23; Ali et al., 2011) as a model. Refinement was performed by a
assay buffer containing 20 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM combination of Refmac5 (Murshudov et al., 1997) and Phenix (Afonine
TCEP, 0.5 mM CHAPS, 100 mM NaCl, and 0.02% casein was
et al., 2010), with manual adjustments to the model in COOT (Emsley
incubated at room temperature for 30 minutes. A 0.2 mM solution of et al., 2010). The quality of the model was monitored using Molprobity
a 36-mer RNA substrate (59/6-FAM rUrUrA rUrCrA rArArU rUrCrU (Chen et al., 2010). Figures were made with PYMOL (Schrödinger,
rUrArU rUrUrG rCrCrC rCrArU rUrUrU rUrUrU rGrGrU rUrUrA
LLC, New York, NY).
BHQ-1/39; Integrated DNA Technologies) was prepared in assay
Detection of XBP 1 Splicing by Real-Time Polymerase
buffer and added to reaction plates containing compounds. The Chain Reaction. Multiple myeloma cancer cell lines were obtained
reaction was initiated with the addition of the RNase L-ATP-2-5A
mixture, and the plates were incubated at room temperature for
from ATCC (Manassas, VA) or DSMZ (Braunschweig, Germany). Cells
were cultured in the appropriate culture medium supplemented with

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Concha et al.
10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO) at 37°C
in humidified incubators under 5% CO2. Cells were seeded into six-
exchange was done with HDExaminer. Tryptic digestion of all the
samples gave a sequence coverage of at least 98% of the amino acid
well plates at a density of 1.5 Â 106 cells/well in the appropriate media sequence.
containing 1% FBS media and were treated with 5 mg/ml tunicamycin
(MP Biomedicals, Newport Beach, CA) for 1 hour before the addition of
GSK2850163 for 3 hours (4 hours total). For studies involving
staurosporine (Sigma-Aldrich) treatment, cells were treated with
staurosporine for 30 minutes, followed by 5 mg/ml tunicamycin for
1 hour. RNA was collected using the Qiagen RNEasy Mini Kit. RNA
was quantitated using a NanoDrop (Thermo Scientific, Philadelphia,
PA) and stored at 280°C. To generate cDNA, the High-Capacity cDNA
Reverse Transcription Kit (Life Technologies) was used. For polymer-
ase chain reaction (PCR), a 25-ml reaction included 12.5 ml of
Master Mix (Promega Go-Taq 2X Master Mix, Madison, WI), 1 ml
each of forward and reverse primers (100 ng/ml), 9.5 ml of water, and
1 ml of cDNA template. Primers for human XBP 1 were forward,
59-CCTGGTTGCTGAAGAGGAGG-39, and reverse, 59-CCATGGGGA-
GATGTTCTGGG-39. Real-time (RT) PCR conditions were 95°C
for 5 minutes, 95°C for 30 seconds, 58°C for 30 seconds, 72°C for
30 seconds, and 72°C for 5 minutes, with 35 cycles of amplification.
After RT-PCR, reactions were run on a 3% agarose gel and visualized
using SYBR Safe DNA gel stain (Life Technologies) and a BioRad
Imager (Hercules, CA).
Western Blot Analysis. RPMI 8226 cells were seeded into six-
well plates at a density of 2.0 Â 106 cells/well in RPMI 1640 media
containing 1% FBS. Cells were treated with the same conditions as
described above for XBP 1 splicing detection by RT-PCR. To harvest
protein lysates, cells were lysed with 60 ml of 1X cell lysis buffer (Cell
Signaling Technologies, Danvers, MA) containing protease and phos-
phatase inhibitors. Cell lysates were quantified using the Pierce BCA
Protein Assay Kit (Thermo Scientific), and samples were read on a
SPECTRAmax 190 instrument (Molecular Devices, Sunnyvale, CA).
Following quantitation, 40 mg of protein was run on 4–12% Bis-Tris
Preparation and Characterization of Compounds 1–24. De-
tails are provided in the Supplemental Methods.
Results
Crystallization of the Novel Inhibitor GSK2850163
Bound to pIRE1a. Because of the relevance of the IRE1a/
XBP 1 pathway in human disease, we sought to identify
small molecules that would inhibit IRE1a RNase activity.
GSK2850163 was discovered as a result of a high-throughput
screening campaign to identify IRE1a-selective inhibitors of
XBP 1 splicing. It is a highly selective inhibitor with dual
activity: it inhibits IRE1a kinase activity (IC50 5 20 nM)
and RNase activity (IC50 5 200 nM) (Fig. 1A; Supplemental
Table 1). In competition kinetic studies, GSK2850163 and a
noncleavable RNA substrate demonstrated mutually exclu-
sive binding to activated IRE1a (Ki 5 200 6 20 nM)
(Supplemental Fig. 1). We hypothesized that this was due to
bound GSK2850163 altering the preferred enzyme struc-
ture for RNA substrate binding (and vice versa) and not due
to a physical overlapping of binding sites.
To investigate the mode of binding and enable structure-
guided optimization of GSK2850163, we determined the coc-
rystal structure of GSK2850163 with the C-terminal portion of
pIRE1a (residues 547–977; PDB ID 4YZ9) (Fig. 1B; Supple-
mental Fig. 2; Table 1). The structure of pIRE1a-GSK2850163
is of a back-to-back dimer, with one inhibitor molecule bound to
gels (Life Technologies), and protein was transferred onto 0.45 mM the kinase domain of each protomer in a pocket next to the
nitrocellulose membranes (Life Technologies) using the BioRad semi-
dry transfer blotting apparatus. Membranes were blocked for 1 hour
using Li-Cor Odyssey Blocking Buffer (Lincoln, NE) and then probed
with the following antibodies overnight: pIRE1a S724 (1:1000,
#ab48187; Abcam, Cambridge, UK), total IRE1a (1:1000, #ab37073;
Abcam), and GAPDH (1:2000, #A300-639A; Bethyl Laboratories,
Montgomery, TX). After washing, blots were incubated with donkey
anti-rabbit IRDye-800CW secondary antibody (Li-Cor), and proteins
were visualized using the Odyssey Imaging System (Li-Cor).
XBP 1 Transcriptional Activity Assay. PANC-1 cells were
seeded into six-well plates at a density of 5.0 Â 103 cells/well in RPMI
1640 media containing 10% FBS. Cells were cotransfected with a
pGL3-5x unfolded protein response element (UPRE)–luciferase re-
porter containing five repetitions of the XBP-1 DNA binding site
(a kind gift from R. Prywes, Columbia University, New York, NY)
and pRL-SV40 (Promega) using the FuGENE6 transfection reagent
(Roche, Indianapolis, IN). Forty-eight hours later, cells were treated
with 2.5 mg/ml tunicamycin for 1 hour, followed by GSK2850163
treatment for 16 hours. Luciferase expression was measured using
Dual-Glo Luciferase Assay kit (Promega) and normalized to Renilla
expression levels.
Hydrogen-Deuterium Exchange. pIRE1a (547–977, 78 mM)
was incubated with 250 mM GSK2850163 or 1 mM staurosporine for
at least 18 hours. Two microliters of protein-inhibitor mixture was
mixed with 18 ml of D2O at room temperature for 1 minute, after which
20 ml of 4 M guanidinium chloride in 1 M glycine buffer, pH 2.5, and
120 ml of formic acid were added and immediately transferred to an ice
cold bath. All subsequent treatment and analysis was done at 2–4°C.
Fifty microliters of this solution was injected into a Waters Enzymate
ethylene bridged hybrid pepsin 2.1 Â 30 mm column for digestion,
then to a C18 column, and into an LTQ XL Orbitrap mass spectrom-
eter (Thermo Scientific). The mass-to-charge values were calculated
with XCalibur (Thermo Scientific) and compared with sequences in
kinase aC helix, approximately 12 Å from the hinge region (Fig.
1C). GSK2850163 adopts a U-shaped conformation, with the
tolyl and dichlorophenyl groups facing the inside of the protein
and the spirodecane core partially solvent exposed with the
piperidine ring (A-ring) in a chair conformation. Two key
interactions with conserved kinase catalytic residues, Glu612
and Lys599, are observed. The urea nitrogen and the pyrroli-
dine nitrogen of GSK2850163 form a hydrogen bond interaction
with the side chain of Glu612, and the carbonyl oxygen of the
urea of GSK2850163 interacts through a hydrogen bond with
the side chain of Lys599.
GSK2850163 displaces the kinase activation loop of
pIRE1a, such that the DFG motif is in the “out” conformation
and is flipped by nearly 180°, occupying the ATP binding site
(Fig. 1C). This clearly contrasts with our resolved structure
of pIRE1a-ADP-Mg21 (PDB ID 4YZD), where the DFG motif
is found in the “in” conformation and Phe712 occupies the
same pocket where the GSK2850163 binds in the pIRE1a-
GSK2850163 structure (Fig. 1D; Table 1). Phe712 makes
a p interaction with Tyr628 in a pocket lined by Val613,
Leu616, Vals625, and Leu679. Superposition of the ADP-
Mg21 and GSK2850163-bound pIRE1a complexes shows that
GSK2850163 does not overlap with the ATP-binding site
(data not shown). The structure of pIRE1a in complex with
Mg21-ADP forms a “face-to-face” dimer across the symmetry
planes with neighboring molecules (Supplemental Fig. 3). Con-
sistent with previously published structures, the kinase active
sites are facing each other and are in an orientation and proximity
favorable for transautophosphorylation [3P23 (Ali et al., 2011)
and 4PL3 (Sanches et al., 2014)]. The present structure may
the MASCOT database. The analysis of the hydrogen-deuterium possibly represent an early postphosphoryl-transfer dimer,

Structure of Human Phosphorylated (Active) IRE1a Dimer
1015
Fig. 1. GSK2850163 binds to human pIRE1a and inhibits XBP 1 splicing. (A) GSK2850163 is a selective inhibitor of pIRE1a kinase activity and RNase
activity. It exhibits no or weak inhibition toward a wide range of the other kinases (shown here is the UPR kinase PERK) and nucleases (shown here is
RNase L, its closest homolog). Refer to Materials and Methods for assay details. (B) Overall structure of the pIRE1a-GSK2850163 (PDB ID 4YZ9) back-to-
back dimer. Chemical structure of GSK2850163 (inset). (C) GSK2850163 (yellow) binds next to the aC helix and displaces the activation loop (DFG-out),
such that it is occupying the ATP-binding site represented by its surface. GSK2850163 makes H-bond interactions with the key kinase catalytic residues
Lys599 (K599) and Glu612 (E612). (D) For comparison, the DFG loop is in the in conformation in the structure of pIRE1a-Mg2+-ADP (PDB ID 4YZD), and
Phe712 (F712) occupies the same pocket where GSK2850163 would bind. For both (C) and (D), * indicates the C-terminal end of the visible portion of the
activation loop.
while the face-to-face structure of dephosphorylated human structure of the complex. Since the activity of the R-enantiomer
IRE1a may represent a state just prior to phosphoryl transfer. is equally potent to the racemic compound and the S-enantiomer
Structure Activity Relationship of GSK2850163. To is not active, the initial SAR study was performed with the ra-
provide supportive evidence for the binding mode of cemic analogs.
GSK2850163 observed in the crystal structure, we performed
Second, the NH of urea nitrogen and the pyrrolidine
structure activity relationship (SAR) studies, whereby we nitrogen are required to make an H bond with Glu612
systematically modified the structure of the ligand and (2.98 Å), and any changes in the GSK2850163 (compound 1)
followed the effect by measuring pIRE1a RNase inhibition. molecule disrupting these specific H-bond interactions
First, we observed that while the S-enantiomer is inactive and resulted in loss of activity. For example, replacement of the
its conformation does not fit in the electron density map, the urea nitrogen (compound 2) or capping the NH with a methyl
R-enantiomer inhibited the RNase activity in vitro, with an group (compound 3) caused a loss of potency (Fig. 2A).
IC50 of 0.2 mM (Fig. 2A). The R-enantiomer is refined in the Similarly, conversion of a basic amine in the pyrrolidine ring

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Concha et al.
TABLE 1
Data collection and refinement statistics (molecular replacement)
Values in parentheses are for the highest-resolution shell. Each dataset was collected from a single crystal.
pIRE1a + GSK2850163
pIRE1a + ADP
pIRE1a + STS
Data collection
Space group
Cell dimensions
a, b, c (Å)
P21212
C2
P212121
244.0, 77.8, 88.3
90, 90, 90
3
2.45 (2.54–2.45)
0.073 (0.592)
22.9 (3.1)
191.8, 122.5, 77.9
90.0, 107.1, 90.0
3
3.14 (3.25–3.10)
0.087 (0.569)
15.2 (2.4)
49.3, 155.3, 155.6
90, 90, 90
a, b, g (°)
Molecules/a.u.
Resolution (Å)
Rsym or Rmerge
I/sI
2
2.62 (2.71–2.62)
0.118 (0.565)
22.7 (5.6)
Completeness (%)
Redundancy
Refinement
Resolution (Å)
Number of reflections
Rwork/Rfree
96.7 (89.7)
6.3 (6.3)
100.0 (100.0)
3.8 (3.8)
100.0 (100.0)
11.8 (11.6)
30.0–2.45
59,654
0.24/0.29
45.6–3.14
60,654
0.20/0.25
40.0–2.62
41,900
0.25/0.29
Number of atoms
Protein
17,853
90
9.822
90
12,041
70
Ligand/ion
Water
81
35
62
B factors
Protein
57
57
51
31
24
10
55
23
48
Ligand/ion
Water
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.007
0.93
0.008
1.09
0.002
1.15
a.u., asymmetric unit.
to a nonbasic amide yielded an inactive analog (compound 4; observed in primary multiple myeloma specimens, and clini-
Fig. 2A). cal studies have associated high levels of spliced XBP 1 mRNA
Third, in the binding mode of GSK2850163 in complex with with poor patient survival (Reimold et al., 2001; Nakamura
pIRE1a described here, the lipophilic groups at both ends of et al., 2006; Bagratuni, et al., 2010). To recapitulate an ER
the molecule (the tolyl and dichlorophenyl groups) are accom- stress-induced environment in cell culture, tunicamycin, an
modated within the lipophilic pockets observed in the core inhibitor of N-linked glycosylation, was used (Yoshida et al.,
crystal structure (Supplemental Fig. 2B) and are critical for 2001). XBP 1 mRNA is found primarily in the unspliced form
the inhibitor’s activity. All analogs with polar functionality, under basal conditions. Upon ER stress stimulation, all cells
such as pyridines, were not active (compounds 5 and 6; Fig. induced varying degrees of XBP 1 splicing, which could be
2B) or much less active (compounds 15–19; Fig. 2C). On the reversed following treatment with GSK2850163 (Fig. 3A). ER
other hand, the lipophilic groups were well tolerated (com- stress also induced increased autophosphorylation of IRE1a,
pounds 8–14 and 20–24) in the hydrophobic pocket. Deletion of which could be reduced in a dose-dependent manner by
one of the chlorines to form 3- or 4-monochloro analogs (7 and GSK2850163 (Fig. 3B). To determine if GSK2850163 could
8, respectively) revealed that the 4-chloro substitution was affect the transcriptional function of XBP 1, cells expressing a
more tolerated than the 3 position in terms of potency (Fig. reporter plasmid under the control of five tandem repeats of
2B). Replacement of 3-chloro with other groups, such as fluoro the UPRE motif were treated with tunicamycin, followed by
(11), methyl (12), or methoxy (13), provided equally potent increasing doses of GSK2850163 (Wang et al., 2000). The
analogs, but the trifluoromethyl group (14) caused a signifi- UPRE sequence contains the binding site found at the pro-
cant loss of potency. The tolyl group at the A-ring urea moter of XBP 1 target genes. Induction of ER stress by
preferred lipophilic substitutions, and polar functionalities tunicamycin significantly increases XBP 1 transcriptional activ-
[e.g., pyridines (15–17) and substituted pyridines (18 and 19)] ity in these cells, yet increasing concentrations of GSK2850163
were not tolerated. Deletion of the methyl group gave the are capable of reducing this activity (Fig. 3C). These data support
simple phenyl analog 20 that maintained RNase activity our biochemical and structural evidence that GSK2850163 is an
(IC50 5 0.40 mM) (Fig. 2B). Replacement of the methyl group inhibitor of IRE1a, which is capable of inhibiting both kinase and
at the 4-position with other lipophilic groups, such as chlorine RNase activities of IRE1a.
(21), methoxy (22), and fluorine (23), was well tolerated. The
Crystallization of Staurosporine Bound to pIRE1a. To
benzodioxole analog 24 exhibited an IC50 value of 100 nM. further understand the mechanism by which the RNase
These SAR study results are consistent with the binding mode activity of IRE1a could be modulated pharmacologically with
of GSK2850163 to pIRE1a observed in the crystal structure, kinase inhibitors, we sought to investigate the effect of the
and indicate that GSK2850163 functions as a kinase and broad-spectrum kinase inhibitor STS on IRE1a. It has been
RNase inhibitor of pIRE1a.
previously shown that STS inhibits IRE1a kinase activity
Cellular Activity of GSK2850163. We used a panel of (Ali et al., 2011) and is likely to bind to the ATP-binding site
eight multiple myeloma cell lines to test the effects of GSK2850163 of IRE1a since it competed with the irreversible IRE1a inhib-
on IRE1a RNase activity. Increased IRE1a activity has been itor 4m8C, preventing the formation of a Schiff base with the

Structure of Human Phosphorylated (Active) IRE1a Dimer
1017
Fig. 2. Selected SAR results of compound 1 (GSK2850163). (A) Replacement of the urea nitrogen with a carbon atom (compound 2) or capping the NH
with a methyl group (compound 3) caused a loss of potency. Conversion of basic amine in pyrrolidine ring to a nonbasic amide yielded an inactive analog
(compound 4). (B and C) The lipophilic groups at the tolyl and dichlorophenyl groups were critical for the compound’s activity. On the other hand, the polar
functionalities, such as pyridines, were not tolerated.
kinase active site residue Lys599 (Cross et al., 2012). We 8226 multiple myeloma cells were treated with STS, followed
found that STS inhibited pIRE1a kinase enzymatic activity by tunicamycin, to measure XBP 1 splicing. STS treat-
(IC50 5 3 nM), but had no effect on pIRE1a RNase activity in ment alone was sufficient to induce XBP 1 splicing simi-
vitro (Fig. 4A). STS exhibited a similar binding affinity lar to levels achieved with tunicamycin (Fig. 4C). Despite
toward phosphorylated IRE1a (IC50 5 30 nM) and dephos- inducing XBP 1 splicing, STS did not increase IRE1a
phorylated IRE1a (IC50 5 50 nM) in a competitive binding autophosphorylation. Instead, STS inhibited tunicamycin-
assay (data not shown). Interestingly, incubation of STS with induced autophosphorylation (Fig. 4D). STS treatment may
dephosphorylated (inactive) IRE1a was capable of activat- also inhibit IRE1a autophosphorylation below basal levels,
ing IRE1a RNase activity (Fig. 4B). To test the effects of but it was difficult to measure this convincingly by Western
STS on IRE1a RNase activity in a cellular context, RPMI blot analysis. These observations, both biochemical and in

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Concha et al.
Fig. 3. Inhibition of IRE1a kinase and RNase activity in cells treated with GSK2850163. (A) Multiple myeloma cell lines were left untreated or treated
with tunicamycin for 1 hour. GSK2850163 (GSK163) was then added for 3 hours at the indicated doses. Total RNA was harvested, and RT-PCR was
performed using human-specific XBP 1 primers flanking the splice site that distinguish between unspliced (XBP 1u) and spliced (XBP 1s) XBP 1 mRNA.
(B) RPMI 8226 cells were left untreated or treated with tunicamycin and/or GSK2850163 as described in (A). Changes in IRE1a phosphorylation were
detected by Western blot using a phosphospecific antibody raised against Ser 724 in the kinase activation loop. (C) XBP 1 transcriptional activity is
reduced in cells treated with GSK2850163. PANC-1 cells were transfected with a 5X UPRE luciferase reporter and pRL-SV40 Renilla constructs. Forty-
eight hours later, cells were left untreated or treated with tunicamycin for 1 hour, followed by GSK2850163 treatment of 16 hours. UPRE luciferase
expression was measured and normalized to Renilla expression levels.
cells, suggest that the effects of STS on IRE1a phosphor- Taylor, 2010). Attempts to crystallize dephosphorylated IRE1a
ylation and XBP 1 splicing could be due to direct bind- with STS were not successful.
ing of STS to IRE1a and not just due to general stress
that could occur due to the broad-spectrum nature of the Protein Dynamics. To test the pIRE1a dimer interactions
compound. observed in the structures, we determined the hydrogen-
Comparison of pIRE1a Dimer Interactions and
To understand the structural basis of the activation of the deuterium exchange of pIRE1a in the presence or absence of
RNase activity of IRE1a by staurosporine, we determined the either GSK2850163 or STS in solution (Supplemental Fig. 5).
crystal structure of the human pIRE1a-STS complex (PDB ID Changes in the labeling pattern can be related to changes in
4YZC; Table 1). The pIRE1a-STS complex forms a back-to- the protein structure or dynamics resulting from a ligand-
back dimer, with one STS molecule bound per protomer (Fig. binding event (Englander et al., 2003; Percy et al., 2012). By
4E; Supplemental Fig. 4A). STS binds in the ATP-binding site mapping the observed labeling onto the two cocrystal struc-
of the kinase domain and interacts with the hinge residues tures, we observed three areas where most of the structural
Glu643, Cys645, and His692 (Supplemental Fig. 4B). The changes occurred. The first region is in the vicinity of the
activation loop containing the phosphorylated Ser residues ligand-binding site (Supplemental Fig. 5A). Increased solvent
(pSer724, pSer726, and pSer729) that was previously disor- exposure of the activation loop and disruption of the conserved
dered in both the pIRE1a-GSK2850163 and pIRE1a-ADP salt bridge Arg627-Asp620 by GSK2850163 are both consis-
complexes is well defined in the pIRE1a-STS complex (Sup- tent with the displacement of the activation loop to the DFG-
plemental Fig. 4C). pSer724 interacts with Asn750 via two H out conformation and the changes in the aC helix to the
bonds, pSer726 is involved in an H-bond interaction with inactive conformation (Supplemental Fig. 5B). Second, in the
Arg722 (3.1 Å) and Arg728 (3.3 Å), and pS729 is interacting presence of STS, the residues at the kinase-RNase intra-
through an H bond with Lys716 (2.7 Å) and Arg687 (2.5 Å). A molecular domain interface show increased solvent exposure
network of H bonds extends from the serines in the activation (Supplemental Fig. 5C). The loosening of the intramolecular
loop to the aC helix and into the active site DFG motif. Binding interactions is consistent with the reorientation of the pIRE1a
of STS to pIRE1a locks the kinase domain in a conformation, dimer that leads to the dimerization of the RNase domains.
in which the 1) activation loop is in the DFG (711–713)-in Simultaneously, the decrease in overall labeling of the RNase
conformation; 2) the DFG-aspartate interacts with His689 domains occurs in concert with the conditions under which
in the HRD motif in the catalytic loop; 3) the conserved Leu616 RNase domains dimerize (Fig. 5, A and D). Third, in the
in the aC helix interacts with the DFG phenylalanine in the presence of bound STS, there is an increase in solvent
regulatory spine of the kinase; and 4) Arg687 of the HRD motif exposure of the residues 900–916 (Supplemental Fig. 5D).
interacts with pSer729 (Supplemental Fig. 4C). These are the These residues form a helix-loop element that interact directly
signatures of a kinase in the active conformation (Kornev and with the RNA substrate and include the catalytically essential

Structure of Human Phosphorylated (Active) IRE1a Dimer
1019
Fig. 4. Structure and activity of the human pIRE1a-STS complex. (A) STS inhibits pIRE1a kinase activity, but not RNase activity. (B) Titration curve
showing that the nuclease activity of dephosphorylated IRE1a [IRE1a (-P)] is stimulated by STS in the RNase assay. (C) STS induces XBP 1 splicing in
RPMI 8826 cells. Cells were treated with STS for 30 minutes, followed by tunicamycin (tuni) (5 mg/ml) treatment in half of the cells for 1 hour. Total RNA
was harvested, and RT-PCR was performed using human-specific XBP 1 primers flanking the splice site that distinguish between unspliced (XBP 1u) and
spliced (XBP 1s) XBP 1 mRNA. (D) STS inhibits tunicamycin-induced IRE1a phosphorylation. RPMI 8226 cells were treated as described in (C). Changes
in IRE1a phosphorylation were detected by Western blot using a phosphospecific antibody raised against Ser 724 in the kinase activation loop. (E)
pIRE1a-STS dimer (PDB ID 4YZC) in the back-to-back configuration, with STS bound in the ATP-binding site.
His910 (Dong et al., 2001; Korennykh et al., 2009). This average of 4.4 6 1.0 Å (range of 3.03–6.40 Å on superposition of
apparent increased dynamic state of the helix-loop catalytic the aC helix carbon atoms) as compared with the position
element may be related to the catalytic readiness favored by observed in the STS complex (Fig. 5B). While the centers of
STS but opposed by GSK2850163.
mass of the kinase N domains (21.5–21.9 Å) and C domains
Comparison of pIRE1a Cocrystal Structures. A com- (43.1–43.6 Å) remain constant, a nearly 20° rotation of the
parison between the pIRE1a-GSK2850163 and pIRE1a-STS domains relative to one another is observed in the two
complexes shows that both structures are back-to-back dimers structures. A differential gap of nearly 4 Å between the RNase
(Fig. 5A). Binding of GSK2850163 causes the kinase aC helix domains in the structures of pIRE1a-GSK2850163 (29.7 Å)
(residues 610–619) to move to the inactive conformation by an and pIRE1a-STS (26.1 Å) is reflected in a different set of

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Concha et al.
Fig. 5. Conformational changes to pIRE1a upon ligand binding. (A) Increased interactions along the kinase dimer interface and decreased surface area
contact of the RNase domains in pIRE1a-GSK2850163 (left panel). pIRE1a2STS structure, where the interactions are tighter between the RNase
domains (right panel). A cartoon model depicts a rocking motion between inactive (left) and active (right) pIRE1a dimers. (B) Residues 610–619 that make
up the aC helix move 4.4 6 1.0 Å between pIRE1a-STS (green) and pIRE1a-GSK2850163 (salmon). The structures of the kinase domains were
superimposed using Ca atoms of the residues forming the hinge region. (C) Bottom-up view of the pIRE1a-GSK2850163 RNase domains. There is one pair
of residues interacting across the RNase dimer interface, which covers a contact area of 269 Å2. Glu913 on chain A (green) and Arg905 on chain B (cyan)
form a salt bridge, but the equivalent interaction (i.e., Glu913 of chain B and Arg905 of chain A) is not possible because the side chain of Glu913 in chain B
is disordered. (D) Bottom-up view of the pIRE1a-STS RNase domains. There are two sets of interdigitating H-bond interactions. The first set involves
His909, Asp847, and Arg905 (3.2–3.5 Å), and the second is between Arg955, Glu836, and Asp927 (3.1–3.5 Å). (E) Calculated buried surface area of contact
for the two pIRE1a dimers and individual domains.
interactions across the RNase dimer interfaces. Hence, the
In the pIRE1a-STS complex, the RNase domains form a
RNase dimer interface in the pIRE1a-GSK2850163 complex is dimer, with two sets of interdigitating H-bond interactions
reduced by 58% (269 Å2) compared with the pIRE1a-STS among His909, Asp847, and Arg905 (3.2–3.5 Å) and among
complex (642 Å2) (Fig. 5E). In pIRE1a-GSK2850163, the RNase Arg955, Glu836, and Asp927 (3.1–3.5 Å), which allow the key
domains are noticeably separated, such that the network of nuclease catalytic residues Tyr892, Arg905, Asn906, and
interactions across the dimer is no longer possible. The interac- His910 to be poised for action (Fig. 5D). Asp847, His909, and
tions between the protomers of the pIRE1a-GSK2850163 dimer Arg905 are highly conserved residues across multiple species
occur mostly between the kinase domains (1734 Å2), with less (Dong et al., 2001). These interactions across the RNase dimer
contact occurring between the RNase domains (269 Å2). This interface are also analogous to those observed previously
separation of RNase domains is consistent with the biochemical for the activation of yeast Ire1 RNase activity by quercetin
and cellular data, showing that GSK2850163 is a potent (3LJ0; Wiseman et al., 2010). In the structure of pIRE1a-
inhibitor of pIRE1a RNase activity.
GSK2850163, the RNase domains are separated; thus, the

Structure of Human Phosphorylated (Active) IRE1a Dimer
1021
network of H bonds between the RNase domains observed in pyrazin-1-yl]naphthalen-1-yl)-3-[3-(trifluoromethyl)phenyl]urea
the active pIRE1a-STS dimer does not exist (Fig. 5C). Hence, (KIRA6) and several close analogs inhibit IRE1a kinase and
the data from the cocrystal structures presented here suggest RNase activities and possibly prevent dimer association (Wang
that the formation of interdigitating H bonds and the stabi- et al., 2012; Ghosh et al., 2014). While cocrystal structures for
lization of the RNase dimer are critical for the active RNase KIRA6 are not available, a close analog was recently cocrystal-
conformation and its enzymatic activity.
lized with the c-Src kinase domain, demonstrating a shift in the
aC helix out to the inactive conformation (PDB ID 3QLF). Thus,
it is possible that KIRA6 invokes a similar conformational
change when bound to IRE1a. Similarly, another inhibitor
Discussion
Recent work has implicated the UPR in a number of recently described by Amgen (Thousand Oaks, CA) potently
diseases, most notably in cancer, where UPR activation has inhibited IRE1a kinase and RNase activity and was cocrystal-
been shown to function as a survival mechanism in cancer, lized with a dephosphorylated IREa monomer (Harrington
promoting tumor growth, regulating angiogenesis, and facil- et al., 2014). The lack of IRE1a dimer structure here could be
itating adaptation to hypoxia (Ma and Hendershot, 2004; due to the compound either preventing dimerization or stabiliz-
Feldman et al., 2005; Chen et al., 2014). Specifically, the ing a monomeric form of IRE1a. While it was previously
IRE1a/XBP 1 pathway has shown to be overexpressed in a suggested that dimerization/oligomerization occurs after auto-
variety of human cancers, and activation of the pathway has phosphorylation, recent crystal structures of dephosphorylated
been shown to be essential for the survival of highly secretory human IRE1a dimers demonstrate that dimerization can pre-
multiple myeloma cells, where the protein load is high (Feldman cede phosphorylation in both face-to-face and back-to-back
et al., 2005; Koong et al., 2006; Carrasco et al., 2007). These configurations (Ali et al., 2011; Joshi et al., 2015). It should also
key findings, coupled with the possibility to modulate IRE1a be noted that this molecule shifted the aC-helix out. Hence, one
activity via targeting two potentially druggable domains, has commonality between this molecule, GSK2850163, and the
made IRE1a a very attractive target in drug discovery (Hetz KIRA6 analogs is that all these inhibitors shift the aC helix to
et al., 2013; Harrington et al., 2014; Maly and Papa, 2014; an inactive conformation, which may likely be a requirement for
Sanches et al., 2014; Joshi et al., 2015).
IRE1a RNase inactivation.
An increasing body of evidence indicates that IRE1a
Aside from kinase domain inhibitors, two other classes of
inhibitors bound to the kinase domain invariably inhibit its IRE1a inhibitors have been characterized that target other
ATPase activity, yet some kinase inhibitors inhibit the RNase potential drug pockets in the IRE1a protein: quercetin and
activity, while others activate its RNase activity. GSK2850163 salicylaldehyde-based inhibitors. Quercetin binds uniquely to
was discovered in an attempt to identify IRE1a-selective yeast Ire1 in a pocket at the enzyme-dimer interface termed
inhibitors of XBP 1 splicing that could regulate multiple the Q site, thereby stabilizing Ire1 in an active conformation
myeloma cancer cells. It is a highly selective kinase inhibitor; that augments RNase activity (Wiseman et al., 2010). While
a panel of 284 kinases was assayed to determine the specificity quercetin has been shown to be a broad spectrum kinase
of GSK2850163, and only two additional kinases were weakly inhibitor and may potentially target the nucleotide binding
inhibited by GSK2850163: Ron (IC50 5 4.4 mM) and FGFR1 site of Ire1, it did not inhibit Ire1 autophosphorylation. In-
V561M (IC50 5 17 mM) (Supplemental Table 1). GSK2850163 dependently, a considerable amount of work has been con-
inhibits IRE1a RNase activity due to the unique way the ducted to target IRE1a RNase activity directly (Maly and Papa,
molecule binds in the kinase domain active site. GSK2850163 2014; Sanches et al., 2014). These salicylaldehyde-based inhib-
binds deep in a pocket next to the kinase aC helix, approxi- itors contain a reactive electrophile that most likely covalently
mately 12 Å from the hinge region, which is clearly distinct modifies Lys907 at the IRE1a RNase active site. It is believed
from the ADP binding site. The molecule adopts a U-shape that these inhibitors should not affect IRE1a autophosphor-
conformation when bound to pIRE1a that could only be ylation or dimerization. GSK2850163 is the first molecule that
achieved with the R-stereoisomer (Supplemental Fig. 2A). inhibits both kinase and RNase activity by binding to phos-
Modeling of the S-stereoisomer places the difluorobenzene phorylated IRE1a and inducing a unique long-range conforma-
outside the electron density and clashes with the protein. tion change that alters the preferred enzyme structure for RNA
Similarly, flipping the U-shaped molecule to the reverse substrate binding.
orientation places the spirodecane within the electron density,
The wealth of data on the dimeric/oligomeric state of IRE1
but the four-bond length between the spirodecane and the as a function of phosphorylation and the effects of various
tolyl group leads to a clash with Leu616, and the two-bond classes of kinase inhibitors do not fully explain the details of
length is too short and causes a clash between the difluor- the conformational changes required to cause modulation of
obenzene and Lys599 instead of the H bond when in the IRE1a RNase activity. The hydrogen-deuterium exchange
modeled orientation. Based on this and the SAR studies rate, which is dependent on both structural and conforma-
performed, it is clear that only the R-stereoisomer in the tional changes, is very well suited to reveal these details
modeled conformation and orientation is capable of fitting the and has the resolution necessary to pinpoint the required
electron density, avoiding clashes, and engaging with Glu612 conformational changes in the solution state. Our hydrogen-
and Lys599.
deuterium exchange data are consistent with the model of
The GSK2850163 mode of binding differs from classic RNase inhibition suggested by the pIRE1a-GSK2850163
ATP-competitive inhibitors [reviewed by Dar and Shokat cocrystal structure. The differences observed in labeling over-
(2011)]. Inhibitors exemplified by APY29 (DFG-in and the lap with the changes observed in the crystal state. The critical
aC helix in the active conformation) activate IRE1a RNase observation of the conserved salt bridge between Arg627 and
activity (Korennykh et al., 2009; Wang et al., 2012). In con- Asp620 in the kinase domain being disrupted by GSK2850163
trast, inhibitors like 1-(4-[8-amino-3-tert-butylimidazo(1,5-a) is highly consistent with the changes in the aC helix to the out

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Concha et al.
(inactive) conformation and the reorientation of the pIRE1a the inhibition of pIRE1a kinase and RNase activity. The
dimer to the inactive form. By the same analysis, the reverse is comparisons made between known structures across various
true in the activation of the RNase.
species and with inhibitors that have differential effects on
A comparison of the yeast Ire1 back-to-back dimers with the IRE1a RNase activity should provide insights into the molecu-
human pIRE1a-STS dimer demonstrates that the kinase lar mechanisms responsible for activation and inhibition of
domain DFG-loop is in the in conformation, the aC helix is IRE1a RNase activity. These new data should also provide a
in the active conformation, and the RNase domains are feasible path to enable the design and use of pharmacological
engaged through a network of H-bond interactions in these agents that differentially affect IRE1a/XBP 1 signaling.
structures, which coincide with conditions of RNase activa-
Coordinates for the pIRE1a-GSK2850163, pIRE1aADP-
tion. Minor differences are observed between the pIRE1a-STS MG2+, and pIRE1a-STS cocrystal structures have been de-
dimer and the three yeast Ire1 structures (PDB ID 2RIO, posited to the Protein Data Bank with the respective accession
3FBV, and 3LJ0) (r.m.s. on Ca 5 1.9–2.1 Å); however, the codes: PDB ID 4YZ9, 4YZD, and 4YZC.
differences are even greater when comparisons are made with
the pIRE1a-GSK2850163 dimer (r.m.s. on Ca 5 3.3–3.4 Å).
GSK2850163 binding results in shifting the structure of the
kinase domain to the DFG-out, aC helix inactive conforma-
tion, and the RNase domains of the dimer are rotated away
from each other. This indicates that the RNase active forms of
yeast and human IRE1 are markedly different from the RNase
inactive pIRE1a-GSK2850163 dimer. While the key interac-
tions in the RNase domains are not mediated by identical
residues across species, they are essential. Mutagenesis stud-
ies have shown that these interfacial residues are critical for
maintaining RNase activity (Lee et al., 2008).
Structural similarities are also observed between the human
phosphorylated (4YZD) and dephosphorylated (3P23) IRE1a-
ADP-Mg21 structures (r.m.s. on Ca 5 0.5 Å). In both cases, the
dimers are face to face with the RNase domains separated in an
inactive conformation. Likewise, pIRE1a-ADP-Mg21 and mouse
IRE1a complexed with the RNase domain inhibitor MKC9989
(4PL3) are similar in dimeric structure (r.m.s on Ca 5 0.8 Å).
The largest differences are located in a loop formed by residues
654–659 in the kinase domain. This stretch of amino acids is
engaged in intermolecular contacts within the neighboring
molecule of the asymmetric unit in the ADP complex. It is worth
noting that previous in vitro studies have shown that ADP
bound to IRE1a can affect dimerization and/or reduce RNase
activity of both mouse and human IRE1a (Prischi et al., 2014;
Sanches et al., 2014).
The pIRE1a-GSK2850163 and pIRE1a-STS cocrystal struc-
tures were also compared with two recently reported human
back-to-back dimers of IRE1a: one in the apo form (4Z7G) and
one in the inhibitor-bound form (4Z7H). In these two struc-
tures, the RNase domains are not engaging with each other
across the dimer interface and more closely resemble the
pIRE1a-GSK2850163 dimer (overall dimer r.m.s. on Ca 5 1.6 Å)
than the pIRE1a-STS dimer (overall dimer r.m.s. on Ca 5 2.7 Å).
Despite this, the imidazopyridine molecule, compound 3, inter-
acts with the hinge and the activation loop (DFG-in) in a similar
fashion as staurosporine (r.m.s. on Ca 5 0.9 Å) and behaves as a
type I kinase inhibitor (Joshi et al., 2015). GSK2850163 and
compound 3 do not occupy overlapping pockets. This structural
observation is at odds with the other type I inhibitor–bound IRE1
structures. A comparison of the IRE1a apo and IRE1a-imidazo-
pyridine complex indicates that they are nearly identical (r.m.s.
on Ca 5 0.6 A). This discrepancy may be due to the way the
experiments were performed, namely, that the inhibitor-bound
form was resolved by soaking into the preformed apo crystals as
opposed to performing cocrystallization.
Acknowledgments
We would like to thank the members of the IRE1a drug discov-
ery program and the Computational and Structural Chemistry De-
partment for fruitful discussions and insightful comments on the
manuscript.
Authorship Contributions
Participated in research design: Evans, Heerding, Buser, Su,
DeYoung.
Conducted experiments: Concha, Smallwood, Bonnette, Totoritis,
Federowicz, Campobasso, Choudhry, Shuster, Evans, Su, DeYoung.
Contributed new reagents or analytic tools: Qi, Chen, Sweitzer,
Shuster, Evans, Ralph.
Performed data analysis: Concha, Smallwood, Totoritis, Zhang,
Yang, Choudhry, Heerding, Su, DeYoung.
Wrote or contributed to the writing of the manuscript: Concha,
Buser, Su, DeYoung.
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Address correspondence to: M. Phillip DeYoung, GlaxoSmithKline,
Oncology R&D, 1250 S. Collegeville Road, UP1450, Collegeville, PA 19426.
E-mail: maurice.p.deyoung@gsk.com or Nestor O. Concha, GlaxoSmithKline,
Oncology R&D, 1250 S. Collegeville Road, UP12-205, Collegeville, PA 19426.
E-mail: Nestor.O.Concha@gsk.com