Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1a Endoribonuclease Activity s

Activation of the inositol-requiring enzyme-1 alpha (IRE1a) 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 conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1a dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1aselective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1a. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1a RNase activity appears to be caused by a conformational change, whereby the aC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1a, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1a RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors. Introduction Cellular stresses, such as accumulation of unfolded proteins, hypoxia, glucose deprivation, depletion of endoplasmic reticulum (ER) calcium levels, and changes in ER redox status activate the unfolded protein response (UPR), an intracellular signal transduction network involved in restoring protein homeostasis [reviewed by Walter and Ron (2011)]. To alleviate these types of stress responses, the UPR responds by halting protein translation, activating transcription of UPRassociated target genes, and degrading misfolded proteins (Harding, et al., 2002; Ron, 2002; Feldman et al., 2005). UPR signaling also regulates cell survival bymodulating apoptosis and autophagy and can induce cell death under prolongedER stress if the misfolded protein burden is too high (Ma and Hendershot, 2004; Rouschop et al., 2010; Woehlbier and Hetz, 2011). 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 andKaufman, 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 transautophosphorylation, 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. s This article has supplemental material available at molpharm. aspetjournals.org. 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-(4methylbenzyl)-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. 1011 http://molpharm.aspetjournals.org/content/suppl/2015/10/05/mol.115.100917.DC1 Supplemental material to this article can be found at: at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org 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m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D 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Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from in activation of the RNase domains (Ali et al., 2011). Phosphorylation within the kinase activation loop is an essential step for RNase activation (Prischi et al., 2014). Mammalian IRE1a excises a 26–base pair intron from the mRNA of X-box binding protein 1 (XBP 1), which causes a translational frame shift downstream of the splice site to produce XBP 1s, the active form of the transcription factor (Yoshida et al., 2001; Calfon et al., 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 machinery (Lee at al., 2003). Activation of IRE1a is also reported to result in the induction of regulated IRE1a-dependent degradation of a subset of mRNAs encoding secretory proteins or the induction of apoptosis via IRE1a signaling through its kinase domain anddownstreameffectorsASK1, JNK1, andCaspase-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 diseases, 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 end, an increasing body of work has been performed to identify potent and selective molecules of IRE1a and better understand how these molecules bind and affect IRE1a activation mechanisms. Crystal structures of the C-terminal region of phosphorylated (active) yeast Ire1 were the first to be characterized 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 formed the basis of a rod-shaped helical structure, representing a high-order oligomeric Ire1 structure in complex with the kinase inhibitor 2-N-(3H-benzimidazol-5-yl)-4-N-(5cyclopropyl-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 IRE1aMg-ADP complex, possibly representing an early state prior to phosphoryl transfer (PDB ID 3P23; Ali et al., 2011). In this structure, the kinase active sites are facing each other and are in a suitable orientation and proximity for transautophosphorylation, 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 phosphorylated (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 IRE1amonomer (PDB ID 4U6R; Harrington et al., 2014). Here, no dimers were found with the inhibitor-bound structure, suggesting that the compound may 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 that this process is conserved evolutionarily. Here, we present a proposed structure of the final state of a human phosphorylated (active) IRE1a dimer that is cocrystallized with two kinase inhibitors that have opposing effects to the RNase activity of the protein. 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 (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 baculovirusinfected 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 10 baculovirusinfected insect cells/10 l culture, and incubated at 27°C. Cell paste was harvested at 66–72 hours postinfection. A human phosphorylated or 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 30minutes at 4°Cwas first captured in 20ml 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 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 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. Phosphorylated IRE1a RNase Activity Assay. The nuclease enzymatic activity of phosphorylated IRE1a (pIRE1a) was measured 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 changewas followed using an Envision plate reader (PerkinElmer, Waltham, MA). 1012 Concha et al. at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from To identify inhibitors of IRE1a nuclease activity, pIRE1a was screened against the GlaxoSmithKline compound collection. The RNA oligomer substrate was added to the assay plates containing 10 mM of compound. The reaction was initiated immediately by the addition of the enzyme, and the plates were centrifuged for 1 minute at 500 rpm. The final reaction mixture contained 10 nM pIRE1a, 25 nM RNA 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 terminatedwith 0.015%SDS in 20mMHepes, pH 7.5. The plateswere centrifuged for 3 minutes at 1000 rpm prior to measuring product formation using the Viewlux imager (PerkinElmer). pIRE1a Kinase Assays. In the ADP-Glo assay, a compound’s potency toward pIRE1a kinase activity wasmeasured as its inhibition of an intrinsic, slow ATP hydrolysis activity. One hundred nanoliters of dimethylsulfoxide solution of (R)-2-(3,4-dichlorobenzyl)-N-(4methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide (GSK2850163) at various concentrations was added into a white Greiner low volume 384-well plate. The reaction was carried out with 5 nM pIRE1a and 60 mM ATP in 10 ml of 50 mM Hepes buffer, pH 7.5, containing 30 mM NaCl, 10 mMMgCl2, 1 mM DTT, 0.02% Chaps, and 0.01 mg/ml bovine serum albumin. The reaction was stopped after 2 hours by adding 5 ml of ADP-Glo reagent I, which also depletes the remaining ATP. Following a 1-hour incubation, 5 ml of ADP-Glo reagent II was added into the reaction, which converts the ADP product into ATP to serve as the substrate for the coupled luciferin/luciferase reaction. After 30 minutes, the plate was read on a Wallac ViewLux microplate imager (PerkinElmer). 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 (GSK369716A), and GSK2851063 at various concentrations. After the addition of reagents, the plate was incubated at room temperature for 15 minutes and then read on an Analyst GT multimode reader (Molecular Devices, Sunnyvale, CA) at an excitation/emission of 485/ 530 nm, with a dichroic of 505 nm. The IC50 values were calculated using the data fitting software GraFit (Erithacus Software, Horley, UK). PERK Kinase Assay. Recombinant PERK protein (Life Technologies) was purchased for use in a selectivity assay to test the pIRE1a inhibitors. A solution of PERK (0.40 nM) in assay buffer containing 10mMHEPES, pH7.5, 2mMCHAPS, 5mMMgCl2, and1mMDTTwas 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 mMbiotinylated 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 detection/quench mixture. The detection/quench mixture contained 15 mM EDTA, 4 nM eukaryotic translation initiation factor 2a phosphoantibody (Millipore, Billerica, MA), 4 nM europium-labeled anti-rabbit antibody (PerkinElmer), and 40 nM streptavidin APC (PerkinElmer). The plateswere incubated for 10minutes at room temperature prior to measuring the homogeneous time resolved fluorescence signal (excitation at 610–640 nm and emission at 660 nm) using the Viewlux imager (PerkinElmer). RNase L Activity Assay. A solution of 0.1 nM RNase L, 1 mM ATP, and 8 nM2-5A [29,59-linked oligoadenylate, p1–3(A29p59A)n$ 2] in assay buffer containing 20 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM TCEP, 0.5 mM CHAPS, 100 mM NaCl, and 0.02% casein was incubated at room temperature for 30 minutes. A 0.2 mM solution of a 36-mer RNA substrate (59/6-FAM rUrUrA rUrCrA rArArU rUrCrU rUrArU rUrUrG rCrCrC rCrArU rUrUrU rUrUrU rGrGrU rUrUrA BHQ-1/39; Integrated DNA Technologies) was prepared in assay buffer and added to reaction plates containing compounds. The reaction was initiated with the addition of the RNase L-ATP-2-5A mixture, and the plates were incubated at room temperature for 90 minutes. Following the incubation, the reaction was terminated with 0.02% SDS solution in nuclease-free water. The plates were centrifuged for 3 minutes at 1000 rpm prior to measuring product formation using the Viewlux imager (PerkinElmer). Competitive Inhibition Studies. In single compound inhibition studies, the concentrations of the substrate or noncleavable substrate (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 velocities to different models (competitive, uncompetitive, and noncompetitive) using Grafit software (Erithacus Software). In a double inhibition experiment, the concentration of the first inhibitor was varied at several different concentrations of the second inhibitor, and the concentrations of the enzyme and substrate were kept constant at 10 and 100 nM, respectively. The reactions were monitored kinetically, and the initial reaction velocities were analyzed using the Yonetani-Theorell equation. Protein Crystallization and Structure Determination. Crystals of pIRE1a (547–977) with GSK2850163 (PDB ID 4YZ9) were prepared bymixing pIRE1awith 0.5mMGSK2850163 and incubating overnight on ice. The crystals were grown at 20°C by vapor diffusion in sitting drops containing 2 ml of protein (13 mg/ml in 50 mMHepes, pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.5 mM GSK2850163, and 0.25% dimethylsulfoxide) and 2 ml of reservoir solution containing polyethylene glycol (PEG) 3350 (16–22%), 100 mMHepes, pH 7.0, and 200mMCa acetate. The crystals were thick rods that appeared over 2–5 days and reached full size (0.05 0.025 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. The crystals of pIRE1awithMg-ADP (PDB ID 4YZD)were grown by mixing 2 ml of protein solution [10 mg/ml pIRE1a (547–977) in 50 mMHepes, pH 7.5, 200 mMNaCl, 5 mM DTT, 1 mM EDTA, 1 mM ADP (100 mM ADP stock was pH ∼7.0), and 1 mMMgCl2] with 2 ml of reservoir solution (16% PEG 3350 and 200mMNamalonate, pH 6.0) in sitting drops at room temperature. Seeding was used to initiate crystal growth. Crystals appeared the next day and grew to full size in 3 weeks. For data collection, the crystals were frozen in a solution of 20% ethylene glycol, 22% PEG 3350, and 200 mM Na malonate, pH 6.0, and added to the protein drop before mounting the crystals on the loop. The complex of pIRE1a (547–977) with staurosporine (STS) (PDB ID 4YZC) was prepared by mixing the protein with 0.5 mM staurosporine 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– 40%), 100 mM Hepes, pH 7.5, and 200 mM KCl. Small hexagonal plates appeared over 5–10 days and reached full size (0.05 0.75 0.15 mm) in ∼20 days. The crystals were flash frozen in liquid N2 directly from the crystallization drop. All diffraction data were collected at the Advanced Photon Source, Argonne National Laboratories, Life Sciences Collaborative Access Team, Sector 21. All structures were determined by molecular replacement with Phaser (Afonine et al., 2010), as implemented in CCP4 (Winn et al., 2011), using the humandephosphorylated IRE1aMg-ADP complex (3P23; Ali et al., 2011) as a model. Refinement was performed by a combination of Refmac5 (Murshudov et al., 1997) and Phenix (Afonine et al., 2010), with manual adjustments to the model in COOT (Emsley et al., 2010). The quality of the model was monitored usingMolprobity (Chen et al., 2010). Figures were made with PYMOL (Schrödinger, LLC, New York, NY). Detection of XBP 1 Splicing by Real-Time Polymerase Chain Reaction. Multiple myeloma cancer cell lines were obtained fromATCC (Manassas, VA) or DSMZ (Braunschweig, Germany). Cells were cultured in the appropriate culture medium supplemented with Structure of Human Phosphorylated (Active) IRE1a Dimer 1013 at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO) at 37°C in humidified incubators under 5% CO2. Cells were seeded into sixwell plates at a density of 1.5 10 cells/well in the appropriate media containing 1% FBSmedia and were treated with 5 mg/ml tunicamycin (MPBiomedicals, 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 at280°C. To generate cDNA, theHigh-Capacity cDNA Reverse Transcription Kit (Life Technologies) was used. For polymerase 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-CCATGGGGAGATGTTCTGGG-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 sixwell plates at a density of 2.0 10 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 phosphatase 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 gels (Life Technologies), and protein was transferred onto 0.45 mM nitrocellulose membranes (Life Technologies) using the BioRad semidry 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 10 cells/well in RPMI 1640 media containing 10% FBS. Cells were cotransfected with a pGL3-5x unfolded protein response element (UPRE)–luciferase reporter 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 mixedwith 18ml of D2O at room temperature for 1minute, after which 20 ml of 4 M guanidinium chloride in 1 M glycine buffer, pH 2.5, and 120ml 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 30 mm column for digestion, then to a C18 column, and into an LTQ XL Orbitrap mass spectrometer (Thermo Scientific). The mass-to-charge values were calculated with XCalibur (Thermo Scientific) and compared with sequences in the MASCOT database. The analysis of the hydrogen-deuterium exchange was done with HDExaminer. Tryptic digestion of all the samples gave a sequence coverage of at least 98% of the amino acid sequence. Preparation and Characterization of Compounds 1–24. Details 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 exclusive 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 structure 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 structureguided optimization of GSK2850163, we determined the cocrystal structure of GSK2850163 with the C-terminal portion of pIRE1a (residues 547–977; PDB ID 4YZ9) (Fig. 1B; Supplemental Fig. 2; Table 1). The structure of pIRE1a-GSK2850163 is of a back-to-back dimer, with one inhibitor molecule bound to the kinase domain of each protomer in a pocket next to the kinase aChelix, approximately 12Å 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 pyrrolidine nitrogen of GSK2850163 forma 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-Mg (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 pIRE1aGSK2850163 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 ADPMg 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 Mg-ADP forms a “face-to-face” dimer across the symmetry planes with neighboring molecules (Supplemental Fig. 3). Consistent with previously published structures, the kinase active sites are facing eachother andare inanorientationandproximity favorable for transautophosphorylation [3P23 (Ali et al., 2011) and 4PL3 (Sanches et al., 2014)]. The present structure may possibly represent an early postphosphoryl-transfer dimer, 1014 Concha et al. at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from