1521-0111/88/6/1011–1023$25.00 http://dx.doi.org/10.1124/mol.115.100917 MOLECULAR PHARMACOLOGY 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- tein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic ki- nase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them func- tional 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, IRE1a- selective 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 ki- nase 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. Inactiva- tion 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 be- tween 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 pro- teins, 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 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 (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 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 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. 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-(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. 1011 http://molpharm.aspetjournals.org/content/suppl/2015/10/05/mol.115.100917.DC1 Supplemental material to this article can be found at: at ASPET Journals on January 13, 2018 molpharm.aspetjournals.org Downloaded from