title: Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity.
abstract: 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 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 IRE1α dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1α-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 kinase and RNase activities of IRE1α. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1α RNase activity appears to be caused by a conformational change, whereby the αC 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 IRE1α, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1α 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.
authors.affiliation: 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.) maurice.p.deyoung@gsk.com Nestor.O.Concha@gsk.com.
authors.forename: Nestor O
authors.initials: NO
authors.lastname: Concha
authors.affiliation: 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.).
authors.forename: Angela
authors.initials: A
authors.lastname: Smallwood
authors.affiliation: 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.).
authors.forename: William
authors.initials: W
authors.lastname: Bonnette
authors.affiliation: 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.).
authors.forename: Rachel
authors.initials: R
authors.lastname: Totoritis
authors.affiliation: 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.).
authors.forename: Guofeng
authors.initials: G
authors.lastname: Zhang
authors.affiliation: 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.).
authors.forename: Kelly
authors.initials: K
authors.lastname: Federowicz
authors.affiliation: 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.).
authors.forename: Jingsong
authors.initials: J
authors.lastname: Yang
authors.affiliation: 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.).
authors.forename: Hongwei
authors.initials: H
authors.lastname: Qi
authors.affiliation: 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.).
authors.forename: Stephanie
authors.initials: S
authors.lastname: Chen
authors.affiliation: 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.).
authors.forename: Nino
authors.initials: N
authors.lastname: Campobasso
authors.affiliation: 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.).
authors.forename: Anthony E
authors.initials: AE
authors.lastname: Choudhry
authors.affiliation: 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.).
authors.forename: Leanna E
authors.initials: LE
authors.lastname: Shuster
authors.affiliation: 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.).
authors.forename: Karen A
authors.initials: KA
authors.lastname: Evans
authors.affiliation: 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.).
authors.forename: Jeff
authors.initials: J
authors.lastname: Ralph
authors.affiliation: 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.).
authors.forename: Sharon
authors.initials: S
authors.lastname: Sweitzer
authors.affiliation: 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.).
authors.forename: Dirk A
authors.initials: DA
authors.lastname: Heerding
authors.affiliation: 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.).
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Buser
authors.affiliation: 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.).
authors.forename: Dai-Shi
authors.initials: DS
authors.lastname: Su
authors.affiliation: 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.) maurice.p.deyoung@gsk.com Nestor.O.Concha@gsk.com.
authors.forename: M Phillip
authors.initials: MP
authors.lastname: DeYoung
mesh_terms: D045744:Cell Line, Tumor; D003460:Crystallization; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D011487:Protein Conformation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D047428:Protein Kinase Inhibitors; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases