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Research ArticleArticle

Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1α Endoribonuclease Activity

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
Molecular Pharmacology December 2015, 88 (6) 1011-1023; DOI: https://doi.org/10.1124/mol.115.100917
Nestor O. Concha
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.)
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Angela Smallwood
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.)
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William Bonnette
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.)
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Rachel Totoritis
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.)
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Guofeng Zhang
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.)
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Kelly Federowicz
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.)
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Jingsong Yang
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.)
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Hongwei Qi
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.)
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Stephanie Chen
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.)
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Nino Campobasso
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.)
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Anthony E. Choudhry
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.)
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Leanna E. Shuster
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.)
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Karen A. Evans
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.)
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Jeff Ralph
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.)
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Sharon Sweitzer
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.)
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Dirk A. Heerding
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Carolyn A. Buser
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.)
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Dai-Shi Su
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.)
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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.)
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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.

Footnotes

    • Received July 20, 2015.
    • Accepted September 25, 2015.
  • 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.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

  • Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 88 (6)
Molecular Pharmacology
Vol. 88, Issue 6
1 Dec 2015
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Research ArticleArticle

Structure of Human Phosphorylated (Active) IRE1α Dimer

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
Molecular Pharmacology December 1, 2015, 88 (6) 1011-1023; DOI: https://doi.org/10.1124/mol.115.100917

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Research ArticleArticle

Structure of Human Phosphorylated (Active) IRE1α Dimer

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
Molecular Pharmacology December 1, 2015, 88 (6) 1011-1023; DOI: https://doi.org/10.1124/mol.115.100917
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