Structure and mechanism of action of the hydroxy aryl aldehyde class of IRE 1 endoribonuclease inhibitors

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy aryl aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic To whom correspondence should be addressed: John Patterson, jpatterson@mankindcorp.com, 661-775-5317, Frank Sicheri, sicheri@lunenfeld.ca, 416-586-8471. *these authors contributed equally to this work Author contributions M.S. and M.T. performed the co-structure determinations. N.D., M.T., L.L., and M.C. performed in vitro enzymatic assays and direct inhibitor binding measurements. D.C., N.T., and K.L. performed biophysical analyses and/or generated reagents. I.K. collected synchrotron diffraction data. D.U., R.A., G.P., M.P. and B.W chemically synthesized OICR573 and OICR464. V. T., C.S., A.T., J. L., D. V. and Q. Z performed in cell analyses, enzyme assays and directed the synthesis of MKC9989, MKC3946, MKC3987 and MKC4027. D.D. supervised M.C. F.S and J.P. designed experiments and interpreted results. F.S and J.P wrote the manuscript with contributions from all other authors. Competing Financial Interests The authors declare no competing financial interests. V. T., C. S., A.T., J. L., D. V.,Q. Z and J.P. are past or present employees of MannKind Corp. and F.S. was a paid consultant of MannKind Corp. Accession codes. Final coordinates and structure factors for the IRE1α-MKC9989, IRE1α-OICR573, and IRE1α-OICR464 costructures have been deposited to the PDB with the respective accession codes: 4PL3, 4PL4, and 4PL5. PubMed Central CANADA Author Manuscript / Manuscrit d'auteur Nat Commun. Author manuscript; available in PMC 2015 July 01. Published in final edited form as: Nat Commun. ; 5: 4202. doi:10.1038/ncomms5202. P M C C aada A uhor M anscript P M C C aada A uhor M anscript P M C C aada A uhor M anscript development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a H-bond with Tyr892. Structure activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.

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